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.

A new development in senescence.

Cellular senescence is implicated in several pathological responses in the adult, with important repercussions in tumor suppression, wound healing, and aging. Two studies by Muñoz-Espín et al. and Storer et al. now reveal that senescence contributes to embryonic development, suggesting a primordial role in normal physiology.

Programmed cell senescence during mammalian embryonic development.

Cellular senescence disables proliferation in damaged cells, and it is relevant for cancer and aging. Here, we show that senescence occurs during mammalian embryonic development at multiple locations, including the mesonephros and the endolymphatic sac of the inner ear, which we have analyzed in detail. Mechanistically, senescence in both structures is strictly dependent on p21, but independent of DNA damage, p53, or other cell-cycle inhibitors, and it is regulated by the TGF-ß/SMAD and PI3K/FOXO pathways. Developmentally programmed senescence is followed by macrophage infiltration, clearance of senescent cells, and tissue remodeling. Loss of senescence due to the absence of p21 is partially compensated by apoptosis but still results in detectable developmental abnormalities. Importantly, the mesonephros and endolymphatic sac of human embryos also show evidence of senescence. We conclude that the role of developmentally programmed senescence is to promote tissue remodeling and propose that this is the evolutionary origin of damage-induced senescence.

Retinoic acid signaling is essential for embryonic hematopoietic stem cell development.

Hematopoietic stem cells (HSCs) develop from a specialized subpopulation of endothelial cells known as hemogenic endothelium (HE). Although the HE origin of HSCs is now well established in different species, the signaling pathways that control this transition remain poorly understood. Here, we show that activation of retinoic acid (RA) signaling in aorta-gonad-mesonephros-derived HE ex vivo dramatically enhanced its HSC potential, whereas conditional inactivation of the RA metabolizing enzyme retinal dehydrogenase 2 in VE-cadherin expressing endothelial cells in vivo abrogated HSC development. Wnt signaling completely blocked the HSC inductive effects of RA modulators, whereas inhibition of the pathway promoted the development of HSCs in the absence of RA signaling. Collectively, these findings position RA and Wnt signaling as key regulators of HSC development and in doing so provide molecular insights that will aid in developing strategies for their generation from pluripotent stem cells.

Mapping human haematopoietic stem cells from haemogenic endothelium to birth.

The ontogeny of human haematopoietic stem cells (HSCs) is poorly defined owing to the inability to identify HSCs as they emerge and mature at different haematopoietic sites1. Here we created a single-cell transcriptome map of human haematopoietic tissues from the first trimester to birth and found that the HSC signature RUNX1+HOXA9+MLLT3+MECOM+HLF+SPINK2+ distinguishes HSCs from progenitors throughout gestation. In addition to the aorta-gonad-mesonephros region, nascent HSCs populated the placenta and yolk sac before colonizing the liver at 6 weeks. A comparison of HSCs at different maturation stages revealed the establishment of HSC transcription factor machinery after the emergence of HSCs, whereas their surface phenotype evolved throughout development. The HSC transition to the liver marked a molecular shift evidenced by suppression of surface antigens reflecting nascent HSC identity, and acquisition of the HSC maturity markers CD133 (encoded by PROM1) and HLA-DR. HSC origin was tracked to ALDH1A1+KCNK17+ haemogenic endothelial cells, which arose from an IL33+ALDH1A1+ arterial endothelial subset termed pre-haemogenic endothelial cells. Using spatial transcriptomics and immunofluorescence, we visualized this process in ventrally located intra-aortic haematopoietic clusters. The in vivo map of human HSC ontogeny validated the generation of aorta-gonad-mesonephros-like definitive haematopoietic stem and progenitor cells from human pluripotent stem cells, and serves as a guide to improve their maturation to functional HSCs.

A multistep computational approach reveals a neuro-mesenchymal cell population in the embryonic hematopoietic stem cell niche.

The first hematopoietic stem and progenitor cells (HSPCs) emerge in the Aorta-Gonad-Mesonephros (AGM) region of the mid-gestation mouse embryo. However, the precise nature of their supportive mesenchymal microenvironment remains largely unexplored. Here, we profiled transcriptomes of laser micro-dissected aortic tissues at three developmental stages and individual AGM cells. Computational analyses allowed the identification of several cell subpopulations within the E11.5 AGM mesenchyme, with the presence of a yet unidentified subpopulation characterized by the dual expression of genes implicated in adhesive or neuronal functions. We confirmed the identity of this cell subset as a neuro-mesenchymal population, through morphological and lineage tracing assays. Loss of function in the zebrafish confirmed that Decorin, a characteristic extracellular matrix component of the neuro-mesenchyme, is essential for HSPC development. We further demonstrated that this cell population is not merely derived from the neural crest, and hence, is a bona fide novel subpopulation of the AGM mesenchyme.

An interactive resource of molecular signalling in the developing human haematopoietic stem cell niche.

The emergence of definitive human haematopoietic stem cells (HSCs) from Carnegie Stage (CS) 14 to CS17 in the aorta-gonad-mesonephros (AGM) region is a tightly regulated process. Previously, we conducted spatial transcriptomic analysis of the human AGM region at the end of this period (CS16/CS17) and identified secreted factors involved in HSC development. Here, we extend our analysis to investigate the progression of dorso-ventral polarised signalling around the dorsal aorta over the entire period of HSC emergence. Our results reveal a dramatic increase in ventral signalling complexity from the CS13-CS14 transition, coinciding with the first appearance of definitive HSCs. We further observe stage-specific changes in signalling up to CS17, which may underpin the step-wise maturation of HSCs described in the mouse model. The data-rich resource is also presented in an online interface enabling in silico analysis of molecular interactions between spatially defined domains of the AGM region. This resource will be of particular interest for researchers studying mechanisms underlying human HSC development as well as those developing in vitro methods for the generation of clinically relevant HSCs from pluripotent stem cells.

Defining the Hoxb8 cell lineage during murine definitive hematopoiesis.

Previously, we have demonstrated that a subpopulation of microglia, known as Hoxb8 microglia, is derived from the Hoxb8 lineage during the second wave (E8.5) of yolk sac hematopoiesis, whereas canonical non-Hoxb8 microglia arise from the first wave (E7.5). Hoxb8 microglia have an ontogeny distinct from non-Hoxb8 microglia. Dysfunctional Hoxb8 microglia cause the acquisition of chronic anxiety and an obsessive-compulsive spectrum-like behavior, trichotillomania, in mice. The nature and fate of the progenitors generated during E8.5 yolk sac hematopoiesis have been controversial. Herein, we use the Hoxb8 cell lineage reporter to define the ontogeny of hematopoietic cells arising during the definitive waves of hematopoiesis initiated in the E8.5 yolk sac and aorta-gonad-mesonephros (AGM) region. Our murine cell lineage analysis shows that the Hoxb8 cell lineage reporter robustly marks erythromyeloid progenitors, hematopoietic stem cells and their progeny, particularly monocytes. Hoxb8 progenitors and microglia require Myb function, a hallmark transcription factor for definitive hematopoiesis, for propagation and maturation. During adulthood, all immune lineages and, interestingly, resident macrophages in only hematopoietic/lymphoid tissues are derived from Hoxb8 precursors. These results illustrate that the Hoxb8 lineage exclusively mirrors murine definitive hematopoiesis.

The genesis of human hematopoietic stem cells.

Developmental hematopoiesis consists of multiple, partially overlapping hematopoietic waves that generate the differentiated blood cells required for embryonic development while establishing a pool of undifferentiated hematopoietic stem cells (HSCs) for postnatal life. This multilayered design in which active hematopoiesis migrates through diverse extra and intraembryonic tissues has made it difficult to define a roadmap for generating HSCs vs non-self-renewing progenitors, especially in humans. Recent single-cell studies have helped in identifying the rare human HSCs at stages when functional assays are unsuitable for distinguishing them from progenitors. This approach has made it possible to track the origin of human HSCs to the unique type of arterial endothelium in the aorta-gonad-mesonephros region and document novel benchmarks for HSC migration and maturation in the conceptus. These studies have delivered new insights into the intricate process of HSC generation and provided tools to inform the in vitro efforts to replicate the physiological developmental journey from pluripotent stem cells via distinct mesodermal and endothelial intermediates to HSCs.

Concerted morphogenesis of genital ridges and nephric ducts in the mouse captured through whole-embryo imaging.

During development of the mouse urogenital complex, the gonads undergo changes in three-dimensional structure, body position and spatial relationship with the mesonephric ducts, kidneys and adrenals. The complexity of genital ridge development obscures potential connections between morphogenesis and gonadal sex determination. To characterize the morphogenic processes implicated in regulating gonad shape and fate, we used whole-embryo tissue clearing and light sheet microscopy to assemble a time course of gonad development in native form and context. Analysis revealed that gonad morphology is determined through anterior-to-posterior patterns as well as increased rates of growth, rotation and separation in the central domain that may contribute to regionalization of the gonad. We report a close alignment of gonad and mesonephric duct movements as well as delayed duct development in a gonad dysgenesis mutant, which together support a mechanical dependency linking gonad and mesonephric duct morphogenesis.

p57Kip2 regulates embryonic blood stem cells by controlling sympathoadrenal progenitor expansion.

Hematopoietic stem cells (HSCs) are of major clinical importance, and finding methods for their in vitro generation is a prime research focus. We show here that the cell cycle inhibitor p57Kip2/Cdkn1c limits the number of emerging HSCs by restricting the size of the sympathetic nervous system (SNS) and the amount of HSC-supportive catecholamines secreted by these cells. This regulation occurs at the SNS progenitor level and is in contrast to the cell-intrinsic function of p57Kip2 in maintaining adult HSCs, highlighting profound differences in cell cycle requirements of adult HSCs compared with their embryonic counterparts. Furthermore, this effect is specific to the aorta-gonad-mesonephros (AGM) region and shows that the AGM is the main contributor to early fetal liver colonization, as early fetal liver HSC numbers are equally affected. Using a range of antagonists in vivo, we show a requirement for intact ß2-adrenergic signaling for SNS-dependent HSC expansion. To gain further molecular insights, we have generated a single-cell RNA-sequencing data set of all Ngfr+ sympathoadrenal cells around the dorsal aorta to dissect their differentiation pathway. Importantly, this not only defined the relevant p57Kip2-expressing SNS progenitor stage but also revealed that some neural crest cells, upon arrival at the aorta, are able to take an alternative differentiation pathway, giving rise to a subset of ventrally restricted mesenchymal cells that express important HSC-supportive factors. Neural crest cells thus appear to contribute to the AGM HSC niche via 2 different mechanisms: SNS-mediated catecholamine secretion and HSC-supportive mesenchymal cell production.

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.

Role of mesonephric contribution to mouse testicular development revisited.

The role of the mesonephros in testicular development was re-evaluated by growing embryonic day 11.5 (E11.5) mouse testes devoid of mesonephros for 8-21 days in vivo under the renal capsule of castrated male athymic nude mice. This method provides improved growth conditions relative to previous studies based upon short-term (4-7 days) organ culture. Meticulous controls involved wholemount examination of dissected E11.5 mouse testes as well as serial sections of dissected E11.5 mouse testes which were indeed shown to be devoid of mesonephros. As expected, grafts of E11.5 mouse testes with mesonephros attached formed seminiferous tubules and also contained mesonephric derivatives. Grafts of E11.5 mouse testes without associated mesonephros also formed seminiferous tubules and never contained mesonephric derivatives. The consistent absence of mesonephric derivatives in grafts of E11.5 mouse testes grafted alone is further proof of the complete removal of the mesonephros from the E11.5 mouse testes. The testicular tissues that developed in grafts of E11.5 mouse testes alone contained canalized seminiferous tubules composed of Sox9-positive Sertoli cells as well as GENA-positive germ cells. The seminiferous tubules were surrounded by α-actin-positive myoid cells, and the interstitial space contained 3ßHSD-1-positive Leydig cells. Grafts of E11.5 GFP mouse testes into wild-type hosts developed GFP-positive vasculature indicating that E11.5 mouse testes contain vascular precursors. These results indicate that the E11.5 mouse testis contains precursor cells for Sertoli cells, Leydig cells, myoid cells and vasculature whose development and differentiation are independent of cells migrating from the E11.5 mesonephros.

Early development of the human embryonic testis.

Human gonadal development culminating in testicular differentiation is described through analysis of histologic sections derived from 33-day to 20-week human embryos/fetuses, focusing on early development (4-8 weeks of gestation). Our study updates the comprehensive studies of Felix (1912), van Wagenen and Simpson (1965), and Juric-Lekic et al. (2013), which were published in books and thus are unsearchable via PubMed. Human gonads develop from the germinal ridge, a thickening of coelomic epithelium on the medial side of the urogenital ridge. The bilateral urogenital ridges contain elements of the mesonephric kidney, namely the mesonephric duct, mesonephric tubules, and mesonephric glomeruli. The germinal ridge, into which primordial germ cells migrate, is initially recognized as a thickening of coelomic epithelium on the urogenital ridge late in the 4th week of gestation. Subsequently, in the 5th week of gestation, a dense mesenchyme develops sub-adjacent to the epithelium of the germinal ridge, and together these elements bulge into the coelomic cavity forming bilateral longitudinal ridges attached to the urogenital ridges. During development, primordial cells migrate into the germinal ridge and subsequently into testicular cords that form within the featureless dense mesenchyme of the germinal ridge at 6-8 weeks of gestation. The initial low density of testicular cords seen at 8 weeks remodels into a dense array of testicular cords surrounded by α-actin-positive myoid cells during the second trimester. Human testicular development shares many features with that of mice being derived from 4 elements: coelomic epithelium, sub-adjacent mesenchyme, primordial germ cells, and the mesonephros.

Expression patterns of sex steroid receptors in developing mesonephros of the male mouse: three-dimensional analysis.

The androgen pathway via androgen receptor (AR) has received the most attention for development of male reproductive tracts. The estrogen pathway through estrogen receptor (ESR1) is also a major contributor to rete testis and efferent duct formation, but the role of progesterone via progesterone receptor (PGR) has largely been overlooked. Expression patterns of these receptors in the mesonephric tubules (MTs) and Wolffian duct (WD), which differentiate into the efferent ductules and epididymis, respectively, remain unclear because of the difficulty in distinguishing each region of the tracts. This study investigated AR, ESR1, and PGR expressions in the murine mesonephros using three-dimensional (3-D) reconstruction. The receptors were localized in serial paraffin sections of the mouse testis and mesonephros by immunohistochemistry on embryonic days (E) 12.5, 15.5, and 18.5. Specific regions of the developing MTs and WD were determined by 3-D reconstruction using Amira software. AR was found first in the specific portion of the MTs near the MT-rete junction at E12.5, and the epithelial expression showed increasing strength from cranial to the caudal regions. Epithelial expression of ESR1 was found in the cranial WD and MTs near the WD first at E15.5. PGR was weakly positive only in the MTs and cranial WD starting on E15.5. This 3-D analysis suggests that gonadal androgen acts first on the MTs near the MT-rete junction but that estrogen is the first to influence MTs near the WD, while potential PGR activity is delayed and limited to the epithelium.

A common epigenetic mechanism across different cellular origins underlies systemic immune dysregulation in an idiopathic autism mouse model.

Immune dysregulation plays a key role in the pathogenesis of autism. Changes occurring at the systemic level, from brain inflammation to disturbed innate/adaptive immune in the periphery, are frequently observed in patients with autism; however, the intrinsic mechanisms behind them remain elusive. We hypothesize a common etiology may lie in progenitors of different types underlying widespread immune dysregulation. By single-cell RNA sequencing (sc-RNA seq), we trace the developmental origins of immune dysregulation in a mouse model of idiopathic autism. It is found that both in aorta-gonad-mesonephros (AGM) and yolk sac (YS) progenitors, the dysregulation of HDAC1-mediated epigenetic machinery alters definitive hematopoiesis during embryogenesis and downregulates the expression of the AP-1 complex for microglia development. Subsequently, these changes result in the dysregulation of the immune system, leading to gut dysbiosis and hyperactive microglia in the brain. We further confirm that dysregulated immune profiles are associated with specific microbiota composition, which may serve as a biomarker to identify autism of immune-dysregulated subtypes. Our findings elucidate a shared mechanism for the origin of immune dysregulation from the brain to the gut in autism and provide new insight to dissecting the heterogeneity of autism, as well as the therapeutic potential of targeting immune-dysregulated autism subtypes.

Two Cases of Mesonephric-like Carcinoma Arising From Endometriosis: Case Report and Review of the Literature.

Endometriosis is a common condition in reproductive age women that is defined as the presence of endometrial tissue (epithelial and/or stromal) outside the uterine corpus. While not a premalignant lesion, it is a condition with a potential for malignancy, especially in the ovaries. Notable endometriosis-associated neoplasms include clear cell carcinoma and endometrioid adenocarcinoma of the ovaries. There have been recent reports of mesonephric-like adenocarcinoma (MLA) of the ovary, a very rare neoplasm with similar morphologic and immunophenotypic characteristics as mesonephric adenocarcinoma, however, without an association with mesonephric remnants. Some of these cases have been associated with endometriosis. Here, we describe 2 cases of MLA arising directly from endometriosis. In both cases, there was evidence of endometriosis contiguous with the tumor and invasion from other sources was excluded. The immunophenotypes of both tumors were typical of mesonephric adenocarcinoma except PAX-8 was strongly positive suggesting a Mullerian origin. Molecular testing on one of the cases revealed KRAS and P53 mutations. We review published findings of MLA and associated endometriosis. This report describes the sixth and seventh reported cases of MLA associated with endometriosis and the first reported cases of MLA arising directly from endometriosis and associated with other forms of epithelial proliferation within endometriosis. These 2 cases provide potential evidence that MLA should be considered an endometriosis-associated neoplasms.

Ovarian Combined Serous Borderline Tumor/Low-grade Serous Carcinoma and Mesonephric-like Lesion: Report of 2 Cases With New Observations.

Ovarian combined serous borderline tumor/low-grade serous carcinomas (SBT/LGSC) and mesonephric-like adenocarcinomas (MLA) have been previously reported and the presence of identical oncogenic somatic mutations in both components supports the concept that at least some of MLAs arise from a Müllerian origin. We report 2 cases of ovarian combined SBT/LGSC and mesonephric-like lesion. Case 1 was a 70-yr-old woman presented with a liver lesion and omental carcinomatosis. Histologic examination revealed biphasic tumors in bilateral ovaries consisting of conventional SBT and invasive MLA with extraovarian spread. The right ovary also had a component of cribriform variant of SBT/noninvasive LGSC. The SBT/LGSC component was diffusely positive for Pax8, WT-1, and ER, focally positive for PR, and negative for GATA3, while the MLA component was diffusely positive for GATA3 but negative for WT-1, ER, and PR. Molecular analysis revealed a KRAS G12V mutation in both the SBT/LGSC and MLA components, indicating their clonal origin. Case 2 was a 58-yr-old woman who presented with conventional type SBT in both ovaries. In addition, the left ovarian tumor demonstrated a few areas (each <5 mm) of mesonephric-like differentiation/hyperplasia in close proximity to the serous-type epithelium, with an immunophenotype of focal GATA3 expression, luminal pattern of CD10 staining and negative WT-1, ER, and PR staining. This phenomenon has been reported in endometrioid borderline tumor but not in any serous type lesions. The findings in case 1 provide further evidence to demonstrate the clonal relationship between these morphologically and immunophenotypically distinct components. It also supports the theory that, unlike cervical mesonephric carcinomas originating from mesonephric remnants, MLAs are derived from a Müllerian-type lesion with differentiation into mesonephric lineage. The presence of a hyperplastic mesonephric-like lesion/differentiation in case 2 indicates that a precursor lesion in the same lineage with the potential to develop into MLA exists in the ovary.

Immunohistochemical Panels to Evaluate Important Immunophenotypes of Human Mesonephros.

Background. The immunophenotypes and potential excretory function of human mesonephros are not well studied. Methods. Five mesonephros specimens of human embryos from the 6th to 10th weeks of gestation were stained with immunohistochemical markers. Results. PAX8 was universally expressed in all renal tubules, while α-methyacyl-CoA racemase (AMACAR) was positive in proximal tubules and GATA3 was positive in distal tubular mesonephric structures. At the 8th weeks of gestation, the mesonephric glomeruli were characterized by opened glomerular capillary loops with Periodic Acid Schiff (PAS)-positive glomerular basement membranes and GATA3-positive mesangial-like cells. By the 8th week, proximal tubules showed PAS-positive brush borders, indicating reabsorption capacity, and the proximal tubules also demonstrated positivity with kidney injury molecule-1 (KIM-1), representing tubular response to injury. Conclusion. Our overall findings show detailed phenotypes of the glomerular and tubular structures of the mesonephros and indicate that at the 8th week of gestation, the mesonephros may carry out temporary excretory function before metanephros becomes fully functional.

Comparative anatomy on the development of sperm transporting pathway between the testis and mesonephros.

The male genital tract is diverse among vertebrates, but its development remains unclear, especially in the rete region. In this study, we investigated the testis-mesonephros complex of rabbit, chicken, and frog (Xenopus tropicalis) by immunohistochemistry for markers such as Ad4BP/Sf-1 (gonadal somatic and rete cells in mammals) and Pax2 (mesonephric tubules), and performed a three-dimensional reconstruction. In all investigated animals, testis cords were bundled at the mesonephros side. Rete cells positive for Ad4BP/Sf-1 (rabbit) or Pax2 (chicken and frog) were clustered at the border region between the testis and mesonephros. The cluster possessed two types of cords; one connected to the testis cords and the other to the mesonephric tubules. The latter rete cords were contiguous to Bowman's capsules in rabbit and chicken but to nephrostomes in frog. In conclusion, this study showed that mammals, avian species, and frogs commonly develop the bundle between the testis cords (testis canal) and the cluster of rete cells (lateral kidney canal), indicating that these animals share basic morphogenesis in the male genital tract. The connection site between the rete cells and mesonephric tubules is suggested to have changed from the nephrostome to the Bowman's capsule during vertebrate evolution from anamniote to amniote.