Single-Cell RNA Sequencing Resolves Spatiotemporal Development of Pre-thymic Lymphoid Progenitors and Thymus Organogenesis in Human Embryos.

Generation of the first T lymphocytes in the human embryo involves the emergence, migration, and thymus seeding of lymphoid progenitors together with concomitant thymus organogenesis, which is the initial step to establish the entire adaptive immune system. However, the cellular and molecular programs regulating this process remain unclear. We constructed a single-cell transcriptional landscape of human early T lymphopoiesis by using cells from multiple hemogenic and hematopoietic sites spanning embryonic and fetal stages. Among heterogenous early thymic progenitors, one subtype shared common features with a subset of lymphoid progenitors in fetal liver that are known as thymus-seeding progenitors. Unbiased bioinformatics analysis identified a distinct type of pre-thymic lymphoid progenitors in the aorta-gonad-mesonephros (AGM) region. In parallel, we investigated thymic epithelial cell development and potential cell-cell interactions during thymus organogenesis. Together, our data provide insights into human early T lymphopoiesis that prospectively direct T lymphocyte regeneration, which might lead to development of clinical applications.

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.

Layered origins of lymphoid tissue inducer cells.

The Innate Lymphoid Cell (ILC) family is a relatively recently described immune cell family involved in innate immune responses and tissue homeostasis. Lymphoid Tissue Inducer (LTi) cells are part of the type 3 (ILC3) family. The ILC3 family is the main ILC population within the embryo, in which the LTi cells are critically associated with embryonic lymph node formation. Recent studies have shown more insights in ILC origin and residency from local embryonic and tissue resident precursors. Embryonic LTi cells originating from a different hemogenic endothelial source were shown to be replaced by HSC derived progenitors in adult. This review will discuss the layered origin of the ILC3 family with an emphasis on the LTi cell lineage.

Generation of a Wt1 conditional deletion, nuclear red fluorescent protein reporter allele in the mouse.

A Wt1 conditional deletion, nuclear red fluorescent protein (RFP) reporter allele was generated in the mouse by gene targeting in embryonic stem cells. Upon Cre-mediated recombination, a deletion allele is generated that expresses RFP in a Wt1-specific pattern. RFP expression was detected in embryonic and adult tissues known to express Wt1, including the kidney, mesonephros, and testis. In addition, RFP expression and WT1 co-localization was detected in the adult uterine stroma and myometrium, suggesting a role in uterine function. Crosses with Wnt7a-Cre transgenic mice that express Cre in the Müllerian duct epithelium activate Wt1-directed RFP expression in the epithelium of the oviduct but not the stroma and myometrium of the uterus. This new mouse strain should be a useful resource for studies of Wt1 function and marking Wt1-expressing cells.

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.

Molecular tracking of interactions between progenitor and endothelial cells via Raman and FTIR spectroscopy imaging: a proof of concept of a new analytical strategy for in vitro research.

Circulating endothelial cell progenitors originating from the bone marrow are considered to be a powerful tool in the repair of endothelium damage. Due to their unique properties, endothelial progenitors are now broadly investigated to assess their clinical significance in diseases e.g., associated with brain endothelial dysfunction. However, their distinction in terms of the expression of specific markers remains ambiguous. Additionally, endothelial progenitor cells may change their repertoire of markers depending on the microenvironment of the tissue in which they are currently located. Here, we applied the label-free Raman and FTIR imaging to discriminate mice brain endothelium and endothelial progenitors. Cells cultured separately showed distinctly different spectral signatures extracted from the whole cellular interior as well as the detected intracellular compartments (nucleus, cytoplasm, perinuclear area, and lipid droplets). Then, we used these spectroscopic signals to examine the cells co-cultured for 24 h. Principal cluster analysis showed their grouping with the progenitor cells and segregation from brain endothelium at a level of the entire cell machinery (in FTIR images) which resulted from biochemical alternations in the cytoplasm and lipid droplets (in Raman images). The models included in partial least square regression indicated that lipid droplets are the key element for the classification of endothelial progenitor-brain endothelial cells interactions.

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.

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.

A transgenic Alx4-CreER mouse to analyze anterior limb and nephric duct development.

BACKGROUND: Genetic tools to study gene function and the fate of cells in the anterior limb bud are very limited. RESULTS: We describe a transgenic mouse line expressing CreERT2 from the Aristaless-like 4 (Alx4) promoter that induces recombination in the anterior limb. Cre induction at embryonic day 8.5 revealed that Alx4-CreERT2 labeled cells using the mTmG Cre reporter contributed to anterior digits I to III as well as the radius of the forelimb. Cre activity is expanded further along the AP axis in the hindlimb than in the forelimb resulting in some Cre reporter cells contributing to digit IV. Induction at later time points labeled cells that become progressively restricted to more anterior digits and proximal structures. Comparison of Cre expression from the Alx4 promoter transgene with endogenous Alx4 expression reveals Cre expression is slightly expanded posteriorly relative to the endogenous Alx4 expression. Using Alx4-CreERT2 to induce loss of intraflagellar transport 88 (Ift88), a gene required for ciliogenesis, hedgehog signaling, and limb patterning, did not cause overt skeletal malformations. However, the efficiency of deletion, time needed for Ift88 protein turnover, and for cilia to regress may hinder using this approach to analyze cilia in the limb. Alx4-CreERT2 is also active in the mesonephros and nephric duct that contribute to the collecting tubules and ducts of the adult nephron. Embryonic activation of the Alx4-CreERT2 in the Ift88 conditional line results in cyst formation in the collecting tubules/ducts. CONCLUSION: Overall, the Alx4-CreERT2 line will be a new tool to assess cell fates and analyze gene function in the anterior limb, mesonephros, and nephric duct.

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.

Estrogens and development of the rete testis, efferent ductules, epididymis and vas deferens.

Estrogen has always been considered the female hormone and testosterone the male hormone. However, estrogen's presence in the testis and deleterious effects of estrogen treatment during development have been known for nearly 90 years, long before estrogen receptors (ESRs) were discovered. Eventually it was learned that testes actually synthesize high levels of estradiol (E2) and sequester high concentrations in the reproductive tract lumen, which seems contradictory to the overwhelming number of studies showing reproductive pathology following exogenous estrogen exposures. For too long, the developmental pathology of estrogen has dominated our thinking, even resulting in the "estrogen hypothesis" as related to the testicular dysgenesis syndrome. However, these early studies and the development of an Esr1 knockout mouse led to a deluge of research into estrogen's potential role in and disruption of development and function of the male reproductive system. What is new is that estrogen action in the male cannot be divorced from that of androgen. This paper presents what is known about components of the estrogen pathway, including its synthesis and target receptors, and the need to achieve a balance between androgen- and estrogen-action in male reproductive tract differentiation and adult functions. The review focuses on what is known regarding development of the male reproductive tract, from the rete testis to the vas deferens, and examines the expression of estrogen receptors and presence of aromatase in the male reproductive system, traces the evidence provided by estrogen-associated knockout and transgenic animal models and discusses the effects of fetal and postnatal exposures to estrogens. Hopefully, there will be enough here to stimulate discussions and new investigations of the androgen:estrogen balance that seems to be essential for development of the male reproductive tract.

Unique and ancestral features in trunk kidney microanatomy and ultrastructure of omul Coregonus migratorius.

This study presents novel data on the microanatomy and ultrastructure of the omul Coregonus migratorius trunk kidney. Adult individuals of C. migratorius were sampled in the Barguzin Bay of Lake Baikal. Active leuko- and erythropoiesis were found in the interstitium of the mesonephros. For the first time, cells with radially arranged vesicles have been described in the renal interstitium of C. migratorius. The quantitative characteristics of blood cells and ultrastructural parameters of leukocytes reflected the functioning of the non-specific defence system in the organism. The share of the renal interstitium, morphological diversity of the epithelial cells of the nephron tubules, the ultrastructural features of the renal corpuscles and nephron tubules and the number of mitochondria in leukocytes and ion-transporting cells were typical for representatives of the whitefish Coregonus lavaretus complex and thus considered ancestral features of the present-day C. migratorius population reflecting its adaptive potential to living in an ultra-deep Lake Baikal.

Gata3 targets Runx1 in the embryonic haematopoietic stem cell niche.

Runx1 is an important haematopoietic transcription factor as stressed by its involvement in a number of haematological malignancies. Furthermore, it is a key regulator of the emergence of the first haematopoietic stem cells (HSCs) during development. The transcription factor Gata3 has also been linked to haematological disease and was shown to promote HSC production in the embryo by inducing the secretion of important niche factors. Both proteins are expressed in several different cell types within the aorta-gonads-mesonephros (AGM) region, in which the first HSCs are generated; however, a direct interaction between these two key transcription factors in the context of embryonic HSC production has not formally been demonstrated. In this current study, we have detected co-localisation of Runx1 and Gata3 in rare sub-aortic mesenchymal cells in the AGM. Furthermore, the expression of Runx1 is reduced in Gata3 -/- embryos, which also display a shift in HSC emergence. Using an AGM-derived cell line as a model for the stromal microenvironment in the AGM and performing ChIP-Seq and ChIP-on-chip experiments, we demonstrate that Runx1, together with other key niche factors, is a direct target gene of Gata3. In addition, we can pinpoint Gata3 binding to the Runx1 locus at specific enhancer elements which are active in the microenvironment. These results reveal a direct interaction between Gata3 and Runx1 in the niche that supports embryonic HSCs and highlight a dual role for Runx1 in driving the transdifferentiation of haemogenic endothelial cells into HSCs as well as in the stromal cells that support this process.

Growing a new human kidney.

There are 3 reasons to generate a new human kidney. The first is to learn more about the biology of the developing and mature organ. The second is to generate tissues with which to model congenital and acquired kidney diseases. In particular, growing human kidneys in this manner ultimately should help us understand the mechanisms of common chronic kidney diseases such as diabetic nephropathy and others featuring fibrosis, as well as nephrotoxicity. The third reason is to provide functional kidney tissues that can be used directly in regenerative medicine therapies. The second and third reasons to grow new human kidneys are especially compelling given the millions of persons worldwide whose lives depend on a functioning kidney transplant or long-term dialysis, as well as those with end-stage renal disease who die prematurely because they are unable to access these treatments. As shown in this review, the aim to create healthy human kidney tissues has been partially realized. Moreover, the technology shows promise in terms of modeling genetic disease. In contrast, barely the first steps have been taken toward modeling nongenetic chronic kidney diseases or using newly grown human kidney tissue for regenerative medicine therapies.

Endothelial-to-haematopoietic transition: an update on the process of making blood.

The first definitive blood cells during embryogenesis are derived from endothelial cells in a highly conserved process known as endothelial-to-haematopoietic transition (EHT). This conversion involves activation of a haematopoietic transcriptional programme in a subset of endothelial cells in the major vasculature of the embryo, followed by major morphological changes that result in transitioning cells rounding up, breaking the tight junctions to neighbouring endothelial cells and adopting a haematopoietic fate. The whole process is co-ordinated by a complex interplay of key transcription factors and signalling pathways, with additional input from surrounding tissues. Diverse model systems, including mouse, chick and zebrafish embryos as well as differentiation of pluripotent cells in vitro, have contributed to the elucidation of the details of the EHT, which was greatly accelerated in recent years by sophisticated live imaging techniques and advances in transcriptional profiling, such as single-cell RNA-Seq. A detailed knowledge of these developmental events is required in order to be able to apply it to the generation of haematopoietic stem cells from pluripotent stem cells in vitro - an achievement which is of obvious clinical importance. The aim of this review is to summarise the latest findings and describe how these may have contributed towards achieving this goal.

Wnt signaling orients the proximal-distal axis of chick kidney nephrons.

The nephron is the fundamental structural and functional unit of the kidney. Each mature nephron is patterned along a proximal-distal axis, with blood filtered at the proximal end and urine emerging from the distal end. In order to filter the blood and produce urine, specialized structures are formed at specific proximal-distal locations along the nephron, including the glomerulus at the proximal end, the tubule in the middle and the collecting duct at the distal end. The developmental processes that specify these different nephron segments are not fully understood. Wnt ligands, which are expressed in the nephric duct and later in the nascent nephron itself, are well-characterized inducers of nephrons, and are both required and sufficient for initiation of nephron formation from nephrogenic mesenchyme. Here, we present evidence that Wnt signaling also patterns the proximal-distal nephron axis. Using the chick mesonephros as a model system, a Wnt ligand was ectopically expressed in the coelomic lining, thereby introducing a source of Wnt signaling that is at right angles to the endogenous Wnt signal of the nephric duct. Under these conditions, the nephron axis was re-oriented, such that the glomerulus was always located at a position farthest from the Wnt sources. This re-orientation occurred within hours of exposure to ectopic Wnt signaling, and was accompanied initially by a repression of the early glomerular podocyte markers Wt1 and Pod1, followed by their re-emergence at a position distant from the Wnt signals. Activation of the Wnt signaling pathway in mesonephric explant cultures resulted in strong and specific repression of early and late glomerular markers. Finally, cytoplasmic ß-catenin, indicative of active canonical Wnt signaling, was found to be enriched in the distal as compared with the proximal region of the forming nephron. Together, these data indicate that Wnt signaling patterns the proximal-distal axis of the nephron, with glomeruli differentiating in regions of lowest Wnt signaling.

Multiple origins of embryonic and tadpole myeloid cells in Xenopus laevis.

Rabbit anti-serum against a myeloid-cell-specific peroxidase (Mpo) of Xenopus laevis was generated to identify myeloid cells in adult and larval animals. Smears of blood samples from adult hematopoietic organs were co-stained with Mpo and with XL-2, a mouse monoclonal antibody against a leukocyte common antigen. Lymphocytes found in the thymus and spleen were XL-2+Mpo- and granulocytes found in peripheral blood cells and the spleen were XL-2+Mpo+, indicating that double-staining with these two antibodies allowed classification of the leukocyte lineages. Immunohistochemical analysis of larval organs showed that XL-2+Mpo- cells were scattered throughout the liver, whereas XL-2+Mpo+ cells were present mainly in the cortex region. Interestingly, a cluster of XL-2+Mpo+ cells was found in the region of the larval mesonephric rudiment. The ratio of XL-2+Mpo+ cells to XL-2+ cells in the mesonephric region was approximately 80%, which was much higher than that found in other hematopoietic organs. In order to elucidate the embryonic origin of the myeloid cells in the tadpole mesonephros, grafting experiments between X. laevis and X. borealis embryos were performed to trace the X. borealis cells as donor cells. Among the embryonic tissues examined, the tailbud tissue at the early neurula stage contributed greatly to the myeloid cluster in the mesonephric region at stage 48. Therefore, at least four independent origins of the myeloid cell population can be traced in the Xenopus embryo.

Thrombopoietin contributes to the formation and the maintenance of hematopoietic progenitor-containing cell clusters in the aorta-gonad-mesonephros region.

In the midgestation mouse embryo, hematopoietic cell clusters containing hematopoietic stem/progenitor cells arise in the aorta-gonad-mesonephros (AGM) region. We have previously reported that forced expression of the Sox17 transcription factor in CD45lowc-Kithigh AGM cells, which are the hematopoietic cellular component of the cell clusters, and subsequent coculture with OP9 stromal cells in the presence of three cytokines, stem cell factor (SCF), interleukin-3 (IL-3), and thrombopoietin (TPO), led to the formation and the maintenance of cell clusters with cells at an undifferentiated state in vitro. In this study, we investigated the role of each cytokine in the formation of hematopoietic cell clusters. We cultured Sox17-transduced AGM cells with each of the 7 possible combinations of the three cytokines. The size and the number of Sox17-transduced cell clusters in the presence of TPO, either alone or in combination, were comparable to that observed with the complete set of the three cytokines. Expression of TPO receptor, c-Mpl was almost ubiquitously expressed and maintained in Sox17-transduced hematopoietic cell clusters. In addition, the expression level of c-Mpl was highest in the CD45lowc-Kithigh cells among the Sox17-transduced cell clusters. Moreover, c-Mpl protein was highly expressed in the intra-aortic hematopoietic cell clusters in comparison with endothelial cells of dorsal aorta. Finally, stimulation of the endothelial cells prepared from the AGM region by TPO induced the production of hematopoietic cells. These results suggest that TPO contributes to the formation and the maintenance of hematopoietic cell clusters in the AGM region.

Antennas of organ morphogenesis: the roles of cilia in vertebrate kidney development.

Cilia arose early during eukaryotic evolution, and their structural components are highly conserved from the simplest protists to complex metazoan species. In recent years, the role of cilia in the ontogeny of vertebrate organs has received increasing attention due to a staggering correlation between human disease and dysfunctional cilia. In particular, the presence of cilia in both the developing and mature kidney has become a deep area of research due to ciliopathies common to the kidney, such as polycystic kidney disease (PKD). Interestingly, mutations in genes encoding proteins that localize to the cilia cause similar cystic phenotypes in kidneys of various vertebrates, suggesting an essential role for cilia in kidney organogenesis and homeostasis as well. Importantly, the genes so far identified in kidney disease have conserved functions across species, whose kidneys include both primary and motile cilia. Here, we aim to provide a comprehensive description of cilia and their role in kidney development, as well as highlight the usefulness of the zebrafish embryonic kidney as a model to further understand the function of cilia in kidney health.