Progressive divergence of definitive haematopoietic stem cells from the endothelial compartment does not depend on contact with the foetal liver

The yolk sac and the para-aortic splanchnopleura/aorta-genital ridges-mesonephros (P-Sp/AGM) region are the main sites of haematopoietic activity in the mouse embryo at the pre-liver stage of development. By day 11.5 of gestation, the AGM region is capable of autonomous initiation and expansion of definitive haematopoietic stem cells (HSCs). By day 12.5, HSC activity in the AGM region is reduced whilst a second wave of HSCs begins to emerge in the yolk sac. We show here that HSCs emerging in both locations are marked by co-expression of the endothelial-specific marker VE-cadherin and the pan-leukocyte antigen CD45. Phenotypic characterisation using CD31, TIE2, FLK1, Ac-LDL receptors, and CD34 markers demonstrated significant similarities between this VE-cadherin+CD45+ ;double-positive' population and endothelial cells suggesting a common origin for these cells. The double-positive fraction also expressed the stem cell markers Kit, Sca1 and AA4.1. Long-term transplantation experiments demonstrated that the double-positive population, which constituted less than 0.05% of the day 11.5 AGM region and the day 12.5 yolk sac, is highly enriched for HSCs. In vitro assays showed that this population is also enriched for myeloid progenitors. During foetal liver colonization, circulating HSCs remained within the VE-cadherin+ cell fraction, although their phenotypic similarity with endothelial cells became less prominent. Upon liver colonisation the majority of HSCs downregulated VE-cadherin, expression of which was completely lost in the adult bone marrow. Partial loss of VE-cadherin expression in HSCs can be observed extra hepatically in the advanced AGM region by E12.5. Similarly, the CD34+KIT+ population in the placenta, recently identified as a reservoir of HSCs, partly lose VE-cadherin expression by E12.5. By culturing isolated E11.5 AGM region and E12.5 yolk sac we show that the developmental switch from a ;primary' VE-cadherin+CD45+ to a more ;advanced' VE-cadherin-CD45+ phenotype does not require contact of HSCs with the liver and is probably a function of developmental time.     

Hematopoiesis-dependent expression of CD44 in murine hepatic progenitor cells

The fetal liver serves as the predominant hematopoietic organ until birth. However, the mechanisms underlying this link between hematopoiesis and hepatogenesis are unclear. Previously, we reported the isolation of a monoclonal antibody (anti-Liv8) that specifically recognizes an antigen (Liv8) present in murine fetal livers at embryonic day 11.5 (E11.5). Liv8 is a cell surface molecule expressed by hematopoietic cells in both fetal liver and adult mouse bone marrow. Here, we report that Liv8 is also transiently expressed by hepatoblasts at E11.5. Using protein purification and mass spectrometry, we have identified Liv8 as the CD44 protein. Interestingly, the expression of Liv8/CD44 in fetal liver was completely lost in AML1^−/− murine embryos, which lack definitive hematopoiesis. These results show that hepatoblasts change from Liv8/CD44-negative to Liv8/CD44-positive status in a hematopoiesis-dependent manner by E11.5, and indicate that Liv8/CD44 expression is an important link between hematopoiesis and hepatogenesis during fetal liver development.     

Estrogen dynamics in the female rhesus monkey

The metabolic clearance rates (MCR) and interconversions [( rho]BB) values for estrone (E1) and estradiol (E2) in female rhesus (Macaca mulatta) monkeys on Days 9, 14, and 23 of the menstrual cycle were measured using constant infusions of [3H] estradiol and [14C] estrone. The menstrual cycles in these monkeys were reproduced by using Silastic capsules of E2 and progesterone after bilateral ovariectomy. The serum levels of E2 and progesterone were measured by radioimmunoassay and were similar to those for the intact menstrual cycle. The MCR of E2 on Day 14 (52.8 +/- 6.8 l/day/kg) was significantly greater (p less than 0.05) than that measured on Day 9 (31.1 +/- 3.6 l/day/kg) or Day 23 (35.4 +/- 2.1 l/day/kg). The MCR of E1 was also different (p less than 0.05) on Day 14 (77.6 +/- 14.9 l/day/kg) compared to the values on Days 9 and 23 (50.2 +/- 4.9 and 48.2 +/- 3.9 l/day/kg, respectively. There was no change in percentage of free E2, percentage of albumin-bound E2, or sex hormone-binding globulin levels on those 3 days of the cycle. The interconversions between E2 and E1 were not influenced by the day of the cycle. We conclude that the high levels of E2 occurring at the time of the E2 peak result in increases in the MCRs of both E2 and E1 that are not associated with changes in the pattern of protein-binding or in the activity of the 17 beta-hydroxy steroid dehydrogenase.     

The thyroid hormone receptor-associated protein TRAP220 is required at distinct embryonic stages in placental, cardiac, and hepatic development

Recent work indicates that thyroid hormone receptor-associated protein 220 (TRAP220), a subunit of the multiprotein TRAP coactivator complex, is essential for embryonic survival. We have generated TRAP220 conditional null mice that are hypomorphic and express the gene at reduced levels. In contrast to TRAP220 null mice, which die at embryonic d 11.5 (E11.5), hypomorphic mice survive until E13.5. The reduced expression in hypomorphs results in hepatic necrosis, defects in hematopoiesis, and hypoplasia of the ventricular myocardium, similar to that observed in TRAP220 null embryos at an earlier stage. The embryonic lethality of null embryos at E11.5 is due to placental insufficiency. Tetraploid aggregation assays partially rescues embryonic development until E13.5, when embryonic loss occurs due to hepatic necrosis coupled with poor myocardial development as observed in hypomorphs. These findings demonstrate that, for normal placental function, there is an absolute requirement for TRAP220 in extraembryonic tissues at E11.5, with an additional requirement in embryonic tissues for hepatic and cardiovascular development thereafter.     

Retinoic acid, hypoxia, and GATA factors cooperatively control the onset of fetal liver erythropoietin expression and erythropoietic differentiation

The cytokine erythropoietin (Epo) is an essential factor promoting the survival, proliferation, and differentiation of erythroid progenitor cells. Epo expression and the initial phase of definitive erythropoietic differentiation in the fetal liver (E9-E12) are compromised in mouse embryos lacking the retinoic acid receptor RXRalpha. Our previous work demonstrated that the Epo gene is a direct target of retinoic acid action, via a retinoic acid receptor binding site in the Epo gene enhancer. However, Epo expression and erythropoietic differentiation become normalized in RXRalpha mutants from E12. In this study, we have investigated the molecular mechanisms underlying the transition in Epo gene regulation from RXRalpha-dependence to RXRalpha-independence. We find that three independent regulatory components are required for high level Epo expression in the early fetal liver: ligand-activated retinoic acid receptors, the hypoxia-regulated factor HIF1, and GATA factors. By E11.5, the fetal liver is no longer hypoxic, and retinoic acid signaling is no longer active; Epo expression from E11.5 onward is enhancer-independent, and is driven instead by basal promoter elements that provide a sufficient level of expression to support further erythropoietic differentiation.     

Purkinje cell fate in staggerer mutants: agenesis versus cell death

Staggerer (sg/sg) is an autosomal recessive mutation in an orphan nuclear hormone receptor gene, RORalpha, that causes a cell-autonomous, lineage-specific block in the development of the Purkinje cell. Purkinje cell number is reduced by about 75-90% in adult mutants, and many of the surviving cells are small and ectopically positioned. To determine whether Purkinje cell numbers are reduced owing to either agenesis or cell death, cohorts of Purkinje cells were labeled with the birth-date marker bromodeoxyuridine (BrdU) at embryonic day (E) 10.5 or E11.5. The total number of BrdU-labeled profiles was then compared between cerebella from wild-type mice, heterozygous staggerer, and staggerer mutants at E17.5 and postnatal day (P)5. There was no significant difference between sg/sg mutants and +/sg or +/+ controls in the number of BrdU-labeled profiles or in cerebellar volumes in the E17 embryos. By P5, however, cerebellar volume was significantly reduced in the sg/sg mutants compared to controls (p <.005) and the number of BrdU-labeled profiles was reduced by 33% following E11.5 BrdU injections (p <.02). The results suggest that Purkinje cell genesis is not affected by the staggerer mutation and that Purkinje cell loss begins some time after E17. RORalpha is highly expressed in Purkinje cells by E14, so the delay between initial RORalpha expression and sg/sg Purkinje cell loss suggests that the staggerer mutation does not directly cause Purkinje cell death.     

Identification of Differentially Expressed Proteins in Murine Embryonic and Postnatal Cortical Neural Progenitors

Background

The central nervous system (CNS) develops from a heterogeneous pool of neural stem and progenitor cells (NSPC), the underlying differences among which are poorly understood. The study of NSPC would be greatly facilitated by the identification of additional proteins that mediate their function and that would distinguish amongst different progenitor populations.

Methodology/Principal Findings

To identify membrane and membrane-associated proteins expressed by NSPC, we used a proteomics approach to profile NSPC cultured as neurospheres (NS) isolated from the murine cortex during a period of neurogenesis (embryonic day 11.5, E11.5), as compared to NSPC isolated at a peak of gliogenesis (postnatal day 1, P0) and to differentiated E11.5 NS. 54 proteins were identified with high expression in E11.5 NS, including the TrkC receptor, several heterotrimeric G proteins, and the Neogenin receptor. 24 proteins were identified with similar expression in E11.5 and P0 NS over differentiated E11.5 NS, and 13 proteins were identified with high expression specifically in P0 NS compared to E11.5 NS. To illustrate the potential relevance of these identified proteins to neural stem cell biology, the function of Neogenin was further studied. Using Fluorescence Activated Cell Sorting (FACS) analysis, expression of Neogenin was associated with a self-renewing population present in both E11.5 and adult subventricular zone (SVZ) NS but not in P0 NS. E11.5 NS expressed a putative Neogenin ligand, RGMa, and underwent apoptosis when exposed to a ligand-blocking antibody.

Conclusions/Significance

There are fundamental differences between the continuously self-renewing and more limited progenitors of the developing cortex. We identified a subset of differentially expressed proteins that serve not only as a set of functionally important proteins, but as a useful set of markers for the subsequent analysis of NSPC. Neogenin is associated with the continuously self-renewing and neurogenic cells present in E11.5 cortical and adult SVZ NS, and the Neogenin/RGMa receptor/ligand pair may regulate cell survival during development.     

Murine FGF-4 gene expression is spatially restricted within embryonic skeletal muscle and other tissues

Fibroblast growth factors are believed to play many distinct roles in vertebrate development, owing to their ability to stimulate cell growth, prevent cell death, determine cell fate, and inhibit terminal differentiation in a variety of in vitro culture systems. We have used in situ hybridization to localize fibroblast growth factor-4 (FGF-4, also termed HST and K-FGF) gene expression in 7.5 to 16.5 day gestation mouse embryos. Seven discrete sites of gene expression were detected: (1) primitive streak (E7.5–8.5); (2) paraxial presomitic mesoderm in the trunk (E7.5–11.5); (3) primitive neuroectoderm (E8.0–8.5); (4) pharyngeal pouch endoderm (E8.5–9.5); (5) branchial arch ectoderm (E8.5–9.5); (6) limb apical ectoderm (E10.5–12.5), and (7) skeletal myoblast groups (E9.5–13.5). FGF-4 gene expression is spatially restricted within many of these sites. The profile of FGF-4 gene expression among skeletal muscle groups is overlapping, but distinct, from that of FGF-5, thereby revealing myoblast heterogeneity at the molecular level and suggesting distinct roles for multiple FGFs in muscle development.     

Expression of Cre recombinase in early diploid trophoblast cells of the mouse placenta

An increasing number of genes known to be critical for cell cycle control, differentiation, and tumor suppression have been found to impact development of the placenta. To elucidate how these genes contribute to development of embryonic and extra-embryonic lineages, we generated a transgenic mouse in which the Cre transgene is driven by placenta-specific regulatory sequences from the human CYP19 gene. Using ROSA26 conditional reporter mice, we could detect expression of the CYP19-Cre transgene throughout the extra-embryonic ectoderm and in the ectoplacental cone at embryonic day 6.5 (E6.5). By E11.5, recombination of LoxP reporter sites was detected in all derivatives of trophoblast stem cells, including spongiotrophoblast, giant cells, and labyrinth trophoblasts. We conclude that the CYP19-Cre transgenic mouse developed here can be used in combination with conditional alleles to distinguish between embryonic and extra-embryonic gene function, and to begin to map the period of time when gene function is critical during development.     

A novel putative transmembrane protein, IZP6, is expressed in neural cells during embryogenesis

Gene trapping in mouse embryonic stem cells is an efficient method for identifying new genes and examining their functions. This method has been used in an effort to identify some novel genes involved in mouse development. In the present paper, one such gene named IZP6 is reported. Expression of the IZP6 gene, as monitored by beta-galactosidase expression in heterozygous mice, was detected in a developmentally regulated fashion: the expression pattern has two phases during the embryogenesis. In the first phase, from embryonic day 11.5 (E11.5) until E14.5, the reporter gene is mainly expressed in the forebrain. In the second phase, from E15.5 until birth, expression in the forebrain becomes weaker but is still observed in the olfactory bulb and the skin around the eyes, nose, limbs and tail. Thus, IZP6 gene expression changes from the central nervous system (the first phase) to the peripheral tissues (the second phase) during development. The IZP6 gene encodes a protein of 228 amino acids. Analysis of the secondary structure of the IZP6 protein revealed four hydrophobic regions, indicating that the IZP6 protein is a four transmembrane region protein. These results suggest that IZP6 is a transmembrane protein related to neurogenesis in the mouse.