Gucy1α1 specifically marks kidney, heart, lung and liver fibroblasts.

Fibrosis is a common outcome of numerous pathologies, including chronic kidney disease (CKD), a progressive renal function deterioration. Current approaches to target activated fibroblasts, key effector contributors to fibrotic tissue remodeling, lack specificity. Here, we report Gucy1α1 as a specific kidney fibroblast marker. Gucy1α1 levels significantly increased over the course of two clinically relevant murine CKD models and directly correlated with established fibrosis markers. Immunofluorescent (IF) imaging showed that Gucy1α1 comprehensively labelled cortical and medullary quiescent and activated fibroblasts in the control kidney and throughout injury progression, respectively. Unlike traditionally used markers platelet derived growth factor receptor beta (Pdgfrß) and vimentin (Vim), Gucy1α1 did not overlap with off-target populations such as podocytes. Notably, Gucy1α1 labelled kidney fibroblasts in both male and female mice. Furthermore, we observed elevated GUCY1α1 expression in the human fibrotic kidney and lung. Studies in the murine models of cardiac and liver fibrosis revealed Gucy1α1 elevation in activated Pdgfrß-, Vim- and alpha smooth muscle actin (αSma)-expressing fibroblasts paralleling injury progression and resolution. Overall, we demonstrate Gucy1α1 as an exclusive fibroblast marker in both sexes. Due to its multiorgan translational potential, GUCY1α1 might provide a novel promising strategy to specifically target and mechanistically examine fibroblasts.

Single-cell sequencing dissects the transcriptional identity of activated fibroblasts and identifies novel persistent distal tubular injury patterns in kidney fibrosis.

Examining kidney fibrosis is crucial for mechanistic understanding and developing targeted strategies against chronic kidney disease (CKD). Persistent fibroblast activation and tubular epithelial cell (TEC) injury are key CKD contributors. However, cellular and transcriptional landscapes of CKD and specific activated kidney fibroblast clusters remain elusive. Here, we analyzed single cell transcriptomic profiles of two clinically relevant kidney fibrosis models which induced robust kidney parenchymal remodeling. We dissected the molecular and cellular landscapes of kidney stroma and newly identified three distinctive fibroblast clusters with "secretory", "contractile" and "vascular" transcriptional enrichments. Also, both injuries generated failed repair TECs (frTECs) characterized by decline of mature epithelial markers and elevation of stromal and injury markers. Notably, frTECs shared transcriptional identity with distal nephron segments of the embryonic kidney. Moreover, we identified that both models exhibited robust and previously unrecognized distal spatial pattern of TEC injury, outlined by persistent elevation of renal TEC injury markers including Krt8 and Vcam1, while the surviving proximal tubules (PTs) showed restored transcriptional signature. We also found that long-term kidney injuries activated a prominent nephrogenic signature, including Sox4 and Hox gene elevation, which prevailed in the distal tubular segments. Our findings might advance understanding of and targeted intervention in fibrotic kidney disease.

Tissue-resident macrophages specifically express Lactotransferrin and Vegfc during ear pinna regeneration in spiny mice.

The details of how macrophages control different healing trajectories (regeneration vs. scar formation) remain poorly defined. Spiny mice (Acomys spp.) can regenerate external ear pinnae tissue, whereas lab mice (Mus musculus) form scar tissue in response to an identical injury. Here, we used this dual species system to dissect macrophage phenotypes between healing modes. We identified secreted factors from activated Acomys macrophages that induce a pro-regenerative phenotype in fibroblasts from both species. Transcriptional profiling of Acomys macrophages and subsequent in vitro tests identified VEGFC, PDGFA, and Lactotransferrin (LTF) as potential pro-regenerative modulators. Examining macrophages in vivo, we found that Acomys-resident macrophages secreted VEGFC and LTF, whereas Mus macrophages do not. Lastly, we demonstrate the requirement for VEGFC during regeneration and find that interrupting lymphangiogenesis delays blastema and new tissue formation. Together, our results demonstrate that cell-autonomous mechanisms govern how macrophages react to the same stimuli to differentially produce factors that facilitate regeneration.

Single-cell sequencing dissects the transcriptional identity of activated fibroblasts and identifies novel persistent distal tubular injury patterns in kidney fibrosis.

Examining kidney fibrosis is crucial for mechanistic understanding and developing targeted strategies against chronic kidney disease (CKD). Persistent fibroblast activation and tubular epithelial cell (TEC) injury are key CKD contributors. However, cellular and transcriptional landscapes of CKD and specific activated kidney fibroblast clusters remain elusive. Here, we analyzed single cell transcriptomic profiles of two clinically relevant kidney fibrosis models which induced robust kidney parenchymal remodeling. We dissected the molecular and cellular landscapes of kidney stroma and newly identified three distinctive fibroblast clusters with "secretory", "contractile" and "vascular" transcriptional enrichments. Also, both injuries generated failed repair TECs (frTECs) characterized by decline of mature epithelial markers and elevation of stromal and injury markers. Notably, frTECs shared transcriptional identity with distal nephron segments of the embryonic kidney. Moreover, we identified that both models exhibited robust and previously unrecognized distal spatial pattern of TEC injury, outlined by persistent elevation of renal TEC injury markers including Krt8, while the surviving proximal tubules (PTs) showed restored transcriptional signature. Furthermore, we found that long-term kidney injuries activated a prominent nephrogenic signature, including Sox4 and Hox gene elevation, which prevailed in the distal tubular segments. Our findings might advance understanding of and targeted intervention in fibrotic kidney disease.

Single cell transcriptomic analysis of HPV16-infected epithelium identifies a keratinocyte subpopulation implicated in cancer.

Persistent HPV16 infection is a major cause of the global cancer burden. The viral life cycle is dependent on the differentiation program of stratified squamous epithelium, but the landscape of keratinocyte subpopulations which support distinct phases of the viral life cycle has yet to be elucidated. Here, single cell RNA sequencing of HPV16 infected compared to uninfected organoids identifies twelve distinct keratinocyte populations, with a subset mapped to reconstruct their respective 3D geography in stratified squamous epithelium. Instead of conventional terminally differentiated cells, an HPV-reprogrammed keratinocyte subpopulation (HIDDEN cells) forms the surface compartment and requires overexpression of the ELF3/ESE-1 transcription factor. HIDDEN cells are detected throughout stages of human carcinogenesis including primary human cervical intraepithelial neoplasias and HPV positive head and neck cancers, and a possible role in promoting viral carcinogenesis is supported by TCGA analyses. Single cell transcriptome information on HPV-infected versus uninfected epithelium will enable broader studies of the role of individual keratinocyte subpopulations in tumor virus infection and cancer evolution.

IL-1/MyD88-Dependent G-CSF and IL-6 Secretion Mediates Postburn Anemia.

The anemia of critical illness (ACI) is a nearly universal pathophysiological consequence of burn injury and a primary reason burn patients require massive quantities of transfused blood. Inflammatory processes are expected to drive postburn ACI and prevent meaningful erythropoietic stimulation through iron or erythropoietin supplementation, but to this day no specific inflammatory pathways have been identified as a critical mechanism. In this study, we examined whether secretion of G-CSF and IL-6 mediates distinct features of postburn ACI and interrogated inflammatory mechanisms that could be responsible for their secretion. Our analysis of mouse and human skin samples identified the burn wound as a primary source of G-CSF and IL-6 secretion. We show that G-CSF and IL-6 are secreted independently through an IL-1/MyD88-dependent mechanism, and we ruled out TLR2 and TLR4 as critical receptors. Our results indicate that IL-1/MyD88-dependent G-CSF secretion plays a key role in impairing medullary erythropoiesis and IL-6 secretion plays a key role in limiting the access of erythroid cells to iron. Importantly, we found that IL-1α/ß neutralizing Abs broadly attenuated features of postburn ACI that could be attributed to G-CSF or IL-6 secretion and rescued deficits of circulating RBC counts, hemoglobin, and hematocrit caused by burn injury. We conclude that wound-based IL-1/MyD88 signaling mediates postburn ACI through induction of G-CSF and IL-6 secretion.

Fibroblast inflammatory priming determines regenerative versus fibrotic skin repair in reindeer.

Adult mammalian skin wounds heal by forming fibrotic scars. We report that full-thickness injuries of reindeer antler skin (velvet) regenerate, whereas back skin forms fibrotic scar. Single-cell multi-omics reveal that uninjured velvet fibroblasts resemble human fetal fibroblasts, whereas back skin fibroblasts express inflammatory mediators mimicking pro-fibrotic adult human and rodent fibroblasts. Consequently, injury elicits site-specific immune responses: back skin fibroblasts amplify myeloid infiltration and maturation during repair, whereas velvet fibroblasts adopt an immunosuppressive phenotype that restricts leukocyte recruitment and hastens immune resolution. Ectopic transplantation of velvet to scar-forming back skin is initially regenerative, but progressively transitions to a fibrotic phenotype akin to the scarless fetal-to-scar-forming transition reported in humans. Skin regeneration is diminished by intensifying, or enhanced by neutralizing, these pathologic fibroblast-immune interactions. Reindeer represent a powerful comparative model for interrogating divergent wound healing outcomes, and our results nominate decoupling of fibroblast-immune interactions as a promising approach to mitigate scar.

Single-cell multiomics identifies clinically relevant mesenchymal stem-like cells and key regulators for MPNST malignancy.

Malignant peripheral nerve sheath tumor (MPNST), a highly aggressive Schwann cell (SC)-derived soft tissue sarcoma, arises from benign neurofibroma (NF); however, the identity, heterogeneity and origins of tumor populations remain elusive. Nestin+ cells have been implicated as tumor stem cells in MPNST; unexpectedly, single-cell profiling of human NF and MPNST and their animal models reveal a broad range of nestin-expressing SC lineage cells and dynamic acquisition of discrete cancer states during malignant transformation. We uncover a nestin-negative mesenchymal neural crest-like subpopulation as a previously unknown malignant stem-like state common to murine and human MPNSTs, which correlates with clinical severity. Integrative multiomics profiling further identifies unique regulatory networks and druggable targets against the malignant subpopulations in MPNST. Targeting key epithelial-mesenchymal transition and stemness regulators including ZEB1 and ALDH1A1 impedes MPNST growth. Together, our studies reveal the underlying principles of tumor cell-state evolution and their regulatory circuitries during NF-to-MPNST transformation, highlighting a hitherto unrecognized mesenchymal stem-like subpopulation in MPNST disease progression.

Olig2 defines a subset of neural stem cells that produce specific olfactory bulb interneuron subtypes in the subventricular zone of adult mice.

Distinct neural stem cells (NSCs) reside in different regions of the subventricular zone (SVZ) and generate multiple olfactory bulb (OB) interneuron subtypes in the adult brain. However, the molecular mechanisms underlying such NSC heterogeneity remain largely unknown. Here, we show that the basic helix-loop-helix transcription factor Olig2 defines a subset of NSCs in the early postnatal and adult SVZ. Olig2-expressing NSCs exist broadly but are most enriched in the ventral SVZ along the dorsoventral axis complementary to dorsally enriched Gsx2-expressing NSCs. Comparisons of Olig2-expressing NSCs from early embryonic to adult stages using single cell transcriptomics reveal stepwise developmental changes in their cell cycle and metabolic properties. Genetic studies further show that cross-repression contributes to the mutually exclusive expression of Olig2 and Gsx2 in NSCs/progenitors during embryogenesis, but that their expression is regulated independently from each other in adult NSCs. Finally, lineage-tracing and conditional inactivation studies demonstrate that Olig2 plays an important role in the specification of OB interneuron subtypes. Altogether, our study demonstrates that Olig2 defines a unique subset of adult NSCs enriched in the ventral aspect of the adult SVZ.

Cross talk between LAM cells and fibroblasts may influence alveolar epithelial cell behavior in lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a female-specific cystic lung disease in which tuberous sclerosis complex 2 (TSC2)-deficient LAM cells, LAM-associated fibroblasts (LAFs), and other cell types infiltrate the lungs. LAM lesions can be associated with type II alveolar epithelial (AT2) cells. We hypothesized that the behavior of AT2 cells in LAM is influenced locally by LAFs. We tested this hypothesis in the patient samples and in vitro. In human LAM lung, nodular AT2 cells show enhanced proliferation when compared with parenchymal AT2 cells, demonstrated by increased Ki67 expression. Furthermore, nodular AT2 cells express proteins associated with epithelial activation in other disease states including matrix metalloproteinase 7, and fibroblast growth factor 7 (FGF7). In vitro, LAF-conditioned medium is mitogenic and positively chemotactic for epithelial cells, increases the rate of epithelial repair, and protects against apoptosis. In vitro, LAM patient-derived TSC2 null cells cocultured with LAFs upregulate LAF expression of the epithelial chemokine and mitogen FGF7, a potential mediator of fibroblast-epithelial cross talk, in a mechanistic target of rapamycin (mTOR)-dependent manner. In a novel in vitro model of LAM, ex vivo cultured LAM lung-derived microtissues promote both epithelial migration and adhesion. Our findings suggest that AT2 cells in LAM display a proliferative, activated phenotype and fibroblast accumulation following LAM cell infiltration into the parenchyma contributes to this change in AT2 cell behavior. Fibroblast-derived FGF7 may contribute to the cross talk between LAFs and hyperplastic epithelium in vivo, but does not appear to be the main driver of the effects of LAFs on epithelial cells in vitro.

HoxD transcription factors define monosynaptic sensory-motor specificity in the developing spinal cord.

The specificity of monosynaptic connections between proprioceptive sensory neurons and their recipient spinal motor neurons depends on multiple factors, including motor neuron positioning and dendrite morphology, axon projection patterns of proprioceptive sensory neurons in the spinal cord, and the ligand-receptor molecules involved in cell-to-cell recognition. However, with few exceptions, the transcription factors engaged in this process are poorly characterized. Here, we show that members of the HoxD family of transcription factors play a crucial role in the specificity of monosynaptic sensory-motor connections. Mice lacking Hoxd9, Hoxd10 and Hoxd11 exhibit defects in locomotion but have no obvious defects in motor neuron positioning or dendrite morphology through the medio-lateral and rostro-caudal axes. However, we found that quadriceps motor neurons in these mice show aberrant axon development and receive inappropriate inputs from proprioceptive sensory axons innervating the obturator muscle. These genetic studies demonstrate that the HoxD transcription factors play an integral role in the synaptic specificity of monosynaptic sensory-motor connections in the developing spinal cord.

Combinatorial Transcriptional Profiling of Mouse and Human Enteric Neurons Identifies Shared and Disparate Subtypes In Situ.

BACKGROUND & AIMS: The enteric nervous system (ENS) coordinates essential intestinal functions through the concerted action of diverse enteric neurons (ENs). However, integrated molecular knowledge of EN subtypes is lacking. To compare human and mouse ENs, we transcriptionally profiled healthy ENS from adult humans and mice. We aimed to identify transcripts marking discrete neuron subtypes and visualize conserved EN subtypes for humans and mice in multiple bowel regions. METHODS: Human myenteric ganglia and adjacent smooth muscle were isolated by laser-capture microdissection for RNA sequencing. Ganglia-specific transcriptional profiles were identified by computationally subtracting muscle gene signatures. Nuclei from mouse myenteric neurons were isolated and subjected to single-nucleus RNA sequencing, totaling more than 4 billion reads and 25,208 neurons. Neuronal subtypes were defined using mouse single-nucleus RNA sequencing data. Comparative informatics between human and mouse data sets identified shared EN subtype markers, which were visualized in situ using hybridization chain reaction. RESULTS: Several EN subtypes in the duodenum, ileum, and colon are conserved between humans and mice based on orthologous gene expression. However, some EN subtype-specific genes from mice are expressed in completely distinct morphologically defined subtypes in humans. In mice, we identified several neuronal subtypes that stably express gene modules across all intestinal segments, with graded, regional expression of 1 or more marker genes. CONCLUSIONS: Our combined transcriptional profiling of human myenteric ganglia and mouse EN provides a rich foundation for developing novel intestinal therapeutics. There is congruency among some EN subtypes, but we note multiple species differences that should be carefully considered when relating findings from mouse ENS research to human gastrointestinal studies.

Single-Cell Profiling of AKI in a Murine Model Reveals Novel Transcriptional Signatures, Profibrotic Phenotype, and Epithelial-to-Stromal Crosstalk.

BACKGROUND: Current management of AKI, a potentially fatal disorder that can also initiate or exacerbate CKD, is merely supportive. Therefore, deeper understanding of the molecular pathways perturbed in AKI is needed to identify targets with potential to lead to improved treatment. METHODS: We performed single-cell RNA sequencing (scRNA-seq) with the clinically relevant unilateral ischemia-reperfusion murine model of AKI at days 1, 2, 4, 7, 11, and 14 after AKI onset. Using real-time quantitative PCR, immunofluorescence, Western blotting, and both chromogenic and single-molecule in situ hybridizations, we validated AKI signatures in multiple experiments. RESULTS: Our findings show the time course of changing gene expression patterns for multiple AKI stages and all renal cell types. We observed elevated expression of crucial injury response factors-including kidney injury molecule-1 (Kim1), lipocalin 2 (Lcn2), and keratin 8 (Krt8)-and of several novel genes (Ahnak, Sh3bgrl3, and Col18a1) not previously examined in kidney pathologies. AKI induced proximal tubule dedifferentiation, with a pronounced nephrogenic signature represented by Sox4 and Cd24a. Moreover, AKI caused the formation of "mixed-identity cells" (expressing markers of different renal cell types) that are normally seen only during early kidney development. The injured tubules acquired a proinflammatory and profibrotic phenotype; moreover, AKI dramatically modified ligand-receptor crosstalk, with potential pathologic epithelial-to-stromal interactions. Advancing age in AKI onset was associated with maladaptive response and kidney fibrosis. CONCLUSIONS: The scRNA-seq, comprehensive, cell-specific profiles provide a valuable resource for examining molecular pathways that are perturbed in AKI. The results fully define AKI-associated dedifferentiation programs, potential pathologic ligand-receptor crosstalk, novel genes, and the improved injury response in younger mice, and highlight potential targets of kidney injury.

Single-Cell Transcriptomic Analysis Identifies a Unique Pulmonary Lymphangioleiomyomatosis Cell.

Rationale: Lymphangioleiomyomatosis (LAM) is a metastatic neoplasm of reproductive-age women associated with mutations in tuberous sclerosis complex genes. LAM causes cystic remodeling of the lung and progressive respiratory failure. The sources and cellular characteristics of LAM cells underlying disease pathogenesis remain elusive.Objectives: Identification and characterization of LAM cells in human lung and uterus using a single-cell approach.Methods: Single-cell and single-nuclei RNA sequencing on LAM (n = 4) and control (n = 7) lungs, immunofluorescence confocal microscopy, ELISA, and aptamer proteomics were used to identify and validate LAMCORE cells and secreted biomarkers, predict cellular origins, and define molecular and cellular networks in LAM.Measurements and Main Results: A unique cell type termed LAMCORE was identified, which was distinct from, but closely related to, lung mesenchymal cells. LAMCORE cells expressing signature genes included known LAM markers such as PMEL, FIGF, CTSK, and MLANA and novel biomarkers validated by aptamer screening, ELISA, and immunofluorescence microscopy. LAM cells in lung and uterus are morphologically indistinguishable and share similar gene expression profiles and biallelic TSC2 mutations, supporting a potential uterine origin for the LAMCORE cell. Effects of LAM on resident pulmonary cell types indicated recruitment and activation of lymphatic endothelial cells.Conclusions: A unique population of LAMCORE cells was identified in lung and uterus of patients with LAM, sharing close transcriptomic identity. LAM cell selective markers, secreted biomarkers, and the predicted cellular molecular features provide new insights into the signaling and transcriptional programs that may serve as diagnostic markers and therapeutic targets to influence the pathogenesis of LAM.

Physical interactions between Gsx2 and Ascl1 balance progenitor expansion versus neurogenesis in the mouse lateral ganglionic eminence.

The Gsx2 homeodomain transcription factor promotes neural progenitor identity in the lateral ganglionic eminence (LGE), despite upregulating the neurogenic factor Ascl1. How this balance in maturation is maintained is unclear. Here, we show that Gsx2 and Ascl1 are co-expressed in subapical progenitors that have unique transcriptional signatures in LGE ventricular zone (VZ) cells. Moreover, whereas Ascl1 misexpression promotes neurogenesis in dorsal telencephalic progenitors, the co-expression of Gsx2 with Ascl1 inhibits neurogenesis. Using luciferase assays, we found that Gsx2 reduces the ability of Ascl1 to activate gene expression in a dose-dependent and DNA binding-independent manner. Furthermore, Gsx2 physically interacts with the basic helix-loop-helix (bHLH) domain of Ascl1, and DNA-binding assays demonstrated that this interaction interferes with the ability of Ascl1 to bind DNA. Finally, we modified a proximity ligation assay for tissue sections and found that Ascl1-Gsx2 interactions are enriched within LGE VZ progenitors, whereas Ascl1-Tcf3 (E-protein) interactions predominate in the subventricular zone. Thus, Gsx2 contributes to the balance between progenitor maintenance and neurogenesis by physically interacting with Ascl1, interfering with its DNA binding and limiting neurogenesis within LGE progenitors.

SWI/SNF complex heterogeneity is related to polyphenotypic differentiation, prognosis, and immune response in rhabdoid tumors.

BACKGROUND: Rhabdoid tumors (RTs) arise within (atypical teratoid/rhabdoid tumor [AT/RT]) or outside the brain (extra [e]CNS-RT) and are driven mainly by inactivation of the SWItch/sucrose nonfermentable (SWI/SNF) complex subunit SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1 (SMARCB1). A pathognomonic hallmark of RTs is heterogeneous multilineage differentiation, including anomalous neuronal differentiation in some eCNS-RTs. Because remodeling of the SWI/SNF complex regulates differentiation, we hypothesized that SWI/SNF Brahma-associated factors (BAF) and polybromo-associated BAF (PBAF) complex heterogeneity are related to both multilineage differentiation and clinical outcome. METHODS: We performed an integrated analysis of SWI/SNF complex alterations in the developing kidney and cerebellum (most common regions of RT origin) in comparison to eCNS-RT (n = 14) and AT/RT (n = 25) tumors. RT samples were interrogated using immunohistochemistry, DNA methylation, and gene expression analyses. RESULTS: The SWI/SNF BAF paralogs actin-like protein (ACTL)6A and ACTL6B were expressed in a mutually exclusive manner in the developing cerebellum and kidney. In contrast, a subset of eCNS-RTs lost mutual exclusivity and coexpressed both subunits. These tumors showed aberrant DNA methylation of genes that regulate neuronal and renal development and demonstrated immunohistochemical evidence of neuronal differentiation. In addition, low expression of the PBAF subunit polybromo-1 (PBRM1) identified a group of AT/RTs in younger children with better overall prognosis. PBRM1-low AT/RT and eCNS-RTs showed altered DNA methylation and gene expression in immune-related genes. PBRM1 knockdown resulted in lowering immunosuppressive cytokines, and PBRM1 levels in tumor samples showed an inverse relationship with cluster of differentiation (CD)8 cytotoxic T-cell infiltration. CONCLUSIONS: Heterogeneity in SWI/SNF BAF (ACTL6A/ACTL6B) and PBAF (PBRM1) subunits is related to histogenesis, contributes to the immune microenvironment and prognosis in RTs, and may inform opportunities to develop immunotherapies.

Pparg promotes differentiation and regulates mitochondrial gene expression in bladder epithelial cells.

The urothelium is an epithelial barrier lining the bladder that protects against infection, fluid exchange and damage from toxins. The nuclear receptor Pparg promotes urothelial differentiation in vitro, and Pparg mutations are associated with bladder cancer. However, the function of Pparg in the healthy urothelium is unknown. Here we show that Pparg is critical in urothelial cells for mitochondrial biogenesis, cellular differentiation and regulation of inflammation in response to urinary tract infection (UTI). Superficial cells, which are critical for maintaining the urothelial barrier, fail to mature in Pparg mutants and basal cells undergo squamous-like differentiation. Pparg mutants display persistent inflammation after UTI, and Nf-KB, which is transiently activated in response to infection in the wild type urothelium, persists for months. Our observations suggest that in addition to its known roles in adipogegnesis and macrophage differentiation, that Pparg-dependent transcription plays a role in the urothelium controlling mitochondrial function development and regeneration.

Single cell RNA-seq study of wild type and Hox9,10,11 mutant developing uterus.

The uterus is a remarkable organ that must guard against infections while maintaining the ability to support growth of a fetus without rejection. The Hoxa10 and Hoxa11 genes have previously been shown to play essential roles in uterus development and function. In this report we show that the Hoxa9,10,11, Hoxc9,10,11, Hoxd9,10,11 genes play a redundant role in the formation of uterine glands. In addition, we use single cell RNA-seq to create a high resolution gene expression atlas of the developing wild type mouse uterus. Cell types and subtypes are defined, for example dividing endothelial cells into arterial, venous, capillary, and lymphatic, while epithelial cells separate into luminal and glandular subtypes. Further, a surprising heterogeneity of stromal and myocyte cell types are identified. Transcription factor codes and ligand/receptor interactions are characterized. We also used single cell RNA-seq to globally define the altered gene expression patterns in all developing uterus cell types for two Hox mutants, with 8 or 9 mutant Hox genes. The mutants show a striking disruption of Wnt signaling as well as the Cxcl12/Cxcr4 ligand/receptor axis.

Maturation of heart valve cell populations during postnatal remodeling.

Heart valve cells mediate extracellular matrix (ECM) remodeling during postnatal valve leaflet stratification, but phenotypic and transcriptional diversity of valve cells in development is largely unknown. Single cell analysis of mouse heart valve cells was used to evaluate cell heterogeneity during postnatal ECM remodeling and leaflet morphogenesis. The transcriptomic analysis of single cells from postnatal day (P)7 and P30 murine aortic (AoV) and mitral (MV) heart valves uncovered distinct subsets of melanocytes, immune and endothelial cells present at P7 and P30. By contrast, interstitial cell populations are different from P7 to P30. P7 valve leaflets exhibit two distinct collagen- and glycosaminoglycan-expressing interstitial cell clusters, and prevalent ECM gene expression. At P30, four interstitial cell clusters are apparent with leaflet specificity and differential expression of complement factors, ECM proteins and osteogenic genes. This initial transcriptomic analysis of postnatal heart valves at single cell resolution demonstrates that subpopulations of endothelial and immune cells are relatively constant throughout postnatal development, but interstitial cell subpopulations undergo changes in gene expression and cellular functions in primordial and mature valves.

Hedgehog signaling patterns the oral-aboral axis of the mandibular arch.

Development of vertebrate jaws involves patterning neural crest-derived mesenchyme cells into distinct subpopulations along the proximal-distal and oral-aboral axes. Although the molecular mechanisms patterning the proximal-distal axis have been well studied, little is known regarding the mechanisms patterning the oral-aboral axis. Using unbiased single-cell RNA-seq analysis followed by in situ analysis of gene expression profiles, we show that Shh and Bmp4 signaling pathways are activated in a complementary pattern along the oral-aboral axis in mouse embryonic mandibular arch. Tissue-specific inactivation of hedgehog signaling in neural crest-derived mandibular mesenchyme led to expansion of BMP signaling activity to throughout the oral-aboral axis of the distal mandibular arch and subsequently duplication of dentary bone in the oral side of the mandible at the expense of tongue formation. Further studies indicate that hedgehog signaling acts through the Foxf1/2 transcription factors to specify the oral fate and pattern the oral-aboral axis of the mandibular mesenchyme.