Dpc Mouse Embryos Research Articles

Developmental trajectory of prehematopoietic stem cell formation from endothelium

AbstractHematopoietic stem and progenitor cells (HSPCs) in the bone marrow are derived from a small population of hemogenic endothelial (HE) cells located in the major arteries of the mammalian embryo. HE cells undergo an endothelial to hematopoietic cell transition, giving rise to HSPCs that accumulate in intra-arterial clusters (IAC) before colonizing the fetal liver. To examine the cell and molecular transitions between endothelial (E), HE, and IAC cells, and the heterogeneity of HSPCs within IACs, we profiled ∼40 000 cells from the caudal arteries (dorsal aorta, umbilical, vitelline) of 9.5 days post coitus (dpc) to 11.5 dpc mouse embryos by single-cell RNA sequencing and single-cell assay for transposase-accessible chromatin sequencing. We identified a continuous developmental trajectory from E to HE to IAC cells, with identifiable intermediate stages. The intermediate stage most proximal to HE, which we term pre-HE, is characterized by increased accessibility of chromatin enriched for SOX, FOX, GATA, and SMAD motifs. A developmental bottleneck separates pre-HE from HE, with RUNX1 dosage regulating the efficiency of the pre-HE to HE transition. A distal candidate Runx1 enhancer exhibits high chromatin accessibility specifically in pre-HE cells at the bottleneck, but loses accessibility thereafter. Distinct developmental trajectories within IAC cells result in 2 populations of CD45+ HSPCs; an initial wave of lymphomyeloid-biased progenitors, followed by precursors of hematopoietic stem cells (pre-HSCs). This multiomics single-cell atlas significantly expands our understanding of pre-HSC ontogeny.

A novel non-coding RNA within an intron of CDH2 and association of its SNP with non-syndromic cleft lip and palate

BackgroundGenome-wide linkage analysis and whole genome sequencing in a Van der Woude syndrome (VWS) family revealed that the SNP, rs539075, within intron 2 of the cadherin 2 gene (CDH2) co-segregated with the disease phenotype. ResultsA study with nonsyndromic cleft lip with or without cleft palate (NSCL ± P) cases (N = 292) and controls (N = 287) established association of this SNP with NSCL ± P as a risk factor. RT-PCR based expression analysis of the SNP-harbouring region of intron 2 of CDH2 in the clefted lip and/or palate tissues of 16 patients revealed that the mutant allele expressed in all those individuals having it (hetero-/homozygous), whereas the wild type allele expressed in <50% of the samples in which it was present. The intronic transcript was also present in the prospective lip and palate region of 13.5 dpc mouse embryo, detected by RNA in situ hybridization and RT-PCR. ConclusionsThese results including the in silico, characterization of the ~200 nt-intronic transcript showed that conformationally it fits best with noncoding small RNA, possibly a precursor of miRNA. Its function in the orofacial organogenesis remains to be elucidated which will enable us to define the role of this mutant ncRNA in the clefting of lip and palate.

Delivery of mtZFNs into Early Mouse Embryos.

Mitochondrial diseases often result from mutations in the mitochondrial genome (mtDNA). In most cases, mutant mtDNA coexists with wild-type mtDNA, resulting in heteroplasmy. One potential future approach to treat heteroplasmic mtDNA diseases is the specific elimination of pathogenic mtDNA mutations, lowering the level of mutant mtDNA below pathogenic thresholds. Mitochondrially targeted zinc-finger nucleases (mtZFNs) have been demonstrated to specifically target and introduce double-strand breaks in mutant mtDNA, facilitating substantial shifts in heteroplasmy. One application of mtZFN technology, in the context of heteroplasmic mtDNA disease, is delivery into the heteroplasmic oocyte or early embryo to eliminate mutant mtDNA, preventing transmission of mitochondrial diseases through the germline. Here we describe a protocol for efficient production of mtZFN mRNA in vitro, and delivery of these into 0.5 dpc mouse embryos to elicit shifts of mtDNA heteroplasmy.

Complement Component 3 (C3) Is a Mediator of Epithelial-Mesenchymal Transition (EMT)

We have shown that complement component 3 (C3) is expressed in malignant ovarian epithelial cells and enhances cell proliferation in vitro and tumor growth in vivo. C3 is secreted by cancer cells into the tumor microenvironment and promotes tumor growth through an autocrine loop. To understand the mechanism of upregulation of C3 expression in malignant epithelial cells, we studied the transcriptional regulation of C3, and found that TWIST1, a major regulator of EMT, binds to the C3 promoter and regulates C3 transcription. Knockdown of the TWIST1 gene reduced C3 mRNA, and TWIST1 overexpression increased C3 mRNA.TWIST1 promotes epithelial-mesenchymal transition (EMT) during normal development and in metastasis of malignant tumors. An important marker of EMT is a reduction in the surface expression of E-cadherin on cells facilitating migration and invasion of these cells. TWIST1 is a transcriptional repressor of E-cadherin; and because TWIST1 increases C3 expression, we investigated whether C3 is also a negative regulator of E-cadherin expression. We overexpressed C3 in ovarian cancer cells by stable transduction of lentivirus carrying C3 cDNA. Overexpression of C3 was associated with 32% reduction in the expression of E-cadherin resulting in enhanced migration ability of cells by 2.3 folds and invasiveness by 1.75 folds, as compared to control cells transduced with control lentivirus.To investigate whether TWIST1-induced reduction in E-cadherin is C3-mediated or not, we studied the effect of TWIST1 overexpression simultaneous with C3 knockdown in ovarian cancer cells. Overexpression of TWIST1 alone resulted in 70% reduction in E-cadherin mRNA and this was completely reversed after simultaneous C3 knockdown in these cells.To investigate the correlation between C3 and TWIST1 in vivo, we studied the co-expression of these two proteins in mouse embryos (physiologic EMT) and in malignant tumors (pathologic EMT). Given the role of EMT in embryogenesis we immunostained mouse embryos at different stages of development, using antibodies against TWIST1 or C3. Transverse section of 9.5-day post-coitum (9.5dpc) mouse embryos showed co-expression of TWIST1 and C3 in otocyst (ot) and hindbrain (hb) of neural crest. In the whole-mounted 11.5dpc mouse embryos, C3 and TWIST1 were co-expressed in limb buds.Given the role of EMT in malignancy, tumors induced in mice after intraperitoneal injection of murine ovarian cancer cells were resected and immunostained for C3 and TWIST1 proteins. TWIST1 and C3 co-localized at tumor edges, where EMT and tumor cells migration occur. Taken together, these data provide evidence that TWIST1 regulates C3 expression, and C3 promotes EMT through E-cadherin. DisclosuresNo relevant conflicts of interest to declare.

FRA2A is a CGG repeat expansion associated with silencing of AFF3.

Folate-sensitive fragile sites (FSFS) are a rare cytogenetically visible subset of dynamic mutations. Of the eight molecularly characterized FSFS, four are associated with intellectual disability (ID). Cytogenetic expression results from CGG tri-nucleotide-repeat expansion mutation associated with local CpG hypermethylation and transcriptional silencing. The best studied is the FRAXA site in the FMR1 gene, where large expansions cause fragile X syndrome, the most common inherited ID syndrome. Here we studied three families with FRA2A expression at 2q11 associated with a wide spectrum of neurodevelopmental phenotypes. We identified a polymorphic CGG repeat in a conserved, brain-active alternative promoter of the AFF3 gene, an autosomal homolog of the X-linked AFF2/FMR2 gene: Expansion of the AFF2 CGG repeat causes FRAXE ID. We found that FRA2A-expressing individuals have mosaic expansions of the AFF3 CGG repeat in the range of several hundred repeat units. Moreover, bisulfite sequencing and pyrosequencing both suggest AFF3 promoter hypermethylation. cSNP-analysis demonstrates monoallelic expression of the AFF3 gene in FRA2A carriers thus predicting that FRA2A expression results in functional haploinsufficiency for AFF3 at least in a subset of tissues. By whole-mount in situ hybridization the mouse AFF3 ortholog shows strong regional expression in the developing brain, somites and limb buds in 9.5–12.5dpc mouse embryos. Our data suggest that there may be an association between FRA2A and a delay in the acquisition of motor and language skills in the families studied here. However, additional cases are required to firmly establish a causal relationship.

Insights from transcriptional profiling the mouse hindbrain: Novel feedback between retinoids and Hox genes

Adult vertebrate hindbrain regulates critical body functions such as sleep, respiration and heart rate. During embryogenesis hindbrain forms seven transient swellings called rhombomeres, each with unique morphology and gene expression patterns. Rhombomere specification and regulation is controlled by transcription factors including Hox family and Krox20. Disruption of these genes changes the number and properties of rhombomeres and leads to neuronal disorganization causing animal lethality.We are using transcriptional profiling and functional validation to reveal rhombomeric‐specific patterns of gene expression regulated by these key transcription factors. We isolated individual rhombomeres from 9.5 dpc mouse embryos using laser capture microscopy and performed transcriptional profiling on either single rhombomeres from the wild type hindbrains or whole hindbrains of wild type and Hoxa1, Hoxa2, Hoxb1 and Krox20 mutants. Analyses and validation of these results have uncovered several novel targets and pathways associated with hindbrain specification. For example, Hox genes which are induced by retinoids in turn modulate retinoid metabolism. These feedback loops are important for hindbrain patterning.Grant Funding Source: Stowers Institute for Medical Research

Pluripotent genes in avian stem cells

Embryonic stem (ES) cells were first isolated in 1981 in the mouse from the in vitro proliferation of the inner cell mass of a 3.5days post-coitum (dpc) blastocyst. Later on, epiblast stem cells (EpiSC) were identified from in vitro culture of the epiblast of a 6.5dpc mouse embryo, leading to the concept of naïve and primed stem cells. Among non-mammalian species, ES cells have been characterized both in birds and fish; here, we focus on cells derived from chicken and the pluripotent associated markers such as OCT4, SOX2, NANOG, and KLF, previously identified in mammalian cells. In this review, we present both published and original data regarding the involvement of those pluripotent associated genes in the ES cells and early embryo of chicken.

Abstract 237: VEGF-Mediated Phosphorylation of eNOS Regulates Angioblast and Endothelial Cell Proliferation

To evaluate when nitric oxide (NO) is first expressed in the EC lineage, the expression pattern of eNOS, phosphorylated eNOS (P-eNOS), and key proteins that define the endothelial lineage (i.e., Flk-1, TAL-1, CD31) were assessed in 7.0-8.5dpc mouse embryos. Immunohistochemical analyses revealed that embryonic endothelial cells (Flk-1 + /TAL-1 + / CD31 + ) expressed eNOS prior to their investment by smooth muscle cells while isolated angioblasts (Flk-1 + /TAL-1 + / CD31 - ) did not express eNOS. Based on eNOS expression we identified a cell type, transitional angioblasts ( eNOS + / FLK-1 + /TAL-1 + / CD31 - ), intermediate between embryonic endothelial cells (EECs) and angioblasts. Transitional angioblasts are further distinguished from angioblasts by their initiation of cell-cell contacts with other eNOS + Flk-1 + /TAL-1 + /CD31 - cells or with EECs. Analysis of P-eNOS and phospho-histone H3 expression in transitional angioblasts and EECs showed a tight correlation between P-eNOS expression and cell proliferation. This correlation was also observed in cultured human umbilical vein endothelial cells (HUVECs) treated with nocodazole to induce a G2/M cell cycle block. Given that VEGF mediates both EC division and NO production, we evaluated whether VEGF-mediated NO production is critical for neovascular processes (vasculogenesis/angiogenesis). We show using allantoic cultures that VEGF-mediated mitosis in Flk-1 + cells is decreased by the eNOS inhibitors L-NIO and 17-AAG, suggesting that VEGF-mediated eNOS phosphorylation is regulating cell proliferation. Supporting such a role, we found that the alterations in VEGF-mediated vascular patterning we observed in response to eNOS inhibitors were consistent with an overall reduction in the EEC numbers and that VEGF-induced NO production in HUVECs increased the S-nitrosylation of proteins important for the regulation of the G2/M transition, such as cyclin B. Taken together, our findings suggest that VEGF-mediated eNOS phosphorylation regulates transitional angioblast, EEC, and EC proliferation.

Magic-F1 transgene cooperates with Pax 3 during early myogenesis to induce muscular hypertrophy

Met-Activating Genetically Improved Chimeric Factor-1 (Magic-F1) is a human recombinant protein derived from hepatocyte growth factor/scatter factor (HGF/ SF) and consists in two Met-binding domains repeated in tandem and separated by an artificial linker. It has a reduced affinity for Met and, in contrast to HGF, it elicits activation of the AKT but not the ERK signaling pathway. We recently showed that Magic-F1 induces muscle cell hypertrophy but not progenitor cell proliferation, both in vitro and in vivo where a transgenic mouse express the recombinant protein exclusively in skeletal muscle tissue [1]. Here, we examined the temporal and spatial expression pattern of Magic-F1 in comparison with Pax3 (paired box gene 3) transcription factor during embryogenesis [2]. Ranging from 9.5 to 17.5 dpc (days post coitum) mouse embryos were analyzed by in situ hybridization using whole mounts during early stages of development (9.5-10.5-11.5 dpc) and cryostat sections for later stages (11.5-13.5-15.5-17.5 dpc). We found that Magic-F1 is expressed in developing organs and tissues of mesenchymal origin, where Pax3 signal appears to be downregulated respect to the wt embryos. These data suggest that Magic-F1 could be responsible of muscular hypertrophy, cooperating with Pax3 signal pathway in skeletal muscle precursor cells.

Genetic cause of rare disease may be involved in more common birth defects

Genetic cause of rare disease may be involved in more common birth defects

Localization of Magic‐F1 Transgene, Involved in Muscular Hypertrophy, during Early Myogenesis

We recently showed that Magic-F1 (Met-activating genetically improved chimeric factor 1), a human recombinant protein derived from hepatocyte growth factor/scatter factor (HGF/SF) induces muscle cell hypertrophy but not progenitor cell proliferation, both in vitro and in vivo. Here, we examined the temporal and spatial expression pattern of Magic-F1 in comparison with Pax3 (paired box gene 3) transcription factor during embryogenesis. Ranging from 9.5 to 17.5 dpc (days post coitum) mouse embryos were analyzed by in situ hybridization using whole mounts during early stages of development (9.5–10.5–11.5 dpc) and cryostat sections for later stages (11.5–13.5–15.5–17.5 dpc). We found that Magic-F1 is expressed in developing organs and tissues of mesenchymal origin, where Pax3 signal appears to be downregulated respect to the wt embryos. These data suggest that Magic-F1 could be responsible of muscular hypertrophy, cooperating with Pax3 signal pathway in skeletal muscle precursor cells.

Esrrg functions in early branch generation of the ureteric bud and is essential for normal development of the renal papilla

Congenital anomalies of the kidney and urinary tract (CAKUTs) are common disorders of human development affecting the renal parechyma, renal pelvis, ureter, bladder and urethra; they show evidence of shared genetic aetiology, although the molecular basis of this remains unknown in the majority of cases. Breakpoint mapping of a de novo, apparently balanced, reciprocal translocation associated with bilateral renal agenesis has implicated the gene encoding the nuclear steroid hormone receptor ESRRG as a candidate gene for CAKUT. Here we show that the Esrrg protein is detected throughout early ureteric ducts as cytoplasmic/sub-membranous staining; with nuclear localization seen in developing nephrons. In 14.5–16.5 dpc (days post-conception) mouse embryos, Esrrg localizes to the subset of ductal tissue within the kidney, liver and lung. The renal ductal expression becomes localized to renal papilla by 18.5 dpc. Perturbation of function was performed in embryonic mouse kidney culture using pooled siRNA to induce knock-down and a specific small-molecule agonist to induce aberrant activation of Esrrg. Both resulted in severe abnormality of early branching events of the ureteric duct. Mouse embryos with a targeted inactivation of Esrrg on both alleles (Esrrg−/−) showed agenesis of the renal papilla but normal development of the cortex and remaining medulla. Taken together, these results suggest that Esrrg is required for early branching events of the ureteric duct that occur prior to the onset of nephrogenesis. These findings confirm ESRRG as a strong candidate gene for CAKUT.

Reprogramming of melanoma cells by embryonic microenvironments

In recent years, the reversion of the cancer phenotype of human melanoma cells in developing zebrafish and chick embryos has been reported. The aim of this review is to revise these and other related contributions regarding the regulation of embryonic cancer and to provide a framework with which to understand results from our laboratory on the interactions of human melanoma cells with post-implanted mouse embryos cultured in vitro. To this end, we used the A375 human melanoma cell line transfected with the green fluorescent protein (GFP) gene. Labeled cells were transplanted onto the surface of the developing visceral endoderm of 7.5 dpc mouse embryos. Subsequently, we cultured the transplanted embryos for three days and monitored the movements of GFP labeled human melanoma cells by confocal microscopy. Our results show that ectopic melanoma cells internalize and migrate inside the embryo body in a way reminiscent of neural crest cells. The absence of localized tumor growth after 72 hours of in vitro embryo co-culture suggests that malignant phenotype inhibiting factors are active at the gastrulating stage and during early organogenesis. These results complement previous reports of growth regulation of B16 mouse melanoma cells by 10 dpc mouse embryonic skin (Gerschenson et al., 1986). Further research is required to elucidate the final fate of melanoma cells in mammalian embryos and the details of the signaling pathways underlying tumor growth regulation. Understanding regulation of melanoma cells by young embryos could represent a starting point for a developmental theory of the pathogenesis of melanoma, and for future developments of more physiologically-based anticancer therapies for this and indeed, other types of aggressive tumor.

EMB-003 Approach for microdeletion screening in congenital heart defects

s Aspire: ART Paving the way for new frontiers mouse embryonic development. Materials/Methods: Whole-mount in-situ hybridization was carried out as described by Correia and Conlon. Using the cDNA of mouse p19 cells as the template, DNA was amplified with the primers for mouse borealin: forward, 5′-TGCCTTCCATCCAAGAAGAG-3′ and reverse, 5′AGGTGTTGGCAGTGAAGGAC-3′. The amplified DNA was cloned into pGEM-T Easy vector (Promega) and sequenced. Hybridization was performed with a digoxygenin-labelling and detection kit (Roche). Protein extracts were separated by SDS-PAGE and detected by western blotting using the western blot kit (KPL). Human anti-borealin antibody (gift from Dr Earnshaw and Dr Chang) and anti-rabbit IgG (KPL) were used at 1:500 and 1:10,000 dilutions, respectively. Anti-GAPDH antibody, anti-mouse IgG, anti-Oct-4 antibody (Santa) and anti-survivin antibody (Chemicon) were used according to the manufacturer’s protocol. After injection with the anti-borealin antibody or rabbit IgG (Santa), zygotes were brought back into the medium for culture. Indirect immunofluorescence was performed as described previously. After injection, the zygotes in the first mitotic division were immediately fixed in 4% formaldehyde at 4°C for 30 min, followed by incubation with the anti-β-tubulin antibody (Sigma) and fluoresceinconjugated anti-mouse IgG (Sigma). DNA was stained with 0.5 μg/ml DAPI (Chemicon) in 1× PBS. Results: Borealin was highly expressed in the undifferentiated ES cells, the mouse preimplantation embryos and the embryonic brain of 8.5 dpc–9.5 dpc mouse embryo. The mouse embryos were arrested at the 2-cell or 4-cell stage with the Borealin depleted. Conclusion: The authors have indicated for the first time that borealin plays crucial roles in the early development of mouse embryos and in the self-renewal and proliferation of undifferentiated HES cells.

EMB-004 Effect of oxytocin on folliculogenesis and ovulation

s Aspire: ART Paving the way for new frontiers mouse embryonic development. Materials/Methods: Whole-mount in-situ hybridization was carried out as described by Correia and Conlon. Using the cDNA of mouse p19 cells as the template, DNA was amplified with the primers for mouse borealin: forward, 5′-TGCCTTCCATCCAAGAAGAG-3′ and reverse, 5′AGGTGTTGGCAGTGAAGGAC-3′. The amplified DNA was cloned into pGEM-T Easy vector (Promega) and sequenced. Hybridization was performed with a digoxygenin-labelling and detection kit (Roche). Protein extracts were separated by SDS-PAGE and detected by western blotting using the western blot kit (KPL). Human anti-borealin antibody (gift from Dr Earnshaw and Dr Chang) and anti-rabbit IgG (KPL) were used at 1:500 and 1:10,000 dilutions, respectively. Anti-GAPDH antibody, anti-mouse IgG, anti-Oct-4 antibody (Santa) and anti-survivin antibody (Chemicon) were used according to the manufacturer’s protocol. After injection with the anti-borealin antibody or rabbit IgG (Santa), zygotes were brought back into the medium for culture. Indirect immunofluorescence was performed as described previously. After injection, the zygotes in the first mitotic division were immediately fixed in 4% formaldehyde at 4°C for 30 min, followed by incubation with the anti-β-tubulin antibody (Sigma) and fluoresceinconjugated anti-mouse IgG (Sigma). DNA was stained with 0.5 μg/ml DAPI (Chemicon) in 1× PBS. Results: Borealin was highly expressed in the undifferentiated ES cells, the mouse preimplantation embryos and the embryonic brain of 8.5 dpc–9.5 dpc mouse embryo. The mouse embryos were arrested at the 2-cell or 4-cell stage with the Borealin depleted. Conclusion: The authors have indicated for the first time that borealin plays crucial roles in the early development of mouse embryos and in the self-renewal and proliferation of undifferentiated HES cells.

Clonal analysis of the origin of primordial germ cells in the mouse.

Qualitative and quantitative clonal analysis has been used to answer three basic questions about the establishment of the germ cell lineage in the mouse. Where do primordial germ cells originate? What is the size of the founding population at the time of lineage restriction? When and where does lineage restriction occur? Single epiblast cells of 6.0 dpc and 6.5 dpc mouse embryos were injected with a short-term lineage label (lysinated rhodamine dextran, LRDX) and their descendants traced after 40 h embryo culture at neural plate and early somite stages, respectively. An objective matching technique was used to detect the lineage marker in primordial germ cells identified by their characteristic alkaline phosphatase staining. Precursors of the primordial germ cells were found in the proximal epiblast close to the extraembryonic ectoderm in both pregastrulation and early-streak stage embryos. They form part of the presumptive extraembryonic mesoderm and are not lineage restricted while in the epiblast. Quantitative analysis gives a best fit to a model of a founding population of 45 at the time of lineage restriction. The data indicate that the generation time lengthens at the time of allocation. Calculation of clonal histories gives a best fit of 16 h generation time after allocation compared with < 7 h before allocation, with lineage restriction occurring at the early midstreak stage, presumably in the region posterior to the streak in which primordial germ cells are first identifiable. Therefore primordial germ cells are probably allocated early during gastrulation in a group of > 40 cells already segregated in the extraembryonic mesoderm.

Expression of Tsga10 sperm tail protein in embryogenesis and neural development: From cilium to cell division

Tsga10 has been localised in sperm tail as a fibrous sheath protein. In this study, we showed its expression during developmental stages of mouse embryo, in adult mice brain, and in some malignancies. RT-PCR and immunohistochemistry study show that Tsga10 expression starts in 4.5–7.5 dpc mouse embryos and continues throughout embryogenesis. Then we showed that the Tsga10 is expressed in adult brain and in the cells with neural crest origin, olfactory epithelium, and human germ cell tumour. It is expressed with two transcripts in sperm and whole embryos but just with the long transcript in brain embryo as a result of its exon 16 splicing. Our finding of the Tsga10 perinuclear localisation and its expression pattern suggests that it may be involved in active cell division, differentiation, and migrating cells. The results of the experiments in this project hypothesize the presence of Tsga10 protein wherever there is a conserved ciliary structure.

Characterization of Asxl1, a murine homolog of Additional sex combs, and analysis of the Asx-like gene family

The Additional sex combs ( Asx) gene of Drosophila is required to maintain homeotic gene activation and silencing. Here we characterize the three murine homologs of Asx: Additional sex combs-like ( Asxl1, Asxl2, and Asxl3) and identify conserved sequence features. The predicted amino acid sequence of Asxl1 has 16% identity and 40% similarity to Drosophila Asx, and 74% identity and 81% similarity to human ASXL1. Murine Asxl1 contains two regions highly conserved with Drosophila Asx: 1) a conserved amino terminal region of unknown function, termed the ASX homology domain (ASXH) which contains two nuclear receptor (NR) co-regulator binding motifs; and 2) a conserved C-terminal PHD domain. The mammalian Asx-like predicted proteins possess additional conserved sequence features of unknown function not present in Drosophila Asx, including three more NR co-regulator binding motifs. Asxl1and Asxl2 are expressed as multiple transcripts, at varying levels, in adult tissues and in embryonic stem cells analyzed by Northern blot, and exhibit similar expression patterns suggesting they may be co-regulated. Whole mount RNA in situ hybridization revealed that Asxl1 is also expressed in 10.5–11.0 dpc mouse embryos.

Regulation of vertebrate myotome development by the p38 MAP kinase–MEF2 signaling pathway

Biochemical and cell culture studies have characterized the myocyte enhancer factor 2 (MEF2) transcriptional regulatory proteins as obligatory partners for the myogenic regulatory factors (MRFs) in the differentiation of myogenic cells in culture. However, the role of MEF2 activation in somitic myogenesis has not been fully characterized. Here, we report a critical interaction between the p38 mitogen-activated protein kinase (p38 MAPK) and MEF2 in the developing somite myotome. We document expression of MEF2A and p38 MAPK proteins in the somite of 9.5 dpc mouse embryos concurrent with Myf 5 protein expression. We also observed that abrogation of p38 MAPK signaling blocks MEF2 activation using a MEF2 transgenic ‘sensor’ mouse. Inhibition of p38 MAPK signaling concurrently inhibited myogenic differentiation in somite cultures and in embryos in vivo using transplacental injection of a p38 inhibitor (SB203580). Finally, we document that commitment to the myogenic lineage is not appreciably affected by p38 MAPK inhibition since the activation of an early marker of myogenic commitment (Myf 5) occurs normally when p38 MAPK signaling is inhibited. Thus, we present novel evidence indicating a crucial role for p38 MAPK signaling to the MEF2 transcriptional regulators during early mammalian somite development and myotome formation.

Genetic and Functional Characterization of Isolated Stromal Cell Lines from the Aorta-Gonado-Mesonephros (AGM)-Region.

The aorta-gonads-mesonephros (AGM) region autonomously generates adult repopulating hematopoietic stem cells (HSC) in the mouse embryo and provides its own HSC-supportive microenvironment. Stromal cells from adult bone marrow, yolk sac, fetal liver and AGM have been used in coculture systems for analysing growth, maintenance and differentiation of hematopoietic stem cells. We generated >100 cloned stromal cell lines from the AGM of 10.5 dpc mouse embryos. In previous studies, we tested these for support of murine adult and human cord blood (CB) CD34+ cells. We could demonstrate that 25 clones were superior to the MS5 bone marrow stromal cell line in supporting progenitor cell expansion of adult mouse bone marrow both, in 2ndry CFC and CAFC production. In addition we demonstrated that 5 AGM lines promoted in absence of exogenous growth factors the expansion of human CB cells with progenitor (CFC production for at least 5 weeks) and stem cell (repopulation of cocultured cells in NOD/SCID assay) function. Now, we could show that one of the isolated stromal cell lines (AGM-S62) is capable in differentiating undifferentiated murine embryonic stem (mES) cells into cells of the hematopoietic lineage. A sequential coculture of mES-cells with AGM-S62 showed production of CD41+ hematopoietic progenitor cells at day 10 as well as 2ndry CFC and CAFC production of day 10 suspension cells. Hematopoietic cell differentiation was comparable to standard OP9 differentiation assay. With these data, we can describe for the first time, that a stromal cell line other than OP9 can induce hematopoietic differentiation of undifferentiated mES cells. Hematopoietic support occurs independently of M-CSF deficiency, which is the characteristic of OP9 cells, because it is strongly expressed by AGM-S62. To evaluate genes responsible for hematopoietic cell support, we compared a supporting and a non-supporting AGM stromal cell line by microarray analysis. The cell line with hematopoietic support clearly showed a high expression of mesenchymal markers (laminins, thrombospondin-1) as well as characteristic genes for the early vascular smooth muscle phenotype (Eda). Both phenotypes are described for stromal cells with hematopoietic support generated from bone marrow and fetal liver. In addition, the analysed supporting AGM stromal cell line interestingly expressed genes important in early B-cell differentiation (osteoprotegerin, early B-cell factor 1, B-cell stimulating factor 3), which goes in line with data demonstrating early B-cell development in the AGM-region before etablishing of fetal liver hematopoiesis. Further studies will show the significance of single factors found to be expressed in microarray analyses. This unique source of > 100 various cell lines will be of value in elucidating the molecular mechanisms regulating embryonic and adult hematopoiesis in mouse and man.