Migration and proliferation of cultured neural crest cells in W mutant neural crest chimeras.

Chimeric mice, generated by aggregating preimplantation embryos, have been instrumental in the study of the development of coat color patterns in mammals. This approach, however, does not allow for direct experimental manipulation of the neural crest cells, which are the precursors of melanoblasts. We have devised a system that allows assessment of the developmental potential and migration of neural crest cells in vivo following their experimental manipulation in vitro. Cultured C57Bl/6 neural crest cells were microinjected in utero into neurulating Balb/c or W embryos and shown to contribute efficiently to pigmentation in the host animal. The resulting neural crest chimeras showed, however, different coat pigmentation patterns depending on the genotype of the host embryo. Whereas Balb/c neural crest chimeras showed very limited donor cell pigment contribution, restricted largely to the head, W mutant chimeras displayed extensive pigmentation throughout, often exceeding 50% of the coat. In contrast to Balb/c chimeras, where the donor melanoblasts appeared to have migrated primarily in the characteristic dorsoventral direction, in W mutants the injected cells appeared to migrate in the longitudinal as well as the dorsoventral direction, as if the cells were spreading through an empty space. This is consistent with the absence of a functional endogenous melanoblast population in W mutants, in contrast to Balb/c mice, which contain a full complement of melanocytes. Our results suggest that the W mutation disturbs migration and/or proliferation of endogenous melanoblasts. In order to obtain information on clonal size and extent of intermingling of donor cells, two genetically marked neural crest cell populations were mixed and coinjected into W embryos. In half of the tricolored chimeras, no co-localization of donor crest cells was observed, while, in the other half, a fine intermingling of donor-derived colors had occurred. These results are consistent with the hypothesis that pigmented areas in the chimeras can be derived from extensive proliferation of a few donor clones, which were able to colonize large territories in the host embryo. We have also analyzed the development of pigmentation in neural crest cultures in vitro, and found that neural tubes explanted from embryos carrying wt or weak W alleles produced pigmented melanocytes while more severe W genotypes were associated with deficient pigment formation in vitro.     

Pluripotency of cultured rabbit inner cell mass cells detected by isozyme analysis and eye pigmentation of fetuses following injection into blastocysts or morulae

Pluripotency of isolated rabbit inner cell masses (ICMs) and cultured (3 days) inner cell mass (ICM) cells was tested by injecting these donor cells into day 3.5 blastocysts (experiment 1) or day 3 morulae (experiment 2) to produce chimeric embryos. Injected (n = 107) and noninjected (n = 103) embryos were transferred to the opposite uterine horns of the same recipient females. Chimerism was determined by adenosine deaminase (ADA) isozyme analysis on fetal tissue and by eye pigmentation at midgestation. In experiment 1, 53% and 64%, respectively, of blastocysts injected with ICMs or cultured ICM cells developed to midgestation, compared with 52% and 48% for controls. Of these fetuses, four (31%) and one (6%), respectively, had ADA chimerism. In experiment 2,38% and 62%, respectively, of the morulae injected with ICMs or cultured ICM cells developed to midgestation, compared with 46% and 56% for control morulae. Six (43%) chimeric fetuses from morulae injected with ICMs were detected by ADA analysis, but 12 (86%) chimeric fetuses were detected by eye pigmentation, indicating that eye pigmentation was a more sensitive marker for chimerism than our ADA assay. None of the 14 fetuses recovered after injecting morulae with cultured ICM cells were chimeric with either marker. No chimeras developed from control embryos. These studies demonstrate (1) that pregnancy rates are not compromised by injection of blastocysts or morulae with ICMs or cultured ICM cells, (2) that chimeric rabbit fetuses can be produced by injecting ICMs into either blastocysts or morulae, and (3) that cultured ICM cells can contribute to embryonic development when injected into blastocysts. © 1993 Wiley-Liss, Inc.     

Experimental embryology of mammals at the Jastrzebiec Institute of Genetics and Animal Breeding

Our Department of Experimental Embryology originated from The Laboratory of Embryo Biotechnology, which was organized and directed by Dr. Maria Czlonkowska until her premature death in 1991. Proving successful international transfer of frozen equine embryos and generation of an embryonic sheep-goat chimaera surviving ten years were outstanding achievements of her term. In the 1990s, we produced advanced fetuses of mice after reconstructing enucleated oocytes with embryonic stem (ES) cells, as well as mice originating entirely from ES cells by substitution of the inner cell mass with ES cells. Attempts at obtaining ES cells in sheep resulted in the establishment of embryo-derived epithelioid cell lines from Polish Heatherhead and Polish Merino breeds, producing overt chimaeras upon blastocyst injection. Successful re-cloning was achieved from 8-cell rabbit embryos, and healthy animals were born from the third generation of cloned embryos. Recently mice were born after transfer of 8-cell embryonic nuclei into selectively enucleated zygotes, and mouse blastocysts were produced from selectively enucleated germinal vesicle oocytes surrounded by follicular cells, upon their reconstruction with 2-cell nuclei and subsequent activation. Embryonic-somatic chimaeras were born after transfer of foetal fibroblasts into 8-cell embryos (mouse) and into morulae and blastocysts (sheep). We also regularly perform the following applications: in vitro production of bovine embryos from slaughterhouse oocytes or those recovered by ovum pick up; cryopreservation of oocytes and embryos (freezing: mouse, rabbit, sheep, goat; vitrification: rabbit, cow); and banking of somatic cells from endangered wild mammalian species (mainly Cervidae).     

Differentiation and grafting of haemopoietic stem cells from early postimplantation mouse embryos

Haemopoietic stem cells evidently arise in early post-implantation mouse embryos at day 6 of gestation, a day earlier than previously thought (Moore & Metcalf, 1970). Disaggregated embryonic cells were injected into mice given a lethal dose of X-irradiation. The presence of donor haemoglobin (Whitney, 1978) and donor lymphocytic glucose phosphate isomerase (GPI) (Siciliano & Shaw, 1976) to detect donor erythrocytes and lymphocytes, respectively, were monitored by starch gel electrophoresis. The presence of donor cells was also assessed by using donor embryos carrying the T6 marker chromosomes. Decidual cells dissected free of embryos did not colonize any recipients. Disaggregated cells from early mouse embryos first colonized the liver and then repopulated the haemopoietic systems of recipients, producing adult donor haemoglobin within 2-3 days and donor GPI within 3-5 days. 80% of grafted X-irradiated recipients survived and donor markers were found in each of them. All nongrafted controls died within 14 days of X-irradiation and none of them showed donor markers. Disaggregated embryonic cells could be grafted across major histocompatibility barriers unlike adult bone marrow. Haemopoietic stem cells could not be identified in disaggregated cells from embryos aged less than 6 days gestation.     

Developmental potency of mouse primitive ectoderm cells, embryonic ectoderm cells and primordial germ cells after blastocyst injection

Developmental potency of primitive and embryonic ectoderm cells from 4.50-day to 6.25-day post-coitum (p.c.)mouse embryos and primordial germ cells from 12.50-day p.c. male genital ridges of fetal mice were studied by direct introducing them into 3.50-day p.c. blastocysts. Sixteen (61.5%) overt chimaeras out of 26(50%) offsprings were obtained after transfer of 52 blastocysts injected with 4.50-day primitive ectoderm cells; four (16.0%) overt chimaeras were obtained out of 25 (51.0%) offsprings with 4.75-day primitive ectoderm cells from 49 transferred blastocysts. However, no overt chimaera was obtained with either 5.25-day or 6.25day embryonic ectoderm cells or 12.50-day male primordial germ cells. GPI analysis of mid-gestation conceptuses developed from injected blastocysts showed that 5.25-day embryonic ectoderm cells could only contributed to yolk sac of conceptus. Results suggested that implantation acts as a trigger for the determination of primitive ectoderm cells, and their developmental potency becomes limited within a short period of time in normal development.     

Immune presensitization and local intrauterine defences as determinants of success or failure of murine interspecies pregnancies

Putatively immuno-incompetent Mus musculus females exhibited failure to support pregnancy of Mus caroli embryos. These results for M. musculus females (i.e. treated by cyclosporine A, of the nu/nu genotype, and as an interspecies chimaera) can be explained in immunological terms. Mus musculus females possessed pre-sensitized cytotoxic T cells against Mus caroli antigen. Nu/nu mice possessed activated NK cells and macrophages, and selectively discriminated against Mus caroli embryos early in pregnancy unlike normal +/+ females; the requirement for T cells to activate non-specific cytotoxic effector mechanisms was bypassed in nu/nu mice. Mus caroli are not inbred, and interspecies chimaeras which are tolerant of the antigens on the Mus musculus donor strain were not tolerant of cells from unrelated Mus caroli. Interspecies chimaeras also behaved as if they were pre-sensitized to Mus caroli. Our results show that Mus caroli embryos recruit fewer active suppressor cells even when gestating in Mus caroli decidua as compared to Mus musculus embryos in Mus musculus decidua and that the ability of Mus caroli placental cells to directly inhibit cytotoxic effector cell killing was inherently less than the inhibitory activity of placental cells from Mus musculus. Mus caroli embryos therefore appear to be less well defended against maternal immune attack even when gestating in a uterus possessing compatible Mus caroli decidual tissue.     

Studies of Sl/Sld in equilibrium with +/+ mouse aggregation chimaeras. I. Different distribution patterns between melanocytes and mast cells in the skin

In spite of their different origin, both melanocytes and mast cells are deficient in the skin of mutant mice of the Sl/Sld genotype. Since the neural crest and the liver of Sl/Sld embryos contain normal precursors of melanocytes and mast cells, respectively, the deficiency is attributed to a defect in tissue environment necessary for migration and/or differentiation of precursor cells. We investigated whether the tissue environment used for differentiation of melanocytes and mast cells was identical by producing aggregation chimaeras from Sl/Sld and +/+ embryos. Chimaeric mice with apparent pigmented and nonpigmented stripes were obtained. In the nonpigmented stripes of these Sl/Sld in equilibrium with +/+ chimaeras, melanocytes were not detectable in hair follicles but were detectable in the dermis. In contrast, melanocytes were detectable neither in hair follicles nor in the dermis of nonchimaeric Sl/Sld mice. Concentrations of mast cells were comparable in the pigmented and nonpigmented stripes of Sl/Sld in equilibrium with +/+ chimaeras, but the average concentration of mast cells significantly varied in the chimaeras (from 8% to 74% of the value observed in control +/+ mice). The present result suggests that mesodermal cells that support the migration and differentiation of both melanocyte precursors and mast-cell precursors mix homogeneously in the dermis and that ectodermal cells that influence the invasion of differentiating melanocytes into hair follicles make discrete patches.     

Thymus differentiation and T-cell specificity in nu/nu +/+ mouse aggregation chimaeras

Thymus development and T cell differentiation were studied in mouse chimaeras produced by aggregating pre-implantation embryos of thymus-deficient nude BALB/c (nu/nu) and wild-type C57BL/6 (+/+) mice and vice versa. Chimaeras showed mosaic distribution of skin and coat pigmentation, of hair follicles, of glucosephosphate isomerase within all tested organs and of lymphocytes expressing the different major transplantation antigens (H-2). When tested for their capacity to generate vaccinia virus-specific and self-H-2 specific cytotoxic T cells, all chimaeras of BALB/c (nu/nu) H-2d in equilibrium C57BL/6 (+/+) H-2b type generated T cells of one or both parental origins that were specific for virus and for self-H-2 of the +/+ (H-2b) type only. In contrast, some BALB/c (+/+) H-2d in equilibrium C57BL/6 (nu/nu) H-2b chimaeras generated vaccinia virus-specific cytotoxic T cells specific for either H-2d (+/+) type or for H-2b (nu/nu) type. These asymmetrical results can be interpreted to indicate the following: (i) The +/+ thymus part alone is functional, but because of asymmetrical cross-reactivities of anti-self-H-2 specificities, the observed T cell restriction phenotypes differ. (ii) Both nu/nu and +/+ thymus parts are functional but immune response defects may be exaggerated in such chimaeras producing unexpected non-responsiveness to vaccinia virus linked to H-2d in H-2b (+/+) in equilibrium H-2d (nu/nu).     

Experimental embryonic-somatic chimaerism in the sheep confirmed by random amplified polymorphic DNA assay

Developmental potencies of sheep somatic cells (foetal fibroblasts, FFs) in chimaeric animals were analysed. FFs from pigmented Polish Heatherhead (wrzosowka) breed were microsurgically injected into morulae or blastocysts of white Polish Merino breed (5 cells to each embryo). In one experiment the cells were stained with vital fluorescent dye PKH26, and chimaeric blastocysts were cultured in vitro to confirm the presence of fluorescent cells. In the majority of experiments the blastocysts were transferred to synchronized recipient ewes for development until term. Cultured embryonic cells (CEC), earlier known to produce chimaeras, were injected into blastocysts in control experiments. Seven young were born from FF-injected embryos and three were born from CEC-injected ones. All of them were white, but all three control lambs and three experimental lambs showed small areas of skin pigmentation, which indicated Heatherhead CEC or FF contribution. Tissue samples originating from three germ layers were taken from two FFs-originating presumably chimaeric lambs (male and female) at the age of one month for DNA analysis. The random amplified polymorphic DNA-PCR method supplied two markers of chimaerism, which were amplification products of 643 bp and 615 bp long DNA fragments, found in tissues of experimental lambs as well as in FFs, but not in the blood of parents of blastocysts. The 643 bp marker was found in the majority of tissues of both lambs. The 615 bp amplicon was detected in the skin and lungs of the female lamb and in the hooves of the male lamb. Our data show that foetal fibroblasts introduced to sheep blastocysts can participate in development and can contribute to all tissue lineages up to at least one month of age.     

Distribution analysis of transferred donor cells in avian blastodermal chimeras.

Blastodermal chimeras were constructed by transferring quail cells to chick blastoderm. Contribution of donor cells to host were histologically analyzed utilizing an in situ cell marker. Of the embryos produced by injection of stage XI-XIII quail cells into stage XI-2 chick blastoderm, more than 50 percent were definite chimeras. The restriction on the spatial arrangement of donor cells was induced by varying the stage of host. Ectodermal chimerism was limited to the head region and no mesodermal chimerism was shown when the quail cells were injected into stage XI-XIII blastoderm. Mesodermal and ectodermal chimerisms were limited to the trunk, not to the head region, when the quail cells were injected into the stage XIV-2 blastoderm. In these chimeras, however, some of the injected quail cells formed ectopic epidermal cysts. Consequently, the stage XIV-2 blastoderm may become intolerant of the injected cells. Our results suggest that it is possible to obtain chimeras that have chimerism limited to a particular germ layer and region by varying the stage of donor cell injection. Injected quail cells contributed to endodermal tissues and primordial germ cells regardless of the injection site. The quail-chick blastodermal chimeras could be useful in the production of a transgenic chicken and in the investigation of immunological tolerance.     

Interspecific melanocyte chimaeras made by introducing rat cells into postimplantation mouse embryos in utero

Dissociated cells of whole midgestation rat embryos were injected into implanted albino mouse embryos on Day 8.5 of gestation in utero. This successfully produced viable interspecific chimaeras which were found to have pigmented hairs. Two of them had many pigmented hairs covering a large area of their bodies, including a forelimb and a hindlimb. The fact that some of the introduced rat cells differentiated into functional melanocytes suggests that embryonic cells of both species were able to interact with each other normally and that the foreign cells were kept from maternal immunological assault.     

Studies of Sl/Sld in equilibrium with +/+ mouse aggregation chimaeras. II. Effect of the steel locus on spermatogenesis

Mutant mice of Sl/Sld genotype are deficient in melanocytes, erythrocytes, mast cells and germ cells. Deficiency of melanocytes, erythrocytes and mast cells is not attributable to an intrinsic defect in their precursor cells but to a defect in the tissue environment that is necessary for migration, proliferation and/or differentiation. We investigated the mechanism of germ cell deficiency in male Sl/Sld mice by producing aggregation chimaeras from Sl/Sld and +/+ embryos. Chimaeric mice with apparent white stripes were obtained. Two of four such chimaeras were fertile and the phenotypes of resulting progenies showed that some Sl/Sld germ cells had differentiated into functioning sperms in the testis of the chimaeras. In cross sections of the testes of chimaeras, both differentiated and nondifferentiated tubules were observed. However, the proportions of type A spermatogonia to Sertoli cells in both types of tubules were comparable to the values observed in differentiated tubules of normal +/+ mice. We reconstructed the whole length of four tubules from serial sections. Differentiated and nondifferentiated segments alternated in a single tubule. The shortest differentiated segment contained about 180 Sertoli cells and the shortest nondifferentiated segment about 150 Sertoli cells. These results suggest that Sertoli cells of either Sl/Sld or +/+ genotype make discrete patches and that differentiation of type A spermatogonia does not occur in patches of Sl/Sld Sertoli cells.     

Mutant gene expression in mouse aggregation chimeras. 8. The effect of the white gene on coat pigmentation

Aggregation of mouse embryos produced 11 chimaeras Miwh/+C/C----+/+c/c and 8 chimaeras +/+C/C----+/+c/c (control). Chimaerism was detected by mosaicism of coat retinal pigment epithelium and by electrophoretic pattern of glucose phosphate isomerase. All chimaeras showed a common pattern of pigmented and unpigmented hair regions that alternated as stripes of different length and width and extended from spine in lateral-ventral direction. However, white coat color predominated in Miwh/+C/C----+/+c/c chimaeras due to a higher proportion of unpigmented zones as well as to weakening of hair color in pigmented areas. Besides, distal regions of limbs were always unpigmented in Miwh/+C/C----+/+c/c chimaeras and completely or partially pigmented in +/+C/C----+/+c/c chimaeras. Pigmented hair regions are often located on the ventral trunk surface where the Miwh/+ heterozygotes usually had an unpigmented spot. The examination of hairs, taken from the same regions of gray coloration, revealed the presence of pigmented, unpigmented and mosaic hairs. The proportion of unpigmented hairs was much higher in Miwh/+C/C----+/+c/c chimaeras than in +/+C/C----+/+c/c chimaeras. The data obtained indicate that a single Miwh gene dose reduced proliferative activity of melanoblasts which resulted in weakening of coat pigmentation.     

Microinjection of cytoplasm or mitochondria derived from somatic cells affects parthenogenetic development of murine oocytes

Cloned mammals are readily obtained by nuclear transfer using cultured somatic cells; however, the rate of generating live offspring from the reconstructed embryos remains low. In nuclear transfer procedures, varying quantities of donor cell mitochondria are transferred with nuclei into recipient oocytes, and mitochondrial heteroplasmy has been observed. A mouse model was used to examine whether transferred mitochondria affect the development of the reconstructed oocytes. Cytoplasm or purified mitochondria from somatic cells derived from the external ear, skeletal muscle, and testis of Mus spretus mice or cumulus cells of Mus musculus domesticus mice were transferred into M. m. domesticus (B6SJLF1 and B6D2F1) oocytes to observe parthenogenetic development through the morula stage. All B6D2F1 oocytes injected with somatic cytoplasm or mitochondria showed delayed development when compared to oocytes injected with buffer. The developmental rates were not different among injected cell sources, with the exception of testis-derived donor cells injected into B6SJLF1 oocytes (P < 0.01). The developmental rate of B6D2F1 oocytes injected with buffer alone (98.8% survival) was different from those injected with somatic cytoplasm (60.8% survival) or somatic mitochondria (56.5% survival) (P < 0.01). Conversely, injection of ooplasm into B6D2F1 oocytes did not affect parthenogenetic development (100% survival). Our results indicate that injection of somatic cytoplasm or mitochondria affected parthenogenetic development of murine oocytes. These results have further implications for in vitro fertilization protocols employing ooplasmic transfer where primary oocyte failure is not confirmed.     

A comparative investigation on the potency of cells from the inner cell mass and trophectoderm of mouse blastocysts to produce chimeras

The ability of trophectoderm (TE) cells to produce chimeric mice (pluripotency) was compared with that of inner cell mass (ICM) cells. TE and ICM cells of blastocysts and hatching or hatched blastocysts derived from albino mice (CD-1, Gpi-1a/a) were aggregated with zona cut 8- to 16-cell stage embryos or injected into the blastocoele from non-albino mice (C57BL/6 x C3H/He, Gpi-1b/b). After transfer to pseudopregnant female mice, the contribution of the donor cells was examined by glucose phosphate isomerase (GPI) analysis of embryos, membrane and placenta at mid-gestation (Day 10.5 and 12.5) or by the coat color of newborn mice. In contrast to ICM cells, there was no contribution of TE cells in the conceptuses and no coat color chimeric young were obtained. After pre-labeling of TE cells with fluorescent latex microparticles, they were aggregated with embryos and the allocation of TE cells at the compacted morula and blastocyst stages was observed under a fluorescent microscope. Although the TE cells were observed attached onto the surface of the embryos at morula and blastocyst stages, unlike the ICM cells, they were not positively incorporated into the embryos. Thus, the pluripotency of TE cells from mouse blastocysts was not induced by the aggregation and injection methods.     

Germline chimeric chickens from FACS‐sorted donor cells

Germline chimeric chickens can be constructed by injecting donor chicken blastodermal cells (CBCs) into recipient embryos and incubating to hatch. Transgenic chickens can be produced through chimeric intermediates if the donor cells are genetically manipulated; the chance of producing a transgenic chimera would be increased by enriching the donor population in transfected cells. To demonstrate that donor CBCs can be sorted according to the expression of a foreign gene, CBCs in suspension were subjected to transfection with plasmid DNA encoding bacterial β‐galactosidase (β‐gal). Following an overnight incubation, the CBCs were loaded with 5‐dodecanoylaminofluorescein di‐β‐D‐galactopyranoside (C₁₂FDG), which is fluorescent after cleavage by β‐gal. The treated cells were subjected to fluorescence activated cell sorting (FACS) to give “positive” (fluorescent) and “negative” (non‐fluorescent) populations. Almost 100% of the “positive” population showed β‐gal activity. “Positive” cells were cultured on mouse SNL 76/7 fibroblast feeder cells and formed colonies, most of which still stained positively for β‐gal activity after three days. FACS‐sorted cells of Barred Plymouth Rock origin were injected into recipient White Leghorn embryos, resulting in chimeric embryos. Of the 298 embryos injected with sorted cells, 23 (8%; 18 injected with “positive cells, five with “negative”) survived to rearing. Somatic chimerism was seen in 12 of 18 (67%) “positive” and three of five (60%) “negative” birds with the proportion of black pigmentation averaging 19% overall. Twenty birds reached sexual maturity, of which 12 (60%) were somatically chimeric; seven (35%) of these produced donor‐derived chicks. Donor CBCs can, therefore, be sorted by FACS according to the expression of a selectable marker gene without impairing their ability to contribute to germline chimeras; this procedure could be incorporated into a practicable method by which to increase the chances of producing a transgenic chicken. Mol. Reprod. Dev. 52:33–42, 1999. © 1999 Wiley‐Liss, Inc.     

Production of chimeric loach by cell transplantation from genetically pigmented to orange embryos

To establish techniques for chimera formation and to obtain further knowledge of chimerism, chimeric loach were produced using the wild strain as the donor and the orange strain as the recipient by cell transplantation. Transplantation between embryos at two different stages was performed to achieve efficient chimera formation. In the combination of the early-mid-blastula as the donor and the late-blastula as the recipient, 100-150 blastomeres were injected into the blastoderm of the recipient and the rate of chimera formation was 46.2%. On the other hand, in the combination of early-mid-blastula and early-gastrula, only 30 blastomeres were injected and the rate of chimera formation was 80.0%. These results demonstrating the combination of embryonic stages may provide a key for efficient chimera formation. We also compared the number of melanophores on chimeric larvae with that on donor cells labelled with latex beads; it was found that the number of transplanted cells has a profound effect on chimerism, whereas the site of pigmentation is not always in agreement with the site of actual transplantation of donor cells.     

Production of somatic and germline chimeras in the chicken by transfer of early blastodermal cells

Cells were isolated from stage X embryos of a line of Barred Plymouth Rock chickens (that have black pigment in their feathers due to the recessive allele at the I locus) and injected into the subgerminal cavity of embryos from an inbred line of Dwarf White Leghorns (that have white feathers due to the dominant allele at the I locus). Of 53 Dwarf White Leghorn embryos that were injected with Barred Plymouth Rock blastodermal cells, 6 (11.3%) were phenotypically chimeric with respect to feather colour and one (a male) survived to hatching. The distribution of black feathers in the recipients was variable and not limited to a particular region although, in all but one case, the donor cell lineage was evident in the head. The male somatic chimera was mated to several Barred Plymouth Rock hens to determine the extent to which donor cells had been incorporated into his testes. Of 719 chicks hatched from these matings, 2 were phenotypically Barred Plymouth Rocks demonstrating that cells capable of incorporation into the germline had been transferred. Fingerprints of the blood and sperm DNA from the germline chimera indicated that both of these tissues were different from those of the inbred line of Dwarf White Leghorns. Bands that were present in fingerprints of blood DNA from the chimera and not present in those of the Dwarf White Leghorns were observed in those of the Barred Plymouth Rocks.(ABSTRACT TRUNCATED AT 250 WORDS)     

Generation of pigmented stripes in albino mice by retroviral marking of neural crest melanoblasts.

The pigment cells of the skin are derived from melanoblasts which originate in the neural crest. The dorsoventral migration of melanoblasts has been visualized in pigment stripes seen in aggregation chimeras, and the width of these bands has suggested that the entire pigmentation of the coat is derived from a small number of founder cells. We have generated mosaic mice by marking single melanoblasts in utero to gain information on the clonal history of pigment-forming cells. A retroviral vector carrying the human tyrosinase gene was constructed and microinjected into neurulating albino mouse embryos. Albino mice are devoid of pigmentation due to deficiency of tyrosinase. Thus, transduction of the wild-type gene into the otherwise normal melanoblasts should rescue the mutant phenotype, giving rise to patches of pigmentation, which correspond to the area colonized by the mitotic progeny of a marked clone. Mosaic animals derived from the injected embryos indeed showed pigmented bands with a width strikingly similar to the 'standard' stripes seen in aggregation chimeras. These results are consistent with the notion that the unit width bands seen in aggregation chimeras represent the clonal progeny of a single melanoblast and verify Mintz's (1967) conclusion that a few founder melanoblasts give rise to coat pigmentation. The pigment cells of the eye are of dual origin: the melanocytes in choroid and outer layer of the iris are derived from the neural crest and those in the pigment layer of the retina from the neuroepithelium of the optic cup. Marked clones in both lineages were observed in the eyes of many mosaic animals.     

Neurite extension by peripheral and central nervous system neurons in response to substratum-bound fibronectin and laminin

Small groups of blastoderm cells were transplanted from wild-type donor embryos into genetically marked host embryos of the same age. Donor cells were injected either into an homologous or an ectopic region of the recipient, and both donor and recipient embryos were allowed to develop. Donor flies were examined for defects in external structures. Recipients were scored for patches of donor-type marked tissue derived from the injected cells. After ectopic transfer, the donor cells recovered in chimaeric recipients differentiated structures consistent with the donor site of cell removal. No apparent fate change was observed. In the rare cases when both individuals of a donor/host pair survived, a direct correspondence could be made between the deleted region in the donor and the chimaeric patch in the host. The results show that blastoderm cells are stably determined to within a segment.