Midgestation Embryos Research Articles

Juxtaposed Polycomb complexes co-regulate vertebral identity

Best known as epigenetic repressors of developmental Hox gene transcription, Polycomb complexes alter chromatin structure by means of post-translational modification of histone tails. Depending on the cellular context, Polycomb complexes of diverse composition and function exhibit cooperative interaction or hierarchical interdependency at target loci. The present study interrogated the genetic, biochemical and molecular interaction of BMI1 and EED, pivotal constituents of heterologous Polycomb complexes, in the regulation of vertebral identity during mouse development. Despite a significant overlap in dosage-sensitive homeotic phenotypes and co-repression of a similar set of Hox genes, genetic analysis implicated eed and Bmi1 in parallel pathways, which converge at the level of Hox gene regulation. Whereas EED and BMI1 formed separate biochemical entities with EzH2 and Ring1B, respectively, in mid-gestation embryos, YY1 engaged in both Polycomb complexes. Strikingly, methylated lysine 27 of histone H3 (H3-K27), a mediator of Polycomb complex recruitment to target genes, stably associated with the EED complex during the maintenance phase of Hox gene repression. Juxtaposed EED and BMI1 complexes, along with YY1 and methylated H3-K27, were detected in upstream regulatory regions of Hoxc8 and Hoxa5. The combined data suggest a model wherein epigenetic and genetic elements cooperatively recruit and retain juxtaposed Polycomb complexes in mammalian Hox gene clusters toward co-regulation of vertebral identity.

Analysis of Nsdhl-deficient embryos reveals a role for Hedgehog signaling in early placental development

The X-linked Nsdhl gene encodes a sterol dehydrogenase involved in cholesterol biosynthesis. Mutations in this gene cause the male lethal phenotypes in human CHILD syndrome and bare patches (Bpa) mice. Affected male embryos for several mutant Nsdhl alleles die in mid-gestation with a thin and poorly vascularized placental labyrinth. The timing and specific abnormalities noted suggest a defect in one or more developmental signaling pathways as a possible mechanism. Here, we examined the possible involvement of the hedgehog signaling pathway in the placental pathology of Nsdhl mutants using a transgenic mouse line (Ptch1(tm1Mps)) that contains a lacZ reporter under the control of the promoter for Ptch1, the gene that encodes the major hedgehog receptor. We demonstrate expression of Ptch1 in allantoic mesoderm of the placenta from wild-type mid-gestation embryos. The evidence suggests that the signaling is induced by Indian hedgehog that is produced by distal (ectoplacental) visceral endoderm cells that migrate into the allantoic mesoderm before embryonic day 10.0. Using a ubiquitously expressed, X-linked lacZ transgene that undergoes normal X-inactivation, we demonstrate that the placental defects in Nsdhl/+ female embryos are non-cell autonomous. Further, affected placentas from mutant Nsdhl(Bpa-8H) male embryos demonstrate markedly decreased or no Ptch1-lacZ staining and no migration of Ihh expressing cells into the developing placenta. These data strongly implicate the hedgehog signaling pathway in the pathogenesis of the placental defects in NSDHL deficiency and provide evidence for a role for the hedgehog pathway in the development of a functional mammalian placenta.

Expression of the δ-protocadherin gene Pcdh19 in the developing mouse embryo

Protocadherins constitute a large family of transmembrane proteins primarily involved in weak homophilic adhesion in the brain and several other tissues. In a screen for potential regulators of kidney development, we have identified Pcdh19, a poorly characterized member of the δ-protocadherin subfamily. Here, we report the spatio-temporal expression pattern of Pcdh19 during mouse embryonic development. In midgestation embryos, Pcdh19 mRNA was detected in the mesonephros and in the neuroepithelium of the forebrain and midbrain. At later stages, Pcdh19 was expressed in other neural tissues such as the neural retina, nasal epithelium and spinal cord, as well as in the collecting duct and differentiating nephrons of the metanephros, in the glandular stomach, the exocrine pancreas and the hair follicles. Hence, the Pcdh19 gene is developmentally regulated during mouse organogenesis and shows a unique expression profile among protocadherins.

Characterization of Circulating Primitive Erythroid Cells during Embryogenesis Using a Human ε-Globin-GFP Transgenic Mouse Model.

Primitive erythroid cells (EryP) are the first hematopoietic cell type to mature during embryonic development. EryP are characterized by expression of “embryonic” ε- and βh1-globin genes that are not expressed in fetal liver-derived “adult type” or definitive erythroid cells. Although EryP constitute the majority of the blood cells in mid-gestation embryos, EryP maturation remains poorly defined. Here, we utilize a transgenic mouse line in which green fluorescence protein (GFP) expression is driven by the human ε-globin minimal promoter to characterize the development of circulating EryP throughout embryogenesis. ε-globin(+)/GFP(+) EryP appear from 7.5 dpc within blood islands of the yolk sac. As expected, at 9.5 dpc, essentially all circulating blood cells express GFP. By 14.5 dpc, only 50% of the circulating cells express GFP. This sharp decrease in the numbers of circulating ε-globin(+)/GFP(+) cells continues until birth, by which stage fewer than 1% express the reporter gene. To further characterize the maturation of EryP, we analyzed the expression of surface antigens on ε-globin(+)/GFP(+) cells at various stages of embryonic development. From 9.5 dpc onwards, ε-globin(+)/GFP(+) cells express increasing levels of the erythroid marker Ter-119. The transferrin receptor CD71 is also expressed from early stages of development but is down-regulated as ε-globin(+)/GFP(+) cells mature. Increasing Ter-119 expression with concomitant loss of CD71 have previously been reported as hallmarks of maturing fetal liver erythroid cells (EryD) and we now report that ε-globin(+)/GFP(+) EryP demonstrate a similar developmental progression. Therefore, EryP contribute to the Ter119 and CD71 expression reported by others for total embryonic peripheral blood cells. The GPI-anchored surface marker CD24, present on EryD, is also found on ε-globin(+)/GFP(+) EryP. Interestingly, expression of the adhesion molecules CD44 and α4-integrin was upregulated on maturing ε-globin(+)/GFP(+) EryP, perhaps reflecting a requirement for interaction of EryP with other cells. ε-globin(+)/GFP(+) EryP lack expression of surface antigens typical of endothelial cells (Flk1, VCAM1), hematopoietic stem cells (c-kit, Sca1, CXCR4), myeloid (Gr1, Mac1) and lymphoid cells (CD19, CD3). Together, these data help to define the maturation pathway of the primitive erythroid lineage. It was recently shown by immunohistology that murine EryP enucleate, similar to their definitive counterparts. We used the cell permeable DNA-binding dye Draq5 to quantify enucleation in the circulating ε-globin(+)/GFP(+) population by FACS. We show that enucleated ε-globin(+)/GFP(+) cells are Draq5low/neg whereas those bearing nuclei are Draq5high. At 9.5 dpc, ε-globin(+)/GFP(+) cells are Draq5high. The frequency of nucleated EryPs decreases rapidly such that by 14.5 dpc, half of the circulating e-globin(+)/GFP(+) cells are Draq5low/neg. Shortly before birth, almost all EryP have enucleated. This system will allow us to separate nucleated from enucleated EryP by cell sorting and will help us assess changes in surface antigen expression during EryP maturation and enucleation. The human ε-globin-GFP transgenic mouse model is therefore a useful system for defining, at the cellular and molecular level, the developmental pathways of the primitive erythroid lineage.

In situ analysis of Raidd-beta-galactosidase fusion gene expression in transgenic mouse midgestation embryos

Apoptosis and differentiation are tightly intertwined processes occurring at organ formation and remodelling during embryonic development. RAIDD (receptor-interacting protein [RIP]-associated ICH-1/CED-3-homologous protein with a death domain), a dual-domain adaptor protein has been shown to mediate the recruitment of CASPASE-2 to tumour necrosis factor receptor-1 (TNF-R1) signalling complex through RIP kinase. However, Raidd overexpression studies suggest that apart from the established role in apoptosis, Raidd may have an additional function in cell differentiation. In this study, we could not generate Raidd null adult mice suggesting that lack of function of Raidd might be embryonic lethal. Thus, to elucidate the role of Raidd during mouse embryogenesis when the processes of organogenesis are most dynamic, we studied the Raidd expression pattern in midgestation mouse embryos. We generated Raidd+/- transgenic mice with a reporter transgene encoding the bacterial Beta-galactosidase (beta-gal) under the control of Raidd promoter. During the midgestation period (E8.5-E12.5), Raidd is expressed in developing organs derived from the ectoderm such as lens, structures of the inner ear and the fourth brain ventricle in regions where differentiation takes place implicating Raidd role in this process. In addition, Raidd expression was found in developing mesenchyme organs like heart and kidney and in the endothelial lining of the midgut at the time when profound morphological changes take place in these organs. In developing heart and kidney Raidd expression patterns overlapped with known zones of cell death suggesting Raidd may be involved in apoptosis-mediated remodelling. The observed lethality of mice targeted at both Raidd alleles and Raidd expression patterns during midgestation period strongly suggest that Raidd plays an important role in mammalian development.

LRP5 and Wnt signaling: a union made for bone.

THE IDENTIFICATION OF mutations in the human lowdensity lipoprotein (LDL) receptor–related protein 5 (LRP5) gene that give rise to conditions of low bone mass and increased bone mass has brought what just a few years ago were two seemingly unrelated fields crashing together, namely bone biology and Wnt signaling. It is now clear that the Wnt signaling pathway is another key pathway involved in the regulation of bone mass. This is not to diminish the importance of any other pathway that is known to play a role; rather, this discovery only highlights how little we probably really know about bone mass regulation. Certainly, a big challenge we now face will be to integrate the Wnt signaling pathway with all of its complexities and subtleties into the already complex nature of the other pathways that have been studied, with the ultimate goal of developing a composite understanding of their collective roles and interactions. In this article, we will summarize the main points of our current understanding of LRP5, Wnt’s and Wnt signaling, and the role they may play in bone. This is by no means comprehensive, and there are more unanswered questions than answers.

Nonviral gene transfer to surface skin of mid-gestational murine embryos by intraamniotic injection and subsequent electroporation.

The surface epithelium of mid-gestational murine embryos is thought to be an attractive target for gene therapy in vivo, due to its visibility and accessibility from the external surface of the maternal uterus. Almost all studies of in utero gene transfer have adopted viral vectors for infection of fetal epithelium, and depended on intraamniotic introduction and simple incubation of vectors, leading to only infection of the surface layer (periderm) of fetal skin. Here we report a simple and convenient method of gene transfer of plasmid DNA into the deeper portion of surface skin of murine mid-gestational fetus. One to two microlitres of a solution containing a lacZ expression plasmid (0.5-1 microg) and trypan blue (0.05%) were placed onto the surface of a fetus (E 14.5) near the eye by a micropipette attached to a mouthpiece. This fetus was immediately electroporated by placing it between tweezer-type electrodes attached to a square-pulse generator. At 1 and 4 days after gene transfer, fetuses were subjected to histochemical staining for lacZ activity in the presence of X-Gal, a substrate for lacZ. Focal reactions were observed in the skin epidermal layers including periderm and basal layer 1 day after DNA introduction. However, lacZ-positive cells were limited to a skin surface layer, the stratum corneum, in the samples obtained 4 days after gene transfer. Similar observation was also made in the transgenic fetuses (carrying a lacZ gene placed immediately downstream of the loxP-flanked sequence) injected with Cre expression vector. These findings suggest rapid movement of fetal epidermal cells toward the surface during late developmental stages. This local gene transfer approach appears to be effective as a method for skin-targeted gene transfer, enabling study of the role of genes of interest and tracing of cell lineage during fetal skin development.

Identification of unique, differentiation stage-specific patterns of expression of the bromodomain-containing genes Brd2, Brd3, Brd4, and Brdt in the mouse testis

The bromodomain, an evolutionarily conserved motif that binds acetyl-lysine on histones, is found in many chromatin-associated proteins, transcription factors, and in nearly all known histone acetyltransferases. The BET subclass of bromodomain-containing proteins contains two bromodomains and one ET domain and consists of at least four members in mouse and human, Brd2, Brd3, Brd4, and Brdt. We isolated mouse cDNAs for these genes and studied their expression patterns with particular focus on the testis. Northern hybridization revealed that Brd3 is most abundant in testis, ovary, placenta, uterus, and brain; that Brd4 is rather ubiquitously expressed but is most abundant in mid-gestation embryo, testis, ovary, and brain; and that Brdt is specifically expressed in testis. In situ hybridization and immunostaining on histological sections of mouse testes revealed a strikingly specific and dynamic change of cellular specificity in the germ line during the progression of spermatogenesis. Brd4 is expressed in spermatogonia, Brdt is only expressed in mid- to late-spermatocytes, Brd2 is expressed in diplotene spermatocytes and round spermatids and at low levels in spermatogonia, and Brd3 is expressed in round spermatids. This unique expression pattern suggests that genes in this subclass are not simply redundant. Rather, their expression is tightly regulated in the male germ cell lineage, suggesting that they likely have specific roles in different developmental stages and/or cell types.

Novel testis- and embryo-specific isoforms of the phosphofructokinase-1 muscle type gene

We have identified novel transcriptional isoforms of the human and mouse genes encoding muscle type phosphofructokinase-1 (PFK-M). These isoforms are expressed specifically in the testis and in the mid-gestation embryo, and have been termed TE-PFK-M (testis- and embryo-specific PFK-M). The 5 ′UTR of TE-PFK-M is composed of three newly identified exons that lie much farther upstream of the PFK-M coding region than the previously characterized 5 ′UTR. In addition, this upstream region encodes a series of small polyadenylated transcripts, some of which share the same exons found in the 5 ′UTR of TE-PFK-M, and which may play some role in regulating TE-PFK-M expression. These findings indicate an even more complex level of control of PFK-M expression than previously thought.

In vivo regeneration of murine prostate from dissociated cell populations of postnatal epithelia and urogenital sinus mesenchyme.

The existence of a postnatal prostate stem cell is supported by several types of evidence. Withdrawal of androgen leads to involution of the gland, but readdition can rapidly stimulate regeneration. Tissue fragments derived from mouse or rat prostatic epithelia from midgestation embryos or adult mice, when combined with tissue fragments from urogenital sinus mesenchyme and grafted under the kidney capsule, can regenerate prostatic structures. Indirect evidence supports that the stem cell population is contained within the basal layer. Purified prostatic stem cell preparations would be useful to define the physical and functional properties required for regeneration and to compare with cells that accumulate during abnormal growth states, like prostate cancer. We have developed a regeneration system using dissociated cell populations of postnatal prostate epithelia and embryonic urogenital sinus mesenchyme. Efficient in vivo regeneration of prostatic structures in the subcapsular space of the kidney was observed within 4-8 wk with as few as 103 epithelial cells from prostates derived from donors 10 d to 6 wk of age. The regenerated structures show a branching tubular epithelial morphology, with expression of a panel of markers consistent with prostate development. Donor epithelial populations can be readily infected with GFP expressing lentiviral vectors to provide integration markers and easy visualization. The cell preparations of urogenital sinus mesenchyme can be expanded in short-term in vitro culture while their inductive capabilities are retained. Further definition of the subpopulation of prostate epithelial cells containing the regeneration activity should be possible with such technologies.

The mouse YAF2 gene generates two distinct transcripts and is expressed in pre-and postimplantation embryos

Mammalian Polycomb group (PcG) proteins are known to function during the maintenance of spatially restricted expression of Hox cluster genes and cellular proliferation. To understand the molecular basis of PcG functions, it is important to identify the components of mammalian PcG complexes. We isolated mouse YAF2 as a protein that interacts with Ring1B, a known constituent of mammalian PcG complexes. We show that the murine YAF2 locus generates two different transcripts, mYAF2-a and mYAF2-b by alternative splicing of the third exons which encode two YAF2 isoforms of 179 and conceptual 60 amino acids, respectively. At least five exons encoding mYAF2 transcripts are mapped on chromosome 15E3 region. Expression of mYAF2 mRNA was observed in both pre- and postimplantation embryos. In mid-gestation embryos, mYAF2 expression is strongly seen in the region close to the surface ectoderm. Finally, biochemical evidence and colocalization studies in tissue culture cells suggest that the product of the mYAF2 gene is involved in PcG complexes together with Ring1B and/or Ring1A.

Connective tissue growth factor coordinates chondrogenesis and angiogenesis during skeletal development.

Coordinated production and remodeling of the extracellular matrix is essential during development. It is of particular importance for skeletogenesis, as the ability of cartilage and bone to provide structural support is determined by the composition and organization of the extracellular matrix. Connective tissue growth factor (CTGF, CCN2) is a secreted protein containing several domains that mediate interactions with growth factors, integrins and extracellular matrix components. A role for CTGF in extracellular matrix production is suggested by its ability to mediate collagen deposition during wound healing. CTGF also induces neovascularization in vitro, suggesting a role in angiogenesis in vivo. To test whether CTGF is required for extracellular matrix remodeling and/or angiogenesis during development, we examined the pattern of Ctgf expression and generated Ctgf-deficient mice. Ctgf is expressed in a variety of tissues in midgestation embryos, with highest levels in vascular tissues and maturing chondrocytes. We confirmed that CTGF is a crucial regulator of cartilage extracellular matrix remodeling by generating Ctgf(-/-) mice. Ctgf deficiency leads to skeletal dysmorphisms as a result of impaired chondrocyte proliferation and extracellular matrix composition within the hypertrophic zone. Decreased expression of specific extracellular matrix components and matrix metalloproteinases suggests that matrix remodeling within the hypertrophic zones in Ctgf mutants is defective. The mutant phenotype also revealed a role for Ctgf in growth plate angiogenesis. Hypertrophic zones of Ctgf mutant growth plates are expanded, and endochondral ossification is impaired. These defects are linked to decreased expression of vascular endothelial growth factor (VEGF) in the hypertrophic zones of Ctgf mutants. These results demonstrate that CTGF is important for cell proliferation and matrix remodeling during chondrogenesis, and is a key regulator coupling extracellular matrix remodeling to angiogenesis at the growth plate.

In utero transplantation of wild-type fetal liver cells rescues factor X-deficient mice from fatal neonatal bleeding diatheses

Factor X (FX)-deficient embryos suffer partial embryonic lethality with approximately 30% of the embryos arresting at midgestation. The remaining animals survive to term but die perinatally mainly from abdominal or intracranial hemorrhage. We have rescued FX-deficient mice by transplanting fetal liver cells from FX+/+, Rosa26 fetuses into midgestation embryos derived from FX+/- heterozygous crosses. FX-/- embryos were born at the expected frequency and approximately 50% of the FX-/- neonates survived longer than 4 months. FX-/- embryos receiving saline injections that survived to term died perinatally similar to untreated FX-deficient mice. The plasma levels of FX in the rescued 16-week-old FX-/- mice were approximately 1-6% of wild-type levels. beta-Galactosidase-staining cells derived from the donor Rosa26 fetal liver cells were detected in 47% of the livers of adult mice. In addition, donor-derived cells were also recovered in the bone marrow, spleen, lung, and occasionally in the brain and testis. These results suggest that in utero cell transplantation could be an effective therapeutic strategy to treat pathologies resulting from the deficiency of hepatic-expressed factors.

The first 3 days of B-cell development in the mouse embryo

B-lineage-committed cells are believed to arise in the liver of mouse embryos at 14 days after coitus (dpc). However, pre-B-specific gene transcripts and DJH gene rearrangements have been detected in earlier, midgestation embryos. We describe here a population of c-kit(+)AA4.1(+)CD19(+)Pax5(+) cells present in the aorta-gonad-mesonephros (AGM) area and in the livers of 11-dpc mouse embryos. In contrast to multipotent c-kit(+)AA4.1(+)CD19(-) hematopoietic stem cells (HSCs), these c-kit(+)AA4.1(+)CD19(+) progenitors differentiated only to B-lineage cells in vitro. We propose that mouse embryonic B lymphopoiesis starts earlier than previously thought, at 10 to 11 dpc, both in liver and extra-liver hematopoietic sites. The B-cell differentiation program is not delayed with respect to the emerging lymphohematopoiesis events in the midgestation mouse embryo (8-9 dpc).

Possible mechanism of gene transfer into early to mid-gestational mouse fetuses by tail vein injection.

Our aim is to develop a simple gene transfer method into egg cylinder and mid-gestational murine embryos. We examined whether plasmid/lipid complexes injected into the tail veins of pregnant transgenic mice can be transferred to fetuses at E 4.5-13.5. When pregnant CETZ-17 mice carrying a transgene consisting of a ubiquitous promoter, floxed EGFP/CAT and the LacZ gene, were injected with a Cre expression vector DNA/lipid complex, Cre-mediated excision of the transgenes, as evaluated by X-gal staining, occurred in 10-50% of fetuses treated at E 11.5-13.5. Although younger embryos remained unstained, PCR analysis revealed low levels of the Cre vector DNA and recombined transgene. To examine the fate of a solution given intravenously, we injected trypan blue or fluorescence-labeled plasmid DNA/lipid complexes into females at E 5.5-11.5 and E 6.5, respectively. Both collected in the visceral endoderm (VE) lineage, but were undetectable in the embryo proper. These findings suggest that substances in maternal blood are delivered to post-implantation embryos via cells of the VE lineage and placenta, but that most are trapped in the VE. If significantly improved, gene transfer to fetuses by injection into the maternal circulation may become a promising tool in fetal gene therapy and embryological studies.

Role of Cbfbin hematopoiesis and perturbations resulting from expression of the leukemogenic fusion gene Cbfb-MYH11

Core-binding factor beta (CBFbeta) and CBFalpha2 form a heterodimeric transcription factor that plays an important role in hematopoiesis. The genes encoding either CBFbeta or CBFalpha2 are involved in chromosomal rearrangements in more than 30% of cases of acute myeloid leukemia (AML), suggesting that CBFbeta and CBFalpha2 play important roles in leukemogenesis. Inv(16)(p13;q22) is found in almost all cases of AML M4Eo and results in the fusion of CBFB with MYH11, the gene encoding smooth muscle myosin heavy chain. Mouse embryos heterozygous for a Cbfb-MYH11 knock-in gene lack definitive hematopoiesis, a phenotype shared by Cbfb(-/-) embryos. In this study we generated a Cbfb-GFP knock-in mouse model to characterize the normal expression pattern of Cbfbeta in hematopoietic cells. In midgestation embryos, Cbfbeta was expressed in populations enriched for hematopoietic stem cells and progenitors. This population of stem cells and progenitors was not present in mouse embryos heterozygous for the Cbfb-MYH11 knock-in gene. Together, these data suggest that Cbfb-MYH11 blocks embryonic hematopoiesis at the stem-progenitor cell level and that Cbfb is essential for the generation of hematopoietic stem and progenitor cells. In adult mice, Cbfbeta was expressed in stem and progenitor cells, as well as mature myeloid and lymphoid cells. Although it was expressed in erythroid progenitors, Cbfbeta was not expressed during the terminal stages of erythropoiesis. Our data indicate that Cbfb is required for myeloid and lymphoid differentiation; but does not play a critical role in erythroid differentiation.

Mouse ribonuclease III. cDNA structure, expression analysis, and chromosomal location.

BackgroundMembers of the ribonuclease III superfamily of double-stranded(ds)-RNA-specific endoribonucleases participate in diverse RNA maturation and decay pathways in eukaryotic and prokaryotic cells. A human RNase III orthologue has been implicated in ribosomal RNA maturation. To better understand the structure and mechanism of mammalian RNase III and its involvement in RNA metabolism we determined the cDNA structure, chromosomal location, and expression patterns of mouse RNase III.ResultsThe predicted mouse RNase III polypeptide contains 1373 amino acids (~160 kDa). The polypeptide exhibits a single C-terminal dsRNA-binding motif (dsRBM), tandem catalytic domains, a proline-rich region (PRR) and an RS domain. Northern analysis and RT-PCR reveal that the transcript (4487 nt) is expressed in all tissues examined, including extraembryonic tissues and the midgestation embryo. Northern analysis indicates the presence of an additional, shorter form of the transcript in testicular tissue. Fluorescent in situ hybridization demonstrates that the mouse RNase III gene maps to chromosome 15, region B, and that the human RNase III gene maps to a syntenic location on chromosome 5p13-p14.ConclusionsThe broad transcript expression pattern indicates a conserved cellular role(s) for mouse RNase III. The putative polypeptide is highly similar to human RNase III (99% amino acid sequence identity for the two catalytic domains and dsRBM), but is distinct from other eukaryotic orthologues, including Dicer, which is involved in RNA interference. The mouse RNase III gene has a chromosomal location distinct from the Dicer gene.

αIIb Integrin Expression during Development of the Murine Hemopoietic System

Integrin αIIb is a cell adhesion molecule expressed in association with β3 by cells of the megakaryocytic lineage, from committed progenitors to platelets. While it is clear that lymphohemopoietic cells differentiating along other lineages do not express this molecule, it has been questioned whether mammalian hemopoietic stem cells (HSC) and various progenitor cells express it. In this study, we detected αIIb expression in midgestation embryo in sites of HSC generation, such as the yolk sac blood islands and the hemopoietic clusters lining the walls of the major arteries, and in sites of HSC migration, such as the fetal liver. Since c-Kit, which plays an essential role in the early stages of hemopoiesis, is expressed by HSC, we studied the expression of the αIIb antigen in the c-Kit-positive population from fetal liver and adult bone marrow differentiating in vitro and in vivo into erythromyeloid and lymphocyte lineages. Erythroid and myeloid progenitor activities were found in vitro in the c-Kit+αIIb+ cell populations from both origins. On the other hand, a T cell developmental potential has never been considered for c-Kit+αIIb+ progenitors, except in the avian model. Using organ cultures of embryonic thymus followed by grafting into athymic nude recipients, we demonstrate herein that populations from murine fetal liver and adult bone marrow contain T lymphocyte progenitors. Migration and maturation of T cells occurred, as shown by the development of both CD4+CD8− and CD4−CD8+ peripheral T cells. Multilineage differentiation, including the B lymphoid lineage, of c-Kit+αIIb+ progenitor cells was also shown in vivo in an assay using lethally irradiated congenic recipients. Taken together, these data demonstrate that murine c-Kit+αIIb+ progenitor cells have several lineage potentialities since erythroid, myeloid, and lymphoid lineages can be generated.

Stromal cell lines from mouse aorta-gonads-mesonephros subregions are potent supporters of hematopoietic stem cell activity

The aorta-gonads-mesonephros (AGM) region autonomously generates the first adult repopulating hematopoietic stem cells (HSCs) in the mouse embryo. HSC activity is initially localized to the dorsal aorta and mesenchyme (AM) and vitelline and umbilical arteries. Thereafter, HSC activity is found in the urogenital ridges (UGs), yolk sac, and liver. As increasing numbers of HSCs are generated, it is thought that these sites provide supportive microenvironments in which HSCs are harbored until the bone marrow microenvironment is established. However, little is known about the supportive cells within these midgestational sites, and particularly which microenvironment is most supportive for HSC growth and maintenance. Thus, to better understand the cells and molecules involved in hematopoietic support in the midgestation embryo, more than 100 stromal cell lines and clones were established from these sites. Numerous stromal clones were found to maintain hematopoietic progenitors and HSCs to a similar degree as, or better than, previously described murine stromal lines. Both the AM and UG subregions of the AGM produced many supportive clones, with the most highly HSC-supportive clone being derived from the UGs. Interestingly, the liver at this stage yielded only few supportive stromal clones. These results strongly suggest that during midgestation, not only the AM but also the UG subregion provides a potent microenvironment for growth and maintenance of the first HSCs.

Cloning and expression of Ventrhoid, a novel vertebrate homologue of the Drosophila EGF pathway gene rhomboid

In Drosophila melanogaster, the seven-pass transmembrane protein Rhomboid (Rho) is a crucial positive modulator of EGF signaling playing a substantial role in patterning of the ventral neuroectoderm and fate specification of neuroblasts. Here, we describe the cloning and expression pattern of Ventrhoid ( Vrho), the novel evolutionarily conserved vertebrate cDNA related to fruit fly rho. Most importantly, like rho in Drosophila, Vrho is also expressed in a spatially restricted manner. Vrho expression is most prominent along the developing ventral neural tube, and is also detectable in the ventral forebrain, prospective diencephalon, otic vesicles, mandibular arches, cranial sensory placodes, last formed pair of somites and hindgut in midgestational mouse embryos.