Kit ligand has a critical role in mouse yolk sac and aorta-gonad-mesonephros hematopoiesis.

Few studies report on the in vivo requirement for hematopoietic niche factors in the mammalian embryo. Here, we comprehensively analyze the requirement for Kit ligand (Kitl) in the yolk sac and aorta-gonad-mesonephros (AGM) niche. In-depth analysis of loss-of-function and transgenic reporter mouse models show that Kitl-deficient embryos harbor decreased numbers of yolk sac erythro-myeloid progenitor (EMP) cells, resulting from a proliferation defect following their initial emergence. This EMP defect causes a dramatic decrease in fetal liver erythroid cells prior to the onset of hematopoietic stem cell (HSC)-derived erythropoiesis, and a reduction in tissue-resident macrophages. Pre-HSCs in the AGM require Kitl for survival and maturation, but not proliferation. Although Kitl is expressed widely in all embryonic hematopoietic niches, conditional deletion in endothelial cells recapitulates germline loss-of-function phenotypes in AGM and yolk sac, with phenotypic HSCs but not EMPs remaining dependent on endothelial Kitl upon migration to the fetal liver. In conclusion, our data establish Kitl as a critical regulator in the in vivoAGM and yolk sac endothelial niche.

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.

Distinct regulatory networks control the development of macrophages of different origins in zebrafish.

Macrophages are key components of the innate immune system and play pivotal roles in immune response, organ development, and tissue homeostasis. Studies in mice and zebrafish have shown that tissue-resident macrophages derived from different hematopoietic origins manifest distinct developmental kinetics and colonization potential, yet the genetic programs controlling the development of macrophages of different origins remain incompletely defined. In this study, we use zebrafish, where tissue-resident macrophages arise from the rostral blood island (RBI) and ventral wall of dorsal aorta (VDA), the zebrafish hematopoietic tissue equivalents to the mouse yolk sac and aorta-gonad-mesonephros for myelopoiesis, to address this issue. We show that RBI- and VDA-born macrophages are orchestrated by distinctive regulatory networks formed by the E-twenty-six (Ets) transcription factors Pu.1 and Spi-b, the zebrafish ortholog of mouse spleen focus forming virus proviral integration oncogene B (SPI-B), and the helix-turn-helix DNA-binding domain containing protein Irf8. Epistatic studies document that during RBI macrophage development, Pu.1 acts upstream of Spi-b, which, upon induction by Pu.1, partially compensates the function of Pu.1. In contrast, Pu.1 and Spi-b act in parallel and cooperatively to regulate the development of VDA-derived macrophages. Interestingly, these two distinct regulatory networks orchestrate the RBI- and VDA-born macrophage development largely by regulating a common downstream gene, Irf8. Our study indicates that macrophages derived from different origins are governed by distinct genetic networks formed by the same repertoire of myeloid-specific transcription factors.

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.

Aristaless related homeobox gene, Arx, is implicated in mouse fetal Leydig cell differentiation possibly through expressing in the progenitor cells.

Development of the testis begins with the expression of the SRY gene in pre-Sertoli cells. Soon after, testis cords containing Sertoli and germ cells are formed and fetal Leydig cells subsequently develop in the interstitial space. Studies using knockout mice have indicated that multiple genes encoding growth factors and transcription factors are implicated in fetal Leydig cell differentiation. Previously, we demonstrated that the Arx gene is implicated in this process. However, how ARX regulates Leydig cell differentiation remained unknown. In this study, we examined Arx KO testes and revealed that fetal Leydig cell numbers largely decrease throughout the fetal life. Since our study shows that fetal Leydig cells rarely proliferate, this decrease in the KO testes is thought to be due to defects of fetal Leydig progenitor cells. In sexually indifferent fetal gonads of wild type, ARX was expressed in the coelomic epithelial cells and cells underneath the epithelium as well as cells at the gonad-mesonephros border, both of which have been described to contain progenitors of fetal Leydig cells. After testis differentiation, ARX was expressed in a large population of the interstitial cells but not in fetal Leydig cells, raising the possibility that ARX-positive cells contain fetal Leydig progenitor cells. When examining marker gene expression, we observed cells as if they were differentiating into fetal Leydig cells from the progenitor cells. Based on these results, we propose that ARX acts as a positive factor for differentiation of fetal Leydig cells through functioning at the progenitor stage.

Hes repressors are essential regulators of hematopoietic stem cell development downstream of Notch signaling.

Previous studies have identified Notch as a key regulator of hematopoietic stem cell (HSC) development, but the underlying downstream mechanisms remain unknown. The Notch target Hes1 is widely expressed in the aortic endothelium and hematopoietic clusters, though Hes1-deficient mice show no overt hematopoietic abnormalities. We now demonstrate that Hes is required for the development of HSC in the mouse embryo, a function previously undetected as the result of functional compensation by de novo expression of Hes5 in the aorta/gonad/mesonephros (AGM) region of Hes1 mutants. Analysis of embryos deficient for Hes1 and Hes5 reveals an intact arterial program with overproduction of nonfunctional hematopoietic precursors and total absence of HSC activity. These alterations were associated with increased expression of the hematopoietic regulators Runx1, c-myb, and the previously identified Notch target Gata2. By analyzing the Gata2 locus, we have identified functional RBPJ-binding sites, which mutation results in loss of Gata2 reporter expression in transgenic embryos, and functional Hes-binding sites, which mutation leads to specific Gata2 up-regulation in the hematopoietic precursors. Together, our findings show that Notch activation in the AGM triggers Gata2 and Hes1 transcription, and next HES-1 protein represses Gata2, creating an incoherent feed-forward loop required to restrict Gata2 expression in the emerging HSCs.

Hematopoietic stem cell development requires transient Wnt/ß-catenin activity.

Understanding how hematopoietic stem cells (HSCs) are generated and the signals that control this process is a crucial issue for regenerative medicine applications that require in vitro production of HSC. HSCs emerge during embryonic life from an endothelial-like cell population that resides in the aorta-gonad-mesonephros (AGM) region. We show here that ß-catenin is nuclear and active in few endothelial nonhematopoietic cells closely associated with the emerging hematopoietic clusters of the embryonic aorta during mouse development. Importantly, Wnt/ß-catenin activity is transiently required in the AGM to generate long-term HSCs and to produce hematopoietic cells in vitro from AGM endothelial precursors. Genetic deletion of ß-catenin from the embryonic endothelium stage (using VE-cadherin-Cre recombinase), but not from embryonic hematopoietic cells (using Vav1-Cre), precludes progression of mutant cells toward the hematopoietic lineage; however, these mutant cells still contribute to the adult endothelium. Together, those findings indicate that Wnt/ß-catenin activity is needed for the emergence but not the maintenance of HSCs in mouse embryos.

Live imaging of the developing mouse mesonephros.

Embryonic development is a highly dynamic process involving complex tissue interactions and movements. Recent progress in cell labeling, image acquisition, and image processing technologies has brought the study of embryo morphogenesis to another level. It is now possible to visualize in real time the dynamic morphogenetic changes occurring in vivo and to reconstitute and quantify them in 4D rendering. However, extended live embryo imaging remains challenging in terms of embryo survival and minimization of phototoxicity. Here, we describe a procedure to image the developing mesonephros for up to 16 h in intact mouse embryos. This method can easily be adapted to the imaging of other structures at similar developmental stages.

Potential contributions of heat shock proteins to temperature-dependent sex determination in the American alligator.

Sex determination in the American alligator depends on the incubation temperature experienced during a thermo-sensitive period (TSP), although sex determination can be 'reversed' by embryonic exposure to an estrogenic compound. Thus, temperature and estrogenic signals play essential roles during temperature-dependent sex determination (TSD). The genetic basis for TSD is poorly understood, although previous studies observed that many of the genes associated with genetic sex determination (GSD) are expressed in species with TSD. Heat shock proteins (HSPs), good candidates because of their temperature-sensitive expression, have not been examined in regard to TSD but HSPs have the ability to modify steroid receptor function. A number of HSP cDNAs (HSP27, DNAJ, HSP40, HSP47, HSP60, HSP70A, HSP70B, HSP70C, HSP75, HSP90alpha, HSP90beta, and HSP108) as well as cold-inducible RNA binding protein (CIRBP) and HSP-binding protein (HSPBP) were cloned, and expression of their mRNA in the gonadal-adrenal-mesonephros complex (GAM) was investigated. Embryonic and neonatal GAMs exhibited mRNA for all of the HSPs examined during and after the TSP. One-month-old GAMs were separated into 3 portions (gonad, adrenal gland, and mesonephros), and sexual dimorphism in the mRNA expression of gonadal HSP27 (male > female), gonadal HSP70A (male < female), and adrenal HSP90 alpha (male > female) was observed. These findings provide new insights on TSD and suggest that further studies examining the role of HSPs during gonadal development are needed.

[Current status of study on embryonic hematopoietic development in aorta-gonad-mesonephros -- review].

Aorta-gonad-mesonephros (AGM) is well known as a main structure that de novo generates hematopoietic primary stem cells (HSC) in mid-gestation mammalian embryos. Hemogenic endothelium, and recently, subendothelial mesenchyme as well as hemangioblast are shown as contributing to blood formation in AGM region. AGM-HSC displays dynamic changes in surface markers, including CD41, CD45 and several endothelial-specific molecules. The novel finding of interleukin-3 as a potent regulator of AGM-HSC seems very interesting. Moreover, zebra fish model reveals PGE2 as a novel stimulator of HSC in AGM and kidney marrow, which is also the case in mouse hematopoietic tissues. Identification of mesenchymal stem cells with significant hematopoietic supporting capacity in AGM region suggests an alternative pathway to explore new molecules governing embryonic and adult hematopoiesis. In this paper, the hemogenic model in AGM region, surface markers on HSCs in AGM region and regulation of HSCs in AGM region were reviewed.

Cellular localization of a putative Na(+)/H (+) exchanger 3 during ontogeny in the pronephros and mesonephros of the Japanese black salamander (Hynobius nigrescens Stejneger).

The cloning of cDNA and an examination of the tissue distribution of Na(+)/H(+) exchanger 3 (NHE3) were carried out in the Japanese black salamander, Hynobius nigrescens. The cellular localization of Hynobius NHE3 was examined by in situ hybridization and immunohistochemistry during ontogeny in the nephron of the pronephros and mesonephros of the salamander. The partial amino acid sequence of Hynobius NHE3 was 81% and 72% identical to rat NHE3 and stingray NHE3, respectively. Hynobius NHE3 mRNA and protein were exclusively expressed along the late portion of the distal tubule to the anterior part of the pronephric duct of premetamorphic larvae (IY stages 43-50). NHE3 mRNA was expressed in the pronephros but not in the external gills in the larvae at the digit differentiation stage (IY stage 50). In the adult, mRNA was strongly expressed in the mesonephros but not in the ventral and dorsal skin. In juvenile and adult specimens, NHE3 immunoreactivity was observed at the apical membrane of the initial parts of the distal tubules of the mesonephric kidney. Immunohistochemical and in situ hybridization studies suggested that Na(+) absorption coupled with H(+) secretion via NHE3 occurred in the distal nephron of the pronephros and mesonephros. This is the first study to indicate NHE3 expression during ontogeny in amphibians.

Characteristics of the compensatory renal growth of the remnant embryonic chick kidneys.

Growth of the remnant embryonic kidney (the mesonephros), as expressed by wet weight, was more rapid in the chick embryos with experimentally induced unilateral renal agenesis compared to controls. The difference was significant between embryonic days 8-12, when the doubled weights of remnant kidneys were increased compared with the weights of paired control kidneys. The excessive growth of the mesonephros ceased on day 14, when the normal physiological regression of the embryonic kidney begins. In the definitive kidney, the metanephros, no significant differences in weights of the control vs. remnant metanephros were found on days 10-14. The characteristics of increased mesonephric growth were evaluated by determination of DNA/protein ratios in homogenates of the kidneys. Significant cellular hypertrophy was found in both the mesonephros and metanephros of the embryos with URA on day 10. Additionally, a non-significant cellular hyperplasia was also revealed in the remnant mesonephros on day 8. This gives evidence that the growth stimuli to the mesonephroi were probably strongest between days 8-10 and that they manifested in the remnant mesonephros only.

Sexually dimorphic expression of secreted frizzled-related (SFRP) genes in the developing mouse Müllerian duct.

In developing male embryos, the female reproductive tract primordia (Müllerian ducts) regress due to the production of testicular anti-Müllerian hormone (AMH). Because of the association between secreted frizzled-related proteins (SFRPs) and apoptosis, their reported developmental expression patterns and the role of WNT signaling in female reproductive tract development, we examined expression of Sfrp2 and Sfrp5 during development of the Müllerian duct in male (XY) and female (XX) mouse embryos. We show that expression of both Sfrp2 and Sfrp5 is dynamic and sexually dimorphic. In addition, the male-specific expression observed for both genes prior to the onset of regression is absent in mutant male embryos that fail to undergo Müllerian duct regression. We identified ENU-induced point mutations in Sfrp5 and Sfrp2 that are predicted to severely disrupt the function of these genes. Male embryos and adults homozygous for these mutations, both individually and in combination, are viable and apparently fertile with no overt abnormalities of reproductive tract development.

Quantitative analysis of embryonic kidney impairment by confocal microscopy and stereology: effect of 1,2-dibromoethane in the chick mesonephros.

1. Chick embryos in ovo were treated with a teratogenic dose of 1,2-dibromoethane (DBE) on embryonic day (ED) 3. On ED 6 and 10, histological sections of whole embryos were prepared for confocal microscopy. In parallel, mesonephroi of 10-d-old embryos were dissected for in situ staining with acridine orange (AO), a fluorescence probe for lysosomes. 2. DBE impaired differentiation of renal vessels which manifested as a delay in rearrangement of primitive renal vascular architecture on ED 6 and a significant reduction of the mesonephric vascularisation on ED 10. This was accompanied by delayed functional maturation of embryonic kidney, as suggested by staining with AO. 3. Renal vessels appeared to be more susceptible to DBE than tubules. Unequal growth of these renal components might be a cause of DBE-induced spatial disorganisation of tubular apparatus. 4. Nephrotoxic effects of DBE during the embryonic period are associated primarily with damage to the renal blood supply. 5. Confocal microscopy, stereological methods and three-dimensional reconstruction of developing tissues are useful tools to investigate pathogenic processes during embryonic development.

Is there a mesonephric cell contribution to the gonadal primordium before sexual differentiation in humans?: An ultrastructural study.

Differentiation of the intermediate mesoderm involves a complex series of events that result in the formation of the rudiment of the entire adult renal system, gonads and gonoducts. This work, using light, transmission, and scanning electron microscopy describes in human embryos of different ages, the development and spatiotemporal organization of the mesonephric nephron, and the development of the gonadal primordium, with the purpose of knowing if and how these two blastemas contribute to the origin of the non germinal cell content of the gonad primordium. Our results show that between Carnegie stage 13 and 20, the mesonephric nephrons facing the gonadal area, are separated by a band of mesenchymal tissue from the gonad primordium and they retain their structural integrities at the level of epithelial wall and their basement membrane. The morphological stability of basement membranes of different nephric structures, as well as the mesonephric duct during the period studied, confirm the previous opinion that the mesonephros is functional. During the same period of time, the structural events underlying gonad development show that primordial germ cells (PGCs) first invade the gonadal area, and thereafter interact both with epithelial coelomic cells and mesenchymal cells. Both types of cells surround PGCs, initiating the growth and differentiation of the gonadal primordium. Therefore, a mesonephric cell contribution to the genesis of the somatic cell components of the gonadal primordium should be discarded in humans. The present work emphasizes the need for more research in this field.

Role of the microenvironment of the embryonic aorta-gonad-mesonephros region in hematopoiesis.

Although various cytokines, growth factors, and chemokines are known to regulate hematopoiesis, expansion of hematopoietic stem cells (HSCs) in vitro with the use of such agents has proved problematic. Stromal cells are major components of the microenvironment that surrounds hematopoietic cells and are thought to play an important role in hematopoiesis in vivo. Co-culture of HSCs with stromal cells promotes hematopoiesis and self-renewal of HSCs. Definitive hematopoietic cells first appear during mammalian embryonic development in the aorta-gonad-mesonephros (AGM) region, and it is therefore thought that the microenvironment of this region plays an important role in HSC ontogeny. We have adopted two approaches to studying the contribution of the AGM microenvironment to hematopoiesis. In the first approach, we have developed an in vitro culture system for mouse AGM explants. Hematopoiesis is enhanced in such cultures by the presence of the combination of stem cell factor (SCF), basic fibroblast growth factor, leukemia inhibitory factor, and oncostatin M (SFLO culture). However, transplantation assays revealed that HSCs capable of long-term reconstitution of the hematopoietic compartment of irradiated mice (LTR-HSCs) do not expand in AGM-SFLO cultures; rather, these cultures appear to provide a favorable microenvironment for hematogenic angioblasts that are precursors of both endothelial and hematopoietic cells. In our second approach, we have established various stromal cell lines from the mouse AGM region. The AGM-S3 cell line supports human and mouse primitive hematopoietic cells as well as mouse LTR-HSCs. Maintenance of LTR-HSCs is mediated by a mechanism other than SCF signaling through its receptor (c-Kit). These two in vitro approaches should prove useful for further elucidation of the mechanisms that underlie hematopoiesis and HSC self-renewal.

A conserved Hox axis in the mouse and human female reproductive system: late establishment and persistent adult expression of the Hoxa cluster genes.

The mammalian female reproductive system arises from the uniform paramesonephric duct. The molecular mechanisms that establish differential development along this axis are unknown. We determined the pattern and timing of genes of the Hoxa axis in the development of the Müllerian tract. Hoxa-9, Hoxa-10, Hoxa-11, and Hoxa-13 are all expressed along the length of the paramesonephric duct in the embryonic mouse. After birth, a spatial Hox axis is established, corresponding to the postnatal differentiation of this organ system in the mouse. Hoxa-9 is expressed in the fallopian tubes, Hoxa-10 in the uterus, Hoxa-11 in the uterus and uterine cervix, and Hoxa-13 in the upper vagina. This expression pattern follows the paradigm of spatial colinearity but is a novel exception to temporal colinearity that has been considered typical of Hox genes. These genes remain expressed in the adult mouse and are expressed in the same pattern in the human. The female reproductive system undergoes dramatic structural and functional changes during the estrous cycle and in pregnancy, retaining a high degree of developmental plasticity. The late establishment of a Hox axis and persistent expression of Hox genes in the adult may play an important role in preserving this plasticity.

Sexual differentiation of the urogenital system of the fetal and neonatal tammar wallaby, Macropus eugenii.

In male tammar wallabies, the scrotum is the first organ to become sexually differentiated, 4-5 days before birth (day 22 of gestation). This is followed by enlargement of the gubernaculum and processus vaginalis one day before birth. However the indifferent gonad does not show any signs of testicular cord formation or androgen production until later, at around the time of birth; this is more pronounced at 2 days post-partum (p.p.), when the testis takes on a characteristic rounded appearance. Primordial germ cells proliferate throughout the testis at this time, although the testis does not become significantly heavier than the ovary until around 80 days p.p.. In females, the appearance of the mammary glands is the first sign of sexual differentiation 4-5 days before birth. The indifferent gonad first shows signs of developing an ovarian cortex and medulla 7 days after birth. The migrating germ cells are confined to the cortex, and first start to enter meiosis about 25 days after birth. The Wolffian (mesonephric) ducts are patent to the urogenital sinus in fetuses at day 21 of gestation. In the female they have started to regress by 10 days p.p. and only rudiments remain by day 25 p.p.. The Müllerian (paramesonephric) ducts develop adjacent to the cranial pole of the mesonephros at about day 25 of gestation and grow caudally to meet the urogenital sinus between days 2 and 7 p.p.. The Müllerian duct of the female develops a prominent ostium abdominale by day 9 p.p., but this structure has completely regressed in males by day 13 p.p.. The testis and ovary both migrate caudally, together with the adjacent mesonephros, at about day 10 p.p.. The ovaries remain around the level of lumbar vertebra 4 after about day 7 p.p., while the testes continue to descend. The testes enter the internal inguinal ring at about day 25 p.p., about the time that prostatic buds first appear in the urogenital sinus, and are in the inguinal canal from days 25 to 36 p.p.. They enter the scrotum at around day 36 p.p., and testicular descent is complete by days 65-72 p.p.. Melanin develops in the tunica vaginalis 72 days after birth. The overall development of the urogenital system in this marsupial is similar to that of eutherians but the sequence of events differs, with some aspects of genital differentiation preceding gonadal differentiation, apparently because they are directly controlled by X-linked genes, rather than indirectly controlled by gonadal steroids.

Differential distribution of laminin chains in the development and sex differentiation of mouse internal genitalia.

The distribution of laminin chains and basement membranes (BMs) in the ontogenesis and sex differentiation of male and female mouse gonads and mesonephros was studied by conventional and immunocytochemical light and electron microscopy. The alpha 1 (synonymous to A) chain was recognized with MAbs against fragment E3, and three chains of laminin with PAbs raised against EHS-laminin. BMs, which formed around the mesonephric duct, the mesonephric tubules, and the paramesonephric duct, contained the laminin alpha 1 chain. The alpha 1 chain appeared with epithelial differentiation in the developing gonads in both sexes. The alpha 1 chain was first evident around the embryonic gonadal cords and remained, after development, in the BMs of the testicular cords and ovarian follicles. The laminin alpha 1 chain was also detected in BMs of the myoid cells around the epithelial rete cords, and transiently in the surface epithelium and in the corpus luteum. Laminin beta-gamma chains were found in many locations where the alpha 1 chain was not detected. These included the mesenchyme of the early mesonephros, the BMs of blood vessels and surface epithelium in the differentiated testis and ovary, between the theca cells in the ovary, and in some corpora lutea. The morphological differentiation of the BMs of the embryonic testicular cords proceeded rapidly. In contrast, the BM of the ovarian cords remained relatively poorly differentiated during the prenatal phases, and developed concomitantly with the differentiation of the follicles. The results show that BMs in the differentiating internal genitalia are heterogeneous with respect to their laminin chains, and suggest that all known laminin chains must be analyzed in the differentiation of gonadal epithelia for a complete role of the BMs in gonadal sex differentiation.