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The study of mammalian organogenesis by mosaic pattern analysis
Cell Differentiation, 21 (1987) 79-91
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Elsevier ScientificPublishers Ireland, Ltd. CDF 00440
Review
The study of mammalian organogenesis by mosaic pattern analysis P.M. I a n n a c c o n e Northwestern University Medical School, Department of Pathology and the Northwestern University Cancer Center, Chicago, IL 60611, U.S.A.
(Received 9 March 1987)
Chimeras are animals derived from more than one zygote and composed of two cell lineages which are distinguishable in some way at the cellular level. Spontaneous mosaic animals are also composed of distinguishable cell lineages but are monozygotic. The tissues of both mono- and multizygotic animals of this type are mosaic arrays in which aggregates of like cells form patches, the size and distribution of which can be useful in the analysis of diverse problems in developmental biology. Both biochemical and in situ methods have been applied to the elucidation of mosaic pattern. Both forms of mosaicism have proven useful in establishing theoretic constructs of the formation and maintenance of mammalian organs. A number of these constructs are discussed: cell fusion as related to myotube formation; mechanisms of coat pigmentation and the cellular origin of melanocytes; and pattern analyses of the retinal pigmented epithelium, the intestine, liver, adrenal cortex and thymus. Pathologic alterations in such animals have also been studied utilizing mosaic pattern analysis. In particular, neoplastic tumors and their associated preneoplastic lesions have been shown to be clonal. Organogenesis; Mammal; Mosaic pattern analysis; Chimeras; Tumorigenesis
Introduction Mammalian chimeras are multizygotic animals produced experimentally by the amalgamation of preimplantation embryos of distinguishable strains or occasionally distinguishable species. These animals, which are mosaics by virtue of the multiple genotypic lineages which comprise their tissues, have been used successfully in many laboratories for almost three decades to resolve many important developmental problems (Mintz, 1974; Correspondence address: Dr. P.M. Iannaccone. Northwestern University Medical School, Department of Pathologyand the Northwestern UniversityCancer Center, 303 E. Chicago Ave., Chicago, IL 60611, U.S.A.
McLaren, 1976; Gardner, 1978; Iannaccone, 1980a; Weinberg et al., 1985b; Iannaccone et al., 1987a, b, c). The most difficult of these problems have involved elucidation of mechanisms of organogenesis. Although the literature is rich in descriptive data surrounding the formation of organs in most species, very little evidence derived from experimental manipulation exists to shed light on the various mechanisms involved. Mosaic pattern analysis has provided a method to overcome this deficiency (Fig. 1),
Patch and clone size One of the consequences of the combination of two cell lineages in developing tissues of an animal
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is the d e v e l o p m e n t of a n array of alternative cells. A n aggregate of like cells in such an array is a patch which m a y be considered in one, two or three d i m e n s i o n s (Whitten, 1978). The patch m a y result from either f r a g m e n t a t i o n of a larger aggregate by adjacent p o s i t i o n i n g of daughter cells of divisions from the other lineage ( I a n n a c c o n e et al., 1987b), from m o v e m e n t of cells d u r i n g organ d e v e l o p m e n t (Lewis, 1973) or as the result of chance adjacency of r a n d o m l y placed clones of similar type (West, 1975). This final alternative requires that cells move r a n d o m l y d u r i n g developm e n t for a finite time, then stop a n d develop clonally (Fig. 2). It has been argued that a relationship exists between the patch size a n d the coherent clone size based o n the p r o p o r t i o n of the two cell types in the array (West, 1975). The biological relevance of the concept of coherent clones, however, is a m a t t e r of some debate. I n chimeric a n i m a l s the array can be visualized if an
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¢htjnus Fig. 1. Chimeras are made in a number of ways, one of which is depicted in this diagram. Animals of distinguishable strains are mated to produce homozygous embryos. These embryos are isolated from the mother at the eight-cell cleavage stage and the zonae pellucidae are removed, usually by brief exposure to acidified solutions. The embryos are then pushed together in the presence of phytohemagglutinin and incubated until the blastomeres are intermingled. The 'double' embryo is then surgically implanted in the uterus of pseudopregnant surrogate mother, generally of a third distinguishable strain (the pregnant stippled rodent in this diagram). Offspring have a striped coat if the original embryos were derived from pigmented and unpigmented strains. If the original embryos were from congenic strains, the chimeric offspring will be morphologically indistinguishable from the parental strains. When the original embryos are from strains which can be distinguished histologically, the mosaicism at the tissue level can be established. The mosaic patterns revealed by this process have led to discussion of morphogenesis in several organs depicted here and discussed in the text: liver; adrenal cortex; thymus; intestine; and retina. The data described are from both mouse and rat.
Fig. 2. The diagram depicts an idealized field of mosaic tissue. CIonal expansion within a small hexagonal area can lead to a more complicated patch pattern by coalescence with adjacent clones which by chance are of the same type. The hexagonal areas made up of individual cells are known as coherent clones while the irregular area formed by the chance coalescence of several coherent clones of the same type is a patch. Patches can become highly complex geometrically and their size in some organs is a function of the proportion of the two types in the mosaic field (lannaccone, 1980a; Iannaccone et al. 1987a, c). Reproduced with permission from the Int. J. Cancer.
81 in situ marker of mosaicism is available (as shown below). Otherwise, the size of the patch (or aggregates of like type cells) must be deduced by estimating the variance in proportion of the two cell types between samples of uniform small size from the mosaic tissue (Iannaccone, 1980a).
Myotube formation in skeletal muscle Skeletal muscle consists of multinucleated myotubes as a fundamental unit of contraction. For many years the mechanism of formation of these myotubes was in controversy. The alternative explanations included cell fusion between many primordial cells or endoreduplication of DNA without cell division. Mintz and Baker (1967) resolved the problem of the origin of multinucleated myotubes in the skeletal muscle of mice in favor of cell fusion rather than acytokinetic mitosis. Chimeras were produced between C3Hf and D B A / 2 or between C57BL/6 and CBA mice which expressed variant electrophoretic isoforms of NADP-dependent isocitrate dehydrogenase (IDH; EC 1.1.1.42). The chimeras were thus Idl a / a o Id-I b/b. Most tissues displayed only two isoenzymic forms of IDH which is a dimeric and dimorphic enzyme. However, skeletal muscle displayed three isoenzymes, the third was a heteropolymeric form created by the combination of subunits of the variant and the non-variant form of the enzyme. This event could have occurred only in a cell which had active genes from both lineages used to form the chimera, and thus must be the result of cell fusion. If the muscle myotubes had been formed by acytokinetic mitosis, then only the two homopolyrneric forms of the enzyme would have been present, as in the other non-muscle tissue of the animals.
Coat pigmentation pattern Chimeras produced between mice with variant coat colors display a striped pattern of color variegation which is consistent with the concept of melanocyte migration from the neural crest of the fetus. If, as was thought for many years, the
melanocyte was derived from epidermal components then a salt and pepper pattern of mosaicism would have been expected. Instead, broad bands or stripes of coat color were always obtained. Moreover, there was a mid-dorsal discontinuity such that stripes on one side of the animal did not match stripes on the other side. This indicated that the two sides had independently established melanocyte clones beginning at the midline. Originally it was thought that each stripe represented a separate clone of melanocytes beginning as a single progenitor in the neural crest (Mintz, 1967). This was later shown to be an oversimplification, and it is now generally held that the banded pattern is the manifestation of 64 randomly arranged progenitor melanocytes (32 on each side) of the two genetically distinct types (Wolpert and Gingell, 1970; West, 1975). In the rat, coat pattern analysis has revealed that the hooded pigmentation pattern (under the control of at least three recessive alleles, hi, h i and h n) is the result of a gene which acts to retard the migration of the melanocyte from the neural crest or prevents its entrance into the hair follicle (Yamamura and Markert, 1981). X-Chromosome inactivation
The analysis of coat color pattern has also been used to help establish the timing of X-chromosome inactivation. In eutherian mammals one X-chromosome of the female is inactivated in each cell present in the early embryo. Amongst inner cell mass cells or their derivatives this process appears random with respect to which X-chromosome is inactivated. The process is generally irreversible, and genetically imprints the inactive X-chromosome so that the same X-chromosome is inactive in all progeny cells. In trophectodermal lineages, however, the paternal X-chromosome is preferentially inactivated, and the process appears to occur earlier. This fundamentally important process is a differentiative act in embryogenesis, and its timing has been studied in a variety of ways (Frels and Chapman, 1979; Frels et al., 1979; Frels and Chapman, 1980; Papaioannou and West, 1981). Coat color analysis of chimeras was elegantly utilized in one such study. Cells from embryos with Cattanch's translocation were injected at 3 and 4 days post-coitum into blasto-
82 cysts from a strain which yields sandy colored progeny. If X-chromosome inactivation had already occurred at the time of injection, the resulting chimeras would either be albino and sandy (i.e., the wild type X-chromosome is inactive) or agouti and sandy (i.e., the wild type X-chromosome is active). If, on the other hand, X-chromosome inactivation had not yet occurred in the injected embryonic cells, the resulting chimeras would have all three colors in their coats (albino, sandy, and agouti). The latter result was obtained for injected cells of both 3-day- and 4-day-old embryos (day of sperm discovery = day 0). Thus, X-chromosome inactivation in the inner cell mass of the mouse occurs after day 4 (Gardner and Lyon, 1971).
Cell fate analysis Coat color analysis also allowed the determination of the number of cells within the inner cell mass which are necessary to form the fetus and give birth to offspring. The mouse blastocyst consists of an inner cell mass and a spherical trophectoderm. The inner cell mass at the blastocyst stage of development is thought to consist of some 15 cells in the mouse. It is not known, however, how many of these are actually needed to form the fetus. Progeny of the trophectodermal cells will develop into extraembryonic tissues. Hexaparental mice and octaparental mice were constructed by aggregating embryos of either three or four strains of mouse with distinguishable coat colors. The resulting animals had coats with one, two, or three separate colors indicating lineage contribution from up to three different strains. Expected probabilities from polynomial expansions were compared with observed frequencies of the various combinations of colors in these animals, and it was possible to conclude that at least three cells in the inner cell mass, but probably not more than three cells, contributed to the formation of the fetus and thus all adult tissues (Markert and Petters, 1978).
Retinal pigmentation pattern Genotypic variation in pigment was used to establish patterns of mosaicism in the retina of
mice made between strains which were pigmented and those which were not. The patch pattern of pigmentation in the retina of such chimeras was examined at the microscopic level, both in one dimension and in two. These studies established a mathematical relationship between the proportion of cells from the two lineages which comprised the mosaic and the size of linear patches of cells of contiguously similar type (West, 1976a). The mean one-dimensional patch length was estimated as 1 / ( 1 - p ) where p is the proportion of the cell type under consideration in the section. The estimated coherent clone length was the observed patch length/expected patch length. The number of cells within the coherent clone was estimated as (mean coherent patch length/mean cell length) 2. The analysis allowed the conclusion that patches within the retina were essentially random in distribution and that the number of coherent clones and distribution of patches were similar between chimeras and X-chromosome-linked mosaics. The relationships were extended to two- and three-dimensional analyses by computer simulation (West, 1975; Whitten, 1978). These mathematical analyses proved the intuitively held notion that a background lattice would quickly form in a two or three dimensional mosaic tissue as the proportion of the major cell type increased. This would create islands of cells of the minor lineage within seas of cells of the major lineage. In highly unbalanced chimeras the pattern of mosaicism was shown to be the result of stochastic rather than deterministic development (Schmidt et al., 1986). In previous experiments, the conclusion that the retina forms by deterministic growth was supported by the contention that patches were distributed in various sectors of the retina in a non-random manner (Mintz and Sanyal, 1970; Sanyal and Zeilmaker, 1977). Further mathematical analysis, however, has shown that the distribution of patches in such animals is random, at least when the mixtures of pigmented and unpigmented cells are highly unbalanced. Patches are geometrically complex both in real and simulated mosaics. This complexity can be appreciated by examining the high degree of scatter within a distribution of patch sizes (Schmidt et al., 1986; Iannaccone et al., 1987b). The conclusion that such patterns of mosaicism
83 would arise as the result of cell mingling is substantiated by a generational computer model developed to study the formation of mosaic pattern in chimeric liver in the rat (shown below).
In situ markers of mosaicism A major limitation of many. of the studies utilizing chimeras and X-chromosome-linked mosaic animals has been the absence of practical markers which allow the distinction of cell lineages in histologic sections and provide the opportunity to analyze tissues in situ. This limitation has lessened dramatically in recent years. Histochemical markers The first usable histologic marker of chimerism was produced by exploiting the histochemical method for discerning the presence of flglucuronidase (EC 3.2.1.31). Since there is a strain of mouse which is deficient in the expression of this enzyme, it was possible to produce chimeras between strains which express this enzyme and those which do not. Mosaicism was then demonstrated by staining frozen tissue sections for the presence of fl-glucuronidase (Condamine et al., 1971). In other studies utilizing this marker, the liver was shown to be comprised of many more coherent clones than was previously thought (West, 1976b; Wegmann, 1970). It should be noted, however, that there was an apparent zonal distribution of the enzyme activity within the liver, suggesting that this metabolic enzyme might be modulated by circulatory events in addition to the distribution of the fl-glucuronidase deficient lineage. A similar enzyme (fl-galactosidase; EC 3.2.1.23) was localized histochemically and revealed a patchy distribution of lineage within the salivary gland (Dewey and Mintz, 1978). This marker also was reported to distinguish lineages in the kidney and in the pancreas. DNA markers A very high resolution histologic marker was developed based on the differences in satellite DNA between different species of Mus. By creating a DNA library, which included clones contain-
ing DNA capable of hybridizing with divergent, highly repetitive sequences of satellite DNA of one species but not the other, a system was developed to allow autoradiographic distinction of any nucleated cell of either species. Interspecific chimeras between Mus musculus and Mus caroli were produced and their tissues subjected to in situ hybridization utilizing a radioactive DNA probe capable of recognizing the satellite sequence of M. musculus. This probe proved valuable for the analysis of cell fate within early embryonic lineages and established that a strict species barrier to implantation of transferred embryos existed at the level of placentation. This marker, however, was less well suited to the study of organ development (Rossant et al., 1983a, b; Siracusa et al., 1983). Immunocytochemical markers It has been evident for some time that a system based on major histocompatibility haplotypes might be useful to establish mosaic pattern between congenic strains of varying haplotype. Several such systems have emerged in recent years. Most notable among these are in mouse and in rat. Congenic strains of mouse which express different class I alloantigens of the MHC were produced and direct immunocytochemical techniques were used to localize cell descendants of the two lineages present within chimeras (Ponder et al., 1983). A similar approach was taken utilizing congenic strains of rat (Weinberg et al., 1983, 1985a; Iannaccone et al., 1984). A library of monoclonal antibodies recognizing various epitopes of these antigens was employed to distinguish cells of these strains of rat. Direct iodinated antibodies were applied to tissue sections from chimeras between these strains. Autoradiograms revealed mosaic patterns in most of the tissues of the animals, since the system works in most visceral tissues of the animals (Weinberg et al., 1985b). Direct immunohistochemical localization has led to a number of observations which have allowed deductions concerning the formation and maintenance of several of the organs of these animals (Iannaccone et al., 1987a; Iannaccone and Weinberg, 1987).
84 The determinant density of the brain substance and neurectodermal derivatives which include peripheral nerves and adrenal medulla in the animals is very low, and the antibodies utilized for these studies do not react with these tissues. Vascular tissues within the brain react strongly, and the specificity of this reaction was established with appropriate absorption studies (Weinberg et al., 1985a).
In situ analysis of mosaicism
Following the production of chimeras between strains with different MHC antigen expression, the application of monoclonal antibodies capable of distinguishing the haplotypes was used for studying mosaic pattern in numerous tissues of the animals.
Intestine epithelium Chimeras between H-2 k and H-2 b strains of mouse were used to show that the epithelium of individual crypts of both the small and large intestine of adult animals is composed exclusively of one or the other of the two cell types that compose the animals. The villi of the intestinal lining, however, frequently demonstrated contributions from both cell lineages, indicating that epithelium had been derived from more than one crypt. The number of patch boundaries to be expected in a population of intestinal epithelial cells was estimated to be approximately 1 boundary per 35 cells as an overall average, thus establishing the very low probability that the crypt populations were composed of cells derived from a single lineage by chance alone (Ponder et al., 1985; Schmidt et al., 1985a, c). Statistical analysis of patch distribution within a field of mosaic tissue was performed in intestine of a highly unbalanced chimera by utilizing histological analysis of a polymorphism of Dolichos biflorus agglutinin (DBA) binding as a marker of mosaicism. The object was to determine whether the patches are randomly distributed or represent the result of some sort of pattern. The results of this analysis showed that at least in this animal the intestinal patch distribution did not appear to
be random (Schmidt et al., 1985b). However, it must be kept in mind that this sort of analysis does not demonstrate that pattern formation in the mosaic field is the result of non-random forces. Particularly in highly unbalanced mosaic fields the random adjacent positioning of daughters from randomly dividing cells would result in 'non-random' placement of patches, since the sites of progeny clones would be relatively restricted by the original placement of the marked cell. This can be demonstrated with computer simulations of the generation of highly unbalanced mosaic fields. These simulations also demonstrate that patch fragmentation can result from probabilistic or stochastic division decisions and does not require extensive cell movement, only random adjacent positioning of the daughters of any randomly chosen dividing cell. This mechanism can operate at any developmental stage in which division of cells is occurring, not just during embryogenesis.
Chimeric fiver In the rat chimeric liver an apparently random pattern of patch distribution was observed. The liver develops concurrently from the endodermal hepatic primordium, which begins as a thickening on the ventral surface of the foregut, and the vascular network, which is derived from the umbilical vein. The hepatoblastic epithelium expands as irregular masses of cells and not as well formed cords. These masses eventually form a single continuous plate, the muralium simplex (Du Bois, 1963). The adult organ is a complex structure organized around a circulatory tree which both brings nutrients and removes metabolites. In this representation, the basic unit of liver morphology is the liver acinus (Rappaport et al., 1983). Another view presents the basic unit of liver morphology as the liver lobule, consisting of interconnecting 'portal tracts' forming a roughly hexagonal area with a central vein in the middle (Beketova and Sekamova, 1983). Mosaic pattern in chimeric rat liver was unrelated to either the acinar or the lobular architecture of the liver (Fig. 3; Iannaccone et al., 1987b). A linear relationship between the proportion of the two cell types and the size of the patches was observed. There is an inverse linear relationship
85 generate a model of the formation of the mosaic tissue. This model was used to predict what sort of decisions might be required to result in the mosaic obtained. This approach led to the conclusion that probabilistic decisions may be all that are required to establish the pattern observed in real chimeric liver tissue. The random placement of the cells in the liver primordium, the random selection of cells within the anlage for division, and the random placement of daughter cells in an adjacent position resulted in a mosaic quantitatively similar to that observed in the real liver tissue (Berkwits and Iannaccone, 1985; Iannaccone et al., 1987b).
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Fig. 3. Computer-generated digital data from autoradiograms of chimeric rat liver sections showing the distribution of patches of one of the lineages present within the mosaic (top). A serial section was pin-registered to the autoradiogram (i.e., the sections were aligned using independent registration marks in the tissue). Several liver lobules are outlined (. . . . . . ). The liver lobule is roughly hexagonal in cut section and is defined by portal spaces (ps) and has a central vein (cv) in the center. The liver acinus is arranged around a circulatory tree defined by the terminal hepatic arteriole. An example of a liver acinus is outlined ( . . . . . . ). Z1, Z2, and Z3 refer to zones of progressively poorer quality arterial blood supply. A sublobular vein (slv) is present in the section. There is no apparent relationship between the distribution of patches and either lobular or acinar architecture. Reproduced with permission from the Company of Biologists, Ltd. (Iannaccone et al., 1987b). between the proportion of the minor cell type and the number of patches of that type in a given area of liver. These relationships were then used to
The adrenal gland has a dual embryological origin. The cortex of the organ first appears as a condensation of mesoblast in coelomic mesoderm at the level of the kidney in the mouse on day 13. Coelomic epithelium lateral to the gastric mesentary divides forming large polyhedral cells which extend as cords into mesoderm medial to the urogenital ridge. Cells from the capsule of the nephric glomerulus migrate to the developing gland, and these cells appear to give rise to the stromal elements. The medulla of the organ, on the other hand, arises from clusters of sympathochromaffin cells which are believed to be derivatives of ganglia in the area. The gland enlarges by the continuous migration of coelomic epithelium into the organ. The cortex of the adrenal gland is known to consist of three zones believed to be different histotypes (Arnold, 1886). More than 100 years ago Gottschau (1883) suggested that cell migration was centripetal through all three histozones. The adrenal cortex demonstrated a striped pattern of mosaicism (Fig. 4). This pattern was consistent with the concept that the gland develops from the clonal proliferation of primordial cells in which random movements are selectively excluded. The parallel rays of different cell types in the cortex may serve as an indication of the number of participating clones in the original primordium. The relationship between the clone size and proportion of the two cell types and between the clone number and the two cell types does not follow the relationship observed in the
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Fig. 4. Diagrammatic representation of the results of step-sectioned adrenal glands in chimeric rats. The diagram summarizes results from autoradiographic analysis of mosaic pattern in a number of sections of several glands from animals which are composed of cells derived from lineages which express different class I major histocompatibility alloantigens. As the depth of section proceeds to the center of the gland, the pattern of mosaicism changes from patches to wedges and finally stripes.
liver (Iannaccone et al., 1987b). Thus, random division and random adjacent placement of the daughter cells in succeeding generations would not be sufficient to explain the mosaic pattern observed in this organ. The pattern is much more consistent with the concept of surface signal recognition and the formation of boundary conditions as has been described for the morphogenesis
in avian feather where cell adhesion molecules may represent a critical surface signal for morphogenesis (Chuong and Edelman, 1985a, b; Crossin et al., 1985; Gallin et al., 1986). There is a change in the observed pattern of mosaicism as one obtains sections from progressively deeper portions of the organ. The outermost surface of the organ shows a patchy distribution of mosaicism while deeper sections display a wedge shaped distribution of the two lineages which comprise the organ. The centermost sections show a parallel array of cords of cells composed exclusively of one or the other lineage (Fig. 5). These patterns are most consistent with a model in which columns composed of cells derived from the minor lineage extend through the entire thickness of the cortex. The corners of the sections from the center of the organ can be informative with regard to the question of the direction of division. Gottschau suggested that the adrenal cortex developed by centripetal division. The concept that division proceeds from the outside toward the inside was suggested by extirpation of the central portion of the organ with subsequent complete renewal from the remaining rim of capsule and zona glomerulosa. Mosaic pattern analysis has shown that parallel cords of cells extend through all of the histogenic zones of the organ and intersect in such a way as to suggest that the divisions required for renewal of the organ if not its formation are occurring from the outside toward the inside (Iannaccone and Weinberg, 1987; Iannaccone et al., 1987a). The role of cell-cell contact and adhesion in organ development has been discussed for many years (Saxrn and Wartiovaara, 1966). One possible explanation of the pattern in the adrenal is that boundary conditions in which cell surface recognition molecules are different in two areas of the tissue and the direction of adjacent positioning of the daughter cells or even the decision to divide are constrained as result. Such a system has been proposed and experimentally supported in avian feather embryogenesis (Gallin et al., 1986). These boundary conditions should not exist in the liver parenchyma because of the random nature of the divisions which are required to produce the mosaic pattern observed.
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Fig. 5. Photomicrographs of unstained autoradiograms of adrenal glands from two different chimeras with widely different proportions of cell types. The gland on the left was 38% PVG cells, while the gland on the right was 9570 PVG cells. The parallel array of cells is evident in both cases. Bar = 0.5 mm. Reproduced with permission from J. Exp. Zool. (lannaccone and Weinberg, 1987).
Thymus The thymus is another organ of dual embryologic origin. The thymus originates from the primitive pharynx as an epitheliomesenchymal rudiment. The epithelial portion is thought to be endodermal in origin. Neural crest cells migrate to the developing thymic buds and contribute to mesenchymal elements (von Gaudecker, 1986). The thymic lymphocytes can arise either by transformation of existing thymic epithelium or by substitution from mesenchymal cells. Extrinsic sources of thymic lymphocytes have been demonstrated in birds, amphibia and mammals, although the precise origin of these cells is not known. The thymic lymphocytes enter the cortical regions of the gland in several waves during fetal development. Adult cortical lymphocytes are processed by the cortex and then move to the medulla for export to the circulation. While some theories of cortical lymphocyte trafficking may suggest that residence time within the cortex is short, the fact that chimeras can tolerate tissues derived from both parental lineages and yet retain immunologic specificity might mean that stem cell proliferation
occurs within the thymic cortex (Howard, 1980). Alternatively, the forbidden clone theory of Burnett argues that clones of cells not recognized as self within a competent immune system would be eliminated as they arise (Deol, 1973). Renewal of intrathymic lymphocytes is a physiologic process occurring throughout the life of the animal. It remains to be determined whether or not the process is continuous or discontinuous. The use of radiation chimeras (produced by sublethal irradiation with subsequent repopulation of the bone marrow with genetically distinct cells) suggests that there are two lineages of cells involved: one which populates both the medulla and the cortex; and one which populates the medulla alone (Ezine et al., 1984). Sections of the thymus from a young adult chimeric rat produced between histologically distinguishable strains revealed a pattern consistent with such oligoclonality (Fig. 6). The histologic analysis of mosaicism in normal development is an area of great potential importance to theories of immune tolerance as well as mechanisms of thymic lymphocyte traffic.
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Mosaic analysis of pathological alterations
mann, 1982). Similar results were obtained with a dimorphism in the mouse that results from Xchromosome inactivation. Such X-chromosomelinked mosaic animals were subjected to a variety of carcinogenic treatments, and a wide range of tumors were shown to be clonal in origin (Deamant and Iannaccone, 1985; Iannaccone et al., 1987c). Inflammatory cells within the tumor confounded the isozyme results in some studies which reported non-clonal origins of carcinogen-induced fibrosarcomas. Some studies utilizing X-chromosomelinked mosaics showed both isozymes present within the tumors indicating a non-clonal origin of these cancers (Reddy and Fialkow, 1979). These animals are mosaic by virtue of heterozygosity at the PGK locus which is on the X-chromosome. One of the two X-chromosomes is randomly, and irreversibly, inactivated in embryonic lineages of mammalian females. In M. musculus ~ M. caroli chimeras (described above), however, the in situ marker revealed that inflammatory cells within the fibrosarcoma were derived from both lineages, but the neoplastic cells were derived from a single lineage (Deamant et al., 1986).
Tumor formation A number of current theories of carcinogenesis require that rare events, such as mutations, are critical to the formation of cancers (Reddy et al., 1982; Shih and Weinberg, 1982; Tabin et al., 1982; Sukumar et al., 1983). Rare affected cells would then, as a result, clonally expand into neoplasms. The clonal origin of chemically induced, physically induced and spontaneous cancers has been established by the use of isozymic markers of mosaicism (Fialkow, 1976; Iannaccone et al., 1987c). Chimeras have been produced between strains of mouse which varied in their expression of glucosephosphate isomerase (GPI-1; EC 5.3.1.9; DeLorenzo and Ruddle, 1969). The electrophoretic patterns of tumors chemically induced in the skin and mesenchymal tissues of such animals showed the tumors to be composed exclusively of cells of one or the other type comprising the normal tissues of the chimeras. Thus, the tumors appear to result from the clonal expansion of single, affected cells (Iannaccone, 1980b; Iannaccone et al., 1978; 1987c; Elbing and Sauer-
Preneoplastic changes It is now generally held that the formation of malignant cancers is a multistep process (Farber and Cameron, 1980). It is possible that if a critical event occurs in rare cells at an early step in carcinogenesis, then all preneoplastic steps leading to the development of the cancer would produce clonal lesions. This appears to be the case. Epidermal cancers produced by chemical induction are preceded by the development of papillomas which are the result of clonal expansion of single affected cells (Iannaccone et al., 1978). Further, the development of hepatocellular carcinoma as a result of chemical carcinogens is generally held to involve a number of preneoplastic steps which result in lesions including: nodular morphological alterations called hyperplastic nodules; areas of altered expression of certain hepatic enzymes; and areas of altered glycogen mobilization. These areas represent phenotypic alterations in hepatocyte populations and are thought to be early steps in the formation of hepatocellular carcinoma (Becker, 1978; Bannasch et al., 1979, 1980). The concept
Fig. 6. Photomicrograph of an unstained autoradiogram of the thymus gland from a young PVG *-*PVG-RT1 a chimeric rat. Several large patches of cells of the PVG lineage (white) are evident within the cortex of the gland. The scalloped interface between the cortex and the medulla may indicate that cortical cells may move to the medullary region on broad fronts, x 22. Reproduced with permission from Cold Spring Harbor Laboratory (Iannaccone et al., 1987a).
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approach has proven useful in establishing theories of organ formation and maintenance. The use of mosaic pattern analysis has also been instrumental in establishing the clonal origin of neoplasms and preneoplastic lesions. With the development of new markers of mosaicism the future should continue to provide insights into the organization of mammalian tissues.
Acknowledgements Fig. 7. Photomicrograph of a frozen section from the liver of a chimeric rat following partial hepatectomy, treatment with the liver carcinogen diethylnitrosamine and promotion with phenobarbital. The section was incubated with an iodinated monoclonal antibody which recognizes cells of the PVG-RT1 a lineage. The patch pattern of the liver is distorted by a nodular lesion which had an altered enzyme expression as described in the text. This carcinogen-induced lesion can be seen to be composed exclusively of one of the two cell types present in normal tissue and distorts the surrounding patch pattern. Thus, it represents the clonal expansion of a single affected cell. Bar = 2.0 mm. Reproduced with permission from Int. J. Cancer (Iannaecone et al., 1987c).
that these lesions are involved in the formation of the cancer is supported by a number of observations from many laboratories over the years. By causing their formation in chimeric liver it was possible to establish the clonal origin of many hundreds of these lesions (Weinberg and Iannaccone, 1986; Weinberg et al., 1987). Areas of altered enzyme expression were phenotypically heterogeneous, exhibiting multiple alterations, and yet all of the cells in such areas were demonstrated to be composed of cells exclusively of one or the other of the two cell lineages present in normal tissues (Fig. 7). These results indicate that the earliest steps of chemical induction of liver cancer are clonal, and that phenotypic heterogeneity and complexity within the preneoplastic nodules are the result of progression and not of multicellular origin of the lesions.
Conclusions Multizygotic animals provide the opportunity to analyze mosaic pattern within organs. This
This work was supported in part by USPHS grants CA29078 and ES03498 and MOD Birth Defects Foundation grant 15-49.
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79
Elsevier ScientificPublishers Ireland, Ltd. CDF 00440
Review
The study of mammalian organogenesis by mosaic pattern analysis P.M. I a n n a c c o n e Northwestern University Medical School, Department of Pathology and the Northwestern University Cancer Center, Chicago, IL 60611, U.S.A.
(Received 9 March 1987)
Chimeras are animals derived from more than one zygote and composed of two cell lineages which are distinguishable in some way at the cellular level. Spontaneous mosaic animals are also composed of distinguishable cell lineages but are monozygotic. The tissues of both mono- and multizygotic animals of this type are mosaic arrays in which aggregates of like cells form patches, the size and distribution of which can be useful in the analysis of diverse problems in developmental biology. Both biochemical and in situ methods have been applied to the elucidation of mosaic pattern. Both forms of mosaicism have proven useful in establishing theoretic constructs of the formation and maintenance of mammalian organs. A number of these constructs are discussed: cell fusion as related to myotube formation; mechanisms of coat pigmentation and the cellular origin of melanocytes; and pattern analyses of the retinal pigmented epithelium, the intestine, liver, adrenal cortex and thymus. Pathologic alterations in such animals have also been studied utilizing mosaic pattern analysis. In particular, neoplastic tumors and their associated preneoplastic lesions have been shown to be clonal. Organogenesis; Mammal; Mosaic pattern analysis; Chimeras; Tumorigenesis
Introduction Mammalian chimeras are multizygotic animals produced experimentally by the amalgamation of preimplantation embryos of distinguishable strains or occasionally distinguishable species. These animals, which are mosaics by virtue of the multiple genotypic lineages which comprise their tissues, have been used successfully in many laboratories for almost three decades to resolve many important developmental problems (Mintz, 1974; Correspondence address: Dr. P.M. Iannaccone. Northwestern University Medical School, Department of Pathologyand the Northwestern UniversityCancer Center, 303 E. Chicago Ave., Chicago, IL 60611, U.S.A.
McLaren, 1976; Gardner, 1978; Iannaccone, 1980a; Weinberg et al., 1985b; Iannaccone et al., 1987a, b, c). The most difficult of these problems have involved elucidation of mechanisms of organogenesis. Although the literature is rich in descriptive data surrounding the formation of organs in most species, very little evidence derived from experimental manipulation exists to shed light on the various mechanisms involved. Mosaic pattern analysis has provided a method to overcome this deficiency (Fig. 1),
Patch and clone size One of the consequences of the combination of two cell lineages in developing tissues of an animal
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is the d e v e l o p m e n t of a n array of alternative cells. A n aggregate of like cells in such an array is a patch which m a y be considered in one, two or three d i m e n s i o n s (Whitten, 1978). The patch m a y result from either f r a g m e n t a t i o n of a larger aggregate by adjacent p o s i t i o n i n g of daughter cells of divisions from the other lineage ( I a n n a c c o n e et al., 1987b), from m o v e m e n t of cells d u r i n g organ d e v e l o p m e n t (Lewis, 1973) or as the result of chance adjacency of r a n d o m l y placed clones of similar type (West, 1975). This final alternative requires that cells move r a n d o m l y d u r i n g developm e n t for a finite time, then stop a n d develop clonally (Fig. 2). It has been argued that a relationship exists between the patch size a n d the coherent clone size based o n the p r o p o r t i o n of the two cell types in the array (West, 1975). The biological relevance of the concept of coherent clones, however, is a m a t t e r of some debate. I n chimeric a n i m a l s the array can be visualized if an
ixtmstirm
¢htjnus Fig. 1. Chimeras are made in a number of ways, one of which is depicted in this diagram. Animals of distinguishable strains are mated to produce homozygous embryos. These embryos are isolated from the mother at the eight-cell cleavage stage and the zonae pellucidae are removed, usually by brief exposure to acidified solutions. The embryos are then pushed together in the presence of phytohemagglutinin and incubated until the blastomeres are intermingled. The 'double' embryo is then surgically implanted in the uterus of pseudopregnant surrogate mother, generally of a third distinguishable strain (the pregnant stippled rodent in this diagram). Offspring have a striped coat if the original embryos were derived from pigmented and unpigmented strains. If the original embryos were from congenic strains, the chimeric offspring will be morphologically indistinguishable from the parental strains. When the original embryos are from strains which can be distinguished histologically, the mosaicism at the tissue level can be established. The mosaic patterns revealed by this process have led to discussion of morphogenesis in several organs depicted here and discussed in the text: liver; adrenal cortex; thymus; intestine; and retina. The data described are from both mouse and rat.
Fig. 2. The diagram depicts an idealized field of mosaic tissue. CIonal expansion within a small hexagonal area can lead to a more complicated patch pattern by coalescence with adjacent clones which by chance are of the same type. The hexagonal areas made up of individual cells are known as coherent clones while the irregular area formed by the chance coalescence of several coherent clones of the same type is a patch. Patches can become highly complex geometrically and their size in some organs is a function of the proportion of the two types in the mosaic field (lannaccone, 1980a; Iannaccone et al. 1987a, c). Reproduced with permission from the Int. J. Cancer.
81 in situ marker of mosaicism is available (as shown below). Otherwise, the size of the patch (or aggregates of like type cells) must be deduced by estimating the variance in proportion of the two cell types between samples of uniform small size from the mosaic tissue (Iannaccone, 1980a).
Myotube formation in skeletal muscle Skeletal muscle consists of multinucleated myotubes as a fundamental unit of contraction. For many years the mechanism of formation of these myotubes was in controversy. The alternative explanations included cell fusion between many primordial cells or endoreduplication of DNA without cell division. Mintz and Baker (1967) resolved the problem of the origin of multinucleated myotubes in the skeletal muscle of mice in favor of cell fusion rather than acytokinetic mitosis. Chimeras were produced between C3Hf and D B A / 2 or between C57BL/6 and CBA mice which expressed variant electrophoretic isoforms of NADP-dependent isocitrate dehydrogenase (IDH; EC 1.1.1.42). The chimeras were thus Idl a / a o Id-I b/b. Most tissues displayed only two isoenzymic forms of IDH which is a dimeric and dimorphic enzyme. However, skeletal muscle displayed three isoenzymes, the third was a heteropolymeric form created by the combination of subunits of the variant and the non-variant form of the enzyme. This event could have occurred only in a cell which had active genes from both lineages used to form the chimera, and thus must be the result of cell fusion. If the muscle myotubes had been formed by acytokinetic mitosis, then only the two homopolyrneric forms of the enzyme would have been present, as in the other non-muscle tissue of the animals.
Coat pigmentation pattern Chimeras produced between mice with variant coat colors display a striped pattern of color variegation which is consistent with the concept of melanocyte migration from the neural crest of the fetus. If, as was thought for many years, the
melanocyte was derived from epidermal components then a salt and pepper pattern of mosaicism would have been expected. Instead, broad bands or stripes of coat color were always obtained. Moreover, there was a mid-dorsal discontinuity such that stripes on one side of the animal did not match stripes on the other side. This indicated that the two sides had independently established melanocyte clones beginning at the midline. Originally it was thought that each stripe represented a separate clone of melanocytes beginning as a single progenitor in the neural crest (Mintz, 1967). This was later shown to be an oversimplification, and it is now generally held that the banded pattern is the manifestation of 64 randomly arranged progenitor melanocytes (32 on each side) of the two genetically distinct types (Wolpert and Gingell, 1970; West, 1975). In the rat, coat pattern analysis has revealed that the hooded pigmentation pattern (under the control of at least three recessive alleles, hi, h i and h n) is the result of a gene which acts to retard the migration of the melanocyte from the neural crest or prevents its entrance into the hair follicle (Yamamura and Markert, 1981). X-Chromosome inactivation
The analysis of coat color pattern has also been used to help establish the timing of X-chromosome inactivation. In eutherian mammals one X-chromosome of the female is inactivated in each cell present in the early embryo. Amongst inner cell mass cells or their derivatives this process appears random with respect to which X-chromosome is inactivated. The process is generally irreversible, and genetically imprints the inactive X-chromosome so that the same X-chromosome is inactive in all progeny cells. In trophectodermal lineages, however, the paternal X-chromosome is preferentially inactivated, and the process appears to occur earlier. This fundamentally important process is a differentiative act in embryogenesis, and its timing has been studied in a variety of ways (Frels and Chapman, 1979; Frels et al., 1979; Frels and Chapman, 1980; Papaioannou and West, 1981). Coat color analysis of chimeras was elegantly utilized in one such study. Cells from embryos with Cattanch's translocation were injected at 3 and 4 days post-coitum into blasto-
82 cysts from a strain which yields sandy colored progeny. If X-chromosome inactivation had already occurred at the time of injection, the resulting chimeras would either be albino and sandy (i.e., the wild type X-chromosome is inactive) or agouti and sandy (i.e., the wild type X-chromosome is active). If, on the other hand, X-chromosome inactivation had not yet occurred in the injected embryonic cells, the resulting chimeras would have all three colors in their coats (albino, sandy, and agouti). The latter result was obtained for injected cells of both 3-day- and 4-day-old embryos (day of sperm discovery = day 0). Thus, X-chromosome inactivation in the inner cell mass of the mouse occurs after day 4 (Gardner and Lyon, 1971).
Cell fate analysis Coat color analysis also allowed the determination of the number of cells within the inner cell mass which are necessary to form the fetus and give birth to offspring. The mouse blastocyst consists of an inner cell mass and a spherical trophectoderm. The inner cell mass at the blastocyst stage of development is thought to consist of some 15 cells in the mouse. It is not known, however, how many of these are actually needed to form the fetus. Progeny of the trophectodermal cells will develop into extraembryonic tissues. Hexaparental mice and octaparental mice were constructed by aggregating embryos of either three or four strains of mouse with distinguishable coat colors. The resulting animals had coats with one, two, or three separate colors indicating lineage contribution from up to three different strains. Expected probabilities from polynomial expansions were compared with observed frequencies of the various combinations of colors in these animals, and it was possible to conclude that at least three cells in the inner cell mass, but probably not more than three cells, contributed to the formation of the fetus and thus all adult tissues (Markert and Petters, 1978).
Retinal pigmentation pattern Genotypic variation in pigment was used to establish patterns of mosaicism in the retina of
mice made between strains which were pigmented and those which were not. The patch pattern of pigmentation in the retina of such chimeras was examined at the microscopic level, both in one dimension and in two. These studies established a mathematical relationship between the proportion of cells from the two lineages which comprised the mosaic and the size of linear patches of cells of contiguously similar type (West, 1976a). The mean one-dimensional patch length was estimated as 1 / ( 1 - p ) where p is the proportion of the cell type under consideration in the section. The estimated coherent clone length was the observed patch length/expected patch length. The number of cells within the coherent clone was estimated as (mean coherent patch length/mean cell length) 2. The analysis allowed the conclusion that patches within the retina were essentially random in distribution and that the number of coherent clones and distribution of patches were similar between chimeras and X-chromosome-linked mosaics. The relationships were extended to two- and three-dimensional analyses by computer simulation (West, 1975; Whitten, 1978). These mathematical analyses proved the intuitively held notion that a background lattice would quickly form in a two or three dimensional mosaic tissue as the proportion of the major cell type increased. This would create islands of cells of the minor lineage within seas of cells of the major lineage. In highly unbalanced chimeras the pattern of mosaicism was shown to be the result of stochastic rather than deterministic development (Schmidt et al., 1986). In previous experiments, the conclusion that the retina forms by deterministic growth was supported by the contention that patches were distributed in various sectors of the retina in a non-random manner (Mintz and Sanyal, 1970; Sanyal and Zeilmaker, 1977). Further mathematical analysis, however, has shown that the distribution of patches in such animals is random, at least when the mixtures of pigmented and unpigmented cells are highly unbalanced. Patches are geometrically complex both in real and simulated mosaics. This complexity can be appreciated by examining the high degree of scatter within a distribution of patch sizes (Schmidt et al., 1986; Iannaccone et al., 1987b). The conclusion that such patterns of mosaicism
83 would arise as the result of cell mingling is substantiated by a generational computer model developed to study the formation of mosaic pattern in chimeric liver in the rat (shown below).
In situ markers of mosaicism A major limitation of many. of the studies utilizing chimeras and X-chromosome-linked mosaic animals has been the absence of practical markers which allow the distinction of cell lineages in histologic sections and provide the opportunity to analyze tissues in situ. This limitation has lessened dramatically in recent years. Histochemical markers The first usable histologic marker of chimerism was produced by exploiting the histochemical method for discerning the presence of flglucuronidase (EC 3.2.1.31). Since there is a strain of mouse which is deficient in the expression of this enzyme, it was possible to produce chimeras between strains which express this enzyme and those which do not. Mosaicism was then demonstrated by staining frozen tissue sections for the presence of fl-glucuronidase (Condamine et al., 1971). In other studies utilizing this marker, the liver was shown to be comprised of many more coherent clones than was previously thought (West, 1976b; Wegmann, 1970). It should be noted, however, that there was an apparent zonal distribution of the enzyme activity within the liver, suggesting that this metabolic enzyme might be modulated by circulatory events in addition to the distribution of the fl-glucuronidase deficient lineage. A similar enzyme (fl-galactosidase; EC 3.2.1.23) was localized histochemically and revealed a patchy distribution of lineage within the salivary gland (Dewey and Mintz, 1978). This marker also was reported to distinguish lineages in the kidney and in the pancreas. DNA markers A very high resolution histologic marker was developed based on the differences in satellite DNA between different species of Mus. By creating a DNA library, which included clones contain-
ing DNA capable of hybridizing with divergent, highly repetitive sequences of satellite DNA of one species but not the other, a system was developed to allow autoradiographic distinction of any nucleated cell of either species. Interspecific chimeras between Mus musculus and Mus caroli were produced and their tissues subjected to in situ hybridization utilizing a radioactive DNA probe capable of recognizing the satellite sequence of M. musculus. This probe proved valuable for the analysis of cell fate within early embryonic lineages and established that a strict species barrier to implantation of transferred embryos existed at the level of placentation. This marker, however, was less well suited to the study of organ development (Rossant et al., 1983a, b; Siracusa et al., 1983). Immunocytochemical markers It has been evident for some time that a system based on major histocompatibility haplotypes might be useful to establish mosaic pattern between congenic strains of varying haplotype. Several such systems have emerged in recent years. Most notable among these are in mouse and in rat. Congenic strains of mouse which express different class I alloantigens of the MHC were produced and direct immunocytochemical techniques were used to localize cell descendants of the two lineages present within chimeras (Ponder et al., 1983). A similar approach was taken utilizing congenic strains of rat (Weinberg et al., 1983, 1985a; Iannaccone et al., 1984). A library of monoclonal antibodies recognizing various epitopes of these antigens was employed to distinguish cells of these strains of rat. Direct iodinated antibodies were applied to tissue sections from chimeras between these strains. Autoradiograms revealed mosaic patterns in most of the tissues of the animals, since the system works in most visceral tissues of the animals (Weinberg et al., 1985b). Direct immunohistochemical localization has led to a number of observations which have allowed deductions concerning the formation and maintenance of several of the organs of these animals (Iannaccone et al., 1987a; Iannaccone and Weinberg, 1987).
84 The determinant density of the brain substance and neurectodermal derivatives which include peripheral nerves and adrenal medulla in the animals is very low, and the antibodies utilized for these studies do not react with these tissues. Vascular tissues within the brain react strongly, and the specificity of this reaction was established with appropriate absorption studies (Weinberg et al., 1985a).
In situ analysis of mosaicism
Following the production of chimeras between strains with different MHC antigen expression, the application of monoclonal antibodies capable of distinguishing the haplotypes was used for studying mosaic pattern in numerous tissues of the animals.
Intestine epithelium Chimeras between H-2 k and H-2 b strains of mouse were used to show that the epithelium of individual crypts of both the small and large intestine of adult animals is composed exclusively of one or the other of the two cell types that compose the animals. The villi of the intestinal lining, however, frequently demonstrated contributions from both cell lineages, indicating that epithelium had been derived from more than one crypt. The number of patch boundaries to be expected in a population of intestinal epithelial cells was estimated to be approximately 1 boundary per 35 cells as an overall average, thus establishing the very low probability that the crypt populations were composed of cells derived from a single lineage by chance alone (Ponder et al., 1985; Schmidt et al., 1985a, c). Statistical analysis of patch distribution within a field of mosaic tissue was performed in intestine of a highly unbalanced chimera by utilizing histological analysis of a polymorphism of Dolichos biflorus agglutinin (DBA) binding as a marker of mosaicism. The object was to determine whether the patches are randomly distributed or represent the result of some sort of pattern. The results of this analysis showed that at least in this animal the intestinal patch distribution did not appear to
be random (Schmidt et al., 1985b). However, it must be kept in mind that this sort of analysis does not demonstrate that pattern formation in the mosaic field is the result of non-random forces. Particularly in highly unbalanced mosaic fields the random adjacent positioning of daughters from randomly dividing cells would result in 'non-random' placement of patches, since the sites of progeny clones would be relatively restricted by the original placement of the marked cell. This can be demonstrated with computer simulations of the generation of highly unbalanced mosaic fields. These simulations also demonstrate that patch fragmentation can result from probabilistic or stochastic division decisions and does not require extensive cell movement, only random adjacent positioning of the daughters of any randomly chosen dividing cell. This mechanism can operate at any developmental stage in which division of cells is occurring, not just during embryogenesis.
Chimeric fiver In the rat chimeric liver an apparently random pattern of patch distribution was observed. The liver develops concurrently from the endodermal hepatic primordium, which begins as a thickening on the ventral surface of the foregut, and the vascular network, which is derived from the umbilical vein. The hepatoblastic epithelium expands as irregular masses of cells and not as well formed cords. These masses eventually form a single continuous plate, the muralium simplex (Du Bois, 1963). The adult organ is a complex structure organized around a circulatory tree which both brings nutrients and removes metabolites. In this representation, the basic unit of liver morphology is the liver acinus (Rappaport et al., 1983). Another view presents the basic unit of liver morphology as the liver lobule, consisting of interconnecting 'portal tracts' forming a roughly hexagonal area with a central vein in the middle (Beketova and Sekamova, 1983). Mosaic pattern in chimeric rat liver was unrelated to either the acinar or the lobular architecture of the liver (Fig. 3; Iannaccone et al., 1987b). A linear relationship between the proportion of the two cell types and the size of the patches was observed. There is an inverse linear relationship
85 generate a model of the formation of the mosaic tissue. This model was used to predict what sort of decisions might be required to result in the mosaic obtained. This approach led to the conclusion that probabilistic decisions may be all that are required to establish the pattern observed in real chimeric liver tissue. The random placement of the cells in the liver primordium, the random selection of cells within the anlage for division, and the random placement of daughter cells in an adjacent position resulted in a mosaic quantitatively similar to that observed in the real liver tissue (Berkwits and Iannaccone, 1985; Iannaccone et al., 1987b).
• I(
3
Adrenal cortex K
H-~
Ps
//---.-P-~ p# // .... ......
i:, • "\ NZI
cvo Z3\ "~
"x\ cvo
\ I
ps
cvo
Fig. 3. Computer-generated digital data from autoradiograms of chimeric rat liver sections showing the distribution of patches of one of the lineages present within the mosaic (top). A serial section was pin-registered to the autoradiogram (i.e., the sections were aligned using independent registration marks in the tissue). Several liver lobules are outlined (. . . . . . ). The liver lobule is roughly hexagonal in cut section and is defined by portal spaces (ps) and has a central vein (cv) in the center. The liver acinus is arranged around a circulatory tree defined by the terminal hepatic arteriole. An example of a liver acinus is outlined ( . . . . . . ). Z1, Z2, and Z3 refer to zones of progressively poorer quality arterial blood supply. A sublobular vein (slv) is present in the section. There is no apparent relationship between the distribution of patches and either lobular or acinar architecture. Reproduced with permission from the Company of Biologists, Ltd. (Iannaccone et al., 1987b). between the proportion of the minor cell type and the number of patches of that type in a given area of liver. These relationships were then used to
The adrenal gland has a dual embryological origin. The cortex of the organ first appears as a condensation of mesoblast in coelomic mesoderm at the level of the kidney in the mouse on day 13. Coelomic epithelium lateral to the gastric mesentary divides forming large polyhedral cells which extend as cords into mesoderm medial to the urogenital ridge. Cells from the capsule of the nephric glomerulus migrate to the developing gland, and these cells appear to give rise to the stromal elements. The medulla of the organ, on the other hand, arises from clusters of sympathochromaffin cells which are believed to be derivatives of ganglia in the area. The gland enlarges by the continuous migration of coelomic epithelium into the organ. The cortex of the adrenal gland is known to consist of three zones believed to be different histotypes (Arnold, 1886). More than 100 years ago Gottschau (1883) suggested that cell migration was centripetal through all three histozones. The adrenal cortex demonstrated a striped pattern of mosaicism (Fig. 4). This pattern was consistent with the concept that the gland develops from the clonal proliferation of primordial cells in which random movements are selectively excluded. The parallel rays of different cell types in the cortex may serve as an indication of the number of participating clones in the original primordium. The relationship between the clone size and proportion of the two cell types and between the clone number and the two cell types does not follow the relationship observed in the
86
Fig. 4. Diagrammatic representation of the results of step-sectioned adrenal glands in chimeric rats. The diagram summarizes results from autoradiographic analysis of mosaic pattern in a number of sections of several glands from animals which are composed of cells derived from lineages which express different class I major histocompatibility alloantigens. As the depth of section proceeds to the center of the gland, the pattern of mosaicism changes from patches to wedges and finally stripes.
liver (Iannaccone et al., 1987b). Thus, random division and random adjacent placement of the daughter cells in succeeding generations would not be sufficient to explain the mosaic pattern observed in this organ. The pattern is much more consistent with the concept of surface signal recognition and the formation of boundary conditions as has been described for the morphogenesis
in avian feather where cell adhesion molecules may represent a critical surface signal for morphogenesis (Chuong and Edelman, 1985a, b; Crossin et al., 1985; Gallin et al., 1986). There is a change in the observed pattern of mosaicism as one obtains sections from progressively deeper portions of the organ. The outermost surface of the organ shows a patchy distribution of mosaicism while deeper sections display a wedge shaped distribution of the two lineages which comprise the organ. The centermost sections show a parallel array of cords of cells composed exclusively of one or the other lineage (Fig. 5). These patterns are most consistent with a model in which columns composed of cells derived from the minor lineage extend through the entire thickness of the cortex. The corners of the sections from the center of the organ can be informative with regard to the question of the direction of division. Gottschau suggested that the adrenal cortex developed by centripetal division. The concept that division proceeds from the outside toward the inside was suggested by extirpation of the central portion of the organ with subsequent complete renewal from the remaining rim of capsule and zona glomerulosa. Mosaic pattern analysis has shown that parallel cords of cells extend through all of the histogenic zones of the organ and intersect in such a way as to suggest that the divisions required for renewal of the organ if not its formation are occurring from the outside toward the inside (Iannaccone and Weinberg, 1987; Iannaccone et al., 1987a). The role of cell-cell contact and adhesion in organ development has been discussed for many years (Saxrn and Wartiovaara, 1966). One possible explanation of the pattern in the adrenal is that boundary conditions in which cell surface recognition molecules are different in two areas of the tissue and the direction of adjacent positioning of the daughter cells or even the decision to divide are constrained as result. Such a system has been proposed and experimentally supported in avian feather embryogenesis (Gallin et al., 1986). These boundary conditions should not exist in the liver parenchyma because of the random nature of the divisions which are required to produce the mosaic pattern observed.
87
Fig. 5. Photomicrographs of unstained autoradiograms of adrenal glands from two different chimeras with widely different proportions of cell types. The gland on the left was 38% PVG cells, while the gland on the right was 9570 PVG cells. The parallel array of cells is evident in both cases. Bar = 0.5 mm. Reproduced with permission from J. Exp. Zool. (lannaccone and Weinberg, 1987).
Thymus The thymus is another organ of dual embryologic origin. The thymus originates from the primitive pharynx as an epitheliomesenchymal rudiment. The epithelial portion is thought to be endodermal in origin. Neural crest cells migrate to the developing thymic buds and contribute to mesenchymal elements (von Gaudecker, 1986). The thymic lymphocytes can arise either by transformation of existing thymic epithelium or by substitution from mesenchymal cells. Extrinsic sources of thymic lymphocytes have been demonstrated in birds, amphibia and mammals, although the precise origin of these cells is not known. The thymic lymphocytes enter the cortical regions of the gland in several waves during fetal development. Adult cortical lymphocytes are processed by the cortex and then move to the medulla for export to the circulation. While some theories of cortical lymphocyte trafficking may suggest that residence time within the cortex is short, the fact that chimeras can tolerate tissues derived from both parental lineages and yet retain immunologic specificity might mean that stem cell proliferation
occurs within the thymic cortex (Howard, 1980). Alternatively, the forbidden clone theory of Burnett argues that clones of cells not recognized as self within a competent immune system would be eliminated as they arise (Deol, 1973). Renewal of intrathymic lymphocytes is a physiologic process occurring throughout the life of the animal. It remains to be determined whether or not the process is continuous or discontinuous. The use of radiation chimeras (produced by sublethal irradiation with subsequent repopulation of the bone marrow with genetically distinct cells) suggests that there are two lineages of cells involved: one which populates both the medulla and the cortex; and one which populates the medulla alone (Ezine et al., 1984). Sections of the thymus from a young adult chimeric rat produced between histologically distinguishable strains revealed a pattern consistent with such oligoclonality (Fig. 6). The histologic analysis of mosaicism in normal development is an area of great potential importance to theories of immune tolerance as well as mechanisms of thymic lymphocyte traffic.
88
Mosaic analysis of pathological alterations
mann, 1982). Similar results were obtained with a dimorphism in the mouse that results from Xchromosome inactivation. Such X-chromosomelinked mosaic animals were subjected to a variety of carcinogenic treatments, and a wide range of tumors were shown to be clonal in origin (Deamant and Iannaccone, 1985; Iannaccone et al., 1987c). Inflammatory cells within the tumor confounded the isozyme results in some studies which reported non-clonal origins of carcinogen-induced fibrosarcomas. Some studies utilizing X-chromosomelinked mosaics showed both isozymes present within the tumors indicating a non-clonal origin of these cancers (Reddy and Fialkow, 1979). These animals are mosaic by virtue of heterozygosity at the PGK locus which is on the X-chromosome. One of the two X-chromosomes is randomly, and irreversibly, inactivated in embryonic lineages of mammalian females. In M. musculus ~ M. caroli chimeras (described above), however, the in situ marker revealed that inflammatory cells within the fibrosarcoma were derived from both lineages, but the neoplastic cells were derived from a single lineage (Deamant et al., 1986).
Tumor formation A number of current theories of carcinogenesis require that rare events, such as mutations, are critical to the formation of cancers (Reddy et al., 1982; Shih and Weinberg, 1982; Tabin et al., 1982; Sukumar et al., 1983). Rare affected cells would then, as a result, clonally expand into neoplasms. The clonal origin of chemically induced, physically induced and spontaneous cancers has been established by the use of isozymic markers of mosaicism (Fialkow, 1976; Iannaccone et al., 1987c). Chimeras have been produced between strains of mouse which varied in their expression of glucosephosphate isomerase (GPI-1; EC 5.3.1.9; DeLorenzo and Ruddle, 1969). The electrophoretic patterns of tumors chemically induced in the skin and mesenchymal tissues of such animals showed the tumors to be composed exclusively of cells of one or the other type comprising the normal tissues of the chimeras. Thus, the tumors appear to result from the clonal expansion of single, affected cells (Iannaccone, 1980b; Iannaccone et al., 1978; 1987c; Elbing and Sauer-
Preneoplastic changes It is now generally held that the formation of malignant cancers is a multistep process (Farber and Cameron, 1980). It is possible that if a critical event occurs in rare cells at an early step in carcinogenesis, then all preneoplastic steps leading to the development of the cancer would produce clonal lesions. This appears to be the case. Epidermal cancers produced by chemical induction are preceded by the development of papillomas which are the result of clonal expansion of single affected cells (Iannaccone et al., 1978). Further, the development of hepatocellular carcinoma as a result of chemical carcinogens is generally held to involve a number of preneoplastic steps which result in lesions including: nodular morphological alterations called hyperplastic nodules; areas of altered expression of certain hepatic enzymes; and areas of altered glycogen mobilization. These areas represent phenotypic alterations in hepatocyte populations and are thought to be early steps in the formation of hepatocellular carcinoma (Becker, 1978; Bannasch et al., 1979, 1980). The concept
Fig. 6. Photomicrograph of an unstained autoradiogram of the thymus gland from a young PVG *-*PVG-RT1 a chimeric rat. Several large patches of cells of the PVG lineage (white) are evident within the cortex of the gland. The scalloped interface between the cortex and the medulla may indicate that cortical cells may move to the medullary region on broad fronts, x 22. Reproduced with permission from Cold Spring Harbor Laboratory (Iannaccone et al., 1987a).
89
approach has proven useful in establishing theories of organ formation and maintenance. The use of mosaic pattern analysis has also been instrumental in establishing the clonal origin of neoplasms and preneoplastic lesions. With the development of new markers of mosaicism the future should continue to provide insights into the organization of mammalian tissues.
Acknowledgements Fig. 7. Photomicrograph of a frozen section from the liver of a chimeric rat following partial hepatectomy, treatment with the liver carcinogen diethylnitrosamine and promotion with phenobarbital. The section was incubated with an iodinated monoclonal antibody which recognizes cells of the PVG-RT1 a lineage. The patch pattern of the liver is distorted by a nodular lesion which had an altered enzyme expression as described in the text. This carcinogen-induced lesion can be seen to be composed exclusively of one of the two cell types present in normal tissue and distorts the surrounding patch pattern. Thus, it represents the clonal expansion of a single affected cell. Bar = 2.0 mm. Reproduced with permission from Int. J. Cancer (Iannaecone et al., 1987c).
that these lesions are involved in the formation of the cancer is supported by a number of observations from many laboratories over the years. By causing their formation in chimeric liver it was possible to establish the clonal origin of many hundreds of these lesions (Weinberg and Iannaccone, 1986; Weinberg et al., 1987). Areas of altered enzyme expression were phenotypically heterogeneous, exhibiting multiple alterations, and yet all of the cells in such areas were demonstrated to be composed of cells exclusively of one or the other of the two cell lineages present in normal tissues (Fig. 7). These results indicate that the earliest steps of chemical induction of liver cancer are clonal, and that phenotypic heterogeneity and complexity within the preneoplastic nodules are the result of progression and not of multicellular origin of the lesions.
Conclusions Multizygotic animals provide the opportunity to analyze mosaic pattern within organs. This
This work was supported in part by USPHS grants CA29078 and ES03498 and MOD Birth Defects Foundation grant 15-49.
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