Physiological Genetics of the Mouse

Physiological Genetics of the Mouse SALOME GLUECKSOHN-WAELSCH Department of Zoology. Columbia Vniversity. New Pork. New Pork CONTENTS

. . .

.....................

I Introduction I1 Analysis of the Yellow Lethal. a Classic Case of a Lethal Mutation I11 Analysis of Gene-Determined Pigment Characters 1 Autonomy of Pigmentation a Mutant Spots b Transplantation 2 Histological Studies of Pigmentation in Different Gene Substitutions 3 Biochemical Studies of Pigmentation in Different Genotypes 4 Correlation of Histological and Biochemical Results 5 Tyrosinase System in the Determination of Pigment Pattern I V Analysis of Gene-Determined Abnormalities of the Blood 1 Siderocytic Anemia in Flexed 2 Macrocytic Anemia in Dominant Spotting V Analysis of Mutat.ions Affecting the Skin and Its Derivatives 1 Transplantation Studies in Waved-2 2 Transplantation Studies in Rhino 3 Rhino and Vitamin A 4 Developmental Studies of Crinkled V I Developmental Studies of Mutations Affecting the Urogenital Sy.stem 1 Kidney Development in Myelencephalic Blebs 2 Kidney Development in Danforth’s Short Tail 3 Inductive Relationship between Ureter and Metanephros 4 Urogenital Syndrome V I I Developmental Studies of Mutations Affecting the Central Nervous System and Sensory Organs 1 Hydrocephalus 2 Congenital Hydrocephalus 3 Pseudencephaly 4 X-Ray-Induced Translocations and Pseudencephaly 5 Shaker Short a Central Nervous System b Ear 6 Effect of Kreisler 011 the Central Nervous System and Ear 7 Eyelees 8 Microphthalmus VIII Analysis of Endocrine Disturbances-Pituitary Dwarfism I X Analyeis of Mutations Affecting the Skeleton 1 Skull a.Harelip 1

.

.

. . . . .

.

.

.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. .

.................. .. . ......... ....

.

. . . . . . . . . . . . . . . . . . .

.

Page 2 5 7

. . . .

. . . .

. . .. . .. . .

.

. . . .

.

. . .

. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .. .

.

.

. . . . ... .

....

..

............ . . . . . ... . . . . . . . ..... ................. . . . . . . . . . . . . . . . . ................... ............... ................... . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .... ................ . . . . . . . . . . . . . . . . . . .. . . . .. . . . ..... . . . . ................ . . . . . . . . . . . . . . . . . .. . . .

7 7

8 8 9 10 11 11 11 12 14 14 15 15 16 17 17 17 18 18 19 19 20 21 22 23 23 24 24 26

27 28 29

29 29

2

SALOME QLUEOKSOHN-WAELSCH

.

.

.

X

.

.

. .

. . .

. .

. .

. .

. . .

. . .

. . . . . .

XI. XI1 XI11

. . .

. .

.

. . .

.

Page 29 29 30 30 30 30 31 31 31 32 32 32 33 33 34 35 35 36 36 37 37

. . . . . . . .. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

b Tooth Abnormalities i Grey-Lethal ii Screw Tail 2 Extremities a L u x M o n g e n i t a l Absence of Tibia b Luxate c Polydactyly i Development ii Polydactyly and Embryonic Blebs iii Polydactyly and Central Nervous System iv Polydactyly and Maternal Age d Grey-Lethal and Failure of Secondary Bone Absorption 3.Sternum a Screw Tail b Short Ear 4 Spinal Column a.Flexed b Shaker Short c. Screw Tail d Undulated e Danforth's Short Tail The Developmental Effects of Mutations in Chromosome I X of the Mouse 1 Brachy6ry-Heterozygous Effect of T 2 Taillessness T/tn 3 Homozygous Effect of T 4 t-Type Mutations-Developmental Effects of t", t', t' 5 Kink-Homozygous Effect of Ei 6. F u s e d 7 Abnormalities of t ' / P Embryos 8 Miscellaneous Abnormalities in Individuals Carrying Mutations of Chromosome I X 9 The Role of Chromosome I X in Embryonic Growth and Differentiation Developmental Changes Induced by X-Rays Concluding Remarks References

. . . . . . . . . . . . . ....................... . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

......................

38 39 40 40 42 42 43 44 44 45 46 47 49

I. INTRODUCTION A review article on the physiological genetics of the mouse might at first sight appear to have chosen too limited a n object for discussion . But there are a number of reasons why just the house mouse (Mus musculus L.) lends itself well to a discussion of problems of physiological genetics. It is true that microorganims. for example. provide a material more accessible to the analysis of biochemical gene effects. It also is true that Drosophila has been analyzed so thoroughly genetically that it might offer better material for the study of the physiological mecha-

PHYSIOLOGICAL GENETICS OF THE MOUSE

3

nisms of gene effects. On the other hand, the problem of gene effect and gene action in a vertebrate organism stands alone in many respects, both from the theoretical and the practical point of view. The vertebrate organism differs from lower organisms in so many obvious ways that they need hardly be enumerated here. I n its structure, physiology, metabolism, endocrinology, etc., gene-controlled mechanisms and processes are operative which in many respects offer a unique material for the physiological analysis of gene effects. The vertebrate organism is, of course, of particular interest from the point of view of the relationship between genes and processes of embryonic and cellular differentiation because of the epigenetic nature of its development, and by virtue of the character and interdependence of its developmental mechanisms which have been analyzed in such detail, particularly in amphibian embryos. But Amphibia do not lend themselves easily to a genetic analysis, so that it has been difficult to use that particular group of vertebrates for extensive studies of the role of genes in development and differentiation. I n the mouse we know of a considerable number of genes with effects on embryonic processes offering a material on which to study the relationship between genes and embryonic differentiation. I n many cases these same genes may actually serve as tools for the causal analysis of embryonic processes and mechanisms which would defy elucidation by other means. Frequently the creation of an abnormal situation is the prerequisite for the causal analysis of the normal mechanism. The mammalian embryo because of technical difficulties has not been a ready subject for an experimental approach similar to that used in other vertebrates. It is, therefore, fortunate that mutations have been discovered which produce abnormal situations in mouse embryos that resemble those observed in other vertebrates resulting from delicate experimental operations. The analysis of such changes in developmental patterns and the examination of the steps preceding the abnormalities frequently make it possible to discover causal connections between processes which could not have been revealed through a study of the normal alone. The mutations whose effects have been studied in detail and which will be reviewed in this article concern different organ systems of the mouse. These include among others, the skeleton, integument, nervous system, pigment-forming system, blood, and developmental systems of the embryo. Different levels of development are affected by the mutations to be discussed. There are those, just mentioned, which interfere with processes of early embryonic organization and with inductive relationships between different parts of the embryo severely enough to cause death of

4

S-4LOME QLUECKSOHN-WAELSCH

the embryo. I n other cases, the interference of a mutation with an inductive effect does not interfere with viability but leads to abnormalities of structure. Mutations will be discussed where an effect on enzyme systems can be demonstrated, such as those affecting pigmentation. Hemoglobin synthesis will be shown to be disturbed in different hematopoietic tissues and in different phases of development by the action of mutational changes with far-reaching effects. One syndrome of abnormalities will be discussed which will be shown to have arisen as the result of a mutational effect on a hormone. The developmental analysis of some abnormalities of the nervous system will demonstrate how the original effect of a mutation may show no direct relation to the eventual symptoms. The analysis of other mutations reveals that their effects are due to changed rates of growth and differentiation of certain tissues. I n general, developmental studies of the type to be discussed here demonstrate the way in which genes express themselves developmentally, where the term “developmental” is used in a broad sense and is not necessarily restricted to the embryonic phase of the organism. The developmental analysis of mutations will be shown frequently to group gene effects which had appeared a t first sight to have quite different manifestations, while separating, on the other hand, gene effects which had originally shown close resemblance. J ust as the analysis of biochemical mutations in microorganisms has contributed considerably to the general knowledge of biochemical mechanisms, so the analysis of mutations in mice has increased our knowledge of normal developmental processes of different types in mammals, and this aspect will be stressed particularly in this review. From the practical point of view there exists, of course, always the hope that the results obtained from the genetic studies on mice may eventually have some bearing on other mammals, particularly man. F or the purposes of this discussion a considerable degree of selection ha5 been practiced, and we have considered only those mutations and those aspects of a particular mutation whose analysis was thought to contribute to the “physiology of heredity’’ i n the sense of Goldschmidt. The problem is not so much what particular effects some mutation may have but rather how the effect is brought about, i.e., to attempt to trace the steps that lead from primary action of the gene to its eventual manifestation. Thus, many mutations will be omitted from the discussion which have only been described and analyzed genetically, while others are going to be discussed incompletely. Gruneberg’s book (1943a) on the genetics of the mouse contains a complete compilation of data on mouse genetics and is a n invaluable

5

PHYSIOLOGICAL GENETICS OF THE MOUSE

help to anybody working in this or related fields. More recent data from the field of mouse genetics are included in Gruneberg’s second book, Animal Genetics and Medicine (1947). Another book with useful information is the Biology of the Laboratory Mouse edited by Snell (1941). This contains a very good chapter on early mouse embryology. For more detailed descriptive information about early mouse development the reader is referred to a series of papers by Sobotta (1895, 1902, 1911).

11. ANALYSISOF

YELLOWLETHAL,A CLASSICCASEohi LETHAL MUTATION

THE

A

One of the classic cases of a lethal mutation is the so-called yellow lethal in the mouse. Its study also represents the earliest attempt of a n analysis of a n embryonic lethal effect in a mammal. The first abnormal symptoms of yellow homozygotes are recognizable a t the time of implantation in the uterus ; this is the earliest stage a t which detailed observations of effects of any mutation have ever been recorded. The yellow mutation at the agouti locus is dominant over all other members of the agouti series. The absence of yellow homozygotes from the offspring of matings between yellow parents was discovered and pointed out by Cuenot (1905). Cuthot assumed that a “yellow” egg could not be fertilized by a “yellow” spermatozoon. The lethal vharacter of the yellow homozygote was ascertained by Castle and Little (1910) who concluded from their breeding experiments of yellow mice “ t h a t a Mendelian class may be formed and afterwards be lost by failure to develop.” I t was demonstrated later by Kirkham (1919) and still later by Robertson (1942) that the yellow homozygous zygote actually does form but dies in early embryogeny. The earliest reports of the findings of remnants of yellow homozygotes came from Ibsen a n d Steigk d e r (1917) and Little (1919). Kirkham (1919), however, made the first more or less complete study of these homozygotes, and reported the first abnormalities to be the indistinct cell boundaries of embryos of’ the morula stage. Blastocysts with a shrunken appearance, small crowded cells and shrunken blastodermic vesicle were assumed to be yellow homozygotes on the basis of their numerical frequency. A more extended descriptive analysis as well as a n experimental attack on the problem of the yellow homozygote was undertaken by Robertson (1942) who examined embryos from matings of yellow by yellow from the cleavage stages on and could not confirm earlier reports of abnormalities in late cleavage stages. The yellow homozygous embryo develops normally through stages of cleavage and blastodermic vesicle formation. It

6

HALOME QLUECKSOHN-WAELSCH

becomes lodged in a decidual crypt and the uterine epithelium surrounding it changes its character from columnar to low cuboidal while the subepithelial uterine connective tissue cells proliferate to a great extent. But the cuboidal epithelial cells do not disappear as they do in the normal implantation process and the further development of the implantation site is arrested, The blastocyst cavity disappears in the abnormal embryos. “Since the resulting cell mass is so small and has no appearance of differentiation into the various blastocyst structures, it is concluded that upon contact with the uterine epithelium the trophectoderm of the homozygous yellow blastocyst collapses and then quickly degenerates, leaving only the inner cell mass the cells of which soon become abnormal. ” I n the next stage, the egg cylinder stage, the decidual crypts of the abnormal embryos.are small and contain only a few scattered cells : the remnants of homozygous yellow embryos. The uterine mucosa is in a progestational phase and uterine epithelial cells are present in cuboidal form, as on the previous day. Death of the homozygous yellow embryo is assumed to occur after the trophectoderm of the blastocyst has come in contact with the uterine epithelium. Robertson (1942) used a very interesting approach to study the development of homozygous yellow embryos in an environment other than that of the heterozygous yellow mother. He transplanted ovaries from yellow mothers to nonyellow agouti hosts whose own ovaries had been removed. About a week after the operation, the host female underwent a test mating. If yellow offspring appeared in a cross to a male not carrying yellow, the graft was considered successful and the host female was mated to a yellow male; embryos were obtained from the female a t different stages. I n cleavage and preimplantation stages no abnormalities are observed, as expected. The appearance of the abnormalities occurs a t the same stage as in the normal environment, i.e., in the yellow mother. But the homozygous yellow embryo in the agouti mother can be clearly distinguished from the homozygotes in the yellow mother by an advance in development which Robertson summarizes as follows: “1) the development of an ectoplacental cone; 2) the more complete differentiation of a n area of the cell mass corresponding to the embryonic region of the normal implanted embryo j 3 ) the further development of the implantation site as exhibited by the disintegration of the uterine epithelium and the relatively greater depth of the implantation crypt ; and 4) the development of a n extra-embryonic membrane which appears to be Reichert ’s membrane. ’ ’ The onset of lethal action in the yellow homozygote is supposed to occur a t the time of implantation and to be due to the absence of a n

PHYSIOLOQICAL QENETICS OF THE MOUSE

7

enzyme normally secreted by the blastocyst roof and responsible for the erosion of the uterine epithelium and successful implantation. The effect of yellow in homozygous condition may be directed on this enzyme. “Since the homozygous yellow mouse embryo develops further in the agouti uterus than in its usual uterine environment, it is evident that the developmental phenomena peculiar to this embryo are a result of the uterine environment of the heterozygous yellow mother as well as the hereditary factors inherent in the embryo itself.”

111. ANALYSISOF GENE-DETERMINED PIQMENT CHARACTERS One of the developmental systems in the mouse most thoroughly studied from the point of view of gene action is that of pigmentation. Pigmentation offers a number of advantages for the stcdy of the chain of events between gene and character. The development of pigmentation may be observed in situ and is a strictly localized process apparently not determined by circulating substances. That this is the case is evident from observations on mutant spots and from transplantation experiments. I n addition to the strict localization and the autonomous behavior of pigmentation there are other features which make the pigment system a most suitable subject for studies of gene expression and its development. Since hair is being shed and replaced frequently during the lifetime of the mouse, the developmental events leading to pigment formation repeat themselves and can thus be observed continuously within the same organism. The hair, with its differences in pigmentation along the hair shaft, is an image of the processes going on in the hair bulb during the formation of pigment as determined by the individual genes of the particular cell. It has been shown (Rawles, 1947) that the pigment-forming cells of the mouse originate from the neural crest and that they make their way into the developing hairs and bring about their pigmentation. The melanophores reach their definitive positions in the skin of the mouse embryo by the twelfth day after fertilization. Determination of pigment pattern is thus completed a t that stage. The deposition of pigment in the hair by melanophores was demonstrated in witro by Hardy (1949) with the help of tissue culture techniques. 1. Autonomy of Pigmentation

a. Mutant Xpots. On the basis of the generally accepted conclusion that the color reaction of a cell is determined chiefly by the cell’s genes, the color mosaicism of a male mouse has been interpreted as being due

8

SALOME QLUECKSOHN-WAELSOH

to the presence of a changed locus in each of the abnormal color spots (Dunn, 1934a). The same male showed gonadal mosaicism, and a mutation in the cell ancestral to the gonadal and the somatic tissue was assumed to have been responsible for the mosaicism observed. The distribution of the patches which originally arose from a common mutant cell was considered an indication of a complex series of differential growth rates or migrations of the epidermal tissues in different directions. The existence and persistence of the mutant spots is an argument in favor of the local determination of pigmentation as against the decisive influence of generally circulating substances. b. Transplantatim. The autonomous behavior of hair pigmentation in mice indicated by the existence of mutant spots has been studied more closely with the help of transplantation experiments. I n a series of papers (Reed, 1938a, b ; Reed and Henderson, 1940) transplantation of skin of newborn mice between genetically different individuals has been reported. In the case of albinism, agouti pigmentation, dominant spotting, and piebald, complete autonomy of the pigment pattern of the transplanted tissues was found which must thus have been determined before birth. In the case of the black-and-tan mutation there seems to be a difference between tissue and cell determination. Tissues from the black-and-tan mutant ( a W ) themselves are autonomous in respect to their dorsal or ventral organization ; however, the individual prospectively “black-and-tan” cells are capable of forming either black or tan pigment, depending on whether they find themselves in an environment, of dorsal or of ventral cells.

2. Histological Studies of Pigmentation in Different Gene Substitutions Since the gene dependency of pigmentation is thus established the next questions concern the way in which the genes exercise their control, and the mechanisms of their effects. The effect of genes on pigmentation seems to be fairly direct, and the chain of processes connecting gene action and expression rela,tively short, making genetic pigmentation differences a most suitable material for the study of gene physiology. A number of studies have attempted to analyze the effect of differ. ent genic substitutions on pigmentation. Dunn and Einsele (1938) investigated the changes in the dark granular melanins of the hair in different combinations of the albino series with black ( B ) and with brown ( b ) . They found a graded reduction in amount of pigment, as measured by weight, in combination with black ( B ) . The changes in quantity of pigment were shown to be due to a decrease in size of pig-

PHYSIOLOGICAL QENETICS OF THE MOUSE

9

ment granules. “Each mutant gene in the c series thus exerts a characteristic effect on granule size.” Systematic histological studies of pigment in the hair cells of the mouse in different gene substitutions were done first by Werneke (1916). Using the same approach E. S. Russell (1946, 1948, 1949a) tried to ascertain the basic natnre of gene action in five of the main allelic series of mouse coat-color genes : agouti, albino, black/brown, intense/pinkeyed dilution, intense/blue dilution. Thirty-six different genotypes were examined in a series of very careful and thorough studies, and a number of different attributes of pigment granules were observed; it could be shown that a large number of different variable pigmentation attributes contributed to the differences in appearance of coat color mutants. They are color, size, shape, number, arrangement of granules, and several others. A certain amount of interdependence among these different pigment attributes makes it possible to separate them into four groups controlled by four key pigment characteristics : nature of granule color, granule size, granular clumping, level of pigmentation. The eventual goal of these studies is to relate the basic action of each of five allelic series (agouti, albino, black/brown, intense/pink-eyed dilution, intense/blue dilution) to variations in these pigment attributes. The nature of the pigment produced at any particular level of hair (i.e., whether i t is of the xanthic or the eumelanotic type) is determined by genes of the agouti series, while the genes of the albino series determine quantitative changes, i.e., degrees of pigmentation, by altering shape, color intensity, number, size, and distal arrangement of pigment granules. The change from dominant B (black) to b (brown) expresses itself mainly in a change in the nature of eumelanin from black to brown, i.e., in a change from the process leading to pigments of the black-fuscous series to one leading to brown pigments, while the quantitative effect is only minor. Size of pigment granules and deposition of pigment are affected by the change from P (full color) to p (pink). I n animals homozygous for d (dilution), the amount of pigment is a t least as high as in DD, but it is disarranged into large granular clumps and it is distributed unevenly. 3.

Biochemical Studies of Pigwentation in Different Genotypes

The biochemical aspects of pigment formation in various genetic backgrounds were studied (Russell and Russell, 1948) with the aim of getting closer to the actual effect of the pigment genes. The behavior of the dopa reaction was examined on frozen sections of mouse skin, a technique which permits a histological localization of dopa-oxidase activity. This method has a number of advantages over others using tissue

10

SALOME C?LTJEUKSOHN-WAELSCH

extracts: it avoids the inclusion of nonpigmented cells which may introduce inhibitors ; the activity of specified parts of a, cell can be determined ; and each section serves as its own control, since autoxidation of dopa would lead to uniform darkening of the entire section instead of differential darkening localized in the hair bulb as produced by enzymatic oxidation. The same genic substitutions were used which had been studied histologically, and the studies were done on animals of 6-7 days of age since it was found that a t that age the follicles produce that part of the hair which had been examined in the histological compaxison of genotypes. The biochemical results could thus be easily correlated with the histological data. It was found that there existed a close parallelism between the amount of yellow pigment as determined histologically and the activity of the dopa reaction in different gene substitutions ; when a gene mutation had a n effect on the natural yellow pigment it would have a parallel effect on the activity of the dopa reaction. This effect was found in the yellow as well as in the corresponding nonagouti types, and from this fact the authors conclude that “the dopa reaction measures some phase of the yellow producing system and that this system is present in sepias as well as in yellows.” The authors propose several interpretations of their results for the nature of the black-yellow differentiation, one of which postulates that ‘‘sepia-yellow differentiating genes act to produce different substrates. ” “ The discovery of another chromogen whose effect paralleled sepia both in yellow and sepia genotypes would prove the postulate that the sepia-yellow difference was one of substrates rather than enzyme systems, and the relation between this chromogen (presumably akin to the sepia substrate) and dopa could provide a. chemical clue as to the action of sepia-yellow differentiating genes.” 4.

Correlation of Histological artd Biochemical Results

Correlating the results of the histological studies with the biochemical results it appeaxs that the activity of the enzyme system measured by the dopa reaction is not affected by the genes of the agouti series. The genes of the albino series, on the other hand, affect the activity of the enzyme system which in turn is responsible for the intensity of the pigmentation reaction. The change of eumelanin from black to brown due to the genic substitution bb is not accompanied by any change in enzyme as measured by the dopa reaction activity. The effect of the pink substitution on size of granules and level of pigmentation appears to be correlated with a slight but probably significant decrease in dopa. oxidase activity. The dd substitution surprisingly results in a n increase of the

PHYSIOLOGICAL GENETICS O F THE MOUSE

11

reaction system, which runs parallel with some increase of natural pigment in dd individuals. Of the five allelic series studied, the genes a t the C and possibly a t the P locus seem to be involved in the control of the enzyme system measured by the dopa reaction which shows a correlation with the natural yellow pigment. 5. Tyrosinase System in the Determimtivn of Pigme& Pattern Some preliminary studies (Foster, 1951) deal with the role of tyrosinase activity in the determination of the pigment pattern of different genotypes in the mouse. The method used for measuring tyrosinase activity was that of determining oxygen consumption in a Warburg respirometer. The enzyme preparations consisted of homogenized skin, suspended in distilled water. Tyrosine was used as a substrate. The results of this study seem to demonstrate the significance of the tyrosinase system for the development of eumelanin pigment, and the presence of tyrosinase inhibitors in the skin of mice. The very slight amount of tyrosinase activity and the presence of a. tyrosinase inhibitor in yellow skin seem to indicate that the yellow pigment is independent of the tyrosinase system. Albino skin seems to be devoid of the enzyme as well as of its inhibitors. These preliminary results may indicate the decisive role of the tyrosinase system in the control of the black (sepia) pigment system which had been assumed on other grounds to differ from the yellow pigment system (cf. section 111-3). IV. ANALYSISOF GENE-DETERMINED ABNORMALITIES OF THE BLOOD There exist a number of well-analyzed genetic conditions in the mouse which involve the hematopoietic system ; the systematic investigation of the effects of these mutations and the embryogeny of the conditions caused by them has contributed much to our knowledge of the formation of the blood in the mouse. 1. Siderocytic Anemia in Flexed

One of the best-analyzed mutations affecting the blood is the flexed mutation ( f l ) . It was reported by Hunt and Permar (1928), and its effect in the blood system was studied by Kamenoff (1935) and by Griineberg (1942a, c). Flexed ( f l ) also has an effect on the tail and produces a belly spot. However, we want to concern ourselves here only with its effect on blood. The first observations on flexed showed that newborn fljl mice were anemic but that this anemia was transitory and

12

SALOME QLUECKSOHN-WAELSCH

the mice recovered soon. I n studies of the blood of flfl embryos it waa possible to place the onset of the anemia at a time before the fourteenth day after fertilization, i.e., preceding the beginning of the hematopoietic function of the bone marrow (16 days) ; later its beginning was traced hack to 13 days after fertilization. Actually, the anemia of the flp mice hegins to improve at the time when the bone marrow starts its normal hematopoietic function. The flexed gene is thus not concerned with the functioning of the bone marrow. This point was confirmed by red cell counts of adults which were practically normal. Apparently the flexed mutation causes some abnormality in the hematopoietic function of the liver, as indicated by the studies of Oruneberg (1942a, c). Bruneberg reports the abundance of siderocytes in flexed mice, i.e., red blood cells whose iron can be demonstrated in the form of granules by means of the Prussian Blue reaction. Oruneberg originally suggested that flexed anemics were unable to synthesize hemoglobin normally but produced only a hemoglobin precursor, the iron of which could be demonstrated. This inability to synthesize hemoglobin would be, however, restricted to the liver since only its hematopoietic function is affected by the flexed mutation. Lately, on the basis of some work indicating that siderocytes are actually aging cells, Qriineberg (1947) has taken the point of view that the anemia of flexed tailed mice arises from an increased hemoglobin breakdown. Of course, this might in turn be considered a consequence of abnormal hemoglobin synthesis. We thus cannot draw any definite conclusions from the embryonic study of the flexed anemia except that the normal allele of flexed must be involved in the control of either hemoglobin synthesis in the liver, or in its structural maintenance in the erythrocytes derived from the liver. The question remains open why the other hematopoietic systems of the animal should be affected mildly a t the most, and be capable either of normal hemoglobin synthesis, or of producing hemoglobin resistant to premature breakdown. One would perhaps expect this synthesis to be equally disturbed throughout the entire organism. A time factor does not seem to be involved since the first red blood cells originating from the yolk sac are fully hemoglobinized and probably normal. The effect of fl seems thus to consist of a localized disturbance of erythrocytes originating from the liver. If some general agent were operative in the flexed mice, one could expect the bone marrow red cells to be equally affected. 2. Mncrocytic Anemia in Dominant #potting A different step in the development of blood is interfered with by the W mutation. Mice homozygous for the W gene are anemic and in-

PEKYSIOLOGICAL GENETICS ON' THE MOUSE

13

viable; they die soon after birth. Their anemia is of the macrocytic type, i.e., these mice have fewer but larger cells than normal mice of the same age. DeAberle (1927) traced back the anemia to the sixteenth day of embryonic life by the observation of markedly pale embryos a t that stage, and Russell, Fondal, and Smith (1950) by establishing the erythrocyte level found WW embryos to be definitely anemic at 14 days after fertilization. There is some increase in the erythrocyte level of the anemics during embryonic life, so that the W anemia is a hypoplastic rather than a n aplastic anemia. The failure of WW to improve during the period when the bone marrow takes over its hematopoietic function clearly shows that the effect of W differs from that of fl. W apparently has a general effect on erythropoiesis of the entire organism and is considered by Russell et d.to cause a deficiency of a substance essential to erythropoiesis from earliest embryonic stages. The normal allele of W thus seems to be involved in the control of hemoglobin synthesis in general. There exists another allele of the W series, W" (Little and Cloudman, 1937)? which, when homozygous, produces a condition intermediate between that of normal and of W W mice. W"W" mice also have a macrocytic anemia but less severe than that of WW mice with a reduction to about one-half the normal number of erythrocytes; they are viable and actually improve their condition during their lifetime. Their anemia is definitely of the hypoplastic type and W" seems to affect hematopoiesis in the same qualitative manner as W , but to a lesser degree. The same substance essential for normal hemoglobin synthesis and presumed to be deficient in the case of WW would perhaps be less deficient in the case of WOW". A peculiar phenomenon is the existence of a mild anemia in the presence of one dose of W " :lower erythrocyte counts and hematocrit readings were established in W"heterozygotes by Griineberg (1942b), while animals heterozygous for W , which in homozygous condition produces a severe anemia, had a completely normal blood picture. Both alleles, W and W", were recognized first by their effects on pigmentation. When present in one dose, W produces a white belly spot, while W u heterozygotes, in addition t o the white belly spot, show a dilution effect of dorsal and ventral pigmentation. Both homozygotes have unpigmented f u r and black eyes. The dominance relationship of the two alleles in their effects on blood and pigment was analyzed most thoroughly by Russell (1949b) , who showed that W had no effect in one dose on either blood or pigment, but a severe effect in two doses. Wv had an effect on both systems in one dose, expressed in a decrease of granule number and size in the case of pigment, and in lower erythro-

14

SALOME GLUECKSOHN-WAELSCH

cyte counts in the blood ; but it had a milder effect than W on the blood in two doses, while the effect on the pigment equalled that of W. Russell points out the clear parallelism between the effects on the two different systems, blood and pigment : whenever one system is affected, the other one is affected too; in addition, there is a parallelism in degree of abnormality of blood and pigment. On the other hand, the reviewer feels that the divergence in the dosage effect of the two alleles should not be overlooked. One dose of W v is more effective than one dose of W, but two doses of W v are less effective than two doses of W. A hypothesis for the explanation of the simultaneous effects of mutations in mice on pigmentation and blood was put forward recently by Serra (1947), who supposes that an effect on copper may link the manifestations of these mutations in two systems. W and W” would, according to this hypothesis, exert an effect on copper compounds involved both in the formation of pigment and blood. I n the absence of any experimental proof, this idea can be regarded as a n interesting hypothesis only.

v.

ANALYSIS O F MUTATIONS AFFECTING THE

SKIN AND

ITSDERIVATIVES

The development of skin and hair in the mouse is dependent on the collaboration of a great many genes as evidenced by the existence of numerous different hair mutations. Quite a number of them have been reported and analyzed genetically (e.g., waved-1, waved-2, rex, naked, hairless, rhino, crinkled) ; we shall concern ourselves here only with those in which an attempt has been made of an analytical study of the mechanisms responsible for the expression of the individual mutations.

1. Transplantation Studies in Waved-2 Transplantations of skin between normal mice and those homozygous for wa-2, whose hair is waved instead of straight, seemed to show that nonwaved tissues or cells became organized to produce waved hair in the waved environment (Reed, 1 9 3 8 ~ ) . These results were interpreted as revealing not only the a.utonomous nature of waved-2 tissues and cells but also their ability to cause genetically nonwaved cells and tissues to form waved hair. The effect of wa-2 was thus assumed to be the following : “waved” cells (wa-2 wa-2) produce a growth-inhibiting substance which is responsible for the effect of wa-2 on body size and hair structure (wa-2 wa-2 mice are smaller than normal). The growth-inhibiting substance diffuses into the normal tissue and there causes nonwaved cells to produce waved hair. This interpretation has become highly doubtful on the basis of some incidental observations in a different series

PHYSIOLOQICAL GENETICS OF THE MOUSE

15

of transplantations between mice with normal hair structure (Fraser, 1946). Here it was noticed that “both graft and host hairs, when situated near the borders of the graft, were often curved or bent, like the hairs of genetically ‘waved’ mice.” The waviness of the hair was ascribed to a n antagonism between several physical factors acting on the growing hair in its abnormal position. Since this “waved” hair could be produced in transplantations between normal mice, it becomes highly doubtful whether the effect of waved on normal cells is actually due to the diffusion of a growth-inhibiting substance from waved into normal tissue.

2. Transplantation Studies in Rhino Another mutation affecting the hair of mice is rhino (hrrh)which produces hypotrichosis. The mode of action of the rhino gene which causes a follicular hyperkeratosis associated with depilation has been studied in transplantation experiments by Fraser (1946). These transplantations were performed with the aim of finding out whether a cellular condition was responsible for the expression of rhino or whether it was the result of some more general change (e.g., endocrine system) in the physiology of the animal. Grafts of normal skin to rhino hosts beha.ved autonomously and grew a normal coat of hair. But the reciprocal experiment gave a different result. Those parts of the rhino graft which were adjacent to the normal epidermis did not develop the rhino characteristics but had normal hair. It could be shown that these hairs were not invasion hairs of the host. Thus it seems that normal skin cells produce a diffusible substance which is “necessary for the normal maintenance of the cutaneous epithelium.” The effect of rhino might consist in the inability to produce this substance although rhino cells are capable of utilizing it when it is supplied from the outside. 3. Rhino and Vitamin. A Some alleviation of the skin defects associated with rhino, such as formation of cysts in the sebaceous glands and follicle ends of the rhino mouse, was obtained in rhino homozygotes by the administration of large doses of Vitamin A (Fraser, 1949). The conclusion was drawn that “the presence of the rhino gene in the homozygous condition is associated with a decreased ability of the skin cells to utilize some metabolite of Vitamin A necessary for their normal maintenance.” Thus it seems, as though perhaps the production of some substance as well as the ability to utilize it might be affected in cells homozygous for rhino.

16

BALOME OLUECKSOHN-WAELSOH

4.

Developmental #t&s

of Crinkled

A recent study of the effects and of the development of another mutation (crinkled) which affects coat texture has shed some light on the normal events taking place during the development of hair (Falconer Fraser, and King, 1951). Crinkled is a recessive mutation with a number of effects among which that on the fur of the animal is the most obvious. While the normal coat of a mouse consists of 3 types of hair, guard hairs, awls, zigzags, that of a crinkled mouse has one type of hair only, and they resemble awls, the short straight type of hair of the under fur. The question asked in the analysis of the mode of action of crinkled was whether crinkled merely inhibited the differentiation of a n as yet undifferentiated hair type into the three types and directed it into the channel of awl formation only without affecting the total number of hairs formed, or whether it suppressed the formation of guard hairs and zigzags leaving awl hairs only. I n the former case, one might suppose that the three hair types were not developmentally distinct entities, while in the second case the developmentally distinct chasacter of the three types would be strongly indicated. Normally there exist three periods of hair follicle formation in the embryo, which might correspond to the three types of hair in the coat. But while the study of normal hair development was not able to establish critical evidence for the connection between periods of hair formation and type of hair formed, the study of crinkled made it very probable that the formation of guard hairs, awls, and zigzags was linked u p with the first, second, third period of hair follicles formation, respectively. While the first hair follicles form at 14 days in the normal embryo, no follicles are found in the crinkled mouse until 17 days after fertilization when the crinkled embryo forms follicles resembling in general appearance those of the second period in normals. After birth, when the third wave of follicle formation starts in the normal, the crinkled mouse again does not form any follicles. I n the crinkled individual, therefore, the first and third wave of follicle formation seem to be suppressed. During the second wave, hairs are formed resembling awls. The suppression of first and third wave of follicle formation together with the absence of guard hairs and zigzags in crinkled mice supports the hypothesis derived from the study of normal development which postulates that first wave and guard hairs, and third wave and zigzags are connected; the activity of the second wave together with the existence of awls in crinkled mice is strongly in favor of the hypothesis that awls are derived from the second wave of follicle formation.

PHYSIOLOQICAL QENETICS OF THE MOUSE

17

VI. DEVELOPMENTAL STUDIES OF MUTATIONS AFFECTINQ THE UROQENITAL

SYSTEM

The main known genetic factors causing abnormalities of the urogenital system which have been studied from the developmental point of view are: (1) my, myelencephalic blebs; (2) 8d, Danforth’s short tail ; (3) UT, abnormality of the urogenital system. Each one of these three mutations has other effects in addition to those on the urogenital system, and some of these are described in other parts of the review. The embryonic study of two of these mutations haa contributed to our knowledge of the developmental mechanics of the mammalian kidney.

1. Kidney Development in Myelencephalic Blebs The development of kidney abnormalities in the “myelencephalic blebs” strain of mice was studied by Brown (1931) who concluded from her studies that “complete metanephric development is a response to a mutual influence and fusion of two healthy anlagen, the ureter anlage from the lower end of the Wolffian duct and the blastema of the posteriormost part of the nephrogenic mesenchyme. Either one of these anlagen or both may be retarded i n which case no functional kidney is developed and varying degrees of anomaly are produced.”

2. Kidney Development in Danforth’s Bhort Tail The probably decisive role of the ureter in inducing the metanephrogenic blastema to form the secretory elements of the kidney, i.e., secretory tubules and Bowman’s capsule, was revealed by studies of the development of the kidney system in mice heterozygous and homozygous for the dominant mutation Sd (Glluecksohn-Schoenheimer, 1945). Animals heterozygous for Sd show abnormalities of the kidneys which vary from reduction in size to complete absence of one or both kidneys, making the more extreme heterozygotes inviable. The homozygotes have no kidneys and die soon after birth. The extent of the kidney and ureter abnormalities depends on the residual genotype and modifiers play a decisive role in the determination of the anomalies of the urogenital system of both Sd heterozygotes and homozygotes. F o r the study of the development of the abnormal urogenital system and the causal analysis of the developmental processes taking place in normal development, that genotypic background was of course most desirable which permitted a good expression of the urogenital effects of fld. It was found that embryos both heterozygous or homozygous for 8d had

18

SALOME GILUECKSOHN-WAELSCH

a metanephrogenic blastema which in its histological appearance did not differ a t all from those of their normal littermates. On the other hand, a great variability in the condition of the ureters was observed. They were of varying length and the differentiation and branching of the ureter tip into cranial and caudal pole tubules was disturbed or inhibited entirely; sometimes the branching was retarded and fewer than normal pole tubules developed. There seemed to be a correlation between the condition of the pole tubules and that of the differentiation of the metanephrogenic blastema. Secretory elements were always found in conjunction with differentiating pole tubules ; the number of kidney elements and the eventual size of the kidney appeared dependent on the degree of branching of the ureter tip and the number of tubules formed. On the other hand, if the ureter tip did not make contact with the metanephric anlage or failed to branch, absence of the kidney resulted.

3. Inductive Relatiomhip between Ureter and Metanephros Since metanephric differentiation was never observed in the absence of differentiation of the ureter tip, the conclusion seems justified that the metanephros is dependent on the ureter for some inductive stimulus for its normal differentiation. A similar relationship between these two structures had been shown before to exist in other vertebrates, e.g., the chick, where the results of transplantation experiments had led to such a conclusion. Thus, one effect of the Sd mutation appears to be the inhibition of growth and differentiation of the ureter tip, resulting in the failure of a n inductive stimulus to reach the anlage of the metanephros. The existence of this inductive relationship and its significance in mammalian kidney development has been made very probable by the study of the embryonic effects of Sd. This, however, is only one of the effects of fld; its other effect, that on the axial skeleton, is discussed in another part of this review. 4. U r a g e d d flyndrorne The third mutation known to affect the urogenital system ur (Dunn and Oluecksohn-Schoenheimer, 1947) has not been investigated embryologically. Its effects, however, are quite different from those of Sd, cystic, and hydronephrotic kidneys being most prevalent among ur homozygotes ; it thus serves to emphasize the dependence of the development of a n organ on a great many genes all of which are involved in the control of different processes which contribute to the eventual goal of normally developed structure and function.

19

PHPSIOLOQICAL QENETICS OF THE MOUSE

VII.

DEVELOPMENTAL STUDIES OF MUTATIONS AFFECTINQ CENTRAL, NERVOUS SYSTEM AND SENSORY ORQANS

THE

The development of the nervous system offers a great many opportunities for the occurrence of deviations from normal. The complexity of gene-controlled morphological and physiological processes which have to proceed normally in order to give rise to a normally formed and normally functioning nervous system is so great that one is surprised a t the relatively small number of hereditary abnormalities of the central and peripheral nervous system actually observed in the mouse. The great power of regulatory mechanisms operating during vertebrate development is probably part of the explanation for the relative scarcity of nervous abnormalities; on the other hand some of the deviations from normal development are perhaps so drastic that they lead to early embryonic death of the zygote. Only special 'methods would detect such hereditary lethal factors. There exist (uite a number of hereditary functional nervous abnormalities in the house mouse for which a n organic basis has not been established. I n some cases no morphological or histological anomalies have been found in spite of careful search; the mechanism responsible for those abnormalities might be of a physiological or biochemical nature not amenable to investigation for lack of methods. It is of course difficult, if not impossible, to study the embryonic development of such nervous disorders or to use such studies for a causal analysis of normal development of the nervous system in the absence of any morphological manifestations of the anomalies in the adult. But, fortunately, at least some hereditary abnormalities of the nervous system have obvious morphological manifestations and several of these have been examined embryologically and will be discussed now. 1. Hydrocephalus

One phenomenon brought out repeatedly by the embryological study of hereditary abnormalities is the diversity of mechanisms which may lead to the same manifestation in the newborn or adult mouse. As pointed out in other parts of this review, this phenomenon emphasizes the dependence of normal form and function of a n organ or part of it on a great number of different processes for their development. Another example illustrating this situation is the study of two mutations both of which cause hydrocephalus in the house mouse. One of the two types of hydrocephalus has been studied embryologically by Bonnevie and by Brodal (1943, 1944, 1945, 1946). The condition is due to a recessive mutation (hy) and manifests itself a t or

20

SALOYE QLUECKSOHN-WAELSClH

after birth as a hydrocephalus of varying intensity. The mutation was first discovered and studied by Clark (1934)’ who reported an obstruction of the aqueduct of Sylvius as one of the anomalies of hydrocephalus. When Bonnevie studied the embryology of this type of hydrocephalus, she found a number of different abnormalities in various stages of emhryogeny. According to Bonnevie ‘(. . the manifestation of the hy-gene proves to be highly varying, a variation which is primarily attached to the fluid circulation of early embryonic stages.” Abnormalities of the embryo are traced from preimplantation stages, ((irregularities during the formation of the unilaminar trophoblast, ” to early implantation stages, “wounds)) in the trophoblast ((through which maternal material (tissue fluid, cells, blood) from the uterine endometrium will enter the embryonic yolk sac)’; the abnormal content of the yolk sac is supposed to lead to abnormal fluid circulation in the embryo, and an abnormally heavy fluid tension is indicated. The excess of fluid is directed into the hrain ventricles as soon as the activity of the choroid plexuses starts a t about 12 days after fertilization. It now follows the normal circulation of the cerebrospinal fluid, and hydrocephalic dilation of the ventricles is a consequence of the excess of fluid which fails to be reabsorbed. Finally a series of brain abnormalities results from the dilation of the ventricles. One wonders whether this causal connection of “irregularities ’)in the trophoblast and excessive fluid formation is really justified.

.

2. Congenital Hydrocephalus While Bonnevie thus traces the eventual hydrocephalus back to original abnormalities of the trophoblast in very early embryonic stages, Gtriineberg (1943b) finds a quite different “pedigree of causes” underlying another manifestation of hydrocephalus. His congenital hydrocephalus” (ch) is recessive and lethal a t birth. “The most obvious anomaly is a steeply bulging forehead consisting of bilateral protuberances which correspond to the cerebral hemispheres. These bulges are . not protected by flat skull bones; the bulges are somewhat flabby sacs, etc.” I n addition, the eyes are open a t birth, the nose is shortened, the sinus hairs are abnormal, and ossification of the sternum is retarded permanently. Gtriineberg was able to trace all these different anomalies back to a n abnormality of the early cartilage. “This condition seems to be of a transitory kind and does not seriously interfere with the development of the embryo, except a t the base of the skull” where the topographical relations are destroyed permanently so that a brain anomaly results which leads t9 the death of the ch-homozygote. The following figure from Briineberg (1943b) gives a picture of the ((

..

21

PHYSIOLOGICAL GENETICS OF THE MOUSE

“pedigree of causes” involved in the different abnormalities of ch-homozygotes which can all be traced back to an original anomaly of the cartilage consisting in a poorly developed matrix, areas of vacuolization and liquefaction, and retardation of cartilage growth. We thus see that the ch-mutation seems to have a general effect on cartilage development and we may conclude that its normal allele is concerned with the control of normal processes involved in cartilage formation ; another mutation, se (cf. section IX-3-b), was shown to afTect cartilage formation, but judging from its different effects, its normal allele seems to control different steps of the cartilage formation process than does the normal allele of ch.

CARTILAGE ANOMALY

Basicranial cartilage

I

Rletacarpals Metatarsals

Meckel’s cartilage

pituitary and Ganglion Class-

bra1 hemorrhages

Hydrocephalus

flat slrnll bones

of sinus hairs

eri

F’IG. I. Pedigree o f causes of congenital hydraeephalus. (1943b).

L

Death From Qriineberg

3. Pseudencephaly

Still another hereditary abnormality of the nervous system has been studied and traced back to abnormalities of early embryonic processes by Bonnevie (1936a). This is the so-called pseudencephaly, a deformity of the head which carries the dorsally open neural tube like a wig turned inside out. Pseudencephaly is probably due to a recessive let.hal mutation (ps). Embryos with this abnormality rarely survive iintil term; in addition to a certain degree of variability, they have a n i i m her of common characteristics, mainly the wiglike appearance of the brain which bulges out of the skull and whose two halves a.re turned

22

SALOME QLUECKSOHN-WAELSCH

inside out. Furthermore, the neural tube is crooked and shows a number of abnormal curvatures in the neck region. Two critical stages are supposed to occur during the development of pseudencephalic embryos ; the first stage occurs very early, embryos die according to Bonnevie “as gastrulae with unseparated germ layers, ” Those pseudencephalic homozygotes which survive this stage die at the end of the embryonic period after the manifestation of pseudencephaly has been completed. The complete eversion of part of the brain with the defective formation of the cranium and other consequences were all ascribed to a “purely mechanical brain catastrophy,” which in turn is supposed to be connected with the existence of abnormal wrinkles and curvatures in the neck region of the medullary tube. The origin of these curvatures is considered to lie in the fact that the embryonic neural tube is too big to find room in its normal sized mesodermal surrounding. Bonnevie considers an early shift of the growth balance of the otherwise normal anlagen the original manifestation of the ps-mutation, and a “delayed separation of the two primary germ layers” the cause of this shift. This latter failure may be severe enough to cause the death of the embryos in the first “critical phase.’’ The shift in growth balance results in a n increased growth rate of the neural folds which do not find room in the normally growing surrounding and are forced to curve and wrinkle. The connection between the early embryonic manifestation of p s and the later extensive malformations-if corroborated-would make this a particularly interesting case of developmental analysis of a mutation.

X-Ray-IitducedTranslocations and Pseudencephaly Pseudencephaly has also been demonstrated in embryos of mouse 4.

strains carrying X-ray-induced translocations (Snell, Bodeman, and Hollander, 1934; Snell and Picken, 1935). The abnormality was interpreted to be the consequence of chromosomal unbalance of the zygotes and not a result of the action of some specific factor or factors, since “nearly all translocations in mice give rise to rather similar types of abnormal embryos. ” Differences in modifying factors and in the intrauterine development are considered to be responsible for the numerous variations found among the abnormal embryos. The neural folds of the abnormal embryos fail to close anteriorly, and the ectoderm which normally forms the roof of the brain has turned outward, thus exposing the floor of the brain. The abnormal parts of the brain include telencephelon, diencephalon, mesencephalon, and the plexuses. The remarkable resemblance of pseudencephaly as determined either by a single recemive gene, or as the result of chromosomal unbalance, or as a consequence of disturbance of embryonic processes by the action of

PHYSIOLOGICAL GENETICS OF THE MOUSE

23

X-rays (cf. section XI) indicates the great sensitivity of the early developmental period of the neural folds as well as the dependence of their normal development on a great many different factors.

5 . Sh,aker Short a. Central Nervous Systeni. A very striking ahnormality of the nervous system with obvious morphological manifestations which has been examined embryologically is the so-called shaker-short mutation first described by Dunn (1934b). Shaker short is a recessive and the homozygotes show a shortening of the tail, choreic movements a n d deafness. The embryology of these animals has been studied by Bonnevie (1936b, 1940), who found brain hernias with far-reaching destruction of mesencephalon and metencephalon in homozygotes a t or shortly before birth. Bonnevie assumes that some explosive forces must have led to this brain destruction and that “the cooperation of various forces and organ systems is necessary to cause the explosive disturbance of the brain.” I n the search for these “forces and organ systems” Bonnevie reports an inhibition of the development of the brain roof and absence of the foramen of Magendie at around 9-11 days after fertilization. This stage is followed by one in which only rudimentary Plexus chorioidei are found, resulting in a deficiency of cerebrospinal fluid, which, in connection with the absence of the foramen of Magendie, leads to disturbances in the circulation of this fluid. Abnormalities of the meninges and the cranium are a consequence of the decreased distance between brain burface and the surrounding condensations of connective tissue. A sudden change of heart activity, determined from sections of embryonic hearts, is reported to set in at this stage and its slowing down is ascribed to a n abnormally high pressure on vagus and sympathetic nerve centers. Finally, brain hernias develop in the dorsal part of the mesencephalon and metencephalon. This entire complicated “pedigree of causes” was further traced back by Bonnevie to an “abnormal thickening of the epithelial parts of the early embryonic brain roof.” A t 8 days after fertilization Bonnevie reports to have observed a n abnormal thickening of the dorsal closure of the medullary tube in the form of a stringlike mass of cells in intimate contact with the medullary tissue underneath it. Moreover, even the observation of a n ectodermal hypertrophy in implantation stages prior to mesoderm formation has been reported. “The causal connection of this hypertrophy of embryos in stages of implantation with the previously described manifestation of all anomalies characteristic for the shaker-short mice may be considered certain. ” The embryonic abnormalities are striking, and their origin in a n ectodermal hypertrophy in premesodermal stages is so interesting

24

SALOME GLUECKSOHN-WAELSCH

from the point of view of developmental mechanics that one would like to see this study extended. One would, for example, like to see stages of archenteron and mesoderm formation studied and note the detailed effect of the ectodermal hypertrophy on other early embryonic formative processes. The reviewer, therefore, agrees with Criineberg (1947) in his criticism of Bmnevie’s analysis : “The resulting pedigree of causes has several weak points which require further study.” b. Ear. A lateral compression of the fourth ventricle of the brain and ensuing abnormal pressure conditions are considered to be decisive for the abnormal development of the ear vesicles of the shaker-short mouse which remain oval, laterally compressed, and practically undifferentiated. However, in analogy with Hertwig’s (1944) analysis of the “kreisler” mutation (cf. section VII-6), one might perhaps rather assume a disturbance in the developmental interrelationship between ear vesicles on the one hand and brain and surrounding mesenchyme on the other hand to be responsible for the failure of the shaker-short ears to differentiate normally. It is interesting to note that Bonnevie in her developmental analyses of abnormalities, consistently favors the disturbance of mechanical conditions as explanations for successive anomalies, rather than developmental interdependencies of different tissues and structures. Pseudencephaly, for example, according to Bonnevie arises as the result of a “purely mechanical brain catastrophy ” (cf. section VII-3), hydrocephalus is due to excessive fluid circulation (cf. section VII-1), and shakershort, to a deficiency of cerebrospinal fluid connected ultitnately with a n “explosive disturbance of the brain” (cf. section VII-5-a). Furthermore, Polydactyly in “Little and Bagg’s abnormal mouse tribe” (to be discussed later, cf. section IX-2-c-ii) is supposed to be due to localized disturbances of development by blebs originating from an abnormally high quantity of fluid expelled from the fourth ventricle through the foramen anterius and migrating eyer the surface of the embryo,

6 . Eflcct of Kreisler on the Central Nervous System and Ear I n contrast to these purely mechanical explanations of developmental abnormalities, Hertwig in her studies (1944) takes into consideration the epigenetic nature of vertebrate development, and developmental interrelationships. Whenever descriptive methods alone are used in the developmental analysis of a mutation, the causal relationship between a n early embryonic abnormality and the disturbance of a n inductive process can only be indicated ; transplantation or explantation experiments are needed for final proof of such relationships. Frequently, when working

PHYSIOLOQICAL QENETICS OF THE MOUSE

25

with an organism such as a mammal whose developmental mechanics are still largely unknown, the analysis of hereditary embryonic abnormalities may give the first strong indication of the very existence of an inductive relationship between two embryonic structures. A case which has been analyzed along these lines of thought is that of another behavior mutation in the mouse, the “ kreisler” mutation (Hertwig, 1944) where the chain of processes connecting the first visible embryonic abnormality with the eventual neurological symptoms of the young mouse has been worked out and the causal connection between a t least some links in the chain made most probable. The mutation kreisler (kr) is a n X-ray-induced recessive. “Kreislers” may be recognized 10-14 days after birth by their peculiar behavior-they tend to crawl in a circle. Older animals show behavior abnormalities such as dancing and head shaking when disturbed and are deaf ; their mortality is high. Pathological-anatomical examination revealed extensive brain defects. Cysts were demonstrated which arose from the rudimentary ductus cochlearis and extended into the subarachnoid space disturbing brain development. Deafness was shown to be the result of the absence of cochlea and the organ of Corti. The examination of “kreisler” embryos showed that a t 9 days after fertilization the invaginating ear vesicles of “kreislers’ ’ were shifted laterally so that their walls did not touch the neural tube, as is the case in the normal, but were separated from it by a layer of mesenchyme. At a slightly later stage, when the normal ear vesicle was pear-shaped and showed the anlage of the ductus endolymphaticus, no differentiation of the ear vesicle was visible in the abnormal embryos. Still older “kreisler” embryos were easily recognized by the absence of ductus and saccus endolymphaticus and by abnormalities in the semicircular canals. The separation of sacculus and utriculus remained incomplete and the ductus cochlearis did not form a spiral cochlea but remained a wide duct only coiled slightly. The defects of the membranous labyrinth resulted in underdevelopment of the periotic cartilage and consequent absence of part of the cerebellum. The absence of ductus endolymphaticus and thus of its efferent function is considered responsible for an increase in endolymphatic pressure. This in t urn gives rise to an evagination of the epithelium of the ductus cochlearis which forms cysts extending into the subarachnoid space. These cause serious defects in the rhombencephalon, the pons, and even the cerebral hemispheres. Only animals with small or unilateral cysts are able to survive. In search for the primary causes of the malformations of the membranous labyrinth Hertwig compares her descriptive results with data

26

SALOME QLUECKSOHN-WAELSCH

obtained in the analysis of the developmental mechanics of the amphibian ear. There a progressive determination of different parts of the differentiating ear vesicle and a strong dependence on normal surroundings for normal organogenesis have been demonstrated ; in analogy with it, the ear anlage of the mouse embryo is considered to be not fully determined a t the time of invagination and disturbed i n its further differentiation by the abnormal conditions of its surroundings. The greater distance of the neural tube keeps it from exerting its normal inductive influence on the ear anlage. Thus, the ear vesicle remains small and is not able to form endolymphatic duct and normal semicircular canals. The analysis of the developmental behavior of the “kreisler ” mutation has thus made very probable the existence of a developmental interrelationship in the mouse between neural tube, surrounding mesenchyme and ear vesicles j it has furthermore shown that ductus and saccus endolymphaticus are responsible for the regulation of the lymphatic pressure in the normal ear, and the efferent function of the ductus endolymphaticus. The motor disturbances could not be traced back to any definite defect. Of course, the question of the nature of the effect of the “kreisler” mutation in producing the origilral lateral shift of the ear vesicles remains open, and the developmental analysis, while showing a “pedigree of causes” and revealing a number of most interesting embryological phenomena, has not been able to find out anything about the primary action of the gene involved.

7. Eyeless Among a number of mutations which have been reported to affect the eye there is one with particularly good penetrance and expressivity, and this eyeless mutation, ey-I, has been made the subject of a careful embryological investigation (Chase and Chase, 1941). I n the anophthalmic strain studied 90% of all the animals homozygous for eyelessness lack the eyes, and the remaining 10% have abnormal eyes. The investigation of the embryonic development of the eyes of such homozygotes showed that the first evagination of the optic vesicle a t 9 days after fertilization was normal. At about 10 days a n inhibition of the eye vesicle set in which prevented it from growing sufficiently in order to come in contact with the ectoderm and induce a lens. Either no invagination a t all occurred to form an eye cup, or a small and irregular cup formed. The subsequent failure of the choroid fissure to close was considered to be either a direct result of the growth inhibition of optic vesicle and cup or an indirect result, due to the failure

PHPSIOLOQICAL GENETICS OF THE MOUSE

27

of lens formation; organogenesis of the eye did not proceed any further. At 13 days after fertilization, no eye was found in 90% of the mice of the anophthalmic strain, and no optic nerve was formed. Consequently, there was a n underdevelopment of the visual centers of the brain, and a number of abnormalities of the cranial nerves could be traced back to the absence of the eye. The absence of the eye, in turn, was not a result of degeneration but of “failure of definitive organization beyond the eye vesicle stage. ” The developmental study of this eyeless mutation seems to show that the mouse behaves developmentally like that group of species of Amphibia where the lens is completely dependent on the eye cup for its formation and where the prospective lens tissue lacks any potency for self-differentiation. Studies of the developmental mechanics of eye formation have demonstrated the principle of double assurance in the formation of the eye in some species of Amphibia, i.e., the eye cup is able to induce the lens, but the prospective lens tissue is also able to selfdifferentiate to a certain degree in the absence of a n inductive stimulus from the eye cup. I n other species, the lens is completely dependent upon the inductive stimulus from the eye cup for its development, and there exist gradations between these two extreme conditions in still other species of Amphibia. It would be very interesting if one could analyze the developmental potencies of the eye anlage of the mouse along the same lines a s done in Amphibia and thus confirm the conclusions obtained from the descriptive embryological study of “eyeless. ” While thus the abnormalities of eye formation in “eyeless” could be traced back to a primary inhibition of the optic vesicle, the effect of the eyeless mutation in producing this inhibition remains still unknown.

8. Micruphthalmus Another mutation affecting developmental processes in the formation of the eye, is “microphthalmus” ( m i ) which arose in an irradiated (X-ray) strain of mice. The homozygous animals have small eyes with colobomas. A very careful study of Miiller (1950) analyses the developmental steps leading to the eye abnormalities in this mutation and uses these findings for a n interpretation of the normal developmental mechanics of the eye. The first observable deviation from normal consists in shifts of the normal relationships of mitotic rates of nervous and pigment layers of the retina at about 10 days after fertilization. The result is a relatively increased growth rate of the pigment layer, resulting in an abnormally shaped eye cup and failure of the choroid fissure to close. Disturbances in nervous connections of eye and optic nerve follow, also eversions of the retina, abnormalities of the optic nerve, ab-

28

SALOME QLUECKSOHN-WAELSCH

normal pressure conditions in the eye chamber, lens deformities, and disturbances or complete absence of function. The successive appearance of a variety of abnormalities in the eye is utilized for a n analysis of the interdependence of different eye structures in normal development. A shift in growth rates of the different layers of the retina is thus considered to be the first observable effect of “mi” on eye development ; again, the mechanism by which the mutation produces this shift remains unknown.

VIII.

ANALYSISOF ENDOCRINE DISTURBANCES-PITUITARY DWARFISM

While the involvement of an endocrine gland was only indicated in the case of the abnormalities produced by the grey-lethal mutation (cf. section IX-2-d), there exists another mutation where the connection between endocrine disturbance and subsequent anomalies due to the effect of a mutational change could be clearly demonstrated. This is the mutation “pituitary dwarfism, ’’ a recessive analyzed genetically by Snell (1929). Dwarfs can be recognized at the age of 2 weeks when their growth and development cease; for the next 2 weeks they do not gain weight and occasionally even lose weight. After this period some animals start to grow again, others do not. Skeletal growth and differentiation are retarded. Male and female dwarfs are sterile, and their mortality is increased. These abnormalities of the dwarf mice seemed to simulate those of hypophysectomized rats, and furthermore, a histological study of the anterior lobe of the pituitary of dwarf mice showed complete absence of eosinophiles, a reduction in number of chromophobe cells and the appearance of a connective-tissue network. Smith and MacDowell (1930), therefore, tested the hypothesis that a defect of the anterior lobe of the pituitary was responsible for all abnormalities observed in the dwarf mouse. Fresh r a t anterior pituitary lobe was implanted daily into mouse dwarfs and gave positive results : “along with the striking resumption of growth, came a rapid loss of the dwarf traits.” Soon “the dwarfs could not be distinguished from normal mice,” aside from lower body weight. Males began to breed and females showed signs of a n estrous cycle. However, “the anterior lobe of the pituitary remains in the same defective condition as in the untreated dwarfs. ” All abnormalities of the mouse homozygous for pituitary dwarfism could thus be traced back to an effect of the mutated gene on the anterior lobe of the pituitary. Further studies were directed toward an analysis of the particular hor-

PHYSIOLOGICAL GENETICS OF THE MOUSE

29

mone effected by the mutational change, b u t for details of these studies the reader is referred to the individual papers (cf. Gruneberg, 1943a). OF MUTATIONS AFFECTINQ THE Ix. ANALYSIS

SKELETON

1. Skzcll a. Harelip. Throughout this review we have stressed repeatedly how different pathways may lead to the same end result, and that thus similarities of manifestation (lo not necessarily indicate similarities of disturbed development. A good illustration of this point is provided by some studies of harelip, a hereditary condition in mice. This condition has been studied by different authors in strains which were all derived from the same parent strain. The genetics of harelip is rather complicated, and it is not even certain whether harelip is due to one or several genes. The study of the embryonic development of harelip showed that apparently disturbances of different kinds may be responsible for the manifestation of the harelip condition. Reed (1933), in his studies, found that the clefts of the jaw were due to a failure of fusion of the lateral and of the medial nasal processes. The reason for this anomaly is sought for in a retarded growth rate of the maxillary processes whose lateral pressure is needed for the completion of fusion. Retardation of growth is also responsible for the failure of the palatine processes to unite. Tn a n examination of his strain of harelip mice, supposedly with the same genetic basis for harelip, Steiniger (1941) found, in addition to embryos which showed the nonfusion origin of harelip, others where the harelip was clearly the result of a break-through of embryonic cysts into the mouth and nasal cavity, and thus of secondary origin. In view of the fact that different disturbances in the same region seem to be responsible for the existence of the harelip condition in these mice, one might perhaps think of a n increased susceptibility of the mouth and nose region i n prospectively harelip mice. Due to a lowered threshold of resistance to different kinds of embryonic traumata, these embryos might react with retarded growth or with cyst formation in the mouth region to conditions which normally would pass unnoticed by the developing embryo. The case of harelip might be similar to the case of taillessness in the rat (Dunn e t al., 1942) where it was found that no single hereditary factor was responsible for the appearance of taillessness b u t where “the genetic constitution determines the threshold of a reaction which is subject to alteration by minor and more or less random accidents early i n development. ” b. Tooth Abnormalities. (i) Grey lethal. The tooth abnormalities of mice homozygous for the grey-lethal mutation (cf. Gruneberg, 1943a,

30

SALOME QLUECKSOHN-WAELSCH

for details) can be traced back to failure of secondary bone absorption which will be discussed in greater detail below (cf. section IX-2-d). (ii) Screw tail. Abnormalities of the teeth, the mandibles, and the skull are symptoms also of another, the so-called screw-tail, mutation. Mice homozygous for this recessive mutation were first recognized by their tail abnormality. The extensive malformations of the cranium, the mandibles, and the teeth have been described in detail (MacDowell et al., 1942).

2 . Extremities a. Lrc.ccc’--C‘o~~~ywzitul Abvencr of l’ibiil,. I h e to the relative paucity of mutations aflecting the extremities of the mouse not many studies have been made which contribute to the developmental analysis of limb structures. Nevertheless, one of the very first mutations studied from the embryological point of view is one affecting the hind limbs (Hovelacque and Noel, 1923). The mutation is a recessive called lux6 and expresses itself in congenital absence of the tibia. The examination of embryos from homozygous abnormal stock showed mesenchymal blastema in the hind limb bud resembling those which, in normals, would differentiate into two anlagen, that of the tibia, and that of the fibula, and become chondrified and ossified eventually. However, the hind limb buds of the embryos with congenital absence of tibia in spite of the presence of normal-appearing blastema show differentiation of only one cartilagenous structure which later ossifies, narriely, the fibula; the tibial anlagt? develops mostly fibrous tissue and only some cartilage in its upper portion which in comparison with the fibula is retarded in its development. The mutant gene thus affects specifically one part of a general anlage, i.e., the tibial portion of the limb bud mesenchyme, preventing it from developing along the normal path of chondrification and ossification while the neighboring anlage of the fibula in the same blastema shows no disturbance whatsoever of the identical processes. The ramifications of this situation for problems of cellular differentiation, such as the respective roles of inherent cell properties and the cell’s environment, were well recognized by the authors at the time of the embryological description of the condition. b. Luxate. Recently a new niutation has been reported (luxate, Carter, 1949, 1951) which in its edects strikingly resembles “1ux6, ” the so-called congenital absence of tibia discussed before. I n homozygous luxates the tibia is reduced, and in addition there are widespread defects of digits and other parts of the leg, prevalently on the preaxial side of the hind limbs. Heterozygotes are either normal, or show abnormalities of the digits also on the preaxial side of the hind feet. Since the original

PHYSIOLOCIICAL GENETICS OF THE MOUSE

31

“1uxQ” stock is extinct, genetic tests of the identity of the two stocks could not be made. Unpublished results of an embryological study (Carter, 1950, personal communication) indicate, however, the probable identity of lnx6 and luxate. The studies of luxate were able to tracr h w k the abnormality to a ninc.11 earlier stage of embryogeny. Abnormal shape of the hind limb buds was observed in luxate homozygotes of ll$$ days embryonic age ; at this stage the hind limb buds were narrower and more symmetrical than those of normal embryos. Differences in rate of development between preaxial and postaxial halves of the hind limb are considered to be the explanation for the differential effect of the mutation on tibia and fibula. c. Polydactyly. Another aspect of limb development that could be studied ,with the help of methods of developmental genetics is that of the regulation of toe pattern. The appearance of supernumerary toes is a rather frequent phenomenon in mice and the developmental study of those cases where this abnormal condition is determined genetically might reveal some of the mechanisms involved in the control of the normal developmental pattern of toe formation. ( i ) . Development. This point of view was kept in mind especially by Chang (1939) who studied the development of supernumerary toes in Fortuyn’s strain of polydactylous mice, where chiefly the first toe of the right hind foot was more or less completely doubled. Shape differences of the right hind footplate of embryos were found to be the first symptoms of polydactyly. The medial side of the abnormal footplate bulged out more sharply than that on the normal side. In addition to the excessive local growth of the medial side of the posterior limb buds there does not seem to be any abnormality of histogenesis. I n contrast to assumptions that some change in the epidermis might be responsible for subsequent abnormalities of the affected toe, Chang considers the excessive growth of both epidermis and mesenchyme to be caused by interaction of mesodermal and ectodermal factors. Such an interaction is operative in amphibian limb development, as shown by transplantation experiments. Chang’s analysis of polydactylism represents one of the first cases to illustrate the attempt to combine descriptive studies of the development of a mutation in mice with results from transplantation studies in Amphibia, with the aim of a causal analysis of mammalian development. (ii) Polyductyly and embryonic blebs. Different causes have been made responsible for the appearance of supernumerary toes by other workers. Bonnevie (1934), in an investigation of the embryogeny of polydactyly in “Little and Bagg ’s abnormal mouse tribe, ” discovered the presence of blebs migrating over the surface of the embryos where

32

SALOME QLUECKSOHN-WAELSCH

the bleb fluid was supposed to have originated from the medullary tube and to have been expelled through the foramen anterius in abnormally high quantity. The author considers these blebs to be responsible for localized disturbance of development, causing for example, as border blebs, the characteristic polydactyly. However, there are several phenomena wliicli could not be explained by such a n assumption. One of thein is the fact that the appearance of border blebs and original abnormalities of the footplate o m i r s a t least simultaneously, if not in reversed order, i.e., abnormalitiw of shape are recognizable before first appearance of blebs. (iii) Polydactyly and central n e r u m s system. The possibly causal relationship of polydactylism and hypertrophy of the ventral motor horn cell column a t the lnnibrosacral level was investigated by Tsang (1939). IIe fonntl that a correlation cxist,rtl between polydactyly and a hypertrophy of the retrodorsolateral cell column in the homolateral ventral horn of the spinal cord. Froin his results Tsang concluded that polydactyly was the immediate consequcnce of the neural hypertrophy. Ilowever, a reverse order of cause and effect seems more likely in view of experimental evidence in frogs and birds where hyperplasia of neural growth is produced as a consequence of implantation of supernumerary peripheral structures. Furthermore, Chang 's studies reveal, as pointed out by Griineberg (1943a), that the first abnormalities in shape of the footplate appear prior to the time of neural differentiation. (iv) Polydactyly and maternal age. That polydactylism may be influenced in its expression by a number of mutually interdependent developmental factors aside from genetic ones is apparent from studies which showed the effect of maternal age in the manifestation of the polydactyly gene, which decreased with increasing maternal age (Holt, 19-18). d. G'rey-T,ethal and Foilirve of Rccondary Bone Absorption. The extremities of mice show deviations from normal a s the result of the effects of still another mutation which produces a number of additional abnormalities. The mutation is the grey-lethal, discovered and very thoroughly analyzed by Griineberg (1936, 1937, 1938). One of the main abnormalities of mice homozygous for the grey-lethal mutation is the absence of secondary bone absorption which leads to changes in the shape of all the bones, including those of the extremities. No common marrow cavity exists in the long bones, and shape anomalies of the long bones develop. I n the absence of secondary bone absorption the skeleton of the grey-lethal mouse provides very good material for a comparison with the normal slieleton for the purpose of studying normal bone absorption.

PHYSIOLOGICAL GENETICS OF THE MOUSE

33

The cells which are responsible for bone absorption, i.e., osteoclasts, are found in normal number in the grey-lethal. The embryonic effects of the grey-lethal mutation have not been studied. However, reciprocal transplantation experiments between grey-lethal and normal mice (Barnicot, 1941) at least excluded some possibilities of explaining the effect of grey-lethal, without however revealing its actual nature. These reciprocal bone transplants did not behave autonomously, but grey-lethal bones, transplanted into normal hosts, approached normalcy while normal bones, transplanted to grey-lethal hosts, would sometimes assume grey-lethal characteristics although the results were not constant. On the basis of these experiments the effects in grey-lethal mice were ascribed to either the absence of an essential substance o r the presence of an inhibitory substance in the circulation and not to any factor inherent in the bone itself. Barnicot (1945) then attempted to answer the question of whether the grey-lethal affected some endocrine gland and caused a hormonal deficiency. One of the glands to be suspected in the grey-lethal mouse was the parathyroid, since it was known that parathyroid extracts caused bone absorption. The grey-lethal mice differed from normal mice in their reaction to the injection of parathyroid extracts; there was no evidence, however, that the treatment cured the faulty bone absorption, although the skeleton reacted to large doses of the hormone with considerable bone absorption. While the interpretation of these results remains speculative, there seems no question that the parathyroid, or its hormone, is somehow affected by the grey-lethal mutation and that the function of the gland or that of its hormone is disturbed in the grey-lethal mouse. I n addition to its effect on the skeleton, the grey-lethal mutation interferes with pigment formation : animals homozygous for grey-lethal have no yellow pigment in their fur. So far no physiological connection between these different effects of grey-lethal has been discovered.

3. Sternum a. Screw Tail. One of the very striking abnormalities of the screwtailed mouse (cf. section IX-l-b-ii) is the absence of segmentation of the sternum. I n contrast to the normal mouse, where the sternum consists of six separate segments, the sternum of the screw-tailed mutant is unsegmented, and while six centers of ossification exist in the normal, the screw sternum, except for the xiphoid process, arises from a single center of ossification and is considerably shorter than normal. I n addition, there are abnormalities of the ribs. It was hoped that a study of the development of the abnormal sternum of the screw-tailed mouse might reveal some of the mechanisms operating in the development of the nor-

34

SALOME QLUECKSOHN-WAELSCH

ma1 sternum, particularly in respect to the mutual relationship between ribs and sternum. Such a study was undertaken (Bryson, 1945) and indicated the decisive role which the ribs played in the determination of the ossification pattern of the sternum and thus its segmentation. According to the author, (‘the induence of costal cartilages upon the sternum is to delay ossification at the region of contact, both in the mutant and normal structure.” Thus ossification is normally inhibited in those regions where the “opposing zones of inhibition” have come in contact medially as a result of growth of the ribs. I n the screw-tail sternum, however, retarded growth of the ribs has kept the “paired zones of inhibition” apart so that ossification can proceed throughout the entire sternum without inhibition. The screw-tail gene is considered to have a general retarding effect on the axial skeleton of which the retardation of rib growth is only one symptom. I n normal development, ((th e determination of ossification pattern occurs after the union of ribs and sternum and is due to a n inductive effect.’’ b. Short Ear. Another aspect of the development of the sternum was studied by Green and Green (1942) who used the short-ear, se, mutation in the mouse as a material. Animals homozygous for this mutation show abnormalities of the ear, decreased body size, and a malformation of the xiphisternum. Depending on the residual genotype, the xiphisternum may be either reduced, practically absent, or bifurcated. The embryogeny of this abnormality was investigated and compared with normal development ; it was found that the sternal bands appeared shortened at about 13 days after fertilization so that the last rib frequently did not attach to the sternum. When the sternal bands moved together to form the sternum, it appeared that the xiphisternum was either reduced or completely absent in conformity with the shortening of the sternal bands. I n those cases where bifurcation of the xiphisternum occurred, the abnormality did not become apparent till slightly later in embryonic life-at about 15 days the posterior ends of the sternal bands of the animal with a prospectively bifurcated xiphisternum did not unite. Preceding this stage the sternal bands were not as clearly outlined a t their posterior ends as were the normal bands and the mesenchyme appeared less condensed. On the basis of their observations the authors conclude that (‘the condensation of mesenchyme leading to cartilage formation is certainly defective in the xiphoid region of the sternal bands, ” and that thus “defective precartilage is the actual forerunner of the adult departure from normality.’’ The short-ear gene thus seems to have a locally determined effect on certain regions of precartilage.

PHYSIOLOGICAL QENETICS OF T H E MOUSE

35

A more recent paper of M. C. Green (1950) demonstrates a more widespread effect of the mutation in the bony skeleton as well as other cartilages. How f a r all these effects can be attributed to a single cause remains for future elucidation.

Spinal Colwmn The development of the spinal column is dependent on a great number of genetic factors for its normal morphogenesis. Many mutations have been found which frequently have effects on the vertebral column as well as on other organ systems. Actually, the spine and particularly its distal portion, the tail, serve as a good indicator for the detection of mutations in the mouse. As we shall see, a number of mutations with most interesting additional effects, have been recognized first by their effects on the tail. Not all the tail mutations known have been examined developmentally, and in this case the mechanisms responsible for their effects on spine and tail development are quite unknown. We shall not deal here with these mutations, but restrict our discussion to those in which a thorough investigation of the embryology of the tail abnormalities has been made. We shall see that most of the mutations to be discussed have made their appearance in other parts of this article because they all have effects in addition to those on the tail. Their pleiotropic nature is a characteristic phenomenon of these tail mutations. a. Flexed. One of the first tail mutations whose developmental effects were studied thoroughly is “flexed, ” a recessive mutation affecting both blood and skeletal system (Kamenoff, 1935). Flexed homozygotes have stiff segments i n different parts of the tail, and flexures which are due to unilateral fusions between two successive vertebrae. Typical asymmetrical fusions of vertebrae are also found in the thoracic, lumbar, and sacral region of the spine. The flexures are primarily abnormalities of the intervertebral disc where cartilaginous or bony tissue substitutes for the normal felted fibers. Vertebrae in the mouse, as in mammals in general, are formed in the following way. The posterior half of one sclerotome unites with the anterior half of the subsequent sclerotome to form the anlage of the centrum. The first evidence of the intervertebral disc is rapid proliferation of cells from the two half-sclerotomes adjacent to the intervertebral fissure, and subsequently the notochord shows bulgelike thickenings at the site of the discs. Shortly before birth the notochord is present only in the intervertebral masses as the anlage of the nucleus pulposus. The cartilagelike cells of the intervertebral disc lose their matrix, elongate, and gradually become more fibrous in their appearance ; this change proceeds from the periphery of the disc toward the center. At 4.

36

SALOME QLUECKSOHN-WAELSOH

birth, the normal intervertebral disc consists of a central nucleus pulposus surrounded by a mass of fiberlike cells which will give rise to the felted fibers of the adult disc. The first abnormality recognizable in flexed embryos appears a t 14 days after fertilization when the cartilaginous cells of the intervertebral discs fail to be modified into elongated fibrous cells. These cells produce a bridge of cartilage from one vertebra to the next. If this bridge is unilateral, a true flexure will result; if it is bilateral, a straight stiff joint will be found. I n all cases, the original abnormality consists in the failure of the early cartilage of intervertebral discs to differentiate into felted fibers. The effect of “flexed” on the blood has been discussed before (cf. section IV-1) ; flexed homozygotes stiffer from a. transitory anemia, the so-called siderocytic anemia. I n addition, the flexed mutation produces a growth retardation in early embryonic stages as evidenced by shorter lengths of vertebrae. Two hypotheses are advanced to connect the different effects of flexed: (1) that growth retardation is responsible for both anemia and abnormalities of intervertebral discs; (2) that the anemia produces, by reducing the oxygen supply, the more general retardation evident in the axial system. b. Shaker Short. The extensive effects of shaker short on the brain with ensuing abnormalities of hearing and sense of balance have been discussed in another part of this review (cf. section VII-5) ; a “pedigree of causes” traced all these anomalies back to a hypertrophy of the ectodermal germ layer in very early embryonic stages (Bonnevie, 1936a, 1940). The effect of shaker short by which the mutation was originally recognized has not been discussed yet. Homozygous shaker-short mice have short tails. I n her report on the embryogeny of shaker-short mice, Bonnevie refers only briefly to he; studies of the tail development of these mice : “. . u p to about 12 days, the tail of the embryo was quite normal. Now, however, simultaneously with the abortive formations of the plexus (chorioideus), its tip, on account of the blood tension, swells like a blister, resulting in an arrest of development and in a more or less long filamentous thinning out of this tip-known also from other mutations.” It seems thus that circulatory abnormalities may be made responsible for the abnormalities of the tail of the shaker-short mouse, although the details of the abnormal processes do not appear from Bonnevie’s description or illustrations. c. Screw Tail. Another mutation first recognized by its effects on the tail is the screw-tail mutation; its effects on the development of ribs and sternum were discussed before, and it was pointed out that the

.

PHYSIOLOGICAL GENETICS OF THE MOUSE

37

screw-tail gene was supposed to have a general retarding effect on the axial skeleton (section IX-3-a). The tail of the screw-tailed homozygote is coiled a t birth and straightens out later on (MacDowell et al., 1942). Some flexures remain in the caudal, lumbar, and thoracic region of the spine. The centra of some of the vertebrae are shortened, and two or three vertebrae are mising from the tip of the tail. The difference in tail length between normal and screw-tailed mice becomes establirhed a t about 12 days after fertilization and is not due to a decrease in number of soiriites but to a reduction in size of the individual vertebrae. The tail abnormafities are thus considered to be due to a general mesodermal growth deficiency of the axial skeleton. d. [Jnclulatecl. Recently the effects of a previously reported recessive mutation (undulated, Wright, 1947) producing abnormalities in spine and tail have been analyzed in some detail (Griineberg, 1950). Although the critical embryological evidence is still forthcoming, some interesting conclusions have been drawn on the basis of observations on animals between the ages of late fetuses and fully adult individuals. The tail of undulated individuals is shortened and has a number of kinks which are not due to fusions of the vertebrae but to irregularities in the distal ends of cauclal vertebrae. Kyphosis of the lower tlioracic and upper lumbar region may be ohserved. A niimber of anatomical abnormalities of the individual vertehrae of the spine have h e n described. The reduction in size of indivicliial vertebrae is more niarlretl distally than proximally. The undulated syndrome is explained on the following basis : “The mesenchymatous axial hlteletoii of the undulated mouse would differ from that of a normal mouse hy a reduction in size which would bring certain esposed parts below the threshold size necessary for chondrification while other parts, though reduced in size, remain above the threshold and hence chonclrify and ossify normally.’’ There is a n interesting similarity between the effects of screw tail and those of undulated on the asial skeleton, as concluded from the developmental analysis of both of these mutations. T t i both cases, a general mesodermal growth deficiency of the axial skeleton i s considered to h~ produced by the mutations. It wo~ild he interesting to esamine the genetic relationship between scww tail and undulated. This has not been done so far. e. Daiaforth’s Short Tail. The last mutation to be discussed in this group is a dominant mutation with striking effects on the tail and axial skeleton whose urogenital effects were analyzed above (cf. section VI-2) ; it is the Danforth’s short-tail mutation, Sd. Sd heterozygotes have a short tail o r no tail a t all, and sacral as well as remaining caudal verte-

38

SALOME QLUECKSOHN-WAELSCH

brae may be shaped abnormally and fused with each other (QluecksohnSchoenheimer, 1943). The abnormalities of the axial skeleton of the homozygotes are more striking; the tail is always absent, and caudal and sacral vertebrae are missing completely while the few lumbars present are abnormal. The development of the tail abnormalities was studied in heterozygotes and homozygotes (Oluecksohn-Schoenheimer, 1945) ; the tail of hetcrozygotes was found to be normal up to the age of 10 days after fertilization. At 10%-11 days small hematomata are found in the tip of the tail, the somites appear small and irregular, and a progressive degeneration of tail somites in distal-proximal direction may be noted. The hematomata increase in size and sometimes fill one-half to one-third of the entire tail, while all structures in the affected part of the tail such as neural tube, notochord, tail gut, and blood vessels, break down. Progressive cell necrosis may be observed in all affected parts of the tail even prior to the appearance of hematomata. The abnormalities of the tail of the homozygotes are similar in principle but more extensive. Pycnotic granules may be observed in earlier stages in all structures of the tail ; they increase in number as the degenerative process in the tail continues. Eventually all tail structures which had originally formed normally, such as neural tube, notochord, somites, tail gut break down, and, at the end of 12 days, only a filament is left with large hematomata inside. The effect of Sd on the axial skeleton in one and two doses seems to consist in the initiation of a cellular degeneration process which starts out in the mesenchyme of the tail and subsequently attacks notochord, neural tube, and somites. The appearance of hematomata in the abnormal tail is ascribed to a breakdown of the walls of the tail blood vessels rather than to a general circulatory defect in all distal parts of the embryo, because the extremities are always free of such hematomata. Since cell pycnosis is observed even in the absence of hemorrhages, it cannot be a consequence of breakdown of circulation. Cell degeneration is considered to be the first recognizable effect of the Xd-mutation in one or two doses.

EFFECTS OF MUTATIONS IN CHROMOSOME X. THE DEVELOPMENTAL IX OF THE MOUSE From the point of view of the experimental embryologist, the most interesting mutations in the mouse are a group located on chromosome IX. These mutations were first recognized by their effects on the tail of newborn and adult mice; but their main interest lies in their effects

PHYSIOLOGICAL QENETICS OF THE MOUSE

39

on early processes of embryonic growth and differentiation which, in most of the homozygotes of this series, are severe enough to lead to the death of the embryos in early stages. F or details about the genetic behavior of these mutations the reader is referred to earlier papers (cf. e.g., Dunn and Gluecksohn-Schoenheimer, 1950). Suffice it here to say that we are dealing with a group of three closely linked dominant mutations and a number of either allelic or pseudoallelic recessives. Most of these mutations are lethal when homozygous, and animals heterozygous for two of the lethals not showing recombination breed true as a result of a balanced lethal system. The study of the developmental effects of these genes has made it possible to trace the individual steps which lead to the manifestation of the eventual mutant conditions in the axial skeleton of the newborn or the adult. Furthermore, these mutations serve as tools for the study of the normal developmental mechanics of a mammalian organism since it is only through the comparison with the abnormal that we can analyze the processes and events of normal development. Finally, the developmental effects of these genes provide us with material to study the general relationship between genes and embryonic differentiation and thus at least certain aspects of gene action (cf. Gluecksohn-Schoenheimer, 1949b). I n heterozygous condition all the mutations in this group affect thc development of the axial skeleton, and we shall deal first with this aspect of their effects. 1. Brachyury-Heterozygozts

Effect of T

The first mutation whose enibryological effects were analyzed i n detail is a dominant, T, the so-called Brachyury mutation. Animals heterozygous for T have a short tail, and homozygotes die a t about 10 days after fertilization (Chesley, 1935). The tail of the heterozygous embryo is normal up to the age of 11 days after fertilization. At this time a sharp constriction appears in the tail, and the point of constriction determines the end of the tail of the newborn mouse. The part of the tail distal to the constriction becomes smaller and smaller and loses its segmentation. Eventually, only a short filament remains. Histologically, notochordal abnormalities are a characteristic feature of the heterozygote in stages even prior to the onset of external abnormalities. Irregularities of the neural tube are frequent but occur always in conjunction with irregularities of the notochord, indicating the primary character of the notochord anomalies.

40

SALOME QLUECKSOlIN-WAELSCH

2. Taillessness, T / t n The abnormalities of the tail become more extreme when T is combined with one of the recessive mutations of the t-type also located in chromosome IX and behaving like alleles of T. Animals heterozygous for T and one of the t ’ s are usually completely tailless. The embryog eny of such prospectively tailless animals was studied, and it was found that the embryo formed a normal tail which elongated u p to the age of about 11 days after fertilization (Glnecksohn-Schoenheimer, 1938a). At that time a constriction appeared, always at the base of the tail, and subsequently the constricted tail became smaller, lost its segmentation, and eventually only a filament remained. Histologically, the prospectively tailless embryo could be recognized prior to the appearance of the constriction by the absence of a notochord. Neural tube, somites, and tail were formed normally, but were unable to persist and continue differentiation. Actually these striictures dedifferentiated and eventually only undifferentiated mesenchyme was found in their place. We thus see that in T/+ neural tube abnormalities seemed to follow those of the notochord and in T/tn the complete absence of the notochord from the tail preceded the subsequent dedifferentiation of the originally normal tail structures : neural tube, somites, tail gut. An original effect of T alone or in combination with t on the notochord or its precursor is thus one conclusion to be derived from the study of the developmental eflects of T and t, while on the other hand, the decisive role of the notochord in the normal development of the axial skeleton is strongly indicated. More information about the developmental effects of T and some of the t’s and also about the role of the notochord in the developmental mechanics of the mouse embryo was obtained from the study of embryos homozygous for T and for t. 3. Homozygous Effect of 1’

Chesley (1935) reports the absence of any gross abnormalities in the embryo homozygous for T before the age of 8y2 days. A t about 9 days, “small paired or unpaired blebs or small vesicles on either side of the midline” appear. A slight irregularity of the neural tube is observed and the somites are less clear and less regular than in normals. Shortly before death, 1 0 days after fertilization, the homozygous T-embryo shows the following characteristic external features : the posterior region of the body, including posterior limb buds, is missing completely. Normal segmentation is absent but remnants of somites may be recognized. The fore-limb buds are directed dorsad instead of ven-

PHYSIOLOQICAL QENETICS OF THE MOUSE

41

trad. The closure of the neural folds does not occur along one straight mid-dorsal line but deviates laterally and is crooked and twisted. Histologically, the somites are normal in early stages and reduced in number and abnormal in shape in later stages. They are, however, not fused medially. The neural tube shows its twisted shape on sections and is found to send out secondary branches. The notochord forming material appears a t first histologically normal, but the differentiation of the chorda dorsalis is abnormal. By the day during which death occurs, no traces of notochord are present. The notochord is thus the most severely affected structure in the T-homozygote. One of the most interesting conclusions to be drawn from the developmental studies of both T-homozygotes and T-heterozygotes is the one concerning the developmental relationship of notochord and neural tube : “Abnormalities of the neural tube have not been encountered in the absence of those of the notochord while in the lumbosacral region notochordal abnormalities without those of the neural tube are common.” “From the facts reported, supported by experimental evidence from other sources, it is concluded that there is strong indication that abnormality of the notochord is one of the more fundamental of the disorders involved and that the condition of the neural tube is either wholly or in part due to the abnormality of the notochord” (Chesley, 1935). The decisive role of the notochord for normal axial development of the mouse embryo, indicated by the studies of prospectively short-tail or tailless embryos, becomes thus even inore certain as a result of the observations of the development of 7’/T embryos. Ephrussi (1935) explanted tissues from embryos homozygous for T and found that the individual tissues were able to survive far beyond the stage a t which they would have died if left in the embryo. T is thus not a cell lethal and the death of T I T embryos is supposed to be caused by a “disturbance of the normal correlations” and to be due “exclusively to internal factors.” In a n attempt to develop a method by which mouse embryos could be grown under extra-uterine conditions and details of mutant development observed and perhaps attacked experimentally, TIT embryos were explanted into the extra-embryonic coelom of the chick in an early embryonic stage (Gluecksohn-Schoenheimer, 1944). It was shown that the abnormal development of T I T embryos was determined in early egg cylinder stages before becoming apparent morphologically. An incidental observation reported is a severe abnormality of the allantois which prevents the umbilical vessels from developing normally and is responsible for the failure of circulatory connection to become established between mother and embryo. The time of death a t 10 days thus is consid-

42

SALOME QLUECKSOHN-WAELSCH

ered to be determined by the absence of nutrition of the embryo by way of the maternal circulation and the cause of death seems sufficiently explained without any additional assumptions. 4.

t-Type Mutations-Developmental

Effects of to, tl, t4

It has been estimated (Dunn and Gluecksohn-Schoenheimer, 1950) that a t least 14 changes of the t-type have occurred in chromosome I X

of the mouse, i.e., 14 different t-mutations have been detected. Five of these are known to be lethal when homozygous, three are viable, and the behavior of the others in bomozygous condition is not yet known. The first t-type mutation studied in its effects on development in homozygous condition is to (Qluecksohn-Schoenheimer, 1940). Pt0 embryos are reported to be normal u p to a stage soon after implantation in the uterus, the so-called egg cylinder stage. But while the normal embryo now enters a stage of rapid growth and morphogenesis, growth and development of the homozygous embryo cease. It remains in a stage where a n unorganized inner cell mass (=ectoderm) is surrounded by a layer of entoderm (this “inversion of germ layers” is a typical feature of many rodent embryos) until disintegration sets in and the embryo becomes necrotic and is resorbed. During a time when organization of the ectoderm and mesoderm formation characterizn +he development of normal 7 signs of these processes; embryos, the abnormal embryo fails to shou it survives for about 40 hours before it disintegrates. The failure of organization and mesoderm formation is a characteristic feature of totoembryos. Another mutation of the t-type (t’) has been reported to be lethal before implantation (Gluecksohn-Schoenheimer, 1938b). The details of the abnormalities of tltl are not known. Unpublished observations of abnormalities of t4-homozygotes ( Qluecksohn-Waelsch) indicate that the death of these embryos occurs at the age of 7-8 days after fertilization, i.e., a t a stage when embryonic growth and differentiation are most active. 5. Kink-Hmoizygous

Effect of Ki

Still another mutation located on chromosome IX seems to affect processes of early embryonic growth and differentiation. This is the dominant mutation Kink which in heterozygous condition is responsible for the rather typically kinky appearance of the tail. The embryonic events leading to the manifestation of Kink in heterozygous condition have not been analyzed as yet. But the homozygous embryos, which die at about 9 days after fertilization, have been studied in detail (Gluecksohn-Schoenheimer, 1949a). Their abnormalities are typical and strik-

PHYSIOLOQICAL QENETICS OF THE MOUSE

43

ing although varying considerably from one another individually. The feature common to all these abnormal embryos is the presence of duplications of different types and degrees. The duplications reported range from complete or partial duplication of the entire embryonic axis through duplications of organs or parts of them to the presence of only some excess growth in the form of a lump of tissue. On the basis of these observations comparisons are made with results obtained in experiments on amphibian embryos, where constrictions of eggs resulted in different types and degrees of duplications, comparable to those observed in K i / K i embryos. From the existence of these duplications it is concluded that ‘ ‘ the mouse embryo possesses a considerable amount of regulative property in early developmental stages, ’’ and ( ( t ha t organization phenomena and inductive interrelationships between different parts of the embryo known to function in amphibians and birds apparently exist in the mouse as well.’’ I n connection with the duplications in Kink homozygotes a genetically determined duplication of the posterior part of the body of the mouse described by Danforth (1930) is of interest. The details of the genetics of “posterior duplication” are not known; the morphology of the posterior doubling varies considerably from typical duplicitas posterior with ectopic viscera and two complete sets of pelvic organs and hind limbs to those with only a slight degree of doubling. A “pronounced thickening of the ventral tissues a t the posterior end of the embryo” a t 11 days after fertilization is reported as the first observed symptom of the abnormality. ((Th e hypertrophy seems to be initiated in the mesoderm,’’ and “there is evidence of some direct and determining influence of one region on another. ’’

6. Fused The development of Fused, the third dominant tail mutation in chromosome I X in heterozygous or homozygous condition has not been studied in detail but Reed (1937) reports that ( ( a n embryological study of Fused showed the first observable abnormalities to be poor alignment of the notochord and distinct curves and angles of the neural crests,” without, however, giving any evidence for his statement. I n preliminary studies of the embryology of Fused homozygotes we have observed a considerable number of embryos with brain abnormalities resembling pseudencephaly (cf. section VII-3). These embryos are apparently unable to survive until term and die in utero. The effect of Fused in homozygous condition on the development of the anterior part of the neural tube does not seem to be regular, but occurs only in a fraction of the embryos and depends apparently on the residual genotype.

44

SALOME QLUECKSOHN-WAELSCH

We may deal here with another example of the type of developmental reaction discussed above (cf. section IX-1-a). The head abnormalities in Pu/Pw embryos might be due to an increased susceptibility of the prospective head region, or in other words to a lowered threshold of resistance to embryonic traumata which normally would not leave any effect on the developing embryo. The lowered threshold of resistance might in t ur n be due to the presence of the Fused mutation in two doses.

7. A b n m d i t i e s of to/tl Embryos While the main effect of the “tail” mutations of the ninth chromosome of the house mouse a t least when heterozygous seems to be on the posterior part of the body, Fu-homozygotes are not the only individuals in this group of mutations which exhibit abnormalities of the head region. Genetic data from intercrosses of two tailless lines (A and 29) differing in the type of t ( T / t o X T / t l ) had shown that the combination of to with tl produced normal-tailed offspring. However, there seemed to be a deficiency in the number of young of this constitution (to/tl) at birth. Uteri of females supposed to carry to/tl embryos were therefore examined in a search f o r possibly abnormal P/tl embryos unable to survive until term (Dunn and Gluecksohn-Schoenheimer, 1943). Such embryos were discovered and found to have severe abnormalities of the head, such as microcephaly, microphthalmia and anencephaly. Control experiments excluded the possibility that these abnormals were not of the to/tl genotype. Just as in the case of Fused-homozygotes only a fraction of to/tl embryos developed head abnormalities and apparently again the residual genotype played a role in determining the variability of manifestation of the to/tl zygote. But there is no doubt that the anterior part of the axial system may also be affected by the action of these “tail” mutations.

8. Miscellaneous Abnormalities in IncEividuals Carrying Mutations of Chromoame I X A general fundamental disturbance of early embryonic development by mutations on chromosome IX is also indicated by another set of observations ( Gluecksohn-Schoenheimer and Dunn, 1945). Monsters with extreme malformations of the posterior region of the trunk and the extremities (sirenoid malformations), or with striking abnormalities of the head (aprosopi), o r with severe intestinal abnormalities (complete absence of large parts of the intestine) were found among newborn mice which carried different combinations of mutations of chromosome IX. Similar abnormalities had not been observed in numerous other strains at the same laboratory. While they cannot be ascribed to any par-

PHYSIOLOQICAL QENETICS OF THE MOUSE

45

ticular genotype, they seem to owe their origin to the presence of different mutations of chromosome IX. Although these abnormalities have not been traced back embryologically, they are assumed to be caused by severe disturbances of early processes of embryonic growth and differentiation. It seems that the presence of mutations in chromosome IX makes the developmental system of the mouse sufficiently labile so that i t will react with severe disturbances to some embryonic trauma which normally would leave the embryo unharmed. 9. The Role of Chromosome I X in Embryonic Growth and Differentiation Chromosome IX of the mouse thus seems to carry a group of genetic factors all of which are concerned with the control of processes of early embryonic growth and differentiation. No mutation with other effects has been located in this chromosome. This does not mean, o f , course, that chromosome IX plays a n exclusive role in the control of these processes. There is no doubt about the existence of many genetic factors which are involved in the control of the same processes of early growth and differentiation and are located in other chromosomes. The heterozygous effect of several of the tail mutations of chromosome IX has already been shown to be duplicated by tail mutations in other chromosomes of the mouse, as described above. The important point, however, is the grouping of mutations with similar effects in one chromosome regardless of the fact that other such mutations most certainly do exist in other chromosomes as well. The individual mutations in chromosome IX affect either different steps of early embryonic processes, or the same processes in different ways. Evidence for such a n assumption comes first from the fact that individuals heterozygous for two mutations, each of which causes death of the embryo when present in two doses, are viable and sometimes even normal. Furthermore, the embryonic study showed that the developmental changes produced by these mutations were clearly different in the case of the different mutations. Changes in most of these genetic factors lead to abnormalities of derivatives of the notochord-mesoderm material and to phenomena traceable to a n absence of functioning or to abnormal functioning of this same material. Any hypothesis trying to explain the close linkage of genes with closely related effects on embryonic processes has to take into account their peculiar genetic behavior which has been reported in a series of papers (for references cf. Dunn and Qluecksohn-Schoenheimer, 1950). Some speculations in this connection were advanced recently (Gtluecksohn-Schoenheimer, 1949b). The close linkage of mutations with

46

SALOME QLUECKSOHN-WAELSOH

similar embryonic effect was discussed first in the light of Goldschmidt ’s ( 1949) hypothesis about the relation of heterochromatic heredity to processes of early growth and differentiation, and then in connection with Pontecorvo’s ideas (1950) about. the relation between location of genes and the processes they control. The grouping of these genes seems of the greatest significance for problems of gene action and the relation of genes and cell differentiation. XI.

CHANQESINDUCED BY X-RAYS DEVELOPMENTAL

The developpental changes in the mouse embryo discussed so fa r were produced by changes in genes, i.e., mutations. But attempts have been made also to affect developmental processes directly, e.g., with the help of X-rays. Kaven (1938a,b) irradiated pregnant mice with X-rays in different fitages of pregnancy and studied the effect between 7 and 19 days after fertilization. The resulting disturbances of development consisted of brain hernias, hydrocephalus, tail abnormalities, lens turbidity, and fiterility. Since these abnormalities had also been observed as the result of the effect of mutations, it was interesting to compare the sensitive periods during which X-ray irradiation produced a certain type of developmental abnormality with its onset as a result of a mutational change. Kaven found a highly lethal effect as the result of X-ray treatment a t 7 clays, corresponding to the time of manifestation of lethality in embryos carrying mutations which produced changes in early processes of growth and differentiation. Brain abnormalities were induced at 8 days after fertilization, the age at which the first symptoms of genetically determined brain anomalies in mice were observed. X-ray treatment between 9 and 13 days resulted in tail abnormalities, and again it is noteworthy that this is the time at which the first recognizable deviations of tail development could be observed in animals carrying tail mutations. . Recently L. B. Russell (1950) has attacked the same problem on a broader basis, extending the stages of treatment to the very beginning of the gestation period. An analysis of the changes induced by x-ray treatment is expected to reveal “intrinsic patterns of sensitivity in the organism and the discove;y of sensitive periods in particular developmental processes. ’’ These sensitive periods were established throughout the embryonic period by determining prenatal mortality and abnormalities a t birth. A comparison is made which shows a certain degree of parallelism bet ween the abnormalities produced by irradiation and those described as the effects of mutations. However, “the entire set of changes char-

PHYSIOLOGICAL GENETICS OF THE MOUSE

47

acteristic of a particular stage-dose group is usually more inclusive than the set of features characteristic of any parallel mutant.’’ A number of abnormalities described as the result of gene action were not observed in “newborns as a result of irradiation of embryos.” This may, however, be due to the fact that embryos with such abnormalities were unable to survive to term. A combination of results obtained with this method with those arrived at from studies of developmental genetics might prove very fruitful “in bringing analysis of the development of the abnormal character even closer (chronologically) to the original gene action.”

XII. CONCLUDING REMARKS The mutations discussed i n this review have been shown to effect practically every organ system and thus have contributed to the analysis of a great number of different mechanisms involved in the development of form and function of a mammalian organism. A number of general points of interest have emerged from this discussion. When abnormalities observed in the newborn or adult organism are traced back into embryonic stages it frequently appears that the first deviation from normal recognizable in the embryo is not necessarily one in the primordium of the organ which becomes abnormal later on. Such a ease is illustrated, for example, by the analysis of congenital hydrocephalus by Griineberg (1943b) (cf. section VII-2) which could be traced back to an original abnormality of the cartilage and where the effect on the brain was thus quite indirect. Developmental studies of mutations are therefore a prerequisite for the detection of the first visible effects of a mutation before the problem of primary gene action can even be approached. The vast number of genes contributing to the normal development of any organ is reflected in the large number of different mutations which were found to effect any single organ system. Although, as pointed out i n the introduction, only a relatively small and selected number of mutations was chosen for discussion in this review, and actually many more exist, it is evident even so that, e.g., the nervous system, the skeleton, or the skin, are dependent on the collaboration of a great many hereditary factors for normal form arid function. One very interesting phenomenon emerging from developmental studies of mutational effects is the appearance of definite abnormalities as the result of a number of vastly different agencies. Pseudencephaly, for example, was shown to originate as the result of (1) a simple recessive mutation, (2) chromosomal translocations and ensuing chromosomal

48

SALOME GLUECKGOHN-WAELSUH

unbalance, (3) the presence of mutations which lower the threshold of embryonic resistance (cf. section X-6, F u l F u ) , (4 ) X-ray irradiation of embryos. The same syndrome of urogenital abnormalities and those of the posterior trunk region has been shown to result from the action of different mutations (cf. section X-8), and more examples could be cited to illustrate this. phenomenon. The existence of any particular syndrome of abnormalities does therefore not necessarily reflect an identity of hereditary changes or of developmental pathways disturbed. Different mutations and different developmental changes may lead to the same final picture, emphasizing again the interdependence of developmental processes of the vertebrate embryo. Of course all these phenomena cannot be separated from one of the most important problems of gene action, namely that of pleiotropism. Does the gene have one or more than one primary effect P Or are some of its effects subordinated to the primary effect, i.e., a consequence of it 1 The developmental analysis of mutational effects can sometimes help to eliminate the problem, by the discovery, for example, that a number of different adult effects can be traced back to one common embryonic change. However, as the result of such studies, the problem merely ceases to exist in particular cases while it is by no means solved as such. The problem of pleiotropism in a mammalian organism has been discussed repeatedly on the basis of the analysis of different mutations (cf. e.g., Briineberg, 1943b ; Gluecksohn-Schoenheimer, 1945 ; E. S. Russell, 1949b). Briineberg (1943b), as the result of developmental analyses, arrives a t the conclusion that genuine pleiotropism does not exist and that the primary action of the gene is always either cell specific or tissue specific. It has been pointed out before ( Bluecksohn-Schoenheimer, 1949b) that one cannot expect an elucidation of primary gene action from the study of morphological gene effects which in their complexity must be far removed from the original gene effect. The question of whether a gene has one o r more primary functions will have to be approached with different methods. Furthermore (Gluecksohn-Schoenheimer, 1945), it does not seem that we are justified to call the gene’s primary action cell or tissue specific just because the developmental analyses of the gene’s visible effects with the crude morphological methods a t our disposal seem to indicate such a specificity of effect. Russell in her discussion of different effects of W on blood and pigment cells (1949) points out that it is logically possible that “unity of gene action could exist without tissue or cell specificity” and that the same original gene product could be active in two types of cells. She considers the interesting “parallelism of the effects of the entire W

PHYSTOLOGICAL GENETICS OF THE MOUSE

49

series of genes upon the blood and pigment-producing tissues” a n indication of the very close original connection between the two different effects of W and its alleles (cf. section IV-2). It seems that the problem of pleiotropic gene effects will have to wait for further elucidation until a more direct approach to the problem of primary gene action is possible. While prbblems of primary gene action thus cannot be expected to be solved by developmental studies of gene effects of the type discussed here, one of the most significant contributions of such investigations lies in the discovery of normal interdependencies of developmental processes as revealed by the study of the abnormal. A number of such instances have been discussed. I shall only cite again the dependence of the metaneplrros on a n inductive stimulus from the ureter (cf. section VI-3), the dependence of ossification processes in the sternum on normal rib growth (cf. section IX-3-a), the developmental interrelationship between neural tube and ear vesicles (cf. section VIT-s), the significance of the notochord-mesodermal material in mammalian development (cf. section X-3 and 4 ) , the discovery of organizer type phenomena, and of the existence of regulation in early mammalian embryos (cf. section X-5). Most of the early embryonic changes reported concern disturbances of inductive phenomena, while degeneration processes leading to absence of structures are surprisingly rare. It seems that the study of the role of genes in early growth and differentiation should find the mouse embryo a promising object also for future research, due to the existence of organizer and inductive phenomena on the one hand, and of relatively frequent mutations, on the other hand, which interfere with these processes.

XIII. REFERENCES DeAberle, 8. B., 1927, Amer. J . Anat. 40, 219-249. Barnioot, N. A,, 1941, Amer. J . A n d . 68, 497-531. 1945, J . A m t . 79, 83-91. Bonnevie, K., 1934, J . exp. 2001. 67, 443-520. 1936a, Norske Vidsk.-Akad. Oslo Skr. K1. 1, No. 9. 193613, Genetica 18, 105-125. 1940, Handb. Erbbiol. Menschen 1, 73-180. 1943, Norske Viidsk.-Akad. Oslo Skr. K1. 1, No. 4. 1945, Norske Vidsk.-Akad. Oslo Skr. K1. 1, No. 10. Bonnevie, K., and Brodal, A., 1946, Norske 8idsk.-Akad. Oslo Skr. K1. 1, No. 4. Brodal, A., Bonnevie, K., and Harkmark. W., 1944, Norske Vidsk.-Akad. Oslo Skr. Kl. 1, No. 8. Brown, A. L., 1931, Amer. J . Anat. 47, 117-172. Bryson, V., 1945, Anat. Reo. 91, 119-141.

50

SALOME QLUECKSOHN-WAELSOH

Carter, T. C., 1949, Heredity 3, 378-379. 1951, J. Genet. 60, 277-299. Castle, W. E., and Little, C. C., 1910, Science 32, 868-870. Chang, T. K,, 1939, Peking nat. Hist. Bull. 14, 119-132 Chase, H. B., and Chase, E. B., 1941, J. Morph. 68, 279-301. Chesley, P., 1935, J. exp. 2002. 70, 429-459. Clark, F. H., 1934, Anat. Rec. 68, 225-233. CuBnot, L., 1905, Arch. 2001.exp. gSn. 4" series, 3, Nates et Rev. CBXIII-CXXXII. Danforth, C. H., 1930, Am. J. Anat. 46, 275-287. Dunn, L. C., 1934a, J. Genet. 29, 317-326. 1943b, Proc. nat. Acad. Sci., Wash. 20, 230-232. Dunn, L. C., and Einsele, W., 1938, J . Genet. 36, 145-152 Dunn, L. C., and Gluecksohn-Schaenheimer, S., 1943, Gmrtics, 28, 29-40. 1947, J. exp. 2001.104, 25-52. 1950, Proc. nat. Acad. Sci., Wash. 36, 233-237. Dunn, L. C., Oluecksohn-Schoenheimer, S., Curtis, M. R., and Dunning, W. F., 1942, J . Hered. 33, 65-67. Ephrussi, B., 1935, J. ezp. 2002.70, 197-204. Falconer, D. S., Fraser, A. S., and King, J. W. B., 1951, ,7. Genet. 60, 324-344. Foster, Morris, 1951, in press. Fraser, F. C., 1946, Conad. J. Res. D24, 10-25. 1949, Canad. J. Res. 27, 179-185. Gluecksohn-Schoenheimer, S., 1938a, Genetics 23, 573-584. 1938b, Proo. 800. exp. Biol.,N. Y. 39, 267-268. 1940, Genetics 25, 391-400. 1943, Genetws 28, 341-348. 1944, Proc. nat. Acad. Sct., Wash. SO, 134-140. 1945, Genetics, 30, 29-38. 1949a, J. exp. 2002.~110,47-76. 1949b, Growth IX, 163-176. Gluecksohn-Schoenheimer, S., and D u n , L. C., 1945, Anat. Rec. 92, 201-213. Goldschmidt, R. B., 1949, Proc. 8 t h int. Congr. Genet., Hereditas 244-255. Green, E. L., and Green, M. C., 1942, J. Morpb. 70, 1-19. Green, M. C., 1951, J. Morph. 88, 1-21. Griineberg, H., 1936, J. Hered. 27, 105-109. 1937, J. A n d . 71, 236-244. 1938, J. Genet. 36, 153-170. 1942a, J. Genet. 43, 45-68. 194213, J. Genet. 43, 285-293. 1942c, J. Genet. 44, 246-271. 1943a, The Genetics of the Mouse. Cambridge University Press. 1943b, J. Genet. 46, 22-28. 1947, Animal Genetics and Medicine. Paill R. Hoeber, New York. 1950, J. Genet. 60, 142-173. Hardy, M. H., 1949, J. Anat., 83, 364-384. Hertwig, P., 1944, 2. menschl. Vererbungs.-u. Konstitutionslehre 28, 327-354. Holt, S. B., 1948, Ann. of Eugenics 14, 144-157. Hovelacque, A., and Noel, R., 1923, Bull. Biol. France el Belg. 67, 133-142. Hunt, H. R., and Permar, D., 1928, Anat. Rec. 41, 117. Ibsen, R. L., and Steigleder, E., 1917, Amer. N u t . 61, 740-752.

PHYSIOLOGICAL GENETICS OF THE MOUSE

51

Kamenoff, R. J., 1935, J. Y o r p h . 68, 117-155. Kaven, A., 1938a, Z.menschl. Vererbungs.-u. Konstitutionslehre 22, 238-246. 1038b, Z. menschl. Vererbungs.-u. Konstitutionslehre 22, 247-257. Kirkham, W. B., 1919, J. exp. Zool. 28, 125-135. Little, C. C., 1919, Amer. Nat. 63, 185-187. Little, C. C., and Cloudman, A. M., 1937, Proc. nat. Acad. Sci., Wash. 23, 535-537. MacDowell, E. C., Potter, J. S., Laanes, T., and Ward, E. N., 1942, J . Hered. 33, 439-449. Miiller, G., 1950, 2. ntikrosk-anat. Forsch. 66, 520-558. Pontecorvo, G., 1950, Biochem. 800. Symposia N . 4, 40-50. Rawles, M. E., 1947, Physiol. Zo6l. XX, 248-266. Reed, S. C., 1933, Anat. Ilec. 66,101-110. 1937, Genetics 22, 1-13. 1938a, J . exp. Zool. 79, 331-336. 1938b, J. exp. Zool. 79, 337-346. 1938c, J . exp. Zool. 79, 347-354. Reed, S. C., and Henderson, J. M., 1940, J. exp. 2001.86, 409-418. Robertson, G. G., 1942, J . exp. 2002.89, 197-231. Russell, E. S., 1946, Genetics 31, 327-346. 1948, Genetics 33, 228-236. 1949a, Genetics 34, 133-166. 1949b, Genetics 34, 708-723. Russell, E. S., Fondal, E. L., and Smith, L. J., 1950, Rec. Genet. SOC. Amer. 122123. Russell, L. B., 1950, J. exp. Zool. 114, 545-602. Russell, L. B., and Russell, W. L., 1948, Genetics 33, 237-262. Serra, J. A., 1947, Nature, Lond. 169, 504-505. Smith, P. E., and MacDowell, E. C., 1930, Anat. Rec. 46, 249-257. Snell, Gc. D.,1929, Proc. laat. Acad. Sci. W s h . 15, 733-734. 1941, (Ed.) Biology of the Laboratory Mouse. The Blakiston Company, Philadelphia. Snell, G. D., Bodeman, E., and Hollander, W., 1934, J. esp. Zool. 67, 93-104. Snell, G. D.,and Picken, D. I., 1935, J. Genet. 31, 213-235. Sobotta, J., 1895, Arch. mikr. Anat. 46, 15-93. 1902, Arch. mikr. Anat. 61, 274-330. 1911, Arch. mibr. Anat. 78, 271-352. Steiniger, F., 1941, 2. menschl. Vererbungs.-u. Konstitutionslehre 26, 1-27. Tsang, P.-C., 1939, J. comp. Neuro. 70, 1-8. Werneke, F., 1916, Arch. EntwMech. 4 5 72-106. Wright, M. E., 1947, Heredity 1, 137-141.