Expression of Axwnt-8 and Axszl in the urodele, axolotl: comparison with Xenopus.

In both the urodele axolotl and the anuran Xenopus, Wnt-8 is expressed in posterior lateral plate mesoderm (LPM) in neurula and tailbud stages. In contrast to Xenopus, expression in axolotl is more prominent in gastrula endoderm, is not initiated in mesoderm until late gastrulation, and is present in the tailbud and in the brain at tailbud stages. Sizzled is expressed in axolotl in the ventral region, similar to its pattern in Xenopus. In axolotl, the Wnt-8-expressing LPM remains relatively dorsal through tailbud stages, while ventral blood island (VBI) markers appear in a wide ventral arc.

Expression of axolotl DAZL RNA, a marker of germ plasm: widespread maternal RNA and onset of expression in germ cells approaching the gonad.

How germ cell specification occurs remains a fundamental question in embryogenesis. The embryos of several model organisms contain germ cell determinants (germ plasm) that segregate to germ cell precursors. In other animals, including mice, germ cells form in response to regulative mechanisms during development. To investigate germ cell determination in urodeles, where germ plasm has never been conclusively identified, we cloned a DAZ-like sequence from axolotls, Axdazl. Axdazl is homologous to Xdazl, a component of Xenopus germ plasm found in the vegetal pole of oocytes and eggs. Axdazl RNA is not localized in axolotl oocytes, and, furthermore, these oocytes do not contain the mitochondrial cloud that localizes Xdazl and other germ plasm components in Xenopus. Maternal Axdazl RNA is inherited in the animal cap and equatorial region of early embryos. At gastrula, neurula, and tailbud stages, Axdazl RNA is widely distributed. Axdazl first shows cell-specific expression in primordial germ cells (PGCs) approaching the gonad at stage 40, when nuage (germ plasm) appears in PGCs. These results suggest that, in axolotls, germ plasm components are insufficient to specify germ cells.

Establishment of anterior-posterior polarity in avian embryos.

In pre-streak chick embryos, the extraembryonic posterior marginal zone is able to induce an embryonic axis at an ectopic site without contributing cells to the induced primitive streak. This region expresses mesoderm-inducing factors that are capable of inducing an ectopic streak. Downstream of these events, chordin and bone morphogenetic protein acting within the central disc may play mutually opposing roles influencing streak formation. Although extraembryonic regions are important in establishing the embryonic axis, there does not appear to be an anterior region with head-inducing activity similar to that of the anterior visceral endoderm of the mammalian embryo.

Apoptosis in mouse embryos: elevated levels in pregastrulae and in the distal anterior region of gastrulae of normal and mutant mice.

The pattern of apoptotic cell death has been surveyed in prestreak and primitive streak embryos of four strains of mice and in three mutants affecting gastrulation. In C57BL/6 embryos, a high level of cell death occurs in the early egg cylinder stage at embryonic day 5 (E5) to E5.5. In all strains, cell death is elevated shortly before gastrulation, but the level varies four- to fivefold among strains. During gastrulation, cell death declines but is relatively more abundant in the distal and distal anterior regions. Early streak embryos cultured in media with reduced levels of growth factors show increased cell death mainly in the distal region. In three mutants with disturbed function of the proximal visceral endoderm and/or primitive streak, cell death is increased, and the regional pattern seen in normal embryos is intensified. The results strongly suggest that the proximal visceral endoderm and primitive streak region are the principal sites of synthesis of growth factors promoting cell survival. We conclude that localized growth factor supply has an important role in regulating the size of the embryo and of embryonic regions.

Induction of primitive streak and Hensen's node by the posterior marginal zone in the early chick embryo.

In the preprimitive streak chick embryo, the search for a region capable of inducing the organizer, equivalent to the Nieuwkoop Center of the amphibian embryo, has focused on Koller's sickle, the hypoblast and the posterior marginal zone. However, no clear evidence for induction of an organizer without contribution from the inducing tissue has been provided for any of these structures. We have used DiI/DiO labeling to establish the fate of midline cells in and around Koller's sickle in the normal embryo. In the epiblast, the boundary between cells that contribute to the streak and those that do not lies at the posterior edge of Koller's sickle, except at stage X when it lies slightly more posteriorly in the epiblast. Hypoblast and endoblast (a second lower layer formed under the streak) have distinct origins in the lower layer, and goosecoid expression distinguishes between them. We then used anterior halves of chick prestreak embryos as recipients for grafts of quail posterior marginal zone; quail cells can be identified subsequently with a quail-specific antibody. Anterior halves alone usually formed a streak, most often from the posterior edge. Quail posterior marginal zones without Koller's sickle were grafted to the anterior side of anterior halves. These grafts were able to increase significantly the frequency of streaks arising from the anterior pole of stage X-XI anterior halves without contributing to the streak or node. Stage XII anterior halves no longer responded. A goosecoid-expressing hypoblast did not form under the induced streak, indicating that it is not required for streak formation. We conclude that the marginal zone posterior to Koller's sickle can induce a streak and node, without contributing cells to the induced streak.

Kit ligand mediates survival of type A spermatogonia and dividing spermatocytes in postnatal mouse testes.

In the mouse testis, spontaneous death of spermatogonia has a large impact on the output of differentiating spermatids. The tyrosine kinase receptor c-kit is expressed in type A, intermediate, and B spermatogonia, and kit-ligand (KL) is expressed in Sertoli cells. Previous work indicated a depletion of type A spermatogonia after in vivo exposure to an antibody that blocks c-kit function. The present work was undertaken to determine whether blocking c-kit function results in apoptosis of spermatogonia or in an inability of spermatogonia to proliferate. Testes sections were stained by a method that detects apoptotic cells in situ. In testes of 8-day postnatal (P8) males, type A spermatogonia are the predominant germ cell type present. Stained sections from P8 males injected with the c-kit antagonistic antibody ACK2 showed a fivefold higher rate of cell death than uninjected controls. At least a twofold increase was observed in P12 and P30 injected males and in P30 SId/+ males as compared to uninjected controls. Determination of the stage of germ cell development that was affected in P30 males indicated that the frequency of gonial cell death was increased fourfold, but the frequency of death in spermatocytes around the time of the meiotic division was increased 15-fold. It is concluded that KL acts to prevent apoptosis in the testis in vivo, that the membrane bound form of KL may be more effective, and that survival of late meiotic and dividing spermatocytes is regulated by KL through an indirect mechanism probably mediated by Sertoli cells. Thus, KL is an important regulator of spermatid output.

The Wsh and Ph mutations affect the c-kit expression profile: c-kit misexpression in embryogenesis impairs melanogenesis in Wsh and Ph mutant mice.

The receptor tyrosine kinases (RTKs) c-kit and platelet-derived growth factor receptor alpha chain (PDG-FRa) are encoded at the white spotting (W) and patch (Ph) loci on mouse chromosome 5. While W mutations affect melanogenesis, gametogenesis, and hematopoiesis, the Ph mutation affects melanogenesis and causes early lethality in homozygotes. W-sash (Wsh) is an expression mutation and blocks c-kit expression in certain cell types and enhances c-kit expression in others, including at sites important for early melanogenesis. We have determined the effect of Ph on c-kit expression during embryogenesis in Ph heterozygotes. Immunohistochemical analysis revealed enhanced c-kit expression in several cell types, including sites important for early melanogenesis. We propose that in both Wsh and Ph mutant mice c-kit misexpression affects early melanogenesis and is responsible for the pigment deficiency. Moreover, we have defined the organization of the RTKs in the W/Ph region on chromosome 5 and characterized the Wsh mutation by using pulsed-field gel electrophoresis. Whereas the order of the RTK genes was determined as Pdgfra-c-kit-flk1, analysis of the Wsh mutation revealed that the c-kit and Pdgfra genes are unlinked in Wsh, presumably because of an inversion of a small segment of chromosome 5. The Ph mutation consists of a deletion including Pdgfra and the 3' deletion endpoint of Ph lies between Pdgfra and c-kit. Therefore, positive 5' upstream elements controlling c-kit expression in mast cells and some other cell types are affected by the Wsh mutation and negative elements are affected by both the Wsh and the Ph mutation.

mRNAs for activin receptors II and IIB are expressed in mouse oocytes and in the epiblast of pregastrula and gastrula stage mouse embryos.

Activin is a potent inducer of mesoderm in frog embryos. We showed previously that in the mouse, activin beta A is expressed in the uterine decidua near the embryo before and during the first appearance of mesoderm (E4.5-E6.5). Here, using Northern blotting and in situ hybridization, we show that mouse oocytes, E6.5 and E7.5 embryos, and E6.5 and E7.5 decidua contain mRNAs for both activin receptors type II and IIB. The expression of activin receptor type IIB is particularly strong in embryonic ectoderm apparent at E5.5 and continuing through E8.5. These results support the hypothesis that activin derived from the decidua promotes development of mesoderm in the period E5.5-E6.5.

Disruption of the HNF-4 gene, expressed in visceral endoderm, leads to cell death in embryonic ectoderm and impaired gastrulation of mouse embryos.

Expression of HNF-4, a transcription factor in the steroid hormone receptor superfamily, is detected only in the visceral endoderm of mouse embryos during gastrulation and is expressed in certain embryonic tissues from 8.5 days of gestation. To examine the role of HNF-4 during embryonic development, we disrupted the gene in embryonic stem cells and found that the homozygous loss of functional HNF-4 protein was an embryonic lethal. Cell death was evident in the embryonic ectoderm at 6.5 days when these cells normally initiate gastrulation. As assessed by expression of Brachyury and HNF-3 beta, primitive streak formation and initial differentiation of mesoderm do occur, but with a delay of approximately 24 h. Development of embryonic structures is severely impaired. These results demonstrate that the expression of HNF-4 in the visceral endoderm is essential for embryonic ectoderm survival and normal gastrulation.

Expression of transcription factor HNF-4 in the extraembryonic endoderm, gut, and nephrogenic tissue of the developing mouse embryo: HNF-4 is a marker for primary endoderm in the implanting blastocyst.

The expression of HNF-4 (hepatocyte nuclear factor 4) mRNA in postimplantation mouse embryos was analyzed by in situ hybridization. Expression was found in the primary endoderm at embryonic day 4.5 and was restricted to the columnar visceral endoderm cells of the yolk sac from day 5.5 to day 8.5. HNF-4 mRNA was first detected in embryonic tissues at day 8.5, in the liver diverticulum and the hindgut. At later times HNF-4 transcripts were observed in the mesonephric tubules, pancreas, stomach, and intestine and, still later, in the metanephric tubules of the developing kidney. This expression pattern suggests that HNF-4 has a role in the earliest stages of murine postimplantation development as well as in organogenesis.

The ligand of the c-kit receptor promotes oocyte growth.

Both genetic and descriptive studies have implicated the c-kit receptor and its ligand, KL, in the process of oocyte growth in the postnatal mouse ovary. In order to test the hypothesis that KL is an oocyte growth factor, we used an oocyte culture system to study its effects in vitro. Initial experiments established that both ovarian c-kit and KL are biologically active. An immune complex kinase assay demonstrated that ovarian c-kit, found primarily on oocytes, has autophosphorylation activity, and a bone marrow-derived mast cell coculture assay indicated that granulosa cells produce functional KL. The addition of 10 ng/ml KL to growing follicles cultured in collagen gels resulted in a 67% increase in the rate of oocyte growth, and a doubling of the rate was achieved at around 50 ng/ml. ACK2, a monoclonal antibody against c-kit, severely inhibited the growth of late fetal and neonatal oocytes in coculture with ovarian cells and had less effect on growing oocytes cultured in follicles from 10- to 11-day-old mice. Genistein, an inhibitor of tyrosine kinases, including c-kit, blocked oocyte growth and disrupted follicle morphology. In initial studies on the regulation of KL production in granulosa cells, we found that both dibutyryl cyclic AMP and growing oocytes were able to induce increased KL mRNA accumulation in granulosa cell monolayers as assessed by Northern analysis. These studies demonstrate that c-kit and KL are required for maintenance of oocyte growth in vitro.

W-sash affects positive and negative elements controlling c-kit expression: ectopic c-kit expression at sites of kit-ligand expression affects melanogenesis.

The receptor tyrosine kinase c-kit and its cognate ligand KL are encoded at the white spotting (W) and steel (Sl) loci of the mouse, respectively. Mutations at both the W and the Sl locus cause deficiencies in gametogenesis, melanogenesis and hematopoiesis (erythrocytes and mast cells). The W-sash mutation differs from most W mutations in that it affects primarily mast cells and melanogenesis but not other cellular targets of W and Sl mutations. Thus, Wsh/Wsh mice are fertile and not anemic, but they lack mast cells in their skin and intestine and are devoid of coat pigment. Heterozygotes are black with a broad white sash/belt in the lumbar region. In order to determine the basis for the phenotypes of W-sash mice, we investigated c-kit RNA and protein expression patterns in adult Wsh/Wsh mice and during embryonic development. We show that c-kit expression is absent in bone-marrow-derived Wsh/Wsh mast cells, the fetal and the adult lung, and the digestive tract at embryonic day 13 1/2 (E13 1/2), tissues that normally express c-kit. Unexpectedly, in E10 1/2 and 11 1/2d Wsh/Wsh embryos, we found c-kit expression in the dermatome of the somites, the mesenchyme around the otic vesicle and the floorplate of the neural tube, structures known to express the c-kit ligand in wild-type embryos. The ectopic c-kit expression in Wsh homozygous embryos does not affect c-kit ligand expression. The presumed Wsh/Wsh melanoblasts appeared to be normal and, at E10 1/2, similar numbers were found in normal and homozygous mutant embryos. At E13 1/2 +/+ embryos had a graded distribution of melanoblasts from cranial to caudal with a minimum in the lumbar region. Whereas E13 1/2 homozygous Wsh/Wsh embryos essentially lacked c-kit-positive cells in the skin, E13 1/2 heterozygous Wsh/+ embryos had reduced numbers of melanoblasts compared to +/+ with few or none in the lumbar region (future sash). It is proposed that ectopic c-kit expression in the somitic dermatome affects early melanogenesis in a dominant fashion. Molecular analysis of Wsh chromosomal DNA revealed a deletion or rearrangement in the vicinity of the c-kit gene. These results provide an explanation for the Wsh phenotype and have implications for the control of c-kit expression.

The murine steel panda mutation affects kit ligand expression and growth of early ovarian follicles.

Mutations at the murine steel (Sl) locus encoding the ligand for the c-kit receptor result in defects in gametogenesis, hematopoiesis, and melanogenesis. Steel Panda (Slpan) is an allele at the Sl locus obtained by an X-ray mutagenesis protocol. Slpan/Slpan homozygotes are mildly anemic black-eyed whites with pigmented ears and scrotum; females are sterile while males are fertile. To investigate the basis of the phenotype of the Slpan mutation, the coding region of the kit ligand (KL) in Slpan/Slpan animals was characterized and shown to be identical to that from +/+ mice. RNA expression patterns in adult Slpan/Slpan mice were investigated by RNA blot analysis and RNase protection assays. KL RNA expression was shown to be reduced in several tissues including testis, lung, and kidney, to about 60% in heterozygotes and 20% in homozygous mutant mice. Intermediate effects were seen in cerebellum and spleen, while in heart and brain no change was apparent. Therefore, the Slpan mutation affects KL RNA levels in a tissue-specific manner. Histological analysis showed that the number of oocytes in neonatal homozygotes was reduced to 20% of that in heterozygotes, and that in juvenile and adult mice ovarian follicle development was arrested at the one-layered cuboidal stage, with a few exceptions. KL production by central cords of the perinatal ovary was severely reduced as shown by immunohistochemistry. In neonatal testes of homozygotes, the germ cell number was reduced to 30% of that in heterozygotes, but meiotic spermatocytes were produced on schedule in juvenile animals. Therefore, a reduced level of KL in Slpan/Slpan ovary arrests ovarian follicle development, while a similar reduction in testes has relatively little effect on spermatogonial development.

A discrete LINE-1 transcript in mouse blastocysts.

The LINE-1 (L1) repetitive elements of mammalian genomes are retrotransposons lacking LTRs; L1-encoded reverse transcriptase probably mediates an important step in the generation of new copies. Most L1 transcripts are nonspecific, but discrete full length transcripts are present in embryonal carcinoma cells. We report here an abundant L1 transcript in mouse blastocysts but not in oocytes. The transcript is about 8 kb, sense strand, polyadenylated, and includes the 5' end of the two open reading frames. We propose that retrotransposition which generates pseudogenes and mammalian SINES as well as the L1 family occurs around the blastocyst stage of the germ cell cycle.

The expression pattern of the c-kit ligand in gonads of mice supports a role for the c-kit receptor in oocyte growth and in proliferation of spermatogonia.

The tyrosine kinase receptor c-kit and its ligand KL are required for postnatal development of germ cells, in addition to their role in primordial germ cells. To clarify their function, a detailed examination of the pattern of expression of KL in postnatal gonads was undertaken. In ovaries, the expression of KL as seen by RNA blot analysis and by RNase protection assays is relatively high at birth (P0), low from P5 to P8, and high from P12 onward. KL expression is relatively high in testes of all ages. The forms of KL RNA present in the testes suggest that from P5 onward the membrane-bound form of KL predominates, while in the ovary significant amounts of both forms are present. As observed by in situ hybridization and immunohistochemistry, in the newborn ovary KL is highly expressed in central cords whose cells contribute to the formation of central growing follicles. Expression is low in follicle cells of small growing follicles and increases to high levels in three-layered follicles during late oocyte growth. Large amounts of the ligand are found within growing oocytes. After oocyte growth ceases, expression continues only in the outer layers of multilayered follicles. In the testis, from P0 through P9, KL expression is distinct in Sertoli cells, but not in germ cells. Thereafter, the intensity of KL expression declines as the number of spermatogenic cells increases within the tubules. KL in Sertoli cells appears to be concentrated basally at the stage of the cycle of the seminiferous epithelium when it is known to interact with differentiating type A spermatogonia. These data are consistent with a role for KL in oocyte growth and in facilitating proliferation and/or differentiation of type A spermatogonia.

The kit-ligand (steel factor) and its receptor c-kit/W: pleiotropic roles in gametogenesis and melanogenesis.

The c-kit receptor tyrosine kinase belongs to the PDGF/CSF-1/c-kit receptor subfamily. The kit-ligand, KL, also called steel factor, is synthesized from two alternatively spliced mRNAs as transmembrane proteins that can either be proteolytically cleaved to produce soluble forms of KL or can function as cell-associated molecules. The c-kit receptor kinase and KL are encoded at the white spotting (W) and steel (Sl) loci of the mouse, respectively. Mutations at both the W and the Sl locus cause deficiencies in gametogenesis, melanogenesis and hematopoiesis. The c-kit receptor is expressed in the cellular targets of W and Sl mutations, while KL is expressed in their microenvironment. In melanogenesis, c-kit is expressed in melanoblasts from the time they leave the neural crest and expression continues during embryonic development and in the melanocytes of postnatal animals. In gametogenesis c-kit is expressed in primordial germ cells, in spermatogonia, and in primordial and growing oocytes, implying a role at three distinct stages of gametogenesis. Many mutant alleles are known at W and Sl loci and their phenotypes vary in the degree of severity in the different cellular targets of the mutations. While many W and Sl alleles severely affect primordial germ cells (PGC), several mild Sl alleles have weak effects on PGCs and exhibit differential male or female sterility. Steel Panda (Sl(pan)) is a KL expression mutation in which KL RNA transcript levels are reduced in most tissues analyzed. In female Sl(pan)/Sl(pan) mice, ovarian follicle development is arrested at the one layered cuboidal stage as a result of reduced KL expression in follicle cells, indicating a role for c-kit in oocyte growth. Wsh is a c-kit expression mutation, which affects mast cells and melanogenesis. While the mast cell defect results from lack of c-kit expression, the pigmentation deficiency appears to stem from ectopic c-kit receptor expression in the somitic dermatome at the time of migration of melanoblasts from the neural crest to the periphery. It is proposed that the ectopic c-kit expression in Wsh mice affects early melanogenesis in a dominant fashion. The "sash" or white belt of Wsh/+ animals and some other mutant mice is explained by the varying density of melanoblasts along the body axis of wild-type embryos.

c-kit receptor and ligand expression in postnatal development of the mouse cerebellum suggests a function for c-kit in inhibitory interneurons.

The c-kit receptor and its cognate ligand, KL, are encoded at the white spotting locus (W) and the steel locus (Sl) of the mouse, respectively. Sl and W mutations affect the same cellular targets in melanogenesis, gametogenesis and hematopoiesis during embryonic development and in adult life. c-kit is expressed in cellular targets of W and Sl mutations, whereas KL is expressed in the microenvironment of these targets. c-kit and KL, however, are also expressed in tissues and cell types that are not targets of W and Sl mutations, including the brain. The cerebellum contains a small number of neural cell types whose developmental origins, pathways of migration, and synaptic contacts are known. We have investigated the patterns of expression of the c-kit and KL RNA and protein products in postnatal cerebellar development of the mouse. In the adult cerebellum, c-kit RNA and protein expression was evident in basket, stellate, and Golgi neurons. Most strikingly, the c-kit protein is expressed in the basket cell axons that form "basket" and "pinceau" structures entwining the Purkinje cell soma and the initial segment of the Purkinje cell axon. KL RNA expression was found in Purkinje cells, and the KL protein was detected in Purkinje cell bodies and dendrites. Soluble KL protein was also present in c-kit-expressing basket, stellate, and Golgi cells, presumably as a result of internalization of ligand-receptor complexes. During postnatal development, c-kit and KL RNA and protein expression in Golgi and Purkinje neurons, respectively, was evident by day 0 and persisted subsequently. c-kit expression in basket and stellate cells was detected from their time of birth, starting at day 4. These results suggest a role for the c-kit receptor system in postnatal development of the cerebellum.

Expression of activins and TGF beta 1 and beta 2 RNAs in early postimplantation mouse embryos and uterine decidua.

The expression of the mesoderm inducing factors, activins and TGF beta s, was characterized in 5 1/2-9 1/2 day mouse embryos and implantation sites by in situ hybridization. Activin beta A RNA was not detected within the embryo, but is expressed in nearby decidual cells from 5 to 7 days. Thus activin A could play a role within the embyro during gastrulation. Activin beta A is also expressed in more mesometrially located decidual cells from 6 to 9 1/2 days. Activin beta B and inhibin alpha RNAs were not detected, while a control tissue was highly positive. TGF beta 1 is expressed in the secondary decidual zone and in developing endothelial cells in the decidua and embryo. TGF beta 2 is expressed in the mesometrial decidua at 6 1/2 days and in the midline of the cranial neural plate.

Expression of c-kit encoded at the W locus of mice in developing embryonic germ cells and presumptive melanoblasts.

The W locus of mice encodes the c-kit tyrosine kinase receptor. In embryos homozygous for severe W mutations, the number of germ cells does not increase after 8 days of development, melanocytes do not appear, and production of erythrocytes and mast cells is deficient. To gain some insight into the role of the c-kit receptor, we have used in situ hybridization to explore the time period of expression of c-kit transcripts in early germ cells and melanoblasts. At 6 1/2 days of development, expression was not seen in the embryonic cylinder, but did appear in parietal endoderm. Germ cells displayed a low level of c-kit transcripts from their first appearance in the 7 1/2 -day embryo, continuing through early proliferation and migration to the gonad. During migration, surrounding tissues also expressed c-kit. Expression increased in gonia and then ceased as they became nonproliferative. Expression in presumptive melanoblasts was first seen in the cervical region of 10-day embryos and continued as they spread over the surface of the body, entered the epidermis, and differentiated in hair follicles after birth. The effects of mutations of c-kit on germ cells and melanoblasts can be interpreted as an absence of a proliferative signal shortly after their segregation from other cell types. This signal may be required throughout the proliferative phase of early germ cells [and also in postnatal stages of germ cell development (Manova et al. (1990). Development 110, 1057-1069]. In melanoblasts, c-kit may play a role during both proliferation and differentiation.