Non-endothelial expression of Endomucin in the mouse and human choroid.

Endomucin (EMCN) is a 261 AA transmembrane glycoprotein that is highly expressed by venous and capillary endothelial cells where it plays a role in VEGF-mediated angiogenesis and regulation of immune cell recruitment. However, it is better known as a histological marker, where it has become widespread due to the commercial availability of high-quality antibodies that work under a wide range of conditions and in many tissues. The specificity of EMCN staining has been well-validated in retinal vessels, but while it has been used extensively as a marker in other tissues of the eye, including the choroid, the pattern of expression has not been described in detail. Here, in addition to endothelial expression in the choriocapillaris and deeper vascular layers, we characterize a population of EMCN-positive perivascular cells in the mouse choroid that did not co-localize with cells expressing other endothelial markers such as PECAM1 or PODXL. To confirm that these cells represented a new population of EMCN-expressing stromal cells, we then performed single cell RNA sequencing in choroids from adult wild-type mice. Analysis of this new dataset confirmed that, in addition to endothelial cells, Emcn mRNA expression was present in choroidal pericytes and a subset of fibroblasts, but not vascular smooth muscle cells. Besides Emcn , no known endothelial gene expression was detected in these cell populations, confirming that they did not represent endothelial-stromal doublets, a common technical artifact in single cell RNA seq datasets. Instead, choroidal Emcn -expressing fibroblasts exhibited high levels of chemokine and interferon signaling genes, while Emcn -negative fibroblasts were enriched in genes encoding extracellular matrix proteins. Emcn expressing fibroblasts were also detected in published datasets from mouse brain and human choroid, suggesting that stromal Emcn expression was not unique to the choroid and was evolutionarily conserved. Together, these findings highlight unique fibroblast and pericyte populations in the choroid and provide new context for the role of EMCN in angiogenesis and immune cell recruitment.

The transcription factor Tcf21 is required for specifying Foxd1 cells to the juxtaglomerular cell lineage.

Renin is a crucial enzyme involved in the regulation of blood pressure and electrolyte balance. It has been shown that renin expressing cells arise from the Foxd1+ stromal progenitors, however the factors involved in guiding Foxd1+ cells towards the renin-secreting cell fate remain poorly understood. Tcf21, also known as Pod1 or Capsulin, is a bHLH transcription factor that is expressed in the metanephric mesenchyme and plays a crucial role in kidney development. We have previously shown that deletion of Tcf21 in Foxd1+ cells ( Foxd1 Cre/+ ;Tcf21 f/f ) results in paucity of vascular mural cells and in disorganized renal arterial tree with fewer, shorter, and thinner arterioles. Here, we sought to examine the relationship between Tcf21 and renin cells during kidney development and test whether Tcf21 is implicated in the regulation of juxtaglomerular cell differentiation. Immunostaining for renin demonstrated that kidneys of Foxd1 Cre/+ ;Tcf21 f/f have fewer renin-positive spots at E16.5 and E18.5 compared with controls. In-situ hybridization for renin mRNA showed reduced expression in Foxd1 Cre/+ ;Tcf21 f/f kidneys at E14.5, E16.5, and E18.5. Together, these data suggest that stromal expression of Tcf21 is required for the emergence of renin cells. To dissect the role of Tcf21 in juxtaglomerular (JG) cells, we deleted Tcf21 upon renin promoter activation ( Ren1 dCre/+ ;Tcf21 f/f ). Interestingly, the Ren1 dCre/+ ;Tcf21 f/f kidney showed normal arterial tree at E16.5 identical to controls. Furthermore, inactivation of Tcf21 upon renin expression did not alter kidney morphology in two- and four-month-old mice. Finally, expression renin mRNA was similar between Ren1 dCre/+ ;Tcf21 f/f and controls at 2 months. Taken together, these findings suggest that Tcf21 expression in Foxd1+ cells is essential for specifying the fate of these cells into juxtaglomerular cells. However, once renin cell identity is assumed, Tcf21 is dispensable. Uncovering the regulation of Foxd1+ cells and their derivatives, including the JG cell lineage, is crucial for understanding the mechanisms underlying renal vasculature formation.

Oncogenic deubiquitination controls tyrosine kinase signaling and therapy response in acute lymphoblastic leukemia.

Dysregulation of kinase signaling pathways favors tumor cell survival and therapy resistance in cancer. Here, we reveal a posttranslational regulation of kinase signaling and nuclear receptor activity via deubiquitination in T cell acute lymphoblastic leukemia (T-ALL). We observed that the ubiquitin-specific protease 11 (USP11) is highly expressed and associates with poor prognosis in T-ALL. USP11 ablation inhibits leukemia progression in vivo, sparing normal hematopoiesis. USP11 forms a complex with USP7 to deubiquitinate the oncogenic lymphocyte cell-specific protein-tyrosine kinase (LCK) and enhance its activity. Impairment of LCK activity leads to increased glucocorticoid receptor (GR) expression and glucocorticoids sensitivity. Genetic knockout of USP7 improved the antileukemic efficacy of glucocorticoids in vivo. The transcriptional activation of GR target genes is orchestrated by the deubiquitinase activity and mediated via an increase in enhancer-promoter interaction intensity. Our data unveil how dysregulated deubiquitination controls leukemia survival and drug resistance, suggesting previously unidentified therapeutic combinations toward targeting leukemia.

Posttranslational Regulation of the Exon Skipping Machinery Controls Aberrant Splicing in Leukemia.

Splicing alterations are common in diseases such as cancer, where mutations in splicing factor genes are frequently responsible for aberrant splicing. Here we present an alternative mechanism for splicing regulation in T-cell acute lymphoblastic leukemia (T-ALL) that involves posttranslational stabilization of the splicing machinery via deubiquitination. We demonstrate there are extensive exon skipping changes in disease, affecting proteasomal subunits, cell-cycle regulators, and the RNA machinery. We present that the serine/arginine-rich splicing factors (SRSF), controlling exon skipping, are critical for leukemia cell survival. The ubiquitin-specific peptidase 7 (USP7) regulates SRSF6 protein levels via active deubiquitination, and USP7 inhibition alters the exon skipping pattern and blocks T-ALL growth. The splicing inhibitor H3B-8800 affects splicing of proteasomal transcripts and proteasome activity and acts synergistically with proteasome inhibitors in inhibiting T-ALL growth. Our study provides the proof-of-principle for regulation of splicing factors via deubiquitination and suggests new therapeutic modalities in T-ALL. SIGNIFICANCE: Our study provides a new proof-of-principle for posttranslational regulation of splicing factors independently of mutations in aggressive T-cell leukemia. It further suggests a new drug combination of splicing and proteasomal inhibitors, a concept that might apply to other diseases with or without mutations affecting the splicing machinery.This article is highlighted in the In This Issue feature, p. 1241.