A subset of megakaryocytes regulates development of hematopoietic stem cell precursors.

Understanding the regulatory mechanisms facilitating hematopoietic stem cell (HSC) specification during embryogenesis is important for the generation of HSCs in vitro. Megakaryocyte emerged from the yolk sac and produce platelets, which are involved in multiple biological processes, such as preventing hemorrhage. However, whether megakaryocytes regulate HSC development in the embryonic aorta-gonad-mesonephros (AGM) region is unclear. Here, we use platelet factor 4 (PF4)-Cre;Rosa-tdTomato+ cells to report presence of megakaryocytes in the HSC developmental niche. Further, we use the PF4-Cre;Rosa-DTA (DTA) depletion model to reveal that megakaryocytes control HSC specification in the mouse embryos. Megakaryocyte deficiency blocks the generation and maturation of pre-HSCs and alters HSC activity at the AGM. Furthermore, megakaryocytes promote endothelial-to-hematopoietic transition in a OP9-DL1 coculture system. Single-cell RNA-sequencing identifies megakaryocytes positive for the cell surface marker CD226 as the subpopulation with highest potential in promoting the hemogenic fate of endothelial cells by secreting TNFSF14. In line, TNFSF14 treatment rescues hematopoietic cell function in megakaryocyte-depleted cocultures. Taken together, megakaryocytes promote production and maturation of pre-HSCs, acting as a critical microenvironmental control factor during embryonic hematopoiesis.

Murine AGM single-cell profiling identifies a continuum of hemogenic endothelium differentiation marked by ACE.

In vitro generation and expansion of hematopoietic stem cells (HSCs) holds great promise for the treatment of any ailment that relies on bone marrow or blood transplantation. To achieve this, it is essential to resolve the molecular and cellular pathways that govern HSC formation in the embryo. HSCs first emerge in the aorta-gonad-mesonephros (AGM) region, where a rare subset of endothelial cells, hemogenic endothelium (HE), undergoes an endothelial-to-hematopoietic transition (EHT). Here, we present full-length single-cell RNA sequencing (scRNA-seq) of the EHT process with a focus on HE and dorsal aorta niche cells. By using Runx1b and Gfi1/1b transgenic reporter mouse models to isolate HE, we uncovered that the pre-HE to HE continuum is specifically marked by angiotensin-I converting enzyme (ACE) expression. We established that HE cells begin to enter the cell cycle near the time of EHT initiation when their morphology still resembles endothelial cells. We further demonstrated that RUNX1 AGM niche cells consist of vascular smooth muscle cells and PDGFRa+ mesenchymal cells and can functionally support hematopoiesis. Overall, our study provides new insights into HE differentiation toward HSC and the role of AGM RUNX1+ niche cells in this process. Our expansive scRNA-seq datasets represents a powerful resource to investigate these processes further.

Recent Advances in Developmental Hematopoiesis: Diving Deeper With New Technologies.

The journey of a hematopoietic stem cell (HSC) involves the passage through successive anatomical sites where HSCs are in direct contact with their surrounding microenvironment, also known as niche. These spatial and temporal cellular interactions throughout development are required for the acquisition of stem cell properties, and for maintaining the HSC pool through balancing self-renewal, quiescence and lineage commitment. Understanding the context and consequences of these interactions will be imperative for our understanding of HSC biology and will lead to the improvement of in vitro production of HSCs for clinical purposes. The aorta-gonad-mesonephros (AGM) region is in this light of particular interest since this is the cradle of HSC emergence during the embryonic development of all vertebrate species. In this review, we will focus on the developmental origin of HSCs and will discuss the novel technological approaches and recent progress made to identify the cellular composition of the HSC supportive niche and the underlying molecular events occurring in the AGM region.

The embryo of the silky shrew opossum, Caenolestes fuliginosus (Tomes, 1863): First description of the embryo of Paucituberculata.

The development of caenolestid marsupials (order Paucituberculata) is virtually unknown. We provide here the first description of Caenolestes fuliginosus embryos collected in the Colombian Central Andes. Our sample of four embryos comes from a single female caught during a fieldtrip at Río Blanco (Manizales, Caldas), in 2014. The sample was processed for macroscopic description using a Standard Event System and for histological descriptions (sectioning and staining). The grade of development of the lumbar flexure and coelomic closure differed between embryos, two of them being more advanced than the others (similar to McCrady's stages 30 and 29, respectively). The pericardial and peritoneal cavities were present, the hepatic anlage was organized in hepatic cords, the heart was in its final position, and the mesonephros was functional. Compared to other Neotropical marsupials, an early appearance of the frontonasal-maxillary fusion and the cervical growth (thickness) was observed; however, absorption of the pharyngeal arches into the body and lung development was delayed. Besides these differences, embryos were similar to equivalent stages in Didelphis virginiana and Monodelphis domestica. Previous proposals of litter size of four for C. fuliginosus are supported.

We, the developing rete testis, efferent ducts, and Wolffian duct, all hereby agree that we need to connect.

BACKGROUND: The mechanisms by which the rete testis joins the efferent ducts, which joins the Wolffian duct during development, are not known. Mouse and chick models have been helpful in identifying genes that are important for the development of each part, but genes have not been identified as to those that play a role in the joining of each part. Clinical implications of the failure of the male reproductive tract to form a fully functional conduit for spermatozoa are not trivial. Epididymal disjunction, the failure of the efferent ducts to join the testis, is one of several epididymal anomalies that have been observed in some boys who were cryptorchid at birth. OBJECTIVE: A systematic review of studies focusing on the morphogenesis of the mesonephric duct and mesonephric tubules in different species, and identification of clinical issues should there be failure of these tissues to develop. DESIGN: PubMed and GUDMAP databases, and review of books on kidney development were searched for studies reporting on the mechanisms of morphogenesis of the kidney and epididymis. MAIN OUTCOMES MEASURE(S): Gaps in our knowledge were identified, and hypotheses coupled with suggestions for future experiments were presented. RESULTS: A total of 64 papers were identified as relevant, of which 53 were original research articles and 11 were book chapters and reviews covering morphogenesis and clinical issues. Investigators utilized multiple species including, human, mouse, chick, Xenopus, bovine, and sheep. CONCLUSION: Fundamental understanding of the morphogenesis of the male reproductive tract is limited, especially the morphogenesis of the rete testis and efferent ducts. Therefore, it is not surprising that we do not understand how each part unites to form a whole. Only one mechanism of joining of one part of the tract to another was identified: the joining of the Wolffian duct to the cloaca via controlled apoptosis.

Morphometric analyses and gene expression related to germ cells, gonadal ridge epithelial-like cells and granulosa cells during development of the bovine fetal ovary.

Cells on the surface of the mesonephros give rise to replicating Gonadal Ridge Epithelial-Like (GREL) cells, the first somatic cells of the gonadal ridge. Later germ cells associate with the GREL cells in the ovigerous cords, and the GREL cells subsequently give rise to the granulosa cells in follicles. To examine these events further, 27 bovine fetal ovaries of different gestational ages were collected and prepared for immunohistochemical localisation of collagen type I and Ki67 to identify regions of the ovary and cell proliferation, respectively. The non-stromal cortical areas (collagen-negative) containing GREL cells and germ cells and later in development, the follicles with oocytes and granulosa cells, were analysed morphometrically. Another set of ovaries (n = 17) were collected and the expression of genes associated with germ cell lineages and GREL/granulosa cells were quantitated by RT-PCR. The total volume of non-stromal areas in the cortex increased significantly and progressively with ovarian development, plateauing at the time the surface epithelium developed. However, the proportion of non-stromal areas in the cortex declined significantly and progressively throughout gestation, largely due to a cessation in growth of the non-stroma cells and the continued growth of stroma. The proliferation index in the non-stromal area was very high initially and then declined substantially at the time follicles formed. Thereafter, it remained low. The numerical density of the non-stromal cells was relatively constant throughout ovarian development. The expression levels of a number of genes across gestation either increased (AMH, FSHR, ESR1, INHBA), declined (CYP19A1, ESR2, ALDH1A1, DSG2, OCT4, LGR5) or showed no particular pattern (CCND2, CTNNB1, DAZL, FOXL2, GATA4, IGFBP3, KRT19, NR5A1, RARRES1, VASA, WNT2B). Many of the genes whose expression changed across gestation, were positively or negatively correlated with each other. The relationships between these genes may reflect their roles in the important events such as the transition of ovigerous cords to follicles, oogonia to oocytes or GREL cells to granulosa cells.

Turning mesoderm into kidney.

The intermediate mesoderm is located between the somites and the lateral plate mesoderm and gives rise to renal progenitors that contribute to the three mammalian kidney types (pronephros, mesonephros and metanephros). In this review, focusing largely on murine kidney development, we examine how the intermediate mesoderm forms during gastrulation/axis elongation and how it progressively gives rise to distinct renal progenitors along the rostro-caudal axis. We highlight some of the potential signalling cues and core transcription factor circuits that direct these processes, up to the point of early metanephric kidney formation.

Development of urinary organs in domestic cat during the embryonic and fetal periods.

The embryonic origin of the urogenital system came from the intermediate mesoderm. Kidney development involves three successive renal systems with a fast chronological overlap: the pronephro, the mesonephro, and the metanephro. Due to the lack of specific knowledge about this system in cats the present work aimed to describe their urinary organs development, focusing on the structures seen in pronephro, mesonephro, and metanephro during the embryonic and fetal stages of development. The techniques used in this study were: light microscopy, immunohistochemistry, scanning electron microscopy, and transmission electron microscopy. For that, embryos and fetuses from 12 pregnant mixed-breed domestic cats in different gestational stages were used to describe the proposed organs. The pronephro is present at early stages of embryonary development in embryos from 15 to 19 days with the presence of pronephro's corpuscles, ducts and tubules. The mesonephro is found, in general, between days 17 and 37, and contains mesonephric ducts, mesonephric tubules, and glomeruli. The metanephro is seen since 21 days of pregnancy with the presence of glomeruli, proximal and distal contorted tubules and at day 37, the cortex-medullary region is already differentiated. The evaluation of these structures enhances the knowledge about embryology of the urinary system in cats, aiding a better anatomical understanding of the system in the specie allowing the correlation with other species.

Analysis of Runx1 Using Induced Gene Ablation Reveals Its Essential Role in Pre-liver HSC Development and Limitations of an In Vivo Approach.

Hematopoietic stem cells (HSCs) develop in the embryonic aorta-gonad-mesonephros (AGM) region and subsequently relocate to fetal liver. Runx1 transcription factor is essential for HSC development, but is largely dispensable for adult HSCs. Here, we studied tamoxifen-inducible Runx1 inactivation in vivo. Induction at pre-liver stages (up to embryonic day 10.5) reduced erythromyeloid progenitor numbers, but surprisingly did not block the appearance of Runx1-null HSCs in liver. By contrast, ex vivo analysis showed an absolute Runx1 dependency of HSC development in the AGM region. We found that, contrary to current beliefs, significant Cre-inducing tamoxifen activity persists in mouse blood for at least 72 hr after injection. This deferred recombination can hit healthy HSCs, which escaped early Runx1 ablation and result in appearance of Runx1-null HSCs in liver. Such extended recombination activity in vivo is a potential source of misinterpretation, particularly in analysis of dynamic developmental processes during embryogenesis.

Searching for a Cell-Based Therapeutic Tool for Haemophilia A within the Embryonic/Foetal Liver and the Aorta-Gonads-Mesonephros Region.

The development of new strategies based on cell therapy approaches to correct haemophilia A (HA) requires further insights into new cell populations capable of producing coagulation factor VIII (FVIII) and presenting stable engraftment potential. The major producers of FVIII in the adult are liver sinusoidal endothelial cells (LSECs) and in a lesser degree bone marrow-derived cells, both of which have been shown to ameliorate the bleeding phenotype in adult HA mice after transplantation. We have previously shown that cells from the foetal liver (FL) and the aorta-gonads-mesonephros (AGM) haematopoietic locations possess higher LSEC engraftment potential in newborn mice compared with adult-derived LSECs, constituting likely therapeutic targets for the treatment of HA in neonates. However, less is known about the production of FVIII in embryonic locations. Quantitative polymerase chain reaction and Western blot analysis were performed to assess the relative level of FVIII production in different embryonic tissues and at various developmental stages, identifying the FL and AGM region from day 12 (E12) as prominent sources of FVIII. Furthermore, FL-derived VE-cad+CD45-Lyve1+/- endothelial/endothelial progenitor cells, presenting vascular engraftment potential, produced high levels of F8 ribonucleic acid compared with CD45+ blood progenitors or Dlk1+ hepatoblasts. In addition, we show that the E11 AGM explant cultures expanded cells with LSEC repopulation activity, instrumental to further understand signals for in vitro generation of LSECs. Taking into account the capacity for FVIII expression, culture expansion and newborn engraftment potential, these results support the use of cells with foetal characteristics for correction of FVIII deficiency in young individuals.

Adult zebrafish Langerhans cells arise from hematopoietic stem/progenitor cells.

The origin of Langerhans cells (LCs), which are skin epidermis-resident macrophages, remains unclear. Current lineage tracing of LCs largely relies on the promoter-Cre-LoxP system, which often gives rise to contradictory conclusions with different promoters. Thus, reinvestigation with an improved tracing method is necessary. Here, using a laser-mediated temporal-spatial resolved cell labeling method, we demonstrated that most adult LCs originated from the ventral wall of the dorsal aorta (VDA), an equivalent to the mouse aorta, gonads, and mesonephros (AGM), where both hematopoietic stem cells (HSCs) and non-HSC progenitors are generated. Further fine-fate mapping analysis revealed that the appearance of LCs in adult zebrafish was correlated with the development of HSCs, but not T cell progenitors. Finally, we showed that the appearance of tissue-resident macrophages in the brain, liver, heart, and gut of adult zebrafish was also correlated with HSCs. Thus, the results of our study challenged the EMP-origin theory for LCs.

Characterization of migratory primordial germ cells in the aorta-gonad-mesonephros of a 4.5-week-old human embryo: a toolbox to evaluate in vitro early gametogenesis.

STUDY QUESTION: Which set of antibodies can be used to identify migratory and early post-migratory human primordial germ cells (hPGCs)? STUDY FINDING: We validated the specificity of 33 antibodies for 31 markers, including POU5F1, NANOG, PRDM1 and TFAP2C as specific markers of hPGCs at 4.5 weeks of development of Carnegie stage (CS12-13), whereas KIT and SOX17 also marked the intra-aortic hematopoietic stem cell cluster in the aorta-gonad-mesonephros (AGM). WHAT IS KNOWN ALREADY: The dynamics of gene expression during germ cell development in mice is well characterized and this knowledge has proved crucial to allow the development of protocols for the in vitro derivation of functional gametes. Although there is a great interest in generating human gametes in vitro, it is still unclear which markers are expressed during the early stages of hPGC development and many studies use markers described in mouse to benchmark differentiation of human PGC-like cells (hPGCLCs). Early post-implantation development differs significantly between mice and humans, and so some germ cells markers, including SOX2, SOX17, IFITM3 and ITGA6 may not identify mPGCs and hPGCs equally well. STUDY DESIGN, SIZE, DURATION: This immunofluorescence study investigated the expression of putative hPGC markers in the caudal part of a single human embryo at 4.5 weeks of development. PARTICIPANTS/MATERIALS, SETTING, METHODS: We have investigated by immunofluorescence the expression of a set of 33 antibodies for 31 markers, including pluripotency, germ cell, adhesion, migration, surface, mesenchymal and epigenetic markers on paraffin sections of the caudal part, including the AGM region, of a single human embryo (CS12-13). The human material used was anonymously donated with informed consent from elective abortions without medical indication. MAIN RESULTS AND THE ROLE OF CHANCE: We observed germ cell specific expression of NANOG, TFAP2C and PRDM1 in POU5F1+ hPGCs in the AGM. The epigenetic markers H3K27me3 and 5mC were sufficient to distinguish hPGCs from the surrounding somatic cells. Some mPGC-markers were not detected in hPGCs, but marked other tissues; whereas other markers, such as ALPL, SOX17, KIT, TUBB3, ITGA6 marked both POU5F1+ hPGCs and other cells in the AGM. We used a combination of multiple markers, immunostaining different cellular compartments when feasible, to decrease the chance of misidentifying hPGCs. LARGE SCALE DATA: Non-applicable. LIMITATIONS REASONS FOR CAUTION: Material to study early human development is unique and very rare thus restricting the sample size. We have used a combination of antibodies limited by the number of paraffin sections available. WIDER IMPLICATIONS OF THE FINDINGS: Most of our knowledge on early gametogenesis has been obtained from model organisms such as mice and is extrapolated to humans. However, since there is a dedicated effort to produce human artificial gametes in vitro, it is of great importance to determine the expression and specificity of human-specific germ cell markers. We provide a systematic analysis of the expression of 31 different markers in paraffin sections of a CS12-13 embryo. Our results will help to set up a toolbox of markers to evaluate protocols to induce hPGCLCs in vitro. STUDY FUNDING AND COMPETING INTEREST(S): M.G.F. was funded by Fundação para a Ciência e Tecnologia (FCT) [SFRH/BD/78689/2011] and S.M.C.S.L. was funded by the Interuniversity Attraction Poles (IAP, P7/07) and the European Research Council Consolidator (ERC-CoG-725722-OVOGROWTH). The authors declare no conflict of interest.

The hematopoietic stem cell niche: from embryo to adult.

Hematopoietic stem cells (HSCs) develop in discrete anatomical niches, migrating during embryogenesis from the aorta-gonad-mesonephros (AGM) region to the fetal liver, and finally to the bone marrow, where most HSCs reside throughout adult life. These niches provide supportive microenvironments that specify, expand and maintain HSCs. Understanding the constituents and molecular regulation of HSC niches is of considerable importance as it could shed new light on the mechanistic principles of HSC emergence and maintenance, and provide novel strategies for regenerative medicine. However, controversy exists concerning the cellular complexity of the bone marrow niche, and our understanding of the different HSC niches during development remains limited. In this Review, we summarize and discuss what is known about the heterogeneity of the HSC niches at distinct stages of their ontogeny, from the embryo to the adult bone marrow, drawing predominantly on data from mouse studies.

A molecular roadmap of the AGM region reveals BMPER as a novel regulator of HSC maturation.

In the developing embryo, hematopoietic stem cells (HSCs) emerge from the aorta-gonad-mesonephros (AGM) region, but the molecular regulation of this process is poorly understood. Recently, the progression from E9.5 to E10.5 and polarity along the dorso-ventral axis have been identified as clear demarcations of the supportive HSC niche. To identify novel secreted regulators of HSC maturation, we performed RNA sequencing over these spatiotemporal transitions in the AGM region and supportive OP9 cell line. Screening several proteins through an ex vivo reaggregate culture system, we identify BMPER as a novel positive regulator of HSC development. We demonstrate that BMPER is associated with BMP signaling inhibition, but is transcriptionally induced by BMP4, suggesting that BMPER contributes to the precise control of BMP activity within the AGM region, enabling the maturation of HSCs within a BMP-negative environment. These findings and the availability of our transcriptional data through an accessible interface should provide insight into the maintenance and potential derivation of HSCs in culture.

Effects of Cyclosporine A on the Development of Metanephros in the Pregnant BALB/c Mice.

BACKGROUND:: Cyclosporine A (CsA) is a commonly used clinical immunosuppressant. However, CsA exposure in rabbits during the gestation period was shown to cause a postnatal decrease in the number of nephrons, with the effects remaining unknown. In this study, we aimed to explore the effects of CsA on metanephros development in the pregnant BALB/c mice. METHODS:: Pregnant mice were randomly divided into two groups, and CsA (10 mg·kg-1·d-1) was subcutaneously injected from gestation day 10.5 to day 16.5 in the CsA group, whereas a comparable volume of normal saline was given to the control group. All of the mice were sacrificed on gestation day 17.5 and serum CsA concentration was measured. The fetuses were removed and weighed, and their kidneys were prepared for histological assessment and polymerase chain reaction assay. In an in vitro experiment, embryo kidneys of fetal mice on gestation day 12.5 were used, and CsA (10 µmol/L) was added in the culture of the CsA group. The growth pattern of the ureteric bud and nephrons was assessed by lectin staining. RESULTS:: No significant differences in the weight of embryo (4.54 ± 1.22 vs. 3.26 ± 1.09 mg) were observed between the CsA and control groups, the thickness of the cortical (510.0 ± 30.3 vs. 350.0 ± 29.7 µm, P < 0.05) and nephrogenic zone (272.5 ± 17.2 vs. 173.3 ± 24.0 µm, P < 0.05), and the number of glomeruli (36.5 ± 0.7 vs. 27.5 ± 2.1, P < 0.05) were reduced in the CsA group when compared to the control group. The cell proliferation of Ki-67 positive index between control and CsA group (307.0 ± 20.0 vs. 219.0 ± 25.0, P < 0.05) in the nephrogenic zone was decreased with the increase of apoptotic cells (17.0 ± 2.0 vs. 159.0 ± 33.0, P < 0.05). The mRNA expression of WT-1, Pax2, and Pax8 was downregulated by CsA treatment. As for the in vitro CsA group, the branch number of the ureteric bud was decreased in the CsA-treated group with the nephrons missing in contrast to control after the incubation for 24 h and 72 h (all P < 0.0001). CONCLUSION:: Treatment of CsA suppressed metanephros development in the pregnant mice; however, the potential action of mechanism needs to be further investigated.

The first wave of T lymphopoiesis in zebrafish arises from aorta endothelium independent of hematopoietic stem cells.

T lymphocytes are key cellular components of the adaptive immune system and play a central role in cell-mediated immunity in vertebrates. Despite their heterogeneities, it is believed that all different types of T lymphocytes are generated exclusively via the differentiation of hematopoietic stem cells (HSCs). Using temporal-spatial resolved fate-mapping analysis and time-lapse imaging, here we show that the ventral endothelium in the zebrafish aorta-gonad-mesonephros and posterior blood island, the hematopoietic tissues previously known to generate HSCs and erythromyeloid progenitors, respectively, gives rise to a transient wave of T lymphopoiesis independent of HSCs. This HSC-independent T lymphopoiesis occurs early and generates predominantly CD4 Tαß cells in the larval but not juvenile and adult stages, whereas HSC-dependent T lymphopoiesis emerges late and produces various subtypes of T lymphocytes continuously from the larval stage to adulthood. Our study unveils the existence, origin, and ontogeny of HSC-independent T lymphopoiesis in vivo and reveals the complexity of the endothelial-hematopoietic transition of the aorta.

LIM kinase function and renal growth: Potential role for LIM kinases in fetal programming of kidney development.

AIMS: Maternal dietary restriction during pregnancy impairs nephron development and results in offspring with fewer nephrons. Cell turnover in the early developing kidney is altered by exposure to maternal dietary restriction and may be regulated by the LIM-kinase family of enzymes. We set out to establish whether disturbance of LIM-kinase activity might play a role in the impairment of nephron formation. MAIN METHODS: E12.5 metanephric kidneys and HK2 cells were grown in culture with the pharmacological LIM-kinase inhibitor BMS5. Organs were injected with DiI, imaged and cell numbers measured over 48h to assess growth. Cells undergoing mitosis were visualised by pH3 labelling. KEY FINDINGS: Growth of cultured kidneys reduced to 83% of controls after exposure to BMS5 and final cell number to 25% of control levels after 48h. Whilst control and BMS5 treated organs showed cells undergoing mitosis (100±11 cells/field vs 113±18 cells/field respectively) the proportion in anaphase was considerably diminished with BMS5 treatment (7.8±0.8% vs 0.8±0.6% respectively; P<0.01). This was consistent with effects on HK2 cells highlighting a severe impact of BMS5 on formation of the mitotic spindle and centriole positioning. DiI labelled cells migrated in 100% of control cultures vs 0% BMS5 treated organs. The number of nephrogenic precursor cells appeared depleted in whole organs and formation of new nephrons was blocked by exposure to BMS5. SIGNIFICANCE: Pharmacological blockade of LIM-kinase function in the early developing kidney results in failure of renal development. This is likely due to prevention of dividing cells from completion of mitosis with their resultant loss.

Retinoic acid signaling is dispensable for somatic development and function in the mammalian ovary.

Retinoic acid (RA) is a potent inducer of cell differentiation and plays an essential role in sex-specific germ cell development in the mammalian gonad. RA is essential for male gametogenesis and hence fertility. However, RA can also disrupt sexual cell fate in somatic cells of the testis, promoting transdifferentiation of male Sertoli cells to female granulosa-like cells when the male sexual regulator Dmrt1 is absent. The feminizing ability of RA in the Dmrt1 mutant somatic testis suggests that RA might normally play a role in somatic cell differentiation or cell fate maintenance in the ovary. To test for this possibility we disrupted RA signaling in somatic cells of the early fetal ovary using three genetic strategies and one pharmaceutical approach. We found that deleting all three RA receptors (RARs) in the XX somatic gonad at the time of sex determination did not significantly affect ovarian differentiation, follicle development, or female fertility. Transcriptome analysis of adult triple mutant ovaries revealed remarkably little effect on gene expression in the absence of somatic RAR function. Likewise, deletion of three RA synthesis enzymes (Aldh1a1-3) at the time of sex determination did not masculinize the ovary. A dominant-negative RAR transgene altered granulosa cell proliferation, likely due to interference with a non-RA signaling pathway, but did not prevent granulosa cell specification and oogenesis or abolish fertility. Finally, culture of fetal XX gonads with an RAR antagonist blocked germ cell meiotic initiation but did not disrupt sex-biased gene expression. We conclude that RA signaling, although crucial in the ovary for meiotic initiation, is not required for granulosa cell specification, differentiation, or reproductive function.

Differentiation of human embryonic stem cells to HOXA+ hemogenic vasculature that resembles the aorta-gonad-mesonephros.

The ability to generate hematopoietic stem cells from human pluripotent cells would enable many biomedical applications. We find that hematopoietic CD34+ cells in spin embryoid bodies derived from human embryonic stem cells (hESCs) lack HOXA expression compared with repopulation-competent human cord blood CD34+ cells, indicating incorrect mesoderm patterning. Using reporter hESC lines to track the endothelial (SOX17) to hematopoietic (RUNX1C) transition that occurs in development, we show that simultaneous modulation of WNT and ACTIVIN signaling yields CD34+ hematopoietic cells with HOXA expression that more closely resembles that of cord blood. The cultures generate a network of aorta-like SOX17+ vessels from which RUNX1C+ blood cells emerge, similar to hematopoiesis in the aorta-gonad-mesonephros (AGM). Nascent CD34+ hematopoietic cells and corresponding cells sorted from human AGM show similar expression of cell surface receptors, signaling molecules and transcription factors. Our findings provide an approach to mimic in vitro a key early stage in human hematopoiesis for the generation of AGM-derived hematopoietic lineages from hESCs.