Sexual differentiation of chimeric chickens containing ZZ and ZW cells in the germline

The developmental fate of male and female cells in the ovary and testis was evaluated by injecting blastodermal cells from Stage X (Eyal-Gliadi and Kochav, 1976: Dev Biol 49:321–337) chicken embryos into recipients at the same stage of development to form same-sex and mixed-sex chimeras. The sex of the donor was determined by in situ hybridization of blastodermal cells to a probe derived from repetitive sequences in the W chromosome. The sex of the recipient was assigned after determination of the chromosomal composition of erythrocytes from chimeras at 10, 20, 40, and 100 days of age. If the sex chromosome complement of all of the erythrocytes was the same as that of blastodermal cells from the donor, the sex of the recipient was assumed to be the same as that of the donor. Conversely, if the sex-chromosome complement of a portion of the erythrocytes of the chimera differed from that of the donor blastodermal cells, the sex of the recipient was assumed to differ from that of the donor. Injection of male blastodermal cells into female recipients produced both male and female chimeras in equal proportions whereas injection of female cells into male recipients produced only male chimeras. One phenotypically male chimera developed with a left ovotestis and a right testis although sexual differentiation was usually resolved into an unambiguous sexual phenotype during development when ZZ and ZW cells were present in a chimera. Donor cells contributed to the germline of 25–33% of same-sex chimeras whereas 67% of male chimeras produced by injecting male donor cells into female recipients incorporated donor cells into the germline. When ZW cells were incorporated into chimeric males, W-chromosome-specific DNA sequences were occasionally present in DNA extracted from semen. To examine the potential of W-bearing spermatozoa to fertilize ova, males producing ZW-derived offspring and semen in which W-chromosome-specific DNA was detected by Southern analysis were mated to sex-linked albino hens. Since sex-linked albino female progeny were not obtained from this mating, it was concluded that the W-bearing sperm cells were unable to fertilize ova. The production of Z-derived, but not W-derived, offspring from ZW spermatogonia indicates that female primordial germ cells can become spermatogonia in the testes. In the testes, ZW spermatogonia enter meiosis I and produce functional ZZ spermatocytes. The ZZ spermatocytes complete the second meiotic division, continue to differentiate during spermiogenesis, and leave the seminiferous tubules as functional spermatozoa. By contrast, the WW spermatocytes do not appear to complete spermiogenesis and, therefore, spermatozoa bearing the W chromosome are not produced. When cells from male embryos were incorporated into a female chimera, ZZ “oogonia” were included within the ovarian follicles and the chromosome complement of genetically male oogonia was processed normally during meiosis. Following ovulation, the male-derived ova were fertilized and produced normal offspring. This is the first reported evidence that genetically male avian germ cells can differentiate into functional ova and that genetically female germ cells can differentiate into functional sperm. © 1995 wiley-Liss, Inc.     

Primordial germ cell-mediated chimera technology produces viable pure-line Houbara bustard offspring: potential for repopulating an endangered species

Background

The Houbara bustard (Chlamydotis undulata) is a wild seasonal breeding bird populating arid sandy semi-desert habitats in North Africa and the Middle East. Its population has declined drastically during the last two decades and it is classified as vulnerable. Captive breeding programmes have, hitherto, been unsuccessful in reviving population numbers and thus radical technological solutions are essential for the long term survival of this species. The purpose of this study was to investigate the use of primordial germ cell-mediated chimera technology to produce viable Houbara bustard offspring.

Methodology/Principal Findings

Embryonic gonadal tissue was dissected from Houbara bustard embryos at eight days post-incubation. Subsequently, Houbara tissue containing gonadal primordial germ cells (gPGCs) was injected into White Leghorn chicken (Gallus gallus domesticus) embryos, producing 83/138 surviving male chimeric embryos, of which 35 chimeric roosters reached sexual maturity after 5 months. The incorporation and differentiation of Houbara gPGCs in chimeric chicken testis were assessed by PCR with Houbara-specific primers and 31.3% (5/16) gonads collected from the injected chicken embryos showed the presence of donor Houbara cells. A total of 302 semen samples from 34 chimeric roosters were analyzed and eight were confirmed as germline chimeras. Semen samples from these eight roosters were used to artificially inseminate three female Houbara bustards. Subsequently, 45 Houbara eggs were obtained and incubated, two of which were fertile. One egg hatched as a male live born Houbara; the other was female but died before hatching. Genotyping confirmed that the male chick was a pure-line Houbara derived from a chimeric rooster.

Conclusion

This study demonstrates for the first time that Houbara gPGCs can migrate, differentiate and eventually give rise to functional sperm in the chimeric chicken testis. This approach may provide a promising tool for propagation and conservation of endangered avian species that cannot breed in captivity.     

Demonstration of the neural crest origin of type I (APUD) cells in the avian carotid body,using a cytochemical marker system

Summary The biogenic amines present in the carotid body Type 1 cells of two avian species (Japanese quail and chicken) were identified, by microspectrofluorometry of formaldehyde-induced fluorescence, as dopamine and 5-hydroxytryptamine respectively. These and other cytochemical properties establish the cells as members of the APUD series.Grafts of the neural rhombencephalic primordium from 6 to 10-somite quail embryos were implanted in the appropriate region of chick embryos of the same age. After up to 11 days incubation the carotid bodies of the host were freeze-dried and treated with hot formaldehyde vapour. The carotid body Type 1 cells in the chick host were identified, by the presence of dopamine and the absence of 5-HT, as cells from the quail neural crest.The dopamine phenotype in cells of quail origin thus provides a cytochemical marker which may be used for other allograft experiments. The present work confirms earlier findings, using a biological (nuclear chromatin) marker, which showed the avian carotid body to be of neural crest origin.     

Chick embryos can form teratomas from microinjected mouse embryonic stem cells

We examined whether chick embryos are a suitable experimental model for the evaluation of pluripotency of stem cells. Mouse embryonic stem cells (mESCs) expressing the reporter gene, LacZ or GFP were injected into the subgerminal cavity of blastoderms (freshly oviposited) or the marginal vein of chick embryos (2 days of incubation). Injected mESCs were efficiently incorporated into the body and extra‐embryonic tissues of chick embryos and formed small clusters. Increased donor cell numbers injected were positively associated with the efficiency of chimera production, but with lower viability. A single mESC injected into the blastoderm proliferated into 34.7 ± 3.8 cells in 3 days, implying that the chick embryo provides an optimal environment for the growth of xenogenic cells. In the embryo body, mESCs were interspersed as small clustered chimeras in various tissues. Teratomas were observed in the yolk sac and the brain with three germ layers. In the yolk sac, clusters of mESCs gradually increased in volume and exhibited varied morphology such as a water balloon‐like or dark‐red solid mass. However, mESCs in the brain developed into a large soft tissue mass of whitish color and showed a tendency to differentiate into ectodermal lineage cells, including primitive neural ectodermal and neuronal cells expressing the neurofilament protein. These results indicate that chick embryos are useful for the teratoma formation assays of mESCs and have a broad‐range potential as an experimental host model.     

FH3, a v-myc avian retrovirus with limited transforming ability.

We have isolated a new acute avian transforming virus which contains the oncogene myc. This virus, designated FH3, was isolated after injection of a 10-day-old chick embryo with avian leukosis virus. While FH3 shares many properties with other v-myc-containing avian retroviruses, it also has several unique properties. The primary target for transformation in vitro is chicken macrophages; infection of chicken fibroblasts does not lead to complete morphological transformation. FH3 also exhibits a limited host range, in that Japanese quail macrophages and fibroblasts are infected but are not completely transformed. FH3 induces in vivo a limited tumor type if injected into 10-day-old chick embryos; only a cranial myelocytoma, which does not appear to be metastatic, can be detected. The v-myc gene of FH3 is expressed predominantly as a P145 Gag-Myc protein which is encoded by a ca. 8-kilobase genomic RNA. This FH3-encoded polyprotein is localized in the nucleus of all infected cells, whether or not they are transformed.     

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.     

Improved germline transmission in chicken chimeras produced by transplantation of gonadal primordial germ cells into recipient embryos

In the avian species, germline chimera production could be possible by transfer of donor germ cells into the blood vessel of recipient embryos. This study was conducted to establish an efficient transfer system of chicken gonadal primordial germ cells (gPGCs) for producing the chimeras having a high capacity of germline transmission. Gonadal PGCs retrieved from 5.5-day-old embryos (stage 28) of Korean Ogol chicken (KOC with i/i gene) were transferred into the dorsal aorta of 2.5-day-old embryos (stage 17) of White Leghorn chicken (WL with I/I gene). Prospective evaluations of whether culture duration (0, 5, or 10 days) and subsequent Ficoll separation of gPGCs before transfer affected chimera production and germline transmission in the chimeras were made while retrospective analysis was conducted for examining the effect of chimera sexuality. A testcross analysis by artificial insemination of presumptive chimeras with adult KOC was performed for evaluating each treatment effect. First, comparison was made for evaluating whether experimental treatments could improve chimera production, but none of the treatments were significantly (P = 0.6831) influenced (5.1%-14.4%). Second, it was determined whether each treatment could enhance germline transmission in produced chimeras. More (P < 0.0001) progenies with black feathers (i/i) were produced in the germline chimeras derived from the transfer of 10-day-cultured gPGCs than from the transfer of 0- or 5-day-cultured gPGCs (0.6%-7.8% vs. 10.7%-49.7%). Ficoll separation was negatively affected (P < 0.0001), whereas there was no effect in chimera sexuality (P = 0.6011). In conclusion, improved germline transmission of more than a 45% transmission rate was found in chicken chimeras produced by transfer of 10-day-cultured gPGCs being separated without Ficoll treatment.     

转基因DT40细胞导入早期鸡胚后的分布及绿色荧光蛋白表达的研究

目的为了研究经过基因修饰的体细胞导入到禽类胚胎以后,供体细胞及外源基因是否能在受体胚胎中成活并且外源基因是否可以长期表达。方法筛选得到稳定整合绿色荧光蛋白基因的鸡DT40细胞作为外源蛋白的运载工具,通过血管微注射的方法将其导入到于38.5℃温度条件下孵化65～70 h的鸡胚中,并将操作后的鸡胚在原孵化条件下继续孵化。在孵化的不同时期取移植了DT40细胞的嵌合体胚胎在荧光显微镜下观察荧光细胞的存活与分布情况。并通过PCR以及免疫组织化学方法检测供体细胞在受体中的位置以及绿色荧光蛋白的表达情况。结果荧光标记的DT40细胞可以存活于受体不同的组织器官中,包括：脑、心脏、肝脏等。导入胚胎的整合外源基因的DT40细胞可以存活到胚胎出雏之前,并且外源基因能够正常表达。结论可以通过此方法将外源基因导入到受体中,并使目的蛋白在受体胚胎中持续表达,为胚胎期导入外源蛋白诱导免疫耐受的研究以及将转基因细胞移植到动物体内生产目的蛋白的研究提供科学依据和技术平台。     

The fate of female donor blastodermal cells in male chimeric chickens.

In previous experiments in our laboratories, chickens that are chimeric in their gamete, melanocyte, and blood cell populations have been produced by injection of dispersed stage X blastodermal donor cells into the subgerminal cavity of stage X recipient embryos. In some experiments, donor cells were transfected with reporter gene constructs prior to injection as a preliminary step in the production of transgenic birds. Chimerism was assessed by test mating, observation of plumage, and DNA fingerprinting. Methods were sought that would provide a relatively rapid analysis of the spatial distribution of descendants of donor cells in chimeras to assess the efficacy of various methods of chimera construction. To date, the sex of donor and recipient embryos was not known and, therefore, numerous mixed sex chimeras must have been constructed by chance, since donor cells were usually collected from several embryos rather than from individual embryos. The presence of female-derived cells was determined by in situ hybridization using a W-chromosome-specific DNA probe, using smears of washed erythrocytes from 16 phenotypically male chimeric chickens ranging in age from 4 days to 42 months posthatching. The proportion of female cells detected in the erythrocyte samples was zero (eight samples) or very low (0.020-0.083%), although 1% of the erythrocytes from a phenotypically male chick that was killed 4 days after hatch were female-derived. The low proportions of female-derived cells were surprising, considering that most of these chimeras had been produced by the injection of cells pooled from several donor embryos and most recipients had been exposed to gamma irradiation prior to injection, thus dramatically enhancing the level of incorporation of donor cells into the resulting chimeras. By contrast, 0-100% of the erythrocytes were female-derived in blood samples taken at 10 days of incubation from the chorioallantois of seven phenotypically normal male embryos that resulted from the injection of blastodermal cells pooled from five embryos into irradiated recipient embryos. Approximately 70% of the erythrocytes in a blood sample from a phenotypically normal female chimeric embryo were female-derived, and 100% of the erythrocytes examined from an intersex embryo bearing a right testis and a left ovary were female-derived. These results indicate that female-derived cells can contribute to the formation of erythropoietic tissue during the early development of what will become a phenotypically male chimeric embryo. It would appear, therefore, that female-derived cells are blocked in development or destroyed, or certain male-female combinations of cells may be lethal prior to hatching.(ABSTRACT TRUNCATED AT 400 WORDS)     

The Cloning and Characterization of Chick Tyrosinase from a Novel Embryonic cDNA Library

Very little is known about the genes involved in the regulation of avian skin and feather pigmentation. In mammals, two gene families have been identified as being important for the regulation of melanin biosynthesis. To isolate the avian equivalents of these families, we have generated an embryonic chick melanocyte cDNA library. Neural crest cells from 500 black chick embryos were cultured under conditions supportive of melanocyte differentiation and proliferation. A cDNA library was constructed and screened with a mouse tyrosinase cDNA probe. Nineteen clones were obtained, seven of which cross-hybridized to a mouse tyrosinase cDNA on Southern blots. The longest of these clones, B8.3 (1.9 kb), was sequenced and found to share 99.7% nucleotide and 99.8% amino acid sequence homology to a reported chick tyrosinase cDNA. Both Northern blot analysis andin situhybridization demonstrated that clone B8.3 was expressed in the retinal pigment epithelium of chick embryos. Our results suggest therefore that the cDNA library described here may allow the cloning of novel melanogenic genes.     

Differences in the Developmental Fate of Cultured and Noncultured Myoblasts When Transplanted into Embryonic Limbs

Myoblasts from embryonic, fetal, and adult quail and chick muscles were transplanted into limb buds of chick embryos to determine if myoblasts can form muscle fibers in heterochronic limbs and to define the conditions that affect the ability of transplanted cells to populate newly developing limb musculature. Myoblasts from each developmental stage were either freshly isolated and transplanted or were cultured prior to transplantation into limb buds of 4- to 5-day (ED4-5) chick embryos. Transplanted myoblasts, regardless of the age of the donor from which they were derived, formed muscle fibers within embryonic limb muscles. Transplanted cloned myoblasts formed muscle fibers, although there was little evidence that the number of transplanted myoblasts significantly increased following transplantation or that they migrated any distance from the site of injection. The fibers that formed from transplanted clonal myoblasts often did not persist in the host limb muscles until ED10. Diminished fiber formation from myoblasts transplanted into host limbs was observed whether myoblasts were cloned or cultured at high density. However, when freshly isolated myoblasts were transplanted, the fibers they formed were numerous, widely dispersed within the limb musculature, and persisted in the muscles until at least ED10. These results indicate that transplanted myoblasts of embryonic, fetal, and adult origin are capable of forming fibers during early limb muscle formation. They also indicate that even in an embryonic chick limb where proliferation of endogenous myoblasts and muscle fiber formation is rapidly progressing, myoblasts that are cultured in vitro do not substantially contribute to long-term muscle fiber formation after they are transplanted into developing limbs. However, when the same myoblasts are freshly isolated and transplanted without prior cell culture, substantial numbers of fibers form and persist after transplantation into developing limbs. Thus, these studies demonstrate that the extent to which transplanted myoblasts fuse to form fibers which persist in host musculature depends upon whether donor myoblasts are freshly isolated or maintained in vitro prior to injection.     

Primordial germ cell‐specific expression of eGFP in transgenic chickens

PR domain zinc finger protein 14 (PRDM14) plays an essential role in the development of primordial germ cells (PGCs) in mice. However, its functions in avian species remain unclear. In the present study, we used CRISPR/Cas9 to edit the PRDM14 locus in chickens in order to demonstrate its importance in development. The eGFP gene was introduced into the PRDM14 locus of cultured chicken PGCs to knockout PRDM14 and label PGCs. Chimeric chickens were established by a direct injection of eGFP knocked‐in (gene‐trapped) PGCs into the blood vessels of Hamburger–Hamilton stages (HH‐stages) 13–16 chicken embryos. Gene‐trapped chickens were established by crossing a chimeric chicken with a wild‐type hen with very high efficiency. Heterozygous gene‐trapped chickens grew normally and SSEA‐1‐positive cells expressed eGFP during HH‐stages 13–30. These results indicated the specific expression of eGFP within circulating PGCs and gonadal PGCs. At the blastodermal stage, the ratio of homozygous gene‐trapped embryos obtained by crossing heterozygous gene‐trapped roosters and hens was almost normal; however, all embryos died soon afterward, suggesting the important roles of PRDM14 in chicken early development.     

Production of germ-line chimeras in rainbow trout by blastomere transplantation

We describe a technique for producing germ-line chimeric rainbow trout, Oncorhynchus mykiss, by microinjection of the isolated blastomeres. FITC-labeled donor cells and non-labeled recipient embryos at various developmental stages between the early blastula and early gastrula stages were used for cell transplantation. The chimera formation rate and the degree of donor cell distribution in recipient embryos were evaluated at both the late gastrula stage (5 days post fertilization (dpf)) and the 40-somite stage (10 dpf). Among the six combinations of developmental stages of donor and recipient embryos, the combination of midblastula (2.5 dpf) donor cells and early blastula (1.5 dpf) recipient embryos gave the highest chimera formation rate and the best distribution pattern of donor cells. Using this combination, chimeric rainbow trout were produced with donor blastomeres from dominant orange-colored mutant embryos and wild-type recipient embryos. Of the 238 chimeric embryos produced, 28 (12%) hatched normally and 14 of the 28 fry (50%) had donor-derived orange body color. To test for germ-line transmission of donor cells, gametes obtained from the matured chimeras were fertilized with gametes from wild-type fish. Of the 19 matured chimeras, 6 (32%) yielded donor-derived orange-colored progeny, in addition to wild-type siblings. The contribution rates of donor cells in the germ-line ranged from 0.3 to 14%. This technique for producing germ-line chimeras should be a powerful tool for cell-mediated gene transfer in rainbow trout. Especially, if body color mutants are used for either donor cells or the host embryos, it will be possible to easily concentrate F1 transgenic embryos derived from transplanted donor cells by body color screening. Mol. Reprod. Dev. 59: 380-389, 2001.     

同种异体宫内移植小鼠嵌合模型的建立

干细胞宫内移植是一种很有前途的产前治疗方式。为深入研究干细胞移植后的细胞行为,采用宫内移植的方法建立同种异体的嵌合小鼠模型。将雄鼠骨髓单核细胞宫内注射到胎鼠腹腔,在受体鼠出生后检测雌性受体鼠外周血细胞。应用PCR检测外周血是否存在雄性鼠的DNA,并采用定量PCR技术确定其嵌合量;同时用荧光原位杂交（FISH）技术直观观察外周血中雄性来源的细胞。结果表明：共获得4只阳性外周血嵌合小鼠,其中3只稳定嵌合达到6个月以上。应用宫内移植成功建立了外周血中存在异源细胞的小鼠嵌合模型。     

Enriched gonadal migration of donor-derived gonadal primordial germ cells by immunomagnetic cell sorting in birds

This study was conducted to evaluate whether immunomagnetic treatment could improve the retrieval and migration capacity of avian gonadal primordial germ cells (gPGCs) collected from gonads in 5.5-day-old chick and 5-day-old quail embryos, respectively. Collected gPGCs were loaded into a magnetic-activated cell sorter (MACS) after being conjugated with specific gPGC antibodies and either MACS-treated or non-treated cells in each species were subsequently transferred to the recipient embryos. MACS treatment significantly (P < 0.05) increased the population ratio of gPGCs in gonadal cells retrieved (0.74 to 33.4% in the chicken and 2.68 to 45.1% in the quail). This was due to decreased number of non-gPGCs in total cell population. MACS treatment further enhanced gonadal migration of gPGCs transferred in both species (10% vs. 80-85% in the chicken and 10-15% vs. 70-80% in the quail). Increase in the number of microinjected cells up to 600 cells/embryo did not eliminate such promoting effect. In conclusion, MACS treatment greatly increased the population ratio of avian gPGCs in gonadal cells, resulting improved gonadal migration in recipient embryos.     

Mouse-chick neural chimeras

Embryonic chimera production was used to study the developmental processes of the mouse nervous system. The difficulty of performing in situ transplantation experiments of neural primordium of mouse embryo was overcome by isotopic and isochronic grafting of mouse neural tube fragments into chick embryo. Mouse neural tube cells differentiated perfectly in ovo and neural crest cells associated with the grafted neural tube were able to migrate and reach the normal arrest sites of host neural crests. Cranial neural crest cells penetrated into chick facial areas and entered into the development of dental bud structures, participating in vibrissa formation. Depending on graft level, in ovo implanted mouse neural crest cells formed different components of the peripheral nervous system. At trunk level, they located in spinal ganglia and orthosympathetic chains and gave rise to Schwann cells lining the nerves. When implanted into the lumbosacral region, they penetrated into the enteric nervous system. At the precise 18-24 somite level, they colonized host adrenal gland. Mouse neural tube was involved in the mechanisms required to maintain myogenesis in host somites. Furthermore in ovo grafts of mouse cells from genetically modified embryos, in which many mutations induce early death, are particularly useful to investigate cellular events involved in the development of the nervous system and to identify molecular events of embryogenesis.     

Differential localization of mRNAs of collagen types I and II in chick fibroblasts, chondrocytes, and corneal cells by in situ hybridization using cDNA probes

We have employed a highly specific in situ hybridization protocol that allows differential detection of mRNAs of collagen types I and II in paraffin sections from chick embryo tissues. All probes were cDNA restriction fragments encoding portions of the C-propeptide region of the pro alpha-chain, and some of the fragments also encoded the 3'-untranslated region of mRNAs of either type I or type II collagen. Smears of tendon fibroblasts and those of sternal chondrocytes from 17-d-old chick embryos as well as paraffin sections of 10-d-old whole embryos and of the cornea of 6.5-d-old embryos were hybridized with 3H-labeled probes for either type I or type II collagen mRNA. Autoradiographs revealed that the labeling was prominent in tendon fibroblasts with the type I collagen probe and in sternal chondrocytes with the type II collagen probe; that in the cartilage of sclera and limbs from 10-d-old embryos, the type I probe showed strong labeling of fibroblast sheets surrounding the cartilage and of a few chondrocytes in the cartilage, whereas the type II probe labeled chondrocytes intensely and only a few fibroblasts; and that in the cornea of 6.5-d-old embryos, the type I probe labeled the epithelial cells and fibroblasts in the stroma heavily, and the endothelial cells slightly, whereas the type II probe labeled almost exclusively the epithelial cells except for a slight labeling in the endothelial cells. These data indicate that embryonic tissues express these two collagen genes separately and/or simultaneously and offer new approaches to the study of the cellular regulation of extracellular matrix components.     

In vivo analysis of a new lacZ retrovirus vector suitable for cell lineage marking in avian and other species

To obtain a replication-defective retrovirus vector well suited for cell lineage marking in early avian embryos, we have constructed and tested a derivative of the avian spleen necrosis virus (SNV) carrying the marker gene lacZ. Consistently high titers of this virus, designated CXL, were produced from retroviral packaging cells with no evidence of contaminating helper virus even after 12 months of continuous culture. CXL expresses lacZ strongly and stably in avian cells and has a host range that extends to other avian and some mammalian species. We show that CXL has the potential to mark a wide variety of chick embryo cell types by infection in ovo. The high titers obtainable with this virus can provide a significant advantage over alternative lacZ vectors, especially in lineage marking of early stage embryos. As an example of this, we show that CXL can be used to mark cells of the precardiac mesoderm in stage 4-5 chick embryos.     

Differentiation of female chicken primordial germ cells into spermatozoa in male gonads

In avian species, the developmental fate of different-sex germ cells in the gonads is unclear. The present study attempted to confirm whether genetically female germ cells can differentiate into spermatozoa in male gonads using male germline chimeric chickens produced by the transfer of primordial germ cells (PGC), and employing molecular biological methods. As a result of Southern hybridization, specific sequences of the W chromosome (the female specific sex chromosome in birds) were detected in the genomic DNA extracted from one out of four male germline chimeric chickens. When two-color in situ hybridization was conducted on the spermatozoa of this germline chimera, 0.33% (average) of the nuclei of each semen sample showed the fluorescent signal indicating the presence of the W chromosome. The present study shows that female PGC can differentiate into spermatozoa in male gonads in the chicken. However, the ratio of produced W chromosome-bearing (W-bearing) spermatozoa fell substantially below expectations. It is therefore concluded that most of the W-bearing PGC could not differentiate into spermatozoa because of restricted spermatogenesis.