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.

Autophagy regulates the maturation of hematopoietic precursors in the embryo.

An understanding of the mechanisms regulating embryonic hematopoietic stem cell (HSC) development would facilitate their regeneration. The aorta-gonad-mesonephros region is the site for HSC production from hemogenic endothelial cells (HEC). While several distinct regulators are involved in this process, it is not yet known whether macroautophagy (autophagy) plays a role in hematopoiesis in the pre-liver stage. Here, we show that different states of autophagy exist in hematopoietic precursors and correlate with hematopoietic potential based on the LC3-RFP-EGFP mouse model. Deficiency of autophagy-related gene 5 (Atg5) specifically in endothelial cells disrupts endothelial to hematopoietic transition (EHT), by blocking the autophagic process. Using combined approaches, including single-cell RNA-sequencing (scRNA-seq), we have confirmed that Atg5 deletion interrupts developmental temporal order of EHT to further affect the pre-HSC I maturation, and that autophagy influences hemogenic potential of HEC and the formation of pre-HSC I likely via the nucleolin pathway. These findings demonstrate a role for autophagy in the formation/maturation of hematopoietic precursors.

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.

Runx1+ vascular smooth muscle cells are essential for hematopoietic stem and progenitor cell development in vivo.

Hematopoietic stem cells (HSCs) produce all essential cellular components of the blood. Stromal cell lines supporting HSCs follow a vascular smooth muscle cell (vSMC) differentiation pathway, suggesting that some hematopoiesis-supporting cells originate from vSMC precursors. These pericyte-like precursors were recently identified in the aorta-gonad-mesonephros (AGM) region; however, their role in the hematopoietic development in vivo remains unknown. Here, we identify a subpopulation of NG2+Runx1+ perivascular cells that display a sclerotome-derived vSMC transcriptomic profile. We show that deleting Runx1 in NG2+ cells impairs the hematopoietic development in vivo and causes transcriptional changes in pericytes/vSMCs, endothelial cells and hematopoietic cells in the murine AGM. Importantly, this deletion leads also to a significant reduction of HSC reconstitution potential in the bone marrow in vivo. This defect is developmental, as NG2+Runx1+ cells were not detected in the adult bone marrow, demonstrating the existence of a specialised pericyte population in the HSC-generating niche, unique to the embryo.

Cis inhibition of NOTCH1 through JAGGED1 sustains embryonic hematopoietic stem cell fate.

Hematopoietic stem cells (HSCs) develop from the hemogenic endothelium (HE) in the aorta- gonads-and mesonephros (AGM) region and reside within Intra-aortic hematopoietic clusters (IAHC) along with hematopoietic progenitors (HPC). The signalling mechanisms that distinguish HSCs from HPCs are unknown. Notch signaling is essential for arterial specification, IAHC formation and HSC activity, but current studies on how Notch segregates these different fates are inconsistent. We now demonstrate that Notch activity is highest in a subset of, GFI1 + , HSC-primed HE cells, and is gradually lost with HSC maturation. We uncover that the HSC phenotype is maintained due to increasing levels of NOTCH1 and JAG1 interactions on the surface of the same cell (cis) that renders the NOTCH1 receptor from being activated. Forced activation of the NOTCH1 receptor in IAHC activates a hematopoietic differentiation program. Our results indicate that NOTCH1-JAG1 cis-inhibition preserves the HSC phenotype in the hematopoietic clusters of the embryonic aorta.

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.

Generation of a humanized mesonephros in pigs from induced pluripotent stem cells via embryo complementation.

Heterologous organ transplantation is an effective way of replacing organ function but is limited by severe organ shortage. Although generating human organs in other large mammals through embryo complementation would be a groundbreaking solution, it faces many challenges, especially the poor integration of human cells into the recipient tissues. To produce human cells with superior intra-niche competitiveness, we combined optimized pluripotent stem cell culture conditions with the inducible overexpression of two pro-survival genes (MYCN and BCL2). The resulting cells had substantially enhanced viability in the xeno-environment of interspecies chimeric blastocyst and successfully formed organized human-pig chimeric middle-stage kidney (mesonephros) structures up to embryonic day 28 inside nephric-defective pig embryos lacking SIX1 and SALL1. Our findings demonstrate proof of principle of the possibility of generating a humanized primordial organ in organogenesis-disabled pigs, opening an exciting avenue for regenerative medicine and an artificial window for studying human kidney development.

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.

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.

Nupr1 Negatively Regulates Endothelial to Hematopoietic Transition in the Aorta-Gonad-Mesonephros Region.

In the aorta of mid-gestational mouse embryos, a specialized endothelial subpopulation termed hemogenic endothelial cells (HECs) develops into hematopoietic stem and progenitor cells (HSPCs), through a conserved process of endothelial-to-hematopoietic transition (EHT). EHT is tightly controlled by multiple intrinsic and extrinsic mechanisms. Nevertheless, the molecular regulators restraining this process remain poorly understood. Here, it is uncovered that, one of the previously identified HEC signature genes, Nupr1, negatively regulates the EHT process. Nupr1 deletion in endothelial cells results in increased HSPC generation in the aorta-gonad-mesonephros region. Furthermore, single-cell transcriptomics combined with serial functional assays reveals that loss of Nupr1 promotes the EHT process by promoting the specification of hematopoiesis-primed functional HECs and strengthening their subsequent hematopoietic differentiation potential toward HSPCs. This study further finds that the proinflammatory cytokine, tumor necrosis factor α (TNF-α), is significantly upregulated in Nupr1-deficient HECs, and the use of a specific TNF-α neutralizing antibody partially reduces excessive HSPC generation in the explant cultures from Nupr1-deficient embryos. This study identifies a novel negative regulator of EHT and the findings indicate that Nupr1 is a new potential target for future hematopoietic stem cell regeneration research.

Pronephros and mesonephros characterization during the embryonic development of the giant South American river turtle, Podocnemis expansa (Podocnemididae: Testudines).

This study aimed to describe pronephros and mesonephros morphology during the embryonic development of Podocnemis expansa. Eggs were collected on an artificial beach at Balbina, Amazonas, Brazil, during the entire incubation period (mean of 59 days). The kidney-gonad complex was processed using light microscopy and the mesonephros using transmission electron microscopy. The pronephros was present for the first time on stage 4, composed of external glomeruli devoid of a capsule, protruding into the coelomic cavity, and internally composed of a capillary network. The pronephros degenerated after development stage 15. The first sign of the appearance of the mesonephros occurred around stage 8, indicated by the early formation of renal corpuscles. The mesonephros comprised an renal corpuscles, neck segment, proximal tubule, intermediate segment, distal tubule, collector tubule, and collector duct. Ultrastructural analysis of the mesonephros brush border was done in the proximal tubule, and the presence of cells with structural characters indicative of secretory activity was detected in the juxtatubular region. Renal corpuscles and proximal tubules were the main components that underwent morphological alterations during mesonephros degeneration. The pronephros is a transient kidney, and the mesonephros became the functional embryonic kidney in P. expansa. Mesonephros degeneration occurs in the cranial-caudal direction, and histologically, the degeneration is identified by changes in the morphology of the renal corpuscle and proximal tubule. However, the mesonephros is still present after hatching.

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.

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.

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.

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.

Mimicry of embryonic circulation enhances the hoxa hemogenic niche and human blood development.

Precursors of the adult hematopoietic system arise from the aorta-gonad-mesonephros (AGM) region shortly after the embryonic circulation is established. Here, we develop a microfluidic culture system to mimic the primitive embryonic circulation and address the hypothesis that circulatory flow and shear stress enhance embryonic blood development. Embryonic (HOXA+) hematopoiesis was derived from human pluripotent stem cells and induced from mesoderm by small-molecule manipulation of TGF-ß and WNT signaling (SB/CHIR). Microfluidic and orbital culture promoted the formation of proliferative CD34+RUNX1C-GFP+SOX17-mCHERRY+ precursor cells that were released into the artificial circulation from SOX17+ arterial-like structures. Single-cell transcriptomic analysis delineated extra-embryonic (yolk sac) and HOXA+ embryonic blood differentiation pathways. SB/CHIR and circulatory flow enhance hematopoiesis by the formation of proliferative HOXA+RUNX1C+CD34+ precursor cells that differentiate into monocyte/macrophage, granulocyte, erythrocyte, and megakaryocyte progenitors.