The amnionless gene, essential for mouse gastrulation, encodes a visceral-endoderm-specific protein with an extracellular cysteine-rich domain.

Fate-mapping experiments in the mouse have revealed that the primitive streak can be divided into three functional regions: the proximal region gives rise to germ cells and the extra-embryonic mesoderm of the yolk sac; the distal region generates cardiac mesoderm and node-derived axial mesendoderm; and the middle streak region produces the paraxial, intermediate and lateral plate mesoderm of the trunk. To gain insight into the mechanisms that mediate the assembly of the primitive streak into these functional regions, we have cloned and functionally identified the gene disrupted in the amnionless (amn) mouse, which has a recessive, embryonic lethal mutation that interferes specifically with the formation and/or specification of the middle primitive streak region during gastrulation. Here we report that the gene Amn encodes a novel type I transmembrane protein that is expressed exclusively in the extra-embryonic visceral endoderm layer during gastrulation. The extracellular region of the Amn protein contains a cysteine-rich domain with similarity to bone morphogenetic protein (BMP)-binding cysteine-rich domains in chordin, its Drosophila melanogaster homolog (Short gastrulation) and procollagen IIA (ref. 3). Our findings indicate that Amn may direct the production of trunk mesoderm derived from the middle streak by acting in the underlying visceral endoderm to modulate a BMP signaling pathway.

Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth.

The role of bone marrow (BM)-derived precursor cells in tumor angiogenesis is not known. We demonstrate here that tumor angiogenesis is associated with recruitment of hematopoietic and circulating endothelial precursor cells (CEPs). We used the angiogenic defective, tumor resistant Id-mutant mice to show that transplantation of wild-type BM or vascular endothelial growth factor (VEGF)-mobilized stem cells restore tumor angiogenesis and growth. We detected donor-derived CEPs throughout the neovessels of tumors and Matrigel-plugs in an Id1+/-Id3-/- host, which were associated with VEGF-receptor-1-positive (VEGFR1+) myeloid cells. The angiogenic defect in Id-mutant mice was due to impaired VEGF-driven mobilization of VEGFR2+ CEPs and impaired proliferation and incorporation of VEGFR1+ cells. Although targeting of either VEGFR1 or VEGFR2 alone partially blocks the growth of tumors, inhibition of both VEGFR1 and VEGFR2 was necessary to completely ablate tumor growth. These data demonstrate that recruitment of VEGF-responsive BM-derived precursors is necessary and sufficient for tumor angiogenesis and suggest new clinical strategies to block tumor growth.

The docking protein FRS2alpha is an essential component of multiple fibroblast growth factor responses during early mouse development.

The docking protein FRS2alpha is a major mediator of fibroblast growth factor (FGF) signaling. However, the physiological role of FRS2alpha in vivo remains unknown. In this report, we show that Frs2alpha-null mouse embryos have a defect in anterior-posterior (A-P) axis formation and are developmentally retarded, resulting in embryonic lethality by embryonic day 8. We demonstrate that FRS2alpha is essential for the maintenance of self-renewing trophoblast stem (TS) cells in response to FGF4 in the extraembryonic ectoderm (ExE) that gives rise to tissues of the placenta. By analyzing chimeric embryos, we found that FRS2alpha also plays a role in cell movement through the primitive streak during gastrulation. In addition, experiments are presented demonstrating that Bmp4 expression in TS cells is controlled by mitogen-activated protein kinase-dependent FGF4 stimulation. Moreover, both the expression of Bmp4 in ExE and activation of Smad1/5 in epiblasts are reduced in Frs2alpha-null embryos. These experiments underscore the critical role of FRS2alpha in mediating multiple processes during embryonic development and reveal a potential new link between FGF and Bmp4 signaling pathways in early embryogenesis.

VEGF-ablation therapy reduces drug delivery and therapeutic response in ECM-dense tumors.

The inadequate transport of drugs into the tumor tissue caused by its abnormal vasculature is a major obstacle to the treatment of cancer. Anti-vascular endothelial growth factor (anti-VEGF) drugs can cause phenotypic alteration and maturation of the tumor's vasculature. However, whether this consistently improves delivery and subsequent response to therapy is still controversial. Clinical results indicate that not all patients benefit from antiangiogenic treatment, necessitating the development of criteria to predict the effect of these agents in individual tumors. We demonstrate that, in anti-VEGF-refractory murine tumors, vascular changes after VEGF ablation result in reduced delivery leading to therapeutic failure. In these tumors, the impaired response after anti-VEGF treatment is directly linked to strong deposition of fibrillar extracellular matrix (ECM) components and high expression of lysyl oxidases. The resulting condensed, highly crosslinked ECM impeded drug permeation, protecting tumor cells from exposure to small-molecule drugs. The reduced vascular density after anti-VEGF treatment further decreased delivery in these tumors, an effect not compensated by the improved vessel quality. Pharmacological inhibition of lysyl oxidases improved drug delivery in various tumor models and reversed the negative effect of VEGF ablation on drug delivery and therapeutic response in anti-VEGF-resistant tumors. In conclusion, the vascular changes after anti-VEGF therapy can have a context-dependent negative impact on overall therapeutic efficacy. A determining factor is the tumor ECM, which strongly influences the effect of anti-VEGF therapy. Our results reveal the prospect to revert a possible negative effect and to potentiate responsiveness to antiangiogenic therapy by concomitantly targeting ECM-modifying enzymes.

Localization of a human reduced folate carrier protein in the mitochondrial as well as the cell membrane of leukemia cells.

IgG polyclonal antiserum was generated in New Zealand White rabbits immunized with a 16-mer peptide consisting of a specific amino acid sequence at residues corresponding to the sixth to seventh predicted transmembrane domain of the human reduced folate carrier (RFC). Using Western immunoblotting to examine the cytosolic and membrane fractions of the human CCRF-CEM T-cell lymphoblastic leukemia cell line, polyclonal antihuman RFC antiserum recognized two bands in the cytosolic fraction (approximately 60 kDa and approximately 70 kDa) on 10% polyacrylamide gels. In the membrane fraction, an approximately 60-kDa protein was identified. Comparative studies of a panel of human tumor cell lines including the HT1080 fibrosarcoma, 8805 malignant fibrous histiocytoma, and the MCF breast cancer cell lines revealed similar findings. Likewise, a recombinant approximately 60-kDa membrane protein was identified after expression of baculovirus-infected Sf9 insect cells containing cDNA of the human RFC. In the CEM-7A cell line, a variant of the CCRF-CEM cell line that overexpresses the RFC, 21-fold overexpression of the approximately 60-kDa membrane protein (RFC) was shown by Western analysis. To characterize further the cellular distribution of the human RFC, immunohistochemical analyses were performed in CCRF-CEM T-cell lymphoblastic leukemia cells. Predominantly membrane localization of the antibody reacting sites was detected; however, a cytoplasmic component was noted as well. By confocal microscopy and by immunogold electron microscopy, the cytoplasmic expression was found to be largely of mitochondrial origin. These findings were corroborated by Western immunoblotting of mitochondrial membrane isolates from the CCRF-CEM cell line, which demonstrate an approximately 60-kDa protein. The localization of the human RFC to the mitochondrial membrane is a novel finding, and it suggests a role for the mitochondrial membrane in the transport of folates.

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.

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.

Tumor endothelial marker 8 enhances tumor immunity in conjunction with immunization against differentiation Ag.

BACKGROUND: We have previously shown that xenogeneic DNA vaccines encoding rat neu and melanosomal differentiation Ag induce tumor immunity. Others have developed vaccines targeting tumor neovasculature. Tumor endothelial marker 8 (TEM8) is expressed in the neovasculature of human tumors, and in the mouse melanoma B16, but its expression is limited in normal adult tissues. We describe a DNA vaccine combining xenogeneic tumor Ag and TEM8. METHODS: In-situ hybridization was used to detect TEM8 RNA in mouse tumors. Mice transgenic for the rat neu proto-oncogene were immunized with DNA vaccines encoding TEM8 and the extracellular domain of rat neu and challenged with the 233-VSGA1 breast cancer cell line. In parallel experiments, C57BL/6 mice were immunized with TEM8 and human tyrosinase-related protein 1 (hTYRP1/hgp75) and challenged with B16F10 melanoma. RESULTS: TEM8 was expressed in the stroma of transplantable mouse breast and melanoma tumors. In both model systems, TEM8 DNA had no activity as a single agent but significantly enhanced the anti-tumor immunity of neu and hTYRP1/hgp75 DNA vaccines when given in concert. The observed synergy was dependent upon CD8+ T cells, as depletion of this cell population just prior to tumor challenge obviated the effect of the TEM8 vaccine in both tumor models. DISCUSSION: A local immune response to TEM8 may increase inflammation or tumor necrosis within the tumor, resulting in improved Ag presentation of HER2/neu and hTYRP1/hgp75. Alternatively, TEM8 expression in host APC may alter T-cell interactions or homing. In this way, TEM8 may act more as an adjuvant than an immunologic target.

Tumor endothelial marker 8 enhances tumor immunity in conjunction with immnization against differentiation Ag.

BACKGROUND: We have previously shown that xenogenic DNA vaccines encoding rat neu and melanosomal differentiation Ag induce tumor immunity. Others have developed vaccines targeting tumor neovasculature. Tumor endothelial marker 8 (TEM8) is expressed in the neovasculature of human tumors, and in the mouse melanoma B16, but its expression is limited in normal adult tissues. We describe a DNA vaccine combining xenogeneic tumor Ag and TEM8. METHODS: In-situ hybridization was used to detect TEM8 RNA in mouse tumors. Mice transgenic for the rat neu proto-oncogene were immunized with DNA vaccines encoding TEM8 and the extracellular domain of rat neu and challenged with the 233-VSGA1 breast cancer cell line. In parallel experiments, C57BL/6 mice were immunized with TEM8 and human tyrosinase-related protein 1 (hTYRP1/hgp75) and challenged with B16F10 melanoma. RESULTS: TEM8 was expressed in the stroma of transplantable mouse breast and melanoma tumors. In both model systems, TEM8 DNA had no activity as a single agent but significantly enhanced the anit-tumor immunity of neu and hTYRP1/hgp75 DNA vaccines when given in concert. The observed synergy was dependent upon CD8+ T cells, as depletion of this cell population just prior to tumor challenge obviated the effect of the TEM8 vaccine in both tumor models. DISCUSSION: A local immune responses to TEM8 may increase inflammation or tumor necrosis within the tumor, resulting in improved Ag presentation of HER2/neu and hTYRP1/hgp75. Alternatively, TEM8 expression in host APC may act more as an adjuvant than an immunologic target.

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.

Expression patterns of Id1, Id2, and Id3 are highly related but distinct from that of Id4 during mouse embryogenesis.

The murine dominant negative helix-loop-helix (dnHLH) proteins inhibit the activities of bHLH transcription factors in diverse cell lineages (Benezra et al. [1990] Cell 61:49-59; Christy et al [1991] Proc. Natl. Acad. Sci. U.S.A. 88:1815-1819; Sun et al [1991] Mol. Cell Biol. 11: 5603-5611; Riechmann et al. [1994] Nucleic Acids Res. 22:749-755). Currently, there are four members in the dnHLH family, Id1, Id2, Id3, and Id4. In this report, we have performed a detailed comparative in situ hybridization analysis to examine their expression pattern during post-gastrulational mouse development. Id1, 2, and 3 are expressed in multiple tissues, whereas Id4 expression can only be detected in neuronal tissues and in the ventral portion of the epithelium of the developing stomach. The regions where Id1-3 genes are expressed, such as gut, lung, kidney, tooth, whisker, and several glandular structures, are undergoing active morphogenetic activities. The expression patterns of Id1, 2, and 3 overlap in many organs, except in the tissue derived from primitive gut. In the latter, Id1 and Id3 signals are detected in the mesenchyme surrounding the epithelium, whereas Id2 is expressed within the epithelium. The difference in the patterns of expressions of Id2-3 and Id4 suggest that the dominant negative transcriptional activity of these two subclasses of the Id family may have different physiological consequences.

Each member of the Id gene family exhibits a unique expression pattern in mouse gastrulation and neurogenesis.

We have performed a detailed comparative in situ hybridization analysis to examine the patterns of expression of all the members of the Id gene family (Id1-4) during murine gastrulation and neurogenesis. During gastrulation, both Id1 and Id3 are expressed in the tissues derived from the inner cell mass from 5.5 dpc onward, whereas Id2 is expressed in tissues derived from trophoblasts. Id4 expression is absent during this period of development. Embryonic Id1 messages are detected during gastrulation on the proximal side of the embryonic ectoderm, which is the border between the embryo proper and the extraembryonic tissues, and the expression of Id3 is found throughout the entire embryo proper. This unique pattern of expression of the different members of the Id family suggests a nonredundant role for these genes in antagonizing the activity of bHLH transcription factors during very early mouse development. During neurogenesis, the expression of each member of the Id family is present in an unique pattern along the dorsal-ventral axis of the neural tube: In the early stages of spinal cord development, both Id1 and Id2 are expressed in the roof plate, whereas Id3 is expressed both in the roof and the floor plates. As development progresses, the expression of both Id1 and Id3 is detected in the dividing neuroblasts, whereas Id2 and 4 are expressed in presumptive neurons which are undergoing maturation. The expression patterns of all the members of the Id gene family persist throughout the entire CNS, both in the spinal cord and in the brain. In addition, the characteristic expression of Id2 and Id4 in more mature neurons is reiterated both in the PNS and in the neurons of some of the sensory organs. These data suggest that the expression of different subgroups of the Id gene family may have different physiological consequences and thereby contributes in unique ways to specify the differentiation state of neuronal cells during development.

Gonadal expression of c-kit encoded at the W locus of the mouse.

Recently, it has been shown that the c-kit proto-oncogene is encoded at the white spotting (W) locus in mice. Mutations of this gene cause depletion of germ cells, some hematopoietic cells and melanocytes. In order to define further the role of c-kit in gametogenesis, we have examined its expression in late fetal and postnatal ovaries and in postnatal testis. By RNA blot analysis, c-kit transcripts were not detected in late fetal ovaries but appeared at birth. The relative amount reached a maximum in ovaries of juvenile mice, and decreased in adult ovaries. c-kit transcripts were present in increasing amounts in isolated primordial, growing and full-grown oocytes, as well as in ovulated eggs. Little was detected in early 2-cell embryos and none in blastocysts. In situ hybridization revealed c-kit transcripts in a few oocytes of late fetal ovaries and in all oocytes (from primordial to full-grown) in ovaries from juvenile and adult mice. Expression was also observed in ovarian interstitial tissue from 14 days of age onward. Using indirect immunofluorescence, the c-kit protein was detected on the surface of primordial, growing and full-grown oocytes, as well as on embryos at the 1- and 2-cell stages; little remained in blastocysts. In situ hybridization analysis of testes from mice of different ages demonstrated expression in spermatogonia from 6 days of age onward. Using information provided by determining the stage of the cycle of the seminiferous epithelium for a given tubule and by following the age dependence of labeling, it was concluded that the period of expression of c-kit extends from at least as early as type A2 spermatogonia through type B spermatogonia and into preleptotene spermatocytes. Leydig cells were labelled at all ages examined. The expression pattern in oocytes correlates most strongly with oocyte growth and in male germ cells with gonial proliferation.

A phenotype-based screen for embryonic lethal mutations in the mouse.

The genetic pathways that control development of the early mammalian embryo have remained poorly understood, in part because the systematic mutant screens that have been so successful in the identification of genes and pathways that direct embryonic development in Drosophila, Caenorhabditis elegans, and zebrafish have not been applied to mammalian embryogenesis. Here we demonstrate that chemical mutagenesis with ethylnitrosourea can be combined with the resources of mouse genomics to identify new genes that are essential for mammalian embryogenesis. A pilot screen for abnormal morphological phenotypes of midgestation embryos identified five mutant lines; the phenotypes of four of the lines are caused by recessive traits that map to single regions of the genome. Three mutant lines display defects in neural tube closure: one is caused by an allele of the open brain (opb) locus, one defines a previously unknown locus, and one has a complex genetic basis. Two mutations produce novel early phenotypes and map to regions of the genome not previously implicated in embryonic patterning.

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 amn gene product is required in extraembryonic tissues for the generation of middle primitive streak derivatives.

The primitive streak is the defining feature of the gastrulating mouse embryo. Currently, little is known in the mouse about the mechanisms that mediate the assembly of the primitive streak or about the signaling pathways that specify the different types of mesoderm and endoderm generated from the streak. To gain insight into primitive streak assembly and function, we have conducted a detailed phenotypic characterization of amnionless, a transgene-induced insertional mouse mutation that arrests embryonic development during gastrulation. Our histological and molecular analyses, when examined in the context of the mouse gastrula fate map, lead to the model that middle streak formation is specifically impaired in the amnionless mutant. Significantly, these observations argue that the formation of the middle streak is mediated by a pathway that is genetically separable from those that direct the specification of the distal and proximal streak regions. Intriguingly, our findings from wt ES cell left and right arrow amnionless-/- blastocyst chimeras indicate that this proposed separate pathway for middle streak formation is dependent on amnionless gene functions in the visceral endoderm.

Cloning and characterization of a Golgin-related gene from the large-scale polymorphism linked to the PML gene.

Megabase-scale mapping of the PML gene locus revealed the presence of a large-scale insertion-deletion polymorphism located 25 kb downstream of the PML gene. The polymorphism is organized as a head-to-tail tandem 25-kb repeat containing one to five units. Characterization of the first repeat unit downstream of PML revealed the presence of a gene with strong homology to a family of Golgin-related proteins. The gene, designated GLP (for Golgin linked to PML), is strongly expressed as a 6-kb transcript in normal human testis. In situ hybridization of normal human testis demonstrated that the expression of GLP was restricted to late meiotic germ cells. There was weak expression in late pachytene spermatocytes and strong expression in spermatids. GLP is 50% homologous to other Golgin-related proteins including the vesicle docking protein GM130. Southern blot hybridization of genomic DNA with a GLP probe demonstrated numerous homologous bands outside the PML locus. Three of these loci have been mapped by fluorescence in situ hybridization to chromosome loci 9q34.1, 15q11-q13, and 15q22-q24. Hybridization of a GLP cDNA probe to a zoo blot demonstrated multiple signals in nonhuman primates but not in other species and suggested the duplication of an ancestral locus around 20 million years ago.

Purification and characterization of a haloalkane dehalogenase of a new substrate class from a gamma-hexachlorocyclohexane-degrading bacterium, Sphingomonas paucimobilis UT26.

The linB gene product (LinB), 1,3,4,6-tetrachloro-1,4-cyclohexadiene halidohydrolase, which is involved in the degradation of gamma-hexachlorocyclohexane in Sphingomonas paucimobilis UT26 (Y. Nagata, T. Nariya, R. Ohtomo, M. Fukuda, K. Yano, and M. Takagi, J. Bacteriol. 175:6403-6410, 1993), was overproduced in E. coli and purified to homogeneity. The molecular mass of LinB was deduced to be 30 kDa by gel filtration chromatography and 32 kDa by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel, indicating that LiuB is a monomeric enzyme. The optimal pH for activity was 8.2. Not only monochloroalkanes (C3 to C10) but also dichloroalkanes, bromoalkanes, and chlorinated allphatic alcohols were good substrates for LinB, suggesting that LinB shares properties with another haloalkane dehalogenase, DhlA (S. Keuning, D.B. Janssen, and B. Witholt, J. Bacteriol. 163:635-639, 1985), which shows significant similarity to LinB in primary structure (D. B. Janssen, F. Pries, J. van der Ploeg, B. Kazemier, P. Terpstra, and B. Witholt, J. Bacteriol. 171:6791-6799, 1989) but not in substrate specificity. Principal component analysis of substrate activities of various haloalkane dehalogenases suggested that LinB probably constitutes a new substrate specificity class within this group of enzymes.

Chromosome synapsis defects and sexually dimorphic meiotic progression in mice lacking Spo11.

Spo11, a protein first identified in yeast, is thought to generate the chromosome breaks that initiate meiotic recombination. We now report that disruption of mouse Spo11 leads to severe gonadal abnormalities from defective meiosis. Spermatocytes suffer apoptotic death during early prophase; oocytes reach the diplotene/dictyate stage in nearly normal numbers, but most die soon after birth. Consistent with a conserved function in initiating meiotic recombination, Dmc1/Rad51 focus formation is abolished. Spo11(-/-) meiocytes also display homologous chromosome synapsis defects, similar to fungi but distinct from flies and nematodes. We propose that recombination initiation precedes and is required for normal synapsis in mammals. Our results also support the view that mammalian checkpoint responses to meiotic recombination and/or synapsis defects are sexually dimorphic.

RNA helicase A is essential for normal gastrulation.

RNA helicase A (RHA) is the human homologue of the Drosophila maleless protein, an essential factor for the development of male flies. Recently, it was shown that RHA cooperates with the cAMP-responsive element in mediating the cAMP-dependent transcriptional activation of a number of genes. Due to the participation of cAMP as a second messenger in a number of signaling pathways, we examined the function of RHA during mammalian embryogenesis. To examine the role(s) of RHA in mammalian development, RHA knockout mice were generated by homologous recombination. Homozygosity for the mutant RHA allele led to early embryonic lethality. Histological analysis, combined with terminal deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL) reactions of RHA-null embryos, revealed marked apoptotic cell death specifically in embryonic ectodermal cells during gastrulation. RNA in situ analyses of the expression of HNF-3beta and Brachyury, two molecular markers for gastrulation, showed that RHA-null embryos at days 7.5 and 8.5 expressed both HNF-3beta and Brachyury in a pattern similar to those of pre- and early streak stages of embryos, respectively. These observations indicate that RHA is necessary for early embryonic development and suggest the requirement of RHA for the survival and differentiation of embryonic ectoderm.