Single-cell profiling of the developing embryonic heart in Drosophila.

Drosophila is an important model for studying heart development and disease. Yet, single-cell transcriptomic data of its developing heart have not been performed. Here, we report single-cell profiling of the entire fly heart using ∼3000 Hand-GFP embryos collected at five consecutive developmental stages, ranging from bilateral migrating rows of cardiac progenitors to a fused heart tube. The data revealed six distinct cardiac cell types in the embryonic fly heart: cardioblasts, both Svp+ and Tin+ subtypes; and five types of pericardial cell (PC) that can be distinguished by four key transcription factors (Eve, Odd, Ct and Tin) and include the newly described end of the line PC. Notably, the embryonic fly heart combines transcriptional signatures of the mammalian first and second heart fields. Using unique markers for each heart cell type, we defined their number and location during heart development to build a comprehensive 3D cell map. These data provide a resource to track the expression of any gene in the developing fly heart, which can serve as a reference to study genetic perturbations and cardiac diseases.

Cells of the human intestinal tract mapped across space and time.

The cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. Here, to comprehensively map cell lineages, we use single-cell RNA sequencing and antigen receptor analysis of almost half a million cells from up to 5 anatomical regions in the developing and up to 11 distinct anatomical regions in the healthy paediatric and adult human gut. This reveals the existence of transcriptionally distinct BEST4 epithelial cells throughout the human intestinal tract. Furthermore, we implicate IgG sensing as a function of intestinal tuft cells. We describe neural cell populations in the developing enteric nervous system, and predict cell-type-specific expression of genes associated with Hirschsprung's disease. Finally, using a systems approach, we identify key cell players that drive the formation of secondary lymphoid tissue in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. This catalogue of intestinal cells will provide new insights into cellular programs in development, homeostasis and disease.

Regulation of Drosophila Hematopoiesis in Lymph Gland: From a Developmental Signaling Point of View.

The Drosophila hematopoietic system is becoming increasingly attractive for its simple blood cell lineage and its developmental and functional parallels with the vertebrate system. As the dedicated organ for Drosophila larval hematopoiesis, the lymph gland harbors both multipotent stem-like progenitor cells and differentiated blood cells. The balance between progenitor maintenance and differentiation in the lymph gland must be precisely and tightly controlled. Multiple developmental signaling pathways, such as Notch, Hedgehog, and Wnt/Wingless, have been demonstrated to regulate the hematopoietic processes in the lymph gland. Focusing on blood cell maintenance and differentiation, this article summarizes the functions of several classic developmental signaling pathways for lymph gland growth and patterning, highlighting the important roles of developmental signaling during lymph gland development as well as Drosophila larval hematopoiesis.

Intussusceptive lymphangiogenesis in the sinuses of developing human foetal lymph nodes.

A meshwork of intraluminal processes in lymph node (LN) sinuses originates during LN development. Lymph flows through the meshwork, which has an important role in immunology and pathology. However, the formation mechanism of intraluminal processes has not been sufficiently studied. Our objective is to assess whether this mechanism is by intussusception, as occurs in transcapillary pillar formation in blood vessel intussusceptive angiogenesis. For this purpose, LNs with developing intrasinusal processes were used (human foetuses, 13-18GW) for serial histologic sections and immunohistochemical procedures. The studies showed (a) sinuses originating from lymphatic sacs around expanded LN anlagen, (b) intra-sinus structures (lined by anti-podoplanin+, VEGFR3+, Prox-1+, CD31+ lymphatic endothelial cells) with characteristics (in serial sections and 3D images) similar to those considered the hallmarks of intussusceptive angiogenesis, including pillars (≤2.5µm, with a collagen core), interstitial tissue structures (ITSs) or larger pillars (>2.5µm, with a more cellular core) and folds (that form pillars when spanning), and (c) remodelled and fused pillars, ITSs and folds, which formed meshworks, compartmentalizing the sinuses into small intercommunicating spaces (segmentation). In conclusion, intussusception participates in the formation of the meshwork of processes in LN sinuses during LN development. This mechanism is also of interest because it contributes to the general knowledge of intussusceptive lymphangiogenesis (which has received less attention than intussusception in blood vessels), provides a basis for further studies and supports a new role for vessel intussusception (formation of an intraluminal meshwork with known action in fluid filtering, cell interactions and immunology).

Characterization of lymphatic malformations using primary cells and tissue transcriptomes.

Lymphatic malformations (LMs) are disfiguring congenital anomalies characterized by aberrant growth of lymphatic vessels. They are broadly categorized histopathologically as macrocystic and microcystic. Although sclerotherapy has shown some success in the treatment of macrocystic malformations, there has been less progress with developing treatment strategies for microcystic malformations. In this study, we characterized lymphatic endothelial cells isolated from lymphatic and lymphaticovenous malformations. When compared to cells from normal lymphatic vessels, we found that the primary cultured malformed cells are morphologically different and also exhibited differences in binding, proliferation, migration and tube formation. Transcriptome analysis identified several genes whose expression was substantially higher in malformed compared to normal lymphatic endothelium, including DIRAS3 and FOXF1. Further analysis of LM tissue samples revealed distinguishing gene expression patterns that could pave the way to understanding the molecular pathogenesis of LMs. Based on gene expression signatures, we propose a new hypothesis that the subtype of localized LMs could be formed because of disruptions in lymph node development.

Multiple roles of lymphatic vessels in peripheral lymph node development.

The mammalian lymphatic system consists of strategically located lymph nodes (LNs) embedded into a lymphatic vascular network. Mechanisms underlying development of this highly organized system are not fully understood. Using high-resolution imaging, we show that lymphoid tissue inducer (LTi) cells initially transmigrate from veins at LN development sites using gaps in venous mural coverage. This process is independent of lymphatic vasculature, but lymphatic vessels are indispensable for the transport of LTi cells that egress from blood capillaries elsewhere and serve as an essential LN expansion reservoir. At later stages, lymphatic collecting vessels ensure efficient LTi cell transport and formation of the LN capsule and subcapsular sinus. Perinodal lymphatics also promote local interstitial flow, which cooperates with lymphotoxin-ß signaling to amplify stromal CXCL13 production and thereby promote LTi cell retention. Our data unify previous models of LN development by showing that lymphatics intervene at multiple points to assist LN expansion and identify a new role for mechanical forces in LN development.

Expression of Pref-1 and Related Chemokines during theDevelopment of Rat Mesenteric Lymph Nodes.

OBJECTIVE: The aim of this study was to investigate the ability of Pref-1+ adipocyte progenitor cells to mobilize into mesenteric lymph nodes (MLNs) and the dynamic expression of related chemokines during the development of rat MLNs. METHODS: Immunohistochemical analyses were used to detect the expression of Pref-1 and related chemokines. Transmission electron microscopy (TEM) was used to observe the changes in ultrastructure of MLNs. RESULTS: Cells containing lipid droplets were found in all rat MLNs at embryonic day (E) 18.5, 2 and 6 weeks (w) after birth, and they were similar to fibroblastic reticular cells (FRCs) or follicular dendritic cells (FDCs) under TEM. Pref-1+ adipocyte progenitor cells were found in all MLNs. The expression level of Pref-1 was significantly increased at 2 w after birth and decreased at 6 w after birth. The tendency of Cxcl12 expression was consistent with that of Pref-1 and was positively correlated with the expression of Pref-1 (P < 0.01; r = 0.897). At E18.5, Cxcl13, and Ccr7 were significantly expressed in the MLN anlage, but the expression level of Ccl21 was low. The expression level of Cxcl13, Ccr7, and Ccl21 in MLN were significantly increased at 2 w after birth (P < 0.05), while the expression of Ccr7 and Ccl21 were significantly decreased at 6 w after birth (P < 0.05). CONCLUSION: Adipocyte progenitor cells are involved in the rat MLNs development through differentiation into FRC and FDC. The expression of the relevant chemokines during the development of MLNs is dynamic and may be related to the maintenance of lymph nodes self-balance state.

Differential Roles of LTßR in Endothelial Cell Subsets for Lymph Node Organogenesis and Maturation.

Cellular cross-talk mediated by lymphotoxin αß-lymphotoxin ß receptor (LTßR) signaling plays a critical role in lymph node (LN) development. Although the major role of LTßR signaling has long been considered to occur in mesenchymal lymphoid tissue organizer cells, a recent study using a VE-cadherincreLtbrfl/fl mouse model suggested that endothelial LTßR signaling contributes to the formation of LNs. However, the detailed roles of LTßR in different endothelial cells (ECs) in LN development remain unknown. Using various cre transgenic mouse models (Tekcre , a strain targeting ECs, and Lyve1cre , mainly targeting lymphatic ECs), we observed that specific LTßR ablation in Tekcre+ or Lyve1cre+ cells is not required for LN formation. Moreover, double-cre-mediated LTßR depletion does not interrupt LN formation. Nevertheless, TekcreLtbrfl/fl mice exhibit reduced lymphoid tissue inducer cell accumulation at the LN anlagen and impaired LN maturation. Interestingly, a subset of ECs (VE-cadherin+Tekcre-low/neg ECs) was found to be enriched in transcripts related to hematopoietic cell recruitment and transendothelial migration, resembling LN high ECs in adult animals. Furthermore, endothelial Tek was observed to negatively regulate hematopoietic cell transmigration. Taken together, our data suggest that although Tekcre+ endothelial LTßR is required for the accumulation of hematopoietic cells and full LN maturation, LTßR in VE-cadherin+Tekcre-low/neg ECs in embryos might represent a critical portal-determining factor for LN formation.

The mir-7 and bag of marbles genes regulate Hedgehog pathway signaling in blood cell progenitors in Drosophila larval lymph glands.

Hedgehog (Hh) pathway signaling is crucial for the maintenance of blood cell progenitors in the lymph gland hematopoietic organ present in Drosophila third instar larvae. Previous studies from our lab have likewise shown the importance of the mir-7 and bag of marbles (bam) genes in maintaining the progenitor state. Thus, we sought to investigate a possible interaction between the Hh pathway and mir-7/bam in the prohemocyte population within this hematopoietic tissue. Gain of function mir-7 was able to rescue a blood cell progenitor depletion phenotype caused by Patched (Ptc) inhibition of Hh pathway signaling in these cells. Similarly, expression of a dominant/negative version of Ptc was able to rescue the severe reduction of prohemocytes due to bam loss of function. Furthermore, we demonstrated that Suppressor of fused [Su(fu)], another known inhibitor of Hh signaling, likely serves as a translational repression target of the mir-7 miRNA. Our results suggest the mir-7/bam combination regulates the Hh signaling network through repression of Su(fu) to maintain hemocyte progenitors in the larval lymph gland.

Coordinated lymphangiogenesis is critical in lymph node development and maturation.

BACKGROUND: Lymph node (LN) formation requires multiple but coordinated signaling from intrinsic and extrinsic cellular components during embryogenesis. However, the contribution and role of lymphatic vessels (LVs) in LN formation and maturation are poorly defined. Here, using lymphatic-specific reporters, Prox1-GFP mice and Vegfc+/LacZ mice, we analyzed migration, assembly, and ingrowth of lymphatic endothelial cells (LECs) in LNs during pre- and postnatal development. RESULTS: Prox1+ LECs form string-like connections rather than lymph sac-like structures until E14.5, but the LEC coverage around LN anlagen completes before birth. Compared to wild-type littermates, Vegfc+/LacZ mice had markedly smaller LNs in neonates and adults, presumably due to the decrease in LTi cell clusters and migrating Prox1+ LECs during embryogenesis. In addition, Vegfc-haploinsufficiency or inhibition of VEGFR3 signaling led to an impairment of LN LV ingrowth, resulting in a significant decrease in LN volume. These data indicate that VEGF-C/VEGFR3 signaling plays a substantial role in normal LN formation through proper migration and organization of LECs. CONCLUSIONS: Taken together, our results provide compelling evidence that the contribution of LVs through VEGF-C/VEGFR3 signaling is critical in LN development and maturation. Developmental Dynamics 245:1189-1197, 2016. © 2016 Wiley Periodicals, Inc.

Healthy human second-trimester fetal skin is deficient in leukocytes and associated homing chemokines.

The lack of immune cells in mid-gestational fetal skin is often mentioned as a key factor underlying scarless healing. However, the scarless healing ability is conserved until long after the immune system in the fetus is fully developed. Therefore, we studied human second-trimester fetal skin and compared the numbers of immune cells and chemokine levels from fetal skin with adult skin. By using immunohistochemistry, we show that healthy fetal skin contains significant lower numbers of CD68(+) -macrophages, Tryptase(+) -mast cells, Langerin(+) -Langerhans cells, CD1a(+) -dendritic cells, and CD3(+) -T cells compared to adult skin. Staining with an early lineage leukocyte marker, i.e., CD45, verified that the number of CD45(+) -immune cells was indeed significantly lower in fetal skin but that sufficient numbers of immune cells were present in the fetal lymph node. No differences in the vascular network were observed between fetal and adult skin. Moreover, significant lower levels of lymphocyte chemokines CCL17, CCL21, and CCL27 were observed in fetal skin. However, levels of inflammatory interleukins such as IL-6, IL-8, and IL-10 were undetectable and levels of CCL2 were similar in healthy fetal and adult skin. In conclusion, this study shows that second-trimester fetal skin contains low levels of immune cells and leukocyte chemokines compared to adult skin. This immune cell deficiency includes CD45(+) leukocytes, despite the abundant presence of these cells in the lymph node. The immune deficiency in healthy second-trimester fetal skin may result in reduced inflammation during wound healing, and could underlie the scarless healing capacities of the fetal skin.

Differentiation and function of group 3 innate lymphoid cells, from embryo to adult.

Group 3 innate lymphoid cells (ILC3) represent a heterogeneous population of cells that share the nuclear hormone receptor RORγt (retinoic acid receptor-related orphan receptor γt) as a master regulator for differentiation and function. ILC3 can be divided into two major subsets based on the cell surface expression of the natural cytotoxicity receptor (NCR), NKp46. A subset of NCR(-) ILC3 includes the previously known lymphoid-tissue inducer cells that are essential for the embryonic formation of peripheral lymph nodes and Peyer's patches. After birth, the NCR(-) and NCR(+) ILC3 contribute to the maintenance of health but also to inflammation in mucosal tissues. This review will describe the differentiation pathways of ILC3, their involvement in the development of the adaptive immune system and their role in the establishment and maintenance of gut immunity.

[PERIPHERAL LYMPHOID STRUCTURES: FORMATION AND FUNCTION].

The problem of the formation of new lymphoid structures (neolymphogenesis) is quite controversial and widely discussed in the literature. Under normal conditions, lymphoid organs arise only in the process of fetal development (organogenesis), however in long-standing chronic inflammatory processes, nonhealing wounds, autoimmune diseases, oncologic pathology spontaneous formation of new lymphoid structures was noted. The structures of the peripheral lymphoid formations include the lymphocytes arranged singly and in clusters (infiltration), lymphoid nodules and lymph nodes. The morphogenesis of the components of lymphoid tissue and the possibility of creating artificial lymphoid structures, reproducing the function of the natural ones, is demonstrated. Important role in the development of lymphoid structures is played by mediators of inflammation, cytokines of the family of lymphotoxins, tumor necrosis factor. The possibilities of prosthetic substitution of the functions of the lymphoid structures are described for the activation of protective processes in the body.

The chemokine receptors ACKR2 and CCR2 reciprocally regulate lymphatic vessel density.

Macrophages regulate lymphatic vasculature development; however, the molecular mechanisms regulating their recruitment to developing, and adult, lymphatic vascular sites are not known. Here, we report that resting mice deficient for the inflammatory chemokine-scavenging receptor, ACKR2, display increased lymphatic vessel density in a range of tissues under resting and regenerating conditions. This appears not to alter dendritic cell migration to draining lymph nodes but is associated with enhanced fluid drainage from peripheral tissues and thus with a hypotensive phenotype. Examination of embryonic skin revealed that this lymphatic vessel density phenotype is developmentally established. Further studies indicated that macrophages and the inflammatory CC-chemokine CCL2, which is scavenged by ACKR2, are associated with this phenotype. Accordingly, mice deficient for the CCL2 signalling receptor, CCR2, displayed a reciprocal phenotype of reduced lymphatic vessel density. Further examination revealed that proximity of pro-lymphangiogenic macrophages to developing lymphatic vessel surfaces is increased in ACKR2-deficient mice and reduced in CCR2-deficient mice. Therefore, these receptors regulate vessel density by reciprocally modulating pro-lymphangiogenic macrophage recruitment, and proximity, to developing, resting and regenerating lymphatic vessels.

Polycystin signaling is required for directed endothelial cell migration and lymphatic development.

Autosomal dominant polycystic kidney disease is a common form of inherited kidney disease that is caused by mutations in two genes, PKD1 (polycystin-1) and PKD2 (polycystin-2). Mice with germline deletion of either gene die in midgestation with a vascular phenotype that includes profound edema. Although an endothelial cell defect has been suspected, the basis of this phenotype remains poorly understood. Here, we demonstrate that edema in Pkd1- and Pkd2-null mice is likely to be caused by defects in lymphatic development. Pkd1 and Pkd2 mutant embryos exhibit reduced lymphatic vessel density and vascular branching along with aberrant migration of early lymphatic endothelial cell precursors. We used cell-based assays to confirm that PKD1- and PKD2-depleted endothelial cells have an intrinsic defect in directional migration that is associated with a failure to establish front-rear polarity. Our studies reveal a role for polycystin signaling in lymphatic development.

Fetal immune response to chorioamnionitis.

Chorioamnionitis is a frequent cause of preterm birth and is associated with an increased risk for injury responses in the lung, gastrointestinal tract, brain, and other fetal organs. Chorioamnionitis is a polymicrobial nontraditional infectious disease because the organisms causing chorioamnionitis are generally of low virulence and colonize the amniotic fluid often for extended periods, and the host (mother and the fetus) does not have typical infection-related symptoms such as fever. In this review, we discuss the effects of chorioamnionitis in experimental animal models that mimic the human disease. Our focus is on the immune changes in multiple fetal organs and the pathogenesis of chorioamnionitis-induced injury in different fetal compartments. As chorioamnionitis disproportionately affects preterm infants, we discuss the relevant developmental context for the immune system. We also provide a clinical context for the fetal responses.

Development of secondary lymphoid organs in relation to lymphatic vasculature.

Although the initial event in lymphatic endothelial specification occurs slightly before the initiation of lymph node formation in mice, the development of lymphatic vessels and lymph nodes occurs within the same embryonic time frame. Specification of lymphatic endothelial cells starts around embryonic day 10 (E10), followed by endothelial cell budding and formation of the first lymphatic structures. Through lymphatic endothelial cell sprouting these lymph sacs give rise to the lymphatic vasculature which is complete by E15.5 in mice. It is within this time frame that lymph node formation is initiated and the first structure is secured in place. As lymphatic vessels are crucially involved in the functionality of the lymph nodes, the recent insight that both structures depend on common developmental signals for their initiation provides a molecular mechanism for their coordinated formation. Here, we will describe the common developmental signals needed to properly start the formation of lymphatic vessels and lymph nodes and their interdependence in adult life.

Generation of lymph node-fat pad chimeras for the study of lymph node stromal cell origin.

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (Bénézech et al.). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP(+) embryonic adipose tissue and make a LN/ EYFP(+) fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP(+) fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

IL-7-producing stromal cells are critical for lymph node remodeling.

Nonhematopoietic stromal cells of secondary lymphoid organs form important scaffold and fluid transport structures, such as lymph node (LN) trabeculae, lymph vessels, and conduits. Furthermore, through the production of chemokines and cytokines, these cells generate a particular microenvironment that determines lymphocyte positioning and supports lymphocyte homeostasis. IL-7 is an important stromal cell-derived cytokine that has been considered to be derived mainly from T-cell zone fibroblastic reticular cells. We show here that lymphatic endothelial cells (LECs) are a prominent source of IL-7 both in human and murine LNs. Using bacterial artificial chromosome transgenic IL-7-Cre mice, we found that fibroblastic reticular cells and LECs strongly up-regulated IL-7 expression during LN remodeling after viral infection and LN reconstruction after avascular transplantation. Furthermore, IL-7-producing stromal cells contributed to de novo formation of LyveI-positive lymphatic structures connecting reconstructed LNs with the surrounding tissue. Importantly, diphtheria toxin-mediated depletion of IL-7-producing stromal cells completely abolished LN reconstruction. Taken together, this study identifies LN LECs as a major source of IL-7 and shows that IL-7-producing stromal cells are critical for reconstruction and remodeling of the distinct LN microenvironment.

Expression pattern changes and function of RANKL during mouse lymph node microarchitecture development.

Receptor activator of nuclear factor kappa-B ligand (RANKL) expression was examined during the development of mouse fetal peripheral lymphoid organs. A shift in the expression pattern was detected during the transition from lymphoid tissue inducer (LTi) cells to lymphoid tissue organizer (LTo) cells in the lymph node (LN) anlagen but not in the Peyer's patch anlagen. In order to understand the functional impact of these changes in the fetal expression of RANKL, the RANKL function was blocked by a blocking antibody. Excess anti-RANKL antibody was administered to pregnant mice between 13.5 and 16.5 dpc and was found to completely block LN anlagen development, suggesting that RANKL function during this period is critical for LN development. In addition, small amounts of anti-RANKL antibodies were injected directly into the amniotic space at 13.5 dpc, resulting in perturbed B-cell follicle formation and high endothelial venule differentiation after birth. These results suggest that RANKL expression on LTi cells during the early phase of LN development is critical for the development LN microarchitecture.