The HIRA complex mediates deposition of histone H3.3 independent of replication. Its functions in gene regulation in mice remain to be fully understood. Here we analyze mutations of the HIRA complex genes Ubn1 and Ubn2. We observe that Ubn1 mutant mice of both sexes are viable and fertile. In contrast, mutation of Ubn2 causes embryonic lethality with variable penetrance and skewed sex ratio in favor of males. Combined Ubn1 and Ubn2 mutations cause embryonic lethality with complete penetrance, variable developmental arrest before turning, and reduced recovery of female embryos. Consistent with a female specific function of the HIRA complex, reanalysis of the Hira mutation during embryogenesis reveals that previously observed severe and mild phenotypic classes correspond to female and male sex. Mechanistically, we show that mutations of Ubn1, Ubn2, and Hira in mouse embryonic stem cells affect the initiation of X inactivation. Xist mediated gene silencing is impaired to increasing extent by Ubn1, Ubn2, Hira, and combined Ubn1 and Ubn2 mutations. We identify a failure of establishing histone H3 tri-methyl lysine 27 over X-linked genes after induction of Xist expression as earliest molecular defect, whereas deacetylation of lysine 27 by Xist remains largely unaffected by the loss of Ubinucleins. Our study thereby identifies a switch from histone H3 acetyl to tri-methyl lysine 27 at the initiation of X inactivation that depends on HIRA complex function.

Therapies targeting blood vessels hold promise for autosomal dominant polycystic kidney disease (ADPKD), the most common inherited disorder causing kidney failure. However, the onset and nature of kidney vascular abnormalities in ADPKD are poorly defined. Accordingly, we employed a combination of single-cell transcriptomics; three-dimensional imaging with geometric, topological and fractal analyses; and multimodal magnetic resonance imaging with arterial spin labelling to investigate aberrant microvasculature in ADPKD kidneys. Within human ADPKD kidneys with advanced cystic pathology and excretory failure, we identified a molecularly distinct blood microvascular subpopulation, characterised by impaired angiogenic signalling and metabolic dysfunction, differing from endothelial injury profiles observed in non-cystic human kidney diseases. Next, Pkd1 mutant mouse kidneys were examined postnatally, when cystic pathology is well established, but before excretory failure. An aberrant endothelial subpopulation was also detected, concurrent with reduced cortical blood perfusion. Disorganised kidney cortical microvasculature was also present in Pkd1 mutant mouse fetal kidneys when tubular dilation begins. Thus, aberrant features of cystic kidney vasculature are harmonised between human and mouse ADPKD, supporting early targeting of the vasculature as a strategy to ameliorate ADPKD progression.

Lymphatic vessels maintain tissue fluid homeostasis and modulate inflammation, yet their spatial organization and molecular identity in the healthy human kidney, and how these change during chronic transplant rejection, remain poorly defined. Here, we show that lymphatic capillaries initiate adjacent to cortical kidney tubules and lack smooth muscle coverage. These vessels exhibit an organ-specific molecular signature, enriched for CCL14, DNASE1L3, and MDK, with limited expression of canonical immune-trafficking markers found in other organ lymphatics, such as LYVE1 and CXCL8. In allografts with chronic mixed rejection, lymphatics become disorganized and infiltrate the medulla, with their endothelial junctions remodeling from a button-like to a continuous, zipper-like, architecture. Lymphatics in rejecting kidneys localize around and interconnect tertiary lymphoid structures at different maturation stages, with altered intralymphatic and perilymphatic CD4+ T cell distribution. The infiltrating T cells express IFN-γ, which upregulates coinhibitory ligands in lymphatic endothelial cells, including PVR and LGALS9. Simultaneously, lymphatics acquire HLA class II expression and exhibit C4d deposition, consistent with alloantibody binding and complement activation. Together, these findings define the spatial and molecular features of human kidney lymphatics, revealing tolerogenic reprogramming accompanied by structural perturbations during chronic transplant rejection.

The lymphatic vasculature is essential for fluid homeostasis, immune regulation and possesses diverse organ-specific functions. During development, lymphatic endothelial cells (LEC) arise from multiple progenitor sources that form organ-specific lymphatic networks. While the origins of LECs in the heart, skin, and mesentery have been studied, those in the kidney remain unresolved. Here, we combined genetic lineage tracing in mouse embryos with optical clearing and high-resolution three-dimensional imaging to identify two distinct progenitor sources of kidney lymphatics. The majority of kidney LECs originate from a Tie2⁺ endothelial lineage previously linked to venous or capillary vessels. Approximately 15% derive from Osr1⁺ intermediate mesoderm, a lineage that generates kidney nephrons and stroma. Osr1⁺- derived LECs were absent from the heart, mesentery, and skin, indicating a kidney-specific contribution, and arose independently of nephron and stromal lineages. Both Tie2⁺ and Osr1⁺ lineages contributed to vessel sprouting and de novo formation of lymphatic clusters. Revealing a novel cellular origin of LECs and identifying a dual origin for kidney lymphatics, we demonstrate that de novo lymphatic formation can occur from both shared and organ-specific progenitors. This work advances our understanding of how lymphatics assemble during development and provides a framework for targeting kidney lymphatics in disease. ### Competing Interest Statement The authors have declared no competing interest. Wellcome Trust, https://ror.org/029chgv08, 220895/Z/20/Z, 314710/Z/24/Z Kidney Research UK, https://ror.org/02kx7se86, IN\_012\_20190306 Rosetrees Trust, https://ror.org/04e3zg361, PhD2020\100012 Foulkes Foundation, https://ror.org/03hvqcb43 Medical Research Council, https://ror.org/03x94j517, MR/Y008340/1, APP14742 NIHR Great Ormond Street Hospital Biomedical Research Centre

Hallmarks of autosomal dominant polycystic kidney disease (ADPKD), the most common hereditary kidney anomaly, include expanding fluid-filled epithelial cysts, inflammation, and fibrosis. Despite previous work showing the potential of vascular-based therapies, renal microvascular alterations in ADPKD, and their timing, are poorly understood. Using single-cell transcriptomics of human kidney microvasculature, we identify a population of endothelial cells adjacent to cysts in ADPKD. This pericystic endothelium, distinguishable by its expression of osteopontin (SPP1), has a distinct molecular profile compared to the common endothelial cell injury signature in other kidney diseases. SPP1+ pericystic endothelium was also present in an orthologous mouse model of ADPKD before overt kidney functional decline. By interrogating geometric, topological and fractal properties from three-dimensional imaging of early ADPKD mouse kidneys, we show that pericystic endothelium associates with disorganisation and non-uniformity of the renal cortical microvasculature. Concurrently, we detected region-specific reductions in cortical blood flow within ADPKD murine kidneys using arterial spin labelling. We conclude that ADPKD kidneys contain a unique subset of endothelium manifesting with aberrant remodelling and impaired blood perfusion. Its detection, prior to renal functional decline, advocates the vasculature as a therapeutic target to modulate or preserve renal function in early ADPKD. ### Competing Interest Statement The authors have declared no competing interest.

The authors regret that there was an error in the labelling of the axes shown in the graphs in Figs. 1b and 1c. In both cases, the x-axis label should read E9.5 KO / E9.5 WT (Log2) and not E9.5 WT / E9.5 KO WT (Log2) as originally stated. The email address for the corresponding author has been changed to [email protected]. The authors would like to apologise for any inconvenience caused. HIC2 regulates isoform switching during maturation of the cardiovascular systemJournal of Molecular and Cellular CardiologyVol. 114PreviewPhysiological changes during embryonic development are associated with changes in the isoform expression of both myocyte sarcomeric proteins and of erythrocyte haemoglobins. Cell type-specific isoform expression of these genes also occurs. Although these changes appear to be coordinated, it is unclear how changes in these disparate cell types may be linked. The transcription factor Hic2 is required for normal cardiac development and the mutant is embryonic lethal. Hic2 embryos exhibit precocious expression of the definitive-lineage haemoglobin Hbb-bt in circulating primitive erythrocytes and of foetal isoforms of cardiomyocyte genes (creatine kinase, Ckm, and eukaryotic elongation factor Eef1a2) as well as ectopic cardiac expression of fast-twitch skeletal muscle troponin isoforms. Full-Text PDF Open Access

The chemokine SDF-1 (CXCL12) and its receptor CXCR4 control several processes during embryonic development such as the regulation of stem cell proliferation, differentiation, and migration. However, the role of this pathway in the formation of the pituitary gland is not understood. We sought to characterise the expression patterns of CXCR4, SDF-1 and CXCR7 at different stages of pituitary gland development. Our expression profiling revealed that SDF-1 is expressed in progenitor-rich regions of the pituitary anterior lobe, that CXCR4 and CXCR7 have opposite expression domains and that CXCR4 expression is conserved between mice and human embryos. We then assessed the importance of this signalling pathway in the development and function of the murine pituitary gland through conditional deletion of CXCR4 in embryonic pituitary progenitors. Successful and specific ablation of CXCR4 expression in embryonic pituitary progenitors did not lead to observable embryonic nor postnatal defects but allowed the identification of stromal CXCR4+ cells not derived from HESX1+ progenitors. Further analysis of constitutive SDF-1, CXCR7 and CXCR4 mutants of the pathway indicates that CXCR4 expression in HESX1+ cells and their descendants is not essential for normal pituitary development in mice.

Aims Haploinsufficiency of the chromo-domain protein CHD7 underlies most cases of CHARGE syndrome, a multisystem birth defect including congenital heart malformation. Context specific roles for CHD7 in various stem, progenitor, and differentiated cell lineages have been reported. Previously, we showed severe defects when Chd7 is absent from cardiopharyngeal mesoderm (CPM). Here, we investigate altered gene expression in the CPM and identify specific CHD7-bound target genes with known roles in the morphogenesis of affected structures. Methods and results We generated conditional KO of Chd7 in CPM and analysed cardiac progenitor cells using transcriptomic and epigenomic analyses, in vivo expression analysis, and bioinformatic comparisons with existing datasets. We show CHD7 is required for correct expression of several genes established as major players in cardiac development, especially within the second heart field (SHF). We identified CHD7 binding sites in cardiac progenitor cells and found strong association with histone marks suggestive of dynamically regulated enhancers during the mesodermal to cardiac progenitor transition of mESC differentiation. Moreover, CHD7 shares a subset of its target sites with ISL1, a pioneer transcription factor in the cardiogenic gene regulatory network, including one enhancer modulating Fgf10 expression in SHF progenitor cells vs. differentiating cardiomyocytes. Conclusion We show that CHD7 interacts with ISL1, binds ISL1-regulated cardiac enhancers, and modulates gene expression across the mesodermal heart fields during cardiac morphogenesis.

Abstract Aims The adapter protein p130Cas, encoded by the Bcar1 gene, is a key regulator of cell movement, adhesion, and cell cycle control in diverse cell types. Bcar1 constitutive knockout mice are embryonic lethal by embryonic days (E) 11.5–12.5, but the role of Bcar1 in embryonic development remains unclear. Here, we investigated the role of Bcar1 specifically in cardiovascular development and defined the cellular and molecular mechanisms disrupted following targeted Bcar1 deletions. Methods and results We crossed Bcar1 floxed mice with Cre transgenic lines allowing for cell-specific knockout either in smooth muscle and early cardiac tissues (SM22-Cre), mature smooth muscle cells (smMHC-Cre), endothelial cells (Tie2-Cre), second heart field cells (Mef2c-Cre), or neural crest cells (NCC) (Pax3-Cre) and characterized these conditional knock outs using a combination of histological and molecular biology techniques. Conditional knockout of Bcar1 in SM22-expressing smooth muscle cells and cardiac tissues (Bcar1SM22KO) was embryonically lethal from E14.5–15.5 due to severe cardiovascular defects, including abnormal ventricular development and failure of outflow tract (OFT) septation leading to a single outflow vessel reminiscent of persistent truncus arteriosus. SM22-restricted loss of Bcar1 was associated with failure of OFT cushion cells to undergo differentiation to septal mesenchymal cells positive for SMC-specific α-actin, and disrupted expression of proteins and transcription factors involved in epithelial-to-mesenchymal transformation (EMT). Furthermore, knockout of Bcar1 specifically in NCC (Bcar1PAX3KO) recapitulated part of the OFT septation and aortic sac defects seen in the Bcar1SM22KO mutants, indicating a cell-specific requirement for Bcar1 in NCC essential for OFT septation. In contrast, conditional knockouts of Bcar1 in differentiated smooth muscle, endothelial cells, and second heart field cells survived to term and were phenotypically normal at birth and postnatally. Conclusion Our work reveals a cell-specific requirement for Bcar1 in NCC, early myogenic and cardiac cells, essential for OFT septation, myocardialization and EMT/cell cycle regulation and differentiation to myogenic lineages.

To guide the development of therapeutic interventions for acute kidney injury, elucidating the deleterious pathways of this global health problem is highly warranted. Emerging evidence has indicated a pivotal role of endothelial dysfunction in the etiology of this disease. We found that the class III semaphorin SEMA3C was ectopically upregulated with full length protein excreted into the blood and truncated protein secreted into the urine upon kidney injury and hypothesized a role for SEAM3C in acute kidney injury. Sema3c was genetically abrogated during acute kidney injury and subsequent kidney morphological and functional defects in two well-characterized models of acute kidney injury; warm ischemia/reperfusion and folic acid injection were analyzed. Employing a beta actin-dependent, inducible knockout of Sema3c, we demonstrate that in acute kidney injury SEMA3C promotes interstitial edema, leucocyte infiltration and tubular injury. Additionally, intravital microscopy combined with Evans Blue dye extravasation and primary culture of magnetically sorted peritubular endothelial cells identified a novel role for SEMA3C in promoting vascular permeability. Thus, our study points to microvascular permeability as an important driver of injury in acute kidney injury, and to SEMA3C as a novel permeability factor and potential target for therapeutic intervention.

Much of the pathology related to typical malformations associated with 22q11.2DS can be ascribed to problems with the morphogenesis and subsequent dysfunction of pharyngeal arch system derivatives, including the craniofacial structures, thymus, parathyroid glands, aortic arch, and outflow tract of the heart. These structures receive contributions from all three classic germ layers of the embryo—the endoderm, mesoderm, and ectoderm—together with neural crest cells derived from the closing neural tube. To understand these processes, animal models and cellular approaches are required. The mouse has been the main organism used for investigating developmental aspects of the syndrome as its developmental anatomy is similar to that of a human and it is possible to generate individual gene mutations and multigene deletions. The transcription factor gene TBX1 has been revealed as the main player in the haploinsufficiency syndrome, but contributions from other genes are evident.

ABSTRACT Studies of the structural and molecular features of the lymphatic vasculature, which clears fluid, macromolecules and leukocytes from the tissue microenvironment, have largely relied on animal models, with limited information in human organs beyond traditional immunohistochemical assessment. Here, we use three-dimensional imaging and single-cell RNA-sequencing to study lymphatics in the human kidney. We found a hierarchical arrangement of lymphatic vessels within human kidneys, initiating along specialised nephron epithelium in the renal cortex and displaying a distinct, kidney-specific transcriptional profile. In chronic transplant rejection we found kidney allograft lymphatic expansion alongside a loss of structural hierarchy, with human leukocyte antigen-expressing lymphatic vessels infiltrating the medulla, presenting a putative target for alloreactive antibodies. This occurred concurrently with lymphatic vessels invading and interconnecting tertiary lymphoid structures at early stages of lymphocyte colonisation. Analysis of intercellular signalling revealed upregulation of co-inhibitory molecule-mediated CD4 + T cell-lymphatic crosstalk in rejecting kidneys, potentially acting to limit local alloimmune responses. Overall, we delineate novel structural and molecular features of human kidney lymphatics and reveal perturbations to their phenotype and transcriptome in the context of alloimmunity. SUMMARY Lymphatics regulate fluid balance and immune cell accumulation but are under-studied in human organs such as the kidney. Jafree and colleagues profiled human kidney lymphatics using three-dimensional imaging and single-cell RNA-sequencing, revealing structural and transcriptional perturbations in rejecting kidney transplants.

Congenital heart defects are a feature of several genetic haploinsufficiency syndromes, often involving transcriptional regulators. One property of haploinsufficient genes is their propensity for network interactions at the gene or protein level. In this article we took advantage of an online dataset of high throughput screening of mutations that are embryonic lethal in mice. Our aim was to identify new genes where the loss of function caused cardiovascular phenotypes resembling the 22q11.2 deletion syndrome models, that is, heterozygous and homozygous loss of Tbx1. One gene with a potentially haploinsufficient phenotype was identified, Setd5, thought to be involved in chromatin modification. We found murine Setd5 haploinsufficiency to be associated with double outlet right ventricle and perimembranous ventricular septal defect, although no genetic interaction with Tbx1 was detected. Conditional mutagenesis revealed that Setd5 was required in cardiopharyngeal mesoderm for progression of the heart tube through the ballooning stage to create a four-chambered heart.

Lymphatic vessels have critical roles in both health and disease and their study is a rapidly evolving area of vascular biology. The consensus on how the first lymphatic vessels arise in the developing embryo has recently shifted. Originally, they were thought to solely derive by sprouting from veins. Since then, several studies have uncovered novel cellular mechanisms and a diversity of contributing cell lineages in the formation of organ lymphatic vasculature. Here, we review the key mechanisms and cell lineages contributing to lymphatic development, discuss the advantages and limitations of experimental techniques used for their study and highlight remaining knowledge gaps that require urgent attention. Emerging technologies should accelerate our understanding of how lymphatic vessels develop normally and how they contribute to disease.

Cxcl12-null embryos have dysplastic, misaligned, and hyperplastic semilunar valves (SLVs). In this study, we show that CXCL12 signaling via its receptor CXCR4 fulfills distinct roles at different stages of SLV development, acting initially as a guidance cue to pattern cellular distribution within the valve primordia during the endocardial-to-mesenchymal transition (endoMT) phase and later regulating mesenchymal cell proliferation during SLV remodeling. Transient, anteriorly localized puncta of internalized CXCR4 are observed in cells undergoing endoMT. In vitro, CXCR4+ cell orientation in response to CXCL12 requires phosphatidylinositol 3-kinase (PI3K) signaling and is inhibited by suppression of endocytosis. This dynamic intracellular localization of CXCR4 during SLV development is related to CXCL12 availability, potentially enabling activation of divergent downstream signaling pathways at key developmental stages. Importantly, Cxcr7-/- mutants display evidence of excessive CXCL12 signaling, indicating a likely role for atypical chemokine receptor CXCR7 in regulating ligand bioavailability and thus CXCR4 signaling output during SLV morphogenesis.