Immature hematopoietic stem cells undergo maturation in the fetal liver

Hematopoietic stem cells (HSCs), which are defined by their capacity to reconstitute adult conventional mice, are first found in the dorsal aorta after 10.5 days post coitus (dpc) and in the fetal liver at 11 dpc. However, lympho-myeloid hematopoietic progenitors are detected in the dorsal aorta from 9 dpc, raising the issue of their role in establishing adult hematopoiesis. Here, we show that these progenitors are endowed with long-term reconstitution capacity, but only engraft natural killer (NK)-deficient Rag2γc(-/-) mice. This novel population, called here immature HSCs, evolves in culture with thrombopoietin and stromal cells, into HSCs, defined by acquisition of CD45 and MHC-1 expression and by the capacity to reconstitute NK-competent mice. This evolution occurs during ontogeny, as early colonization of fetal liver by immature HSCs precedes that of HSCs. Moreover, organ culture experiments show that immature HSCs acquire, in this environment, the features of HSCs.     

Differential expression of the HMGN family of chromatin proteins during ocular development

The HMGN proteins are a group of non-histone nuclear proteins that associate with the core nucleosome and alter the structure of the chromatin fiber. We investigated the distribution of the three best characterized HMGN family members, HMGN1, HMGN2 and HMGN3 during mouse eye development. HMGN1 protein is evenly distributed in all ocular structures of 10.5 days post-coitum (dpc) mouse embryos however, by 13.5dpc, relatively less HMGN1 is detected in the newly formed lens fiber cells compared to other cell types. In the adult, HMGN1 is detected throughout the retina and lens, although in the cornea, HMGN1 protein is predominately located in the epithelium. HMGN2 is also abundant in all ocular structures of mouse embryos, however, unlike HMGN1, intense immunolabeling is maintained in the lens fiber cells at 13.5dpc. In the adult eye, HMGN2 protein is still found in all lens nuclei while in the cornea, HMGN2 protein is mostly restricted to the epithelium. In contrast, the first detection of HMGN3 in the eye is in the presumptive corneal epithelium and lens fiber cells at 13.5dpc. In the lens, HMGN3 remained lens fiber cell preferred into adulthood. In the cornea, HMGN3 is transiently upregulated in the stroma and endothelium at birth while its expression is restricted to the corneal epithelium in adulthood. In the retina, HMGN3 upregulates around 2 weeks of age and is found at relatively high levels in the inner nuclear and ganglion cell layers of the adult retina. RT-PCR analysis determined that the predominant HMGN3 splice form found in ocular tissues is HMGN3b which lacks the chromatin unfolding domain although HMGN3a mRNA is also detected. These results demonstrate that the HMGN class of chromatin proteins has a dynamic expression pattern in the developing eye.     

Reduced type I collagen utilization: a pathogenic mechanism in COL5A1 haplo-insufficient Ehlers-Danlos syndrome

To examine mechanisms by which reduced type V collagen causes weakened connective tissues in the Ehlers-Danlos syndrome (EDS), we examined matrix deposition and collagen fibril morphology in long-term dermal fibroblast cultures. EDS cells with COL5A1 haplo-insufficiency deposited less than one-half of hydroxyproline as collagen compared to control fibroblasts, though total collagen synthesis rates are near-normal because type V collagen represents a small fraction of collagen synthesized. Cells from patients with osteogenesis imperfecta (OI) and haplo-insufficiency for proalpha1(I) chains of type I collagen also incorporated about one-half the collagen as controls, but this amount was proportional to their reduced rates of total collagen synthesis. Collagen fibril diameter was inversely proportional to type V/type I collagen ratios (EDS > control > OI). However, a reduction of type V collagen, in the EDS derived cells, was associated with the assembly of significantly fewer fibrils compared to control and OI cells. These data indicate that in cell culture, the quantity of collagen fibrils deposited in matrix is highly sensitive to reduction in type V collagen, far out of proportion to type V collagen's contribution to collagen mass.     

Expression of a medaka (Oryzias latipes) Bar homologue in the differentiating central nervous system and retina

Endoglin is an auxiliary receptor for the transforming growth factor-β family of cytokines and is required for angiogenesis and heart development. Endoglin expression during mouse embryogenesis was analysed by monitoring β-galactosidase expression from a lacZ reporter cassette inserted downstream of the endoglin promoter. Expression was first detected at 6.5 days post-coitum (dpc) in the amniotic fold and developing allantois. Between 7.5 and 8.5 dpc, endoglin was expressed in endothelial cells of the yolk sac, dorsal aorta and primitive heart tube, and from 9.5 to 13.5 dpc in endothelial cells throughout the developing vasculature. Interestingly, this pattern of endoglin expression is almost identical to that reported for Alk1.     

Aortic carboxypeptidase-like protein is expressed in collagen-rich tissues during mouse embryonic development

Aortic carboxypeptidase-like protein (ACLP) was originally identified in vascular smooth muscle cells and contains discoidin and catalytically inactive metallocarboxypeptidase domains. ACLP is a secreted protein that associates with the extracellular matrix and is essential for abdominal wall development and contributes to dermal wound healing. Because of these developmental and adult phenotypes, we examined the expression of ACLP by immunohistochemistry throughout mouse embryonic development. ACLP was not detected in 7.5 days post-coitum (dpc) embryos, however at 9.5 dpc low levels of expression were detected in the somites and dorsal aorta. Expression was detected in both the yolk sac and embryonic vasculature at 10.5d pc. ACLP expression increased in both large and small blood vessels at 11.5 and 13.5 dpc and intense expression was detected within the vascular smooth muscle layer in 16.5 dpc embryos. At later developmental time points, discrete areas of ACLP expression were detected in the mesenchymal cells in the dermal layer, developing skeletal structures, connective tissue, and in the umbilical ring and vessels. The predominance of ACLP immunoreactivity localized with collagen-rich regions including tendons and basement membranes. Overall, the developmental expression pattern is consistent with a regulatory or structural role in the abdominal wall, vasculature, and dermis.     

Murine model of the Ehlers-Danlos syndrome. col5a1 haploinsufficiency disrupts collagen fibril assembly at multiple stages

The most commonly identified mutations causing Ehlers-Danlos syndrome (EDS) classic type result in haploinsufficiency of proalpha1(V) chains of type V collagen, a quantitatively minor collagen that co-assembles with type I collagen as heterotypic fibrils. To determine the role(s) of type I/V collagen interactions in fibrillogenesis and elucidate the mechanism whereby half-reduction of type V collagen causes abnormal connective tissue biogenesis observed in EDS, we analyzed mice heterozygous for a targeted inactivating mutation in col5a1 that caused 50% reduction in col5a1 mRNA and collagen V. Comparable with EDS patients, they had decreased aortic stiffness and tensile strength and hyperextensible skin with decreased tensile strength of both normal and wounded skin. In dermis, 50% fewer fibrils were assembled with two subpopulations: relatively normal fibrils with periodic immunoreactivity for collagen V where type I/V interactions regulate nucleation of fibril assembly and abnormal fibrils, lacking collagen V, generated by unregulated sequestration of type I collagen. The presence of the aberrant fibril subpopulation disrupts the normal linear and lateral growth mediated by fibril fusion. Therefore, abnormal fibril nucleation and dysfunctional fibril growth with potential disruption of cell-directed fibril organization leads to the connective tissue dysfunction associated with EDS.     

Developmentally regulated expression of type XV collagen correlates with abnormalities in Col15a1(-/-) mice.

Lack of type XV collagen in mice results in mild skeletal myopathy and increases vulnerability to exercise-induced skeletal muscle and cardiac injury [Proc. Natl. Acad. Sci. USA 98 (2001), 1194]. The expression of type XV collagen was studied during murine fetal development from 10.5 to 18.5 dpc using immunofluorescence. The first sign of type expression was seen in the capillaries of many tissues at 10.5 dpc, some of them showing developmental transitions in the expression. Interestingly, capillaries forming the blood-brain barrier and those of the sinusoidal type were essentially lacking in this collagen. Early expression was also detected in the skeletal muscle and peripheral nerves, while expression in the heart, kidney and lung appeared to be developmentally regulated. In addition, distinct staining was found in the perichondrium of the cartilage. Collectively, the dynamics of its expression during development, its localization in the basement membrane--fibrillar matrix interface and the consequences of its absence in mice suggest a structural role in providing stability at least in skeletal muscle and capillaries. The early prominent expression of type XV collagen in newly forming blood vessels could also indicate a possible role in angiogenic processes.     

Cell-specific in vivo DNA-protein interactions at the proximal promoters of the pro alpha 1(I) and the pro alpha2(I) collagen genes.

We performed in vivo dimethylsulfate footprinting of the 220 bp mouse proximal proalpha1(I) collagen promoter and the 350 bp mouse proximal proalpha2(I) collagen promoter in BALB/3T3 fibroblasts, primary mouse skin fibroblasts, S-194 B cells, NMuLi liver epithelial cells and RAG renal adenocarcinoma cells and in vitro DNase I footprinting of these promoters using nuclear extracts of these different cell types. Whereas proalpha1(I) and proalpha2(I) collagen RNAs were present in BALB/3T3 fibroblasts and primary fibroblasts, these RNAs could not be detected in the three other cell lines. Comparison of in vitro DNase I footprints for each of the two proximal collagen promoters indicated that the patterns of protection were very similar with the different nuclear extracts, suggesting that the DNA binding proteins binding to these promoters were present in all cell types tested. In contrast, in vivo footprints over these proximal promoters were cell-specific, occurring only in fibroblast cells and not in the other three cell types. The in vivo footprints were generally located within the in vitro footprinted regions. Our results suggest that although all cell types tested contained nuclear proteins that can bind to the proximal proalpha1(I) and proalpha2(I) collagen promoters in vitro , it is only in fibroblasts that these proteins bind to their cognate sites in vivo . We discuss possible regulatory mechanisms in type I collagen genes that can contribute to the cell-specific in vivo protein-DNA interactions at the proximal promoters.     

Expression of Cre Recombinase in the developing mouse limb bud driven by a Prxl enhancer

We have used a Prx1 limb enhancer to drive expression of Cre Recombinase in transgenic mice. This regulatory element leads to Cre expression throughout the early limb bud mesenchyme and in a subset of craniofacial mesenchyme. Crossing a murine line carrying this transgene to a reporter mouse harboring a floxed Cre-reporter cassette revealed that recombinase activity is first observed in the earliest limb bud at 9.5 dpc. By early to mid bud stages at 10.5 dpc recombination is essentially complete in all mesenchymal cells in the limb. Expression of the Cre recombinase was never detected in the limb bud ectoderm. The use of Prx1-Cre mice should facilitate analysis of gene function in the developing limb.     

Mutations in the COL5A1 gene are causal in the Ehlers-Danlos syndromes I and II.

The Ehlers-Danlos syndrome (EDS) is a heterogeneous connective-tissue disorder of which at least nine subtypes are recognized. Considerable clinical overlap exists between the EDS I and II subtypes, suggesting that both are allelic disorders. Recent evidence based on linkage and transgenic mice studies suggest that collagen V is causally involved in human EDS. Collagen V forms heterotypic fibrils with collagen I in many tissues and plays an important role in collagen I fibrillogenesis. We have identified a mutation in COL5A1, the gene encoding the pro(alpha)1(V) collagen chain, segregating with EDS I in a four-generation family. The mutation causes the substitution of the most 5' cysteine residue by a serine within a highly conserved sequence of the pro(alpha)1(V) C-propeptide domain and causes reduction of collagen V by preventing incorporation of the mutant pro(alpha)1(V) chains in the collagen V trimers. In addition, we have detected splicing defects in the COL5A1 gene in a patient with EDS I and in a family with EDS II. These findings confirm the causal role of collagen V in at least a subgroup of EDS I, prove that EDS I and II are allelic conditions, and represent a, so far, unique example of a human collagen disorder caused by substitution of a highly conserved cysteine residue in the C-propeptide domain of a fibrillar collagen.     

Expression pattern of <Emphasis Type="Italic">serine protease inhibitor kazal type 3 (Spink3)</Emphasis> during mouse embryonic development

Recent evidence shows that the serine protease inhibitor Kazal type 3 (Spink3) has more diverse functions than expected. To gain insight into its function, we analyzed the spatiotemporal expression profile of Spink3, using in situ hybridization (ISH) and a Spink3 ( +/lacZ ) knock-in mouse, in which lacZ was inserted into the Spink3 locus. Spink3 ( lacZ ) expression was first observed in the foregut, midgut, hindgut and the forebrain/midbrain junction region at 9.5 days post coitus (dpc). In the pancreas, Spink3 mRNA was detected at 11.5 dpc, before formation of the typical shape of the exocrine structure of the pancreas. Acinar cell expression was clearly identified by 13.5 dpc. After differentiation of the intestinal tract, Spink3 ( lacZ ) expression was observed in the large intestine at 11.5 dpc, followed by expression in the small intestine at 13.5 dpc, before appearance of intestinal digestive enzymes. Spink3 mRNA and Spink3 ( lacZ ) activity were also detected in other tissues, including the mesonephric tubules and the urogenital ridge at 11.5 dpc, the genital swelling at 13.5 dpc, the ductus epididymis at 17.5 dpc, and the seminal vesicle at 8 weeks. These data suggest that Spink3 may play important roles in proliferation and/or differentiation of various cell types during development.     

The mesonephric (wolffian) and paramesonephric (müllerian) ducts of golden hamsters express different intermediate-filament proteins during development

We analysed the expression of intermediate-filament proteins in the developing mesonephric duct (the precursor of the male genital ducts) and the paramesonephric duct (the precursor of the female genital ducts) of golden-hamster embryos using immunohistochemical methods. Embryos were investigated from the early stages of duct development, i.e. at 9.5 days post conceptionem (dpc), through sexual differentiation, until birth (15.5 dpc). Monospecific antibodies to vimentin or keratins 7, 8, 18 or 19 as well as two keratin antibodies that are pan-epithelial in human tissues were tested. Both ducts expressed vimentin to some degree from their early stages (mesonephric duct from 9.5 dpc onwards; paramesonephric duct from 10.5 dpc onwards) until birth. No keratins were detectable at these earliest stages. In the mesonephric duct, keratins 7, 18 and 19 appeared simultaneously at 10.5 dpc and persisted until birth. In the paramesonephric duct, only keratin 18 was detectable at first (at 12.0 dpc), with the expression of keratins 7 and 19 being delayed until 14.5 dpc. This feature was irrespective of sexual differentiation, which begins at 11.0 dpc, so that, in males, these keratins appeared on cue, even though the paramesonephric duct was regressing at this time. The expression of keratin 8 could not be demonstrated in either duct using the antibodies tested in our study. By 14.5 dpc, the differentiated male mesonephric duct and the differentiated female paramesonephric duct exhibited the same intermediate-filament protein pattern (weak vimentin expression and strong expression of keratins 7, 18 and 19), in spite of differences in the intermediate-filament protein patterns exhibited by the two ducts during early development. These different programmes of intermediate-filament protein regulation do not support the concept that the mesonephric duct makes a cellular contribution to the paramesonephric duct during the development of the latter.     

Transfer of proalpha2(I) cDNA into cells of a murine model of human Osteogenesis Imperfecta restores synthesis of type I collagen comprised of alpha1(I) and alpha2(I) heterotrimers in vitro and in vivo

The oim mouse is a model of human Osteogenesis Imperfecta (OI) that has deficient synthesis of proalpha2(I) chains. Cells isolated from oim mice synthesize alpha1(I) collagen homotrimers that accumulate in tissues. To explore the feasibility of gene therapy for OI, a murine proalpha2(I) cDNA was inserted into an adenovirus vector and transferred into bone marrow stromal cells isolated from oim mice femurs. The murine cDNA under the control of the cytomegalovirus early promoter was expressed by the transduced cells. Analysis of the collagens synthesized by the transduced cells demonstrated that the cells synthesized stable type I collagen comprised of alpha1(I) and alpha2(I) heterotrimers in the correct ratio of 2:1. The collagen was efficiently secreted and also the cells retained the osteogenic potential as indicated by the expression of alkaline phosphatase activity when the transduced cells were treated with recombinant human bone morphogenetic protein 2. Injection of the virus carrying the murine proalpha2(I) cDNA into oim skin demonstrated synthesis of type I collagen comprised of alpha1 and alpha2 chains at the injection site. These preliminary data demonstrate that collagen genes can be transferred into bone marrow stromal cells as well as fibroblasts in vivo and that the genes are efficiently expressed. These data encourage further studies in gene replacement for some forms of OI and use of bone marrow stromal cells as vehicles to deliver therapeutic genes to bone.     

Stage- and sex-dependent expressions of Usp9x,an X-linked mouse ortholog of Drosophila Fat facets,during gonadal development and oogenesis in mice

During the Drosophila oogenic processes, Fat facets (Faf), an ubiquitin-specific protease essential for normal development of oocyte and eye, becomes localized at the posterior pole and is incorporated into the pole cells. This is dependent on Oskar, a key factor for pole cell determination, and suggests a role for Faf in germ cell differentiation and development. Here we show that Usp9x, an X-linked ortholog of Faf, is predominantly expressed in both germ cell and supporting cell lineages during mouse gonadal development in stage- and sex-dependent manners. Usp9x was first detected in PGCs at 10.5 days post coitum (dpc), and thereafter its expression both at mRNA and protein levels was enhanced in PGCs of both sexes at 11.5-13.5 dpc. In testis, Usp9x expression rapidly decreased to an undetectable level by 15.5 dpc and after birth to adult, no expression was found in any spermatogenic cells, except for weak expression in Sertoli cells. In the ovary, Usp9x expression in embryonic oocytes was also reduced at the newborn stage, its expression reappeared in oocytes at secondary follicle stage, and its products were highly accumulated in the cytoplasm of Graaffian follicles in adults. Although follicular epithelial cells also expressed Usp9x at a moderate level during postnatal development, its expression was downregulated from early secondary follicle stage. Thus, the present study is not only the first to demonstrate a conserved expression of fat facets in PGCs between mouse and fly, but also sex- and stage-dependent changes of a specific component of the deubiquitylation system during mammalian gonadal development.     

Autonomous transition into meiosis of mouse fetal germ cells in vitro and its inhibition by gp130-mediated signaling

Mouse primordial germ cells (PGCs) arrive at the urogenital ridge (UGR) at around 10.5 days postcoitum (dpc). They proliferate until around 13.5 dpc, then enter into meiosis in the female or become mitotically arrested in the male gonads. In this study, meiotic transition of mouse PGCs was examined in vitro. Female PGCs obtained from UGRs or genital ridges at 10.5-11.5 dpc began to express meiosis-specific genes, Scp3 and Dmc1, after dissociation and cultivation on feeder cells for several days. Meiotic transition into the leptotene stage was confirmed by the formation of axial cores. Male PGCs at 10.5-11.5 dpc and migratory PGCs obtained from mesenteries at 10.5 dpc also expressed Scp3 and formed axial cores after several days of culture, supporting the hypothesis that PGCs are capable of entering meiosis before arriving at the UGR. gp130-mediated signaling, known to promote survival/growth of PGCs and also to inhibit the differentiation of embryonic stem cells, suppressed the expression of Scp3 in PGCs and inhibited the following formation of axial cores in vitro. This novel activity of gp130-mediated signaling may provide some clues for the understanding of pluripotency of mammalian germ-line cells and/or the sex differentiation of fetal germ cells.