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.

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.

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.

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.

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.

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.

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.

The Rhesus Macaque Serves As a Model for Human Lateral Branch Nephrogenesis.

BACKGROUND: Most nephrons are added in late gestation. Truncated extrauterine nephrogenesis in premature infants results in fewer nephrons and significantly increased risk for CKD in adulthood. To overcome the ethical and technical difficulties associated with studies of late-gestation human fetal kidney development, third-trimester rhesus macaques served as a model to understand lateral branch nephrogenesis (LBN) at the molecular level. METHODS: Immunostaining and 3D rendering assessed morphology. Single-cell (sc) and single-nucleus (sn) RNA-Seq were performed on four cortically enriched fetal rhesus kidneys of 129-131 days gestational age (GA). An integrative bioinformatics strategy was applied across single-cell modalities, species, and time. RNAScope validation studies were performed on human archival tissue. RESULTS: Third-trimester rhesus kidney undergoes human-like LBN. scRNA-Seq of 23,608 cells revealed 37 transcriptionally distinct cell populations, including naïve nephron progenitor cells (NPCs), with the prior noted marker genes CITED1, MEOX1, and EYA1 (c25). These same populations and markers were reflected in snRNA-Seq of 5972 nuclei. Late-gestation rhesus NPC markers resembled late-gestation murine NPC, whereas early second-trimester human NPC markers aligned to midgestation murine NPCs. New, age-specific rhesus NPCs (SHISA8) and ureteric buds (POU3F4 and TWIST) predicted markers were verified in late-gestation human archival samples. CONCLUSIONS: Rhesus macaque is the first model of bona fide LBN, enabling molecular studies of late gestation, human-like nephrogenesis. These molecular findings support the hypothesis that aging nephron progenitors have a distinct molecular signature and align to their earlier human counterparts, with unique markers highlighting LBN-specific progenitor maturation.

Kidney Single-cell Transcriptomes Predict Spatial Corticomedullary Gene Expression and Tissue Osmolality Gradients.

BACKGROUND: Single-cell transcriptomes from dissociated tissues provide insights into cell types and their gene expression and may harbor additional information on spatial position and the local microenvironment. The kidney's cells are embedded into a gradient of increasing tissue osmolality from the cortex to the medulla, which may alter their transcriptomes and provide cues for spatial reconstruction. METHODS: Single-cell or single-nuclei mRNA sequencing of dissociated mouse kidneys and of dissected cortex, outer, and inner medulla, to represent the corticomedullary axis, was performed. Computational approaches predicted the spatial ordering of cells along the corticomedullary axis and quantitated expression levels of osmo-responsive genes. In situ hybridization validated computational predictions of spatial gene-expression patterns. The strategy was used to compare single-cell transcriptomes from wild-type mice to those of mice with a collecting duct-specific knockout of the transcription factor grainyhead-like 2 (Grhl2CD-/-), which display reduced renal medullary osmolality. RESULTS: Single-cell transcriptomics from dissociated kidneys provided sufficient information to approximately reconstruct the spatial position of kidney tubule cells and to predict corticomedullary gene expression. Spatial gene expression in the kidney changes gradually and osmo-responsive genes follow the physiologic corticomedullary gradient of tissue osmolality. Single-nuclei transcriptomes from Grhl2CD-/- mice indicated a flattened expression gradient of osmo-responsive genes compared with control mice, consistent with their physiologic phenotype. CONCLUSIONS: Single-cell transcriptomics from dissociated kidneys facilitated the prediction of spatial gene expression along the corticomedullary axis and quantitation of osmotically regulated genes, allowing the prediction of a physiologic phenotype.

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.

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.

Psychrophilic proteases dramatically reduce single-cell RNA-seq artifacts: a molecular atlas of kidney development.

Single-cell RNA-seq is a powerful technique. Nevertheless, there are important limitations, including the technical challenges of breaking down an organ or tissue into a single-cell suspension. Invariably, this has required enzymatic incubation at 37°C, which can be expected to result in artifactual changes in gene expression patterns. Here, we describe a dissociation method that uses a protease with high activity in the cold, purified from a psychrophilic microorganism. The entire procedure is carried out at 6°C or colder, at which temperature mammalian transcriptional machinery is largely inactive, thereby effectively 'freezing in' the in vivo gene expression patterns. To test this method, we carried out RNA-seq on 20,424 single cells from postnatal day 1 mouse kidneys, comparing the results of the psychrophilic protease method with procedures using 37°C incubation. We show that the cold protease method provides a great reduction in gene expression artifacts. In addition, the results produce a single-cell resolution gene expression atlas of the newborn mouse kidney, an interesting time in development when mature nephrons are present yet nephrogenesis remains extremely active.

Reduced Abd-B Hox function during kidney development results in lineage infidelity.

Hox genes can function as key drivers of segment identity, with Hox mutations in Drosophila often resulting in dramatic homeotic transformations. In addition, however, they can serve other essential functions. In mammals, the study of Hox gene roles in development is complicated by the presence of four Hox clusters with a total of 39 genes showing extensive functional overlap. In this study, in order to better understand shared core Hox functions, we examined kidney development in mice with frameshift mutations of multiple Abd-B type Hox genes. The resulting phenotypes included dramatically reduced branching morphogenesis of the ureteric bud, premature depletion of nephron progenitors and abnormal development of the stromal compartment. Most unexpected, however, we also observed a cellular level lineage infidelity in nephron segments. Scattered cells within the proximal tubules, for example, expressed genes normally expressed only in collecting ducts. Multiple combinations of inappropriate nephron segment specific marker expression were found. In some cases, cells within a tubule showed incorrect identity, while in other cases cells showed ambiguous character, with simultaneous expression of genes associated with more than one nephron segment. These results give evidence that Hox genes have an overlapping core function at the cellular level in driving and/or maintaining correct differentiation decisions.

Cross-platform single cell analysis of kidney development shows stromal cells express Gdnf.

The developing kidney provides a useful model for study of the principles of organogenesis. In this report we use three independent platforms, Drop-Seq, Chromium 10x Genomics and Fluidigm C1, to carry out single cell RNA-Seq (scRNA-Seq) analysis of the E14.5 mouse kidney. Using the software AltAnalyze, in conjunction with the unsupervised approach ICGS, we were unable to identify and confirm the presence of 16 distinct cell populations during this stage of active nephrogenesis. Using a novel integrative supervised computational strategy, we were able to successfully harmonize and compare the cell profiles across all three technological platforms. Analysis of possible cross compartment receptor/ligand interactions identified the nephrogenic zone stroma as a source of GDNF. This was unexpected because the cap mesenchyme nephron progenitors had been thought to be the sole source of GDNF, which is a key driver of branching morphogenesis of the collecting duct system. The expression of Gdnf by stromal cells was validated in several ways, including Gdnf in situ hybridization combined with immunohistochemistry for SIX2, and marker of nephron progenitors, and MEIS1, a marker of stromal cells. Finally, the single cell gene expression profiles generated in this study confirmed and extended previous work showing the presence of multilineage priming during kidney development. Nephron progenitors showed stochastic expression of genes associated with multiple potential differentiation lineages.

Activated Hedgehog-GLI Signaling Causes Congenital Ureteropelvic Junction Obstruction.

Intrinsic ureteropelvic junction obstruction is the most common cause of congenital hydronephrosis, yet the underlying pathogenesis is undefined. Hedgehog proteins control morphogenesis by promoting GLI-dependent transcriptional activation and inhibiting the formation of the GLI3 transcriptional repressor. Hedgehog regulates differentiation and proliferation of ureteric smooth muscle progenitor cells during murine kidney-ureter development. Histopathologic findings of smooth muscle cell hypertrophy and stroma-like cells, consistently observed in obstructing tissue at the time of surgical correction, suggest that Hedgehog signaling is abnormally regulated during the genesis of congenital intrinsic ureteropelvic junction obstruction. Here, we demonstrate that constitutively active Hedgehog signaling in murine intermediate mesoderm-derived renal progenitors results in hydronephrosis and failure to develop a patent pelvic-ureteric junction. Tissue obstructing the ureteropelvic junction was marked as early as E13.5 by an ectopic population of cells expressing Ptch2, a Hedgehog signaling target. Constitutive expression of GLI3 repressor in Ptch1-deficient mice rescued ectopic Ptch2 expression and obstructive hydronephrosis. Whole transcriptome analysis of isolated Ptch2+ cells revealed coexpression of genes characteristic of stromal progenitor cells. Genetic lineage tracing indicated that stromal cells blocking the ureteropelvic junction were derived from intermediate mesoderm-derived renal progenitors and were distinct from the smooth muscle or epithelial lineages. Analysis of obstructive ureteric tissue resected from children with congenital intrinsic ureteropelvic junction obstruction revealed a molecular signature similar to that observed in Ptch1-deficient mice. Together, these results demonstrate a Hedgehog-dependent mechanism underlying mammalian intrinsic ureteropelvic junction obstruction.

Lung Gene Expression Analysis (LGEA): an integrative web portal for comprehensive gene expression data analysis in lung development.

'LungGENS', our previously developed web tool for mapping single-cell gene expression in the developing lung, has been well received by the pulmonary research community. With continued support from the 'LungMAP' consortium, we extended the scope of the LungGENS database to accommodate transcriptomics data from pulmonary tissues and cells from human and mouse at different stages of lung development. Lung Gene Expression Analysis (LGEA) web portal is an extended version of LungGENS useful for the analysis, display and interpretation of gene expression patterns obtained from single cells, sorted cell populations and whole lung tissues. The LGEA web portal is freely available at http://research.cchmc.org/pbge/lunggens/mainportal.html.

Single cell dissection of early kidney development: multilineage priming.

We used a single cell RNA-seq strategy to create an atlas of gene expression patterns in the developing kidney. At several stages of kidney development, histologically uniform populations of cells give rise to multiple distinct lineages. We performed single cell RNA-seq analysis of total mouse kidneys at E11.5 and E12.5, as well as the renal vesicles at P4. We define an early stage of progenitor cell induction driven primarily by gene repression. Surprising stochastic expression of marker genes associated with differentiated cell types was observed in E11.5 progenitors. We provide a global view of the polarized gene expression already present in the renal vesicle, the first epithelial precursor of the nephron. We show that Hox gene read-through transcripts can be spliced to produce intergenic homeobox swaps. We also identify a surprising number of genes with partially degraded noncoding RNA. Perhaps most interesting, at early developmental times single cells often expressed genes related to several developmental pathways. This provides powerful evidence that initial organogenesis involves a process of multilineage priming. This is followed by a combination of gene repression, which turns off the genes associated with most possible lineages, and the activation of increasing numbers of genes driving the chosen developmental direction.

Uterine inactivation of muscle segment homeobox (Msx) genes alters epithelial cell junction proteins during embryo implantation.

Embryo implantation requires that the uterus differentiate into the receptive state. Failure to attain uterine receptivity will impede blastocyst attachment and result in a compromised pregnancy. The molecular mechanism by which the uterus transitions from the prereceptive to the receptive stage is complex, involving an intricate interplay of various molecules. We recently found that mice with uterine deletion ofMsxgenes (Msx1(d/d)/Msx2(d/d)) are infertile because of implantation failure associated with heightened apicobasal polarity of luminal epithelial cells during the receptive period. However, information on Msx's roles in regulating epithelial polarity remains limited. To gain further insight, we analyzed cell-type-specific gene expression by RNA sequencing of separated luminal epithelial and stromal cells by laser capture microdissection fromMsx1(d/d)/Msx2(d/d)and floxed mouse uteri on d 4 of pseudopregnancy. We found that claudin-1, a tight junction protein, and small proline-rich (Sprr2) protein, a major component of cornified envelopes in keratinized epidermis, were substantially up-regulated inMsx1(d/d)/Msx2(d/d)uterine epithelia. These factors also exhibited unique epithelial expression patterns at the implantation chamber (crypt) inMsx1(f/f)/Msx2(f/f)females; the patterns were lost inMsx1(d/d)/Msx2(d/d)epithelia on d 5, suggesting important roles during implantation. The results suggest thatMsxgenes play important roles during uterine receptivity including modulation of epithelial junctional activity.-Sun, X., Park, C. B., Deng, W., Potter, S. S., Dey, S. K. Uterine inactivation of muscle segment homeobox (Msx) genes alters epithelial cell junction proteins during embryo implantation.

Recombineering-based dissection of flanking and paralogous Hox gene functions in mouse reproductive tracts.

Hox genes are key regulators of development. In mammals, the study of these genes is greatly confounded by their large number, overlapping functions and interspersed shared enhancers. Here, we describe the use of a novel recombineering strategy to introduce simultaneous frameshift mutations into the flanking Hoxa9, Hoxa10 and Hoxa11 genes, as well as their paralogs on the HoxD cluster. The resulting Hoxa9,10,11 mutant mice displayed dramatic synergistic homeotic transformations of the reproductive tracts, with the uterus anteriorized towards oviduct and the vas deferens anteriorized towards epididymis. The Hoxa9,10,11 mutant mice also provided a genetic setting that allowed the discovery of Hoxd9,10,11 redundant reproductive tract patterning function. Both shared and distinct Hox functions were defined. Hoxd9,10,11 play a crucial role in the regulation of uterine immune function. Non-coding non-polyadenylated RNAs were among the key Hox targets, with dramatic downregulation in mutants. We observed Hox cross-regulation of transcription and splicing. In addition, we observed a surprising anti-dogmatic apparent posteriorization of the uterine epithelium. In caudal regions of the uterus, the normal simple columnar epithelium flanking the lumen was replaced by a pseudostratified transitional epithelium, normally found near the more posterior cervix. These results identify novel molecular functions of Hox genes in the development of the male and female reproductive tracts.

SINCERA: A Pipeline for Single-Cell RNA-Seq Profiling Analysis.

A major challenge in developmental biology is to understand the genetic and cellular processes/programs driving organ formation and differentiation of the diverse cell types that comprise the embryo. While recent studies using single cell transcriptome analysis illustrate the power to measure and understand cellular heterogeneity in complex biological systems, processing large amounts of RNA-seq data from heterogeneous cell populations creates the need for readily accessible tools for the analysis of single-cell RNA-seq (scRNA-seq) profiles. The present study presents a generally applicable analytic pipeline (SINCERA: a computational pipeline for SINgle CEll RNA-seq profiling Analysis) for processing scRNA-seq data from a whole organ or sorted cells. The pipeline supports the analysis for: 1) the distinction and identification of major cell types; 2) the identification of cell type specific gene signatures; and 3) the determination of driving forces of given cell types. We applied this pipeline to the RNA-seq analysis of single cells isolated from embryonic mouse lung at E16.5. Through the pipeline analysis, we distinguished major cell types of fetal mouse lung, including epithelial, endothelial, smooth muscle, pericyte, and fibroblast-like cell types, and identified cell type specific gene signatures, bioprocesses, and key regulators. SINCERA is implemented in R, licensed under the GNU General Public License v3, and freely available from CCHMC PBGE website, https://research.cchmc.org/pbge/sincera.html.