Cell lineage in vertebrate development

Cell lineage in vertebrate

development

H.M. Blau and S.M. Hughes Department

of Pharmacology,

Stanford Current

University

Opinion

in Cell

Introduction The generation of the diversity of cell types typically present in tissues requires complex controls. The lineage of a cell or its ‘family tree’, provides insight into when and where the developmental decisions occur that commit that cell to a particular specialized pathway. Commitment to a differentiated state does not necessarily imply irreversible changes; instead, it could require continuous regulation by a combination of cell-intrinsic and extrinsic controls. Once the lineage of a particular dilferentiated cell type is known, it is possible to ask questions regarding the relative contribution of these two forms of regulation and to examine their molecular nature. When do progenitor cells of a given tissue diverge to generate daughters that are distinct from each other? What is the evidence for cell- intrinsic programs of ditferentiation? What is the role of extrinsic factors in modulating differentiation? In this review, we have selected examples of work published in the past year which make particular points that illustrate the usefulness of the cell-lineage approach in enhancing our understanding of differentiation. We make no attempt to be comprehensive and realize that many excellent papers have been omitted. The advent of novel techniques has revolutionized research in this area and their development and application are highlighted throughout.

When

do cells diverge?

In attempting to answer this question, we must examine the origins of a particular cell type. Is a given precursor cell multipotent and capable of generating a large number of diverse cell types or does a precursor cell give rise to cells capable of generating only a limited number of cell types? The answer depends on the setting. There are several examples in which cell precursors are mukipotent. Fraser and colleagues [l] found that all major cell types of the frog retina could be generated from a single cell in the ciliary margin that was marked by microinjection of the fluorescent dye, lysinated rhodamine dextran. The retina is particularly well suited to this type of analysis because a large variety of cell types is generated with distinct morphologies in defined locations. To examine retinal development in the mouse, Cepko and colleagues [2] used a genetic marker: divid-

School Biology

1990,

of Medicine,

Stanford,

California,

USA

2:981-985

ing cells were infected with a defective retroviral vector that could only be transmitted to other cells upon cell division. Because the vector included a gene encoding j3galactosidase, its presence in a cell could be readily detected histochemicajly. In order to visualize and gain access to fetal retina, injections of retroviral vectors were performed on fetuses outside the uterus. Using this ap preach, Cepko and colleagues found that, as in the frog retina, the diverse cell types of the mouse retina were generated from single cells. A third report by Sanes and colleagues [31 used retroviral vectors to show that the development of motoneurons and glia in the chicken spinal cord is similarly plastic. Thus, in these examples, the generation of cell diversity is a late developmental decision. In other cell lineage studies, precursors appear to be limited in the number of cell types to which they give rise. Hartenstein [4] has shown, by injection of horse-radish peroxidase into single Xenopus neural plate cells, that these cells fall into two classes: one gives rise to primary neurons whereas the other gives rise, after extensive proliferation, to secondary neural cell types. Similarly, Kimme1 ef al [5], upon injection of tracer dyes into developing zebrafish, noted that two types of blastoderm cells can be distinguished by the onset of gastrulation: one that arises in the enveloping layer and generates periderm and another that arises in the deep layer and generates all deeper tissues. Stem and Canning [6] have identied randomly distributed cells within the epiblast of the early chick embryo that are destined to form the primitive streak and mesodenn. To monitor cell fate in these experiments, a novel technique was employed: cells were labeled in viva with a monoclonal antibody HNK-1 coupled to colloidal gold. Cells with the appropriate cell surface antigen endocytosed the complex which could be readily identified in their progeny by light microscopy after they divided, migrated and differentiated within the developing embryo. Fraser et al [7] describe a restriction of the fate of cells in specific locations (segments of the chicken hindbrain known as rhombomeres), a compartmentalization which may share properties with that observed in insects. The molecular basis for such boundaries remains unknown. These experiments illustrate that the expression of a differentiated state may not reflect a restriction in cell potential due to the intrinsic program of a cell, but rather a restriction caused by the lack of exposure to extrinsic factors a cell might otherwise encounter in the absence of barriers to migration.

Abbreviations BUdR-S-bromo-2’-deoxyuridine;

HSV-herpes

@ Current

Biology

simplex

virus;

Ud ISSN 095-74

HSV-l-tk-iiSV

thymidine

kinase. 981

982

Cell differentiation

In lineage studies, it is often critical to the analysis that the events being monitored are clonal. For the microinjection of fluorescent dyes, .it is possible to establish clonality with reasonable certainty by inspection of the number of cells labeled within minutes after injection. Indeed, using this approach, a labeled ceU and the progeny to which it gives rise can be followed continuously and monitored live. A particularly elegant application of this approach is shown in the zebrafish where two different dyes, Auorescein and rhodamine dextrans [8], were injected to monitor the fate of sister cells by time-lapse microscopy. However, dye-injection techniques are limited to organ isms in which cells are easily visualized, such as frogs, chickens, and zebrafish, and to developmental events that occur without much growth because the marker is progressively diluted. Genetic marking of mammalian cells and, more recently, chick cells [9] with retroviral vectors, overcomes the problems of dilution. To test for clonality, investigators have generally resorted to titrating the vector, but this is not definitive. Recently, a double-retrotid marking system was developed that can provide confidence that labeled clusters of cells are clones [9,10]. Two different retroviral vectors are mixed, one that targets b-galactosidase to the nucleus and one that targets it to the cytoplasm, and the mixture injected into the tissue. If clusters of labeled cells are the progency of single cells, the clones will contain either nuclear or cytoplasmic labeling but not both. Retroviral vectors have also recently been used to monitor hematopoetic clones that are not localized [ 121. Because retroviral vectors integrate ran domly into genomic DNA members of a clone can be identified in cell mixtures by their common integration site. The development of polymerase chain reaction protocols in situ on single cells would allow the extension of this’approach to an analysis of clones that migrate and encompass relatively large areas. Another caveat of all lineage studies is that marked clones may contain a subset of unlabeled cells which fail to express detectable levels of the marker. As a result, conclusions can only be based on patterns of labeled cells. This is true even for studies using heritable retroviral vectors, because some promoters function better in some ceU types than in others. Two studies [2,11] make this point clearly by directly comparing the labeling patterns obtained for retroviral vectors with different promoters. In addition, in most cases, it is only possible to mark cells genetically if they are actively dividing. As a result, cells that were quiescent at the time of labeling are missed. A recent exception is a study in which a vector based on herpes simplex virus (HSV), was used to infect postmitotic neurons. The development of novel vectors that can infect and express equally weU in all ceU types and stages of the ceU cycle would greatly facilitate studies of cell lineage. In cases where lineage maps reveal points at which cells diversify, what is the basis for that divergence? Is the apparent restriction in ceU fate due to heritable intrinsic dif ferences or are the differences due to the local environment which the ceU and its descendants encounter? The answers cannot be ascertained by simply assessing lin-

cage relationships by the transmission of markers in uiuo. AS described below, three types of experimental manipulations that can provide insight are: analysis of diierentiation in z&-o in clones and well defined cell mixtures; ablation of cells in zhlq and transplantation of cells to other sites within the organism.

Evidence

for intrinsic

differentiation

programs

One way to distinguish the roles of intrinsic and extrinsic influences in determining ceU fate is to isolate individual cells and study them as clones in tissue culture. In a recent study of this type, Temple [ 141 showed that neuroblast clones of the rat forebrain differ in their proliferative behavior: some do not divide, others divide once, and yet others divide many times and give rise to large clones. This initial evidence of heterogeneity was substantiated by evidence that the progeny of clones differed in their differentiation: some clones contained only neurons, some only glia, whereas others contained both ceU types. A similar diversity of ceU types was observed upon clonal analysis of avian neural crest [ 151. Clones grown in tissue culture from isolated cells contained different numbers of cells and gave rise either to committed Schwann cells only, to bipotent precursors, or to two types of multipotent Schwann ceU precursors. As ceU culture conditions were a constant in these experiments, differences among clones were either intrinsic to the cells at the time of plating or arose shortly after plating in a stochastic manner from an initially homogeneous ceU population. Further support for a cell-autonomous programme could be obtained if two daughter cells, separated immediately following their generation in tissue culture, gave rise to similar clones. Thus, evidence that isolated cells exhibit a specific division potential and differentiation capacity in a heritable manner in vitro suggests that intrinsic programs are operating. What types of regulatory mechanisms could maintain an intrinsic program? Tissue-specific regulatory genes that activate their own expression provide a means for perpetuating a given committed cell state. An example is MyoD and its related family members, genes encoding DNA-binding proteins with a helix-loop-helix motif that are expressed early in muscle development (see Emerson, this issue, pp 1065-1076) [ 161. Each member is capable of activating its own expression and crossactivating the expression of other members in fibroblasts, convert ing them to myoblasts [ 171. Such feedback loops provide memory and ensure that the identity of cells is stably maintained in isolation from in vivo environmental influences. However, MyoD alone is not sufficient to induce myogenesis in alI settings. Expression of MyoD in large regions of early Xenopus embryos activates the endogenous muscle actin genes but fails to convert the cells to muscle [ 181. Similarly, MyoD family members cannot induce myogenesis in hepatocytes unless they are combined with other factors present in muscle cells [ 191. These experiments suggest that additional regulators play a central role in establishing a myogenic commitment.

MyoD is a transcription factor. Additional evidence that key control genes can be transcription factors derives from the recent analysis of two recessive mouse mutants that are dwarves [20]. Both had mutations in the pit- 1 (GHF 1) gene which led not only to the absence of expression of that gene but also to an absence of three major ceU types characteristic of the pituitary. It will be interesting to determine whether other tissue-specific transcription factors, mutated by homologous recombination (see below), also play a causal role in ceU commitment during development. Evidence for extrinsic differentiation

regulation

of

Cell-intrinsic programs define a CelLautonomous pathway of differentiation. A key question in development is to what extent such intrinsic programs can be modulated by extrinsic factors. These Include both diffusible factors and cell surface components. The three approaches described below allow an analysis of the influence of cell-cell interactions on ceU fate. Cell-cell

interactions

in vitro

The influence of one ceU type on the properties of another can be analyzed by examining the interaction of weU de&d combinations of marked cells in r&-o. Bunge et al. [21] developed a culture system in which perineurium, a protective cellular sheath that surrounds nerve fiber fascicles, was generated when purified populations of sensory neurons, Schwann cells, and fibroblasts were mixed. Before mixing, the Schwann cells or fibroblasts were labeled by infection with a retroviral construct that encoded P-galactosidase. Because cells that produced the perineurium were labeled only when libroblasts were infected, the perineurium derived from libroblasts, not Schwann cells. Watanabe and Raff [22] examined the relative roles of intrinsic programs and extrinsic regulation in neural retina cell-differentiation mixing 5-bromo-2’-deoxyuridine (BUdR)-labeled cells of embyronic and postnatal stages: the timing of differentiation did not change but the proportion of cells that differentiated to produce rods did. In vitro systems of this type indicate that both cell-autonomous developmental programs and cell-cell interactions can play a role in ceU fate decisions and provide assays for the relevant molecules. An analysis of cell-cell interactions in tissue culture requires the isolation and labeling of pure populations of cells. This can be achieved by marking cells in transgenic animals. For instance, it was shown recently [23] that the 5’.regulatory element of L7, a gene of unknown function, directed P-galactosidase expression only in retinal bipolar cells and cerebellar Purkinje cells. With methods developed by Herzenberg and colleagues (Nolan et al, Proc Nat1 Acad Sci 1988, 85: 2603-26071, it should now be possible to purify this subset of cells using the fluorescence- activated ceU sorter and to analyze their differentiation alone or in well dehned combinations in tissue culture.

Cell

lineage

Cell

ablation

in vertebrate

development

Blau and Hughes

in vivo

Once the lineage of a ceU type is known, methods for ablating that cell type in uivo allow an analysis of its impact on neighboring cells and on the development of the tissue as a whole. Several recent technical advances have made it possible to eliminate cells efficiently. Stem and Canning [6] describe a novel method using antibody and complement lysis to eliminate a specific cell type; when HNKl-positive cells were ablated in developing chickens, no primitive streak or mesodermal structures developed; such embryos were rescued by a graft of primitive streak from untreated animals. A second ablation techique exploits ceU-type-specific ®ulatory sequences in transgenie animals. These sequences can be used to drive the expression of genes encoding either diptheria toxin A or ricin A chains and target specific ceU types for destruction (Behringer et al., Genes Deu 1988, 2:453 461). Drawbacks of this approach are that the timing of ablation cannot be controlled and late phenotypes may be missed because expression early in development is lethal. ‘Conditional ablation’ provides an attractive alternative. In this approach, the gene that is targeted to specific cell types is not toxic until a specific drug is administered. For example, the expression of the HSV thymidine kinase (HSVl-tk) gene under the control of the growth hormone promoter resulted in the absence of both lactotropes and somatotropes in the anterior pituitary showing that they were dependent upon, and possibly descended from, the same growth hormone-expressing precursor ceU [24]. HSV-l- tk, unlike the mammalian enzyme, phosphorylates the drug gancyclovir to form a nucleoside analog that is incorporated into DNA leading to inhibition of DNA synthesis and ceU death. In this approach, ablation is spatially restricted by introducing HSV-1-tk constructs with tissue-specific promoters and enhancers. Ablation is also temporally controlled by timing the administration of the gancyclovir. The degree of ablation can be controlled by the dose and timecourse of treatment. As a result, spectic cell types can be eliminated at spectic times and their impact on the tissue and on ceU neighbors explored. Limitations of this approach are that it is only effective in cells that are actively proliferating at the time the drug is administered and that ceU death is not immediate upon exposure to gancyclovir. Gene ablation by homologous recombination has led to the elimination of ceU types in some cases. In this approach, embryonic stem cells, in which the endogenous gene has been disrupted by homologous recombination, are introduced into mouse blastocysts and contribute to multiple tissues including the germ line. Animals homozygous for the disrupted gene are obtained by appropriate breeding. Application of this approach to the gene, a component of the class I major p@croglobulin histocompatibility complex, resulted in the specific elim& nation of cytotoxic T cells [ 25,261. Elimination by homologous recombination of writ-1 (int-1), which encodes a secreted protein, resulted in impaired development of the midbrain and cerebellum [ 27,281. Perhaps the most striking demonstration of the potential of this approach are the naturally occurring recessive dwarf mouse mu-

983

984

Cell differentiation

tanks with defective pit-l genes described above [20] in which three major pituitary cell types were missing. Cell transplantation

LEBER SM, BREEDLOVE

l

V: Early neurogenesis in Xenopus the spatiotemporal pattern of proliferation and cell Lineages in the embryonic spinal cord. Nezrron 1989, 3:399-411. This paper demonstrates restriction of early neural tube cell fate by cellular injection of horse-radish peroxidase.

4. .

HM~TENSI-EIN

KIMMEL CB, WARGA RM, SCHIUING IT: Origin and organization 5. . of the zebraf%h fate map. Dec~efopmenl 1990, 108:581-594. Determination of the fate map of the eariy embryo. Significant as a prerequisite to the extensive lineage analysis likely to follow.

6.

STERN CD, CANNING DR: Origin of cells giving rise to mesoderm and endoderm in chick embryo. Nature 1990. 343273275. A landmark paper that introduces one new method for in &lo lineage marking and another for lineage ablation. Essential reading. l e

FRASER S, KEYNX R, LLIMSDEN A: Segmentation in the chick embryo hindbrain is defined by ceU lineage restrictions. Nature 1990, 344:431-435. This technical tour de force shows the development of companments and restriction of cell migration during development of hindbrain structures by fluorescent dye injection and confocal microscopy.

7.

prospects

l e

The ability to study regulation of differentiation by intrinsic programs and extrinsic factors in the well defined conditions of tissue culture should allow the isolation and characterization of the relevant molecules. Although the regulators that autoregulate are likely to play key roles in commitment, there are probably additional regulatory mechanisms for establishing memory and maintaining differentiated states. The isolation of pure populations of closely related cell types, identified by lineage analyis, should facilitate the discovery of molecules with MyoDlike functions in other cell types, possibly by subfraction hybridization. The ability to ablate cells and genes in developing mice will allow stringent tests of cell autonomy and the role of putative regulators in vivo. To test the role of potential extrinsic regulators, the genes that encode them can be introduced in vivo at aberrant times or sites, and their effects on normal patterns of development examined. Finally, it should be possible to examine the intrinsic potential (memory) of marked cells characterized in vitro upon transplantation to ectopic sites in vivo. With the array of techniques now in hand, we anticipate that in the next decade our knowledge of vertebrate development will increase dramatically. Annotated reading

SM, SANES JR: Lineage, arrangement, and death of clonally related motoneurons in chick spinal cord. J Neuracci 1990, 10:2451-2462. Cell are not restricted to a lineage in formation of some chick motor and other neurons and glia.

3.

in vivo

Finally, the study of extrinsic regulation of differentiation is transplantation of specific cell types into ectopic sites where their fate can be monitored. Such experiments provide a stringent test of cell commitment. The classic chick-quail chimera system of Le Douarin, which uses nuclear markers to distinguish transplanted cells from recipient cells, has been used recently to monitor migration and map cell fate in the cerebellum [ 291. It should now be possible to use transplantation to assess cell potential in mice. Pure populations of genetically marked cells could either be obtained by labeling cells in tissue culture with retroviral vectors or by isolating them from trans. genie animals [ 23,30,31]. The fate of these marked cells could then be analyzed following transplantation into dif ferent sites within the organism. Future

lineage analysis in the retina of fetal mice using retroviral marking demonstrates that cells are multipotent until after their terminal division.

references

and recommended

8.

WARGA RM, KIMMEL CB: CeU movements during epiboly and gastrulation in zebrafish. Devekpment 1990, 108:569- -580. This paper describes the continuous analysis by time-lapse recording of sister cells injected at different times with two fluorescent dyes. CeU lineage and migration can be charted over time in live cells. l

GAIJLEO DS, GRAY GE, OWENS GC, MAJORS J, SANES JR Neurons and glia arise from a common progenitor in chicken optic tectum: demonstration with two retroviruses and ceU typespecilic antibodies. Proc Nat1 Acad Sci USA 19%l, 87:45%462. The development of novel retroviral vectors that can be used in chickens allows an analysis of neuroepithelial ceU lineage. Two distinguishable vectors are used to ensure clonality. 9. l

10.

HUGHES SM, BL+U HM: Migration of myoblasts across basal lamina during skeletal muscle development. Nature 1990, 345:350-353. In this report, two distinguishable retrotiral vectors that target f%galactosidase to the nucleus or cytoplasm are used to ensure that groups of marked ceUs are clones and to demonstrate migration of satellite cells in normal development.

l

J, BEDDINGTON R5: Pertnissiventks to murine leukemia virus expression during preimplantation and early postimplantation mouse development. De velcpmen~ 1990, 109~655-665. A comparison of retroviral vectors shows that they are not consistently expressed in all cell ms apparently because of differences in promoter specificity. 11.

SAVATER P, MORGENSTERN

l

12.

JORDAN CT, ~hUSCHK.4 IR: Clonal and systemic analysis of long-term hematopoiesis in the mouse. Genes Da, 1990, 4:220-232. Retroviral-integration+ite polymorphism is used to follow clones of hematopoetic stem cells, allowing an analysis of proliferative capacity and differentiation.

l l l e

Of interest Of outstanding

interest

1.

WEITX R, SERBEDZ~JA GN, FRASER SE: Cell lineage analysis reveals multipotent precursors in the diary margin of the fkog retina Deu Eti 1989, 136:254-263. Analysll of frog retinal cell lineage by dye injection into peripheral neuroblasts reveals that cells are multipotent. l

2. l

TURNER DL, SNYDER EY. CEPKO CL: Lineage-independent termination of cell type in the embryonic mouse Neuron 1990, 4:833&i5.

deretina.

13.

GELUR

Al, FREESE A: Infection

of cultured

central

nervous

system neurons with a defective herpes simplex virus 1 vector results in stable expression of Escherichia coli betagtictosidase. Pm Null Acad Sci C&4 1990. 87:114F1153. This constitutes an important step toward the development of lineage markers that can be used to label postmitotic cells. The limited tropism of HSV-1 restricts use to the nervous system at present. l

Cell lineage 14. l

TEMPE S: Division cells in microculture.

and differentiation Naftrre 1989.

of isolated CNS blast 340:471473.

III this report. the heterogeneity of neuroepithelial as clones suggests intrinsic differences. 15. .

D~IPIN

E, BAKOFFIO

A, DLILAC

stem

C. CAMERON

16.

CP.

II

DOUAR~N

N : Schwann-ceU differentiation in clonal cultures of the neural crest, as evidenced by the anti-Schwann cell myelin protein monoclonal antibody. PXX NutI Acad Sci L&l 1990.

SASOON D. LYONS G, WRIGHT H, BUC~~NCHAM M: Expression

factors Nulure

WE, Lti% V, L%SAR 4 of two myogenic

myogenin and MyoDl 1989, j4 1:303-307.

during

mouse

l

cells analyzed

&37:111~1123. This repon extends previous work elegantly illustrating the heterogene. ity of early neural crest clones with respect to proliferative capacity and differentiation potential. It provides evidence for intrinsic programs.

.

23.

WEINIRA~JB regulatory

24.

BORREUJ E, Transgenic 339:53a541. A transgenic animal drive expression of ablation of expressing temporal control of . .

25.

in which tissue-specific HSV thymidine kinase cells. This conditional ceU elimination during

KOUER BH, MARRACK P, mal development of mice

MHC

class

I proteins,

17.

26.

THAYER MJ, TAPSCOTT WEWIRAUB H: Positive

termination Evidence is provided cell memory molecule. 18. l

SJ, DAVIS RI, WRIGHT WE, IA.%AR AB, autoregulation of the myogenic de-

gene

MyoDl.

for

postitive

Cell 1989, feedback

58:241-248. regulation

HOPWOOD ND, GURDON JB: Activation of muscle genes withauf myogenesis by ectopic expression of MyoD in frog embryo cells. Nature 1990, 347:197-200.

l

27. l

Homologous velopmental

19.

SCHAEFER BW,

E5ect

l

of ceU history on myogenic regulators.

28. .

DAFUINGTON

GJ. BEAU HM:

to heUx-loop-helix 1990, 344:454--158.

family

of

This report shows that myogenic regulators in addition fo MyoD family members are required for myogenic conversion in some cell types.

20. l *

IA S, CRENSHAW EB, RAWSON EJ, SlhlMONS DM, SWANSON LW, RO~ENFELLI MG: Dwarf locus mutants lacking three pituitary

ceU types result from mutations pit-l. Nafure 1990, 347:528-533.

in the

POU-

domain

gene

This

remarkable report describes the discovery that mutants lacking known as GHF-I), a tissue-specilic transcription factor, lack the cell types that normally express it.

Pit-l (also

21. l

BUNGE MB, WOOD PM, TYNAN LB, BATES ML. SANF.S JR: Perineurium originates from libroblasts: demonstration in vitro with a retroviraI marker. Science 1989, 243:22%231.

One of three possible ceU types that forms the protective neural or perineurium, in tissue culture is identified as the responsible retroviral marking.

22. l

WATANABE T, RAFF MC: Rod photoreceptor vitro: intrinsic properties of proliferating cells change as development proceeds Neuron 1990, 4:461467.

ablation

KAPPLER YJV,

deficient and CDS+

of &

sheath, ceU by

development in neuroepithelial in the rat retina.

Evidence is provided of both intrinsic control of timing of dilferentia, tion and extrinsic control of ditferentiation pathway by mixing BUdR. labeled retinal cells in vim

PE, EVANS RM: Nurure 1989,

allows

spatial and

Snunlw

0:

Nor-

in p-2 microglobulin, T cells. Science 1990,

microglobulin

leads

to ablation

of

LORING JM, RAULET DH, deficient mice lack 1990, 344~742-746. of & microgiob-

McMAHoN AP, BRADIEY A: The writ-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain. Cell 1990, 62:10731085.

MyoD

response Nature

965

development.

ZLJLSTRA M, BK M, SIMLSER NE, JAENECH R: Beta 2microglobulin CD4-8+ cytolytic T cells. Nature

This report shows that the expression of the transcription factor is not sufficient for myogenic conversion in some cell types. BLAKELY BT,

Hughes

c®ulatory sequences and permit drug induced

Same results as [ 251 that homologous recombination ulin leads to ablation of cytotoxic T cells.

of a candidate

and

HF~~~AN R& ARVCS C. SAWCHENKO mice with inducible dwarlism.

248:1227-1230. Homologous recombination cytotoxic T ceils.

l

Blau

OBERDICK J. SMEYNE RJ. MANN JR, ZACKSON S, MORGAN Jl: A promater that drives transgene expression in cerebellar Purkinje and retinal bipolar neurons. Science 1390, 248:223226.

of the myogenic regulatory gene family are tierentialty expressed in developing somites and limbs. These are currently the best candidates for commitment genes.

Members

development

An example of how the promoter of a gene of unknown function can be used to direct expression of transgenes in particular ceU types allowing for their subsequent isolation and analysis in tissue culture.

l

embryogenesis.

in vertebrate

recombination signal.

is used

fo create

a null mutation

in a de-

THOMAS KR, CAPECCHI MR: Targeted disruption of the murine int-1 proto-oncogene resulting in severe abnormalities in midbrain and cerebellar development. Nature 1990, 346B47-850.

Same results as [ 271: homologous recombination is used fo create a nuU mutation in a developmental signal. The mechanism of lesion formation remains unknow-n.

29. l

HAILONET ME, TEILET MA, LE DOUARIN N: A new approach to the development of the cerebellum provided by the quail-chick marker system. Devehpmenf 1990, 108:19-31.

CeU migration is analyzed in cerebellar duced by the transplantation of quail

30. .

l

Gene

pro-

BEDDINGTON RS, MORGERNSERN J, LAND H, HOGAN A: An in situ transgenic enzyme marker for the midgestation mouse embryo and the visualization of inner ceU mass clones during early organogenesis. Development 1989, 106:374.

In this paper, a transgenic mouse press &@actosidase, a rich source eage analysis. 31.

development in chimaeras cells into chicken embtyos.

is produced of genetically

in which marked

all cells excells for lin-

expression of EDW~ RH, RL~~-~ER v, HANAHAN D: Directed NGF fo pancreatic beta cells in transgenic mice leads fo selective hyperinnetvation of the islets. cell 1989, 58:161-170. augmentation in viw leads to e5ects on neighboring cells

through cell-cell interactions. Although not direcdy concerned with ceU lineage this paper shows how transgenic mice can be used to perturb in vivo development and test gene function.