Rationale Vascular tubulogenesis is essential to cardiovascular development. Within initial vascular cords of endothelial cells (ECs), apical membranes are established and become cleared of cell-cell junctions, thereby allowing continuous central lumens to open. Rasip1 is required for apical junction clearance, as well as for regulation of Rho GTPase activity. However, it remains unknown how activities of different Rho GTPases are coordinated by Rasip1 to direct tubulogenesis. Objective The aim of this study is to determine the mechanisms downstream of Rasip1 that drive vascular tubulogenesis. Methods and Results Using conditional mouse mutant models and pharmacological approaches, we dissect GTPase pathways downstream of Rasip1. We show that clearance of EC apical junctions during vascular tubulogenesis depends on Ras interacting protein 1 (Rasip1), as well as the GTPase Cdc42 and the kinase Pak4. Genetic deletion of Rasip1 or Cdc42, or inhibition of Pak4, all block EC tubulogenesis. By contrast, inactivation of RhoA signaling leads to vessel overexpansion, implicating actomyosin contractility in control of lumen diameter. Interestingly, blocking activity of NMII either prior to, or after, lumen morphogenesis results in dramatically different tubulogenesis phenotypes, suggesting time-dependent roles. Conclusions Rasip1 controls different pools of GTPases, which in turn regulate different pools of NMII to coordinate junction clearance (remodeling) and actomyosin contractility during vascular tubulogenesis. Rasip1 promotes activity of Cdc42 to activate Pak4, which in turn activates NMII, clearing apical junctions. Once lumens open, Rasip1 suppresses actomyosin contractility via inhibition of RhoA by Arhgap29, allowing controlled expansion of vessel lumens during embryonic growth. These findings elucidate the stepwise processes regulated by Rasip1 through downstream Rho GTPases and NMII.
The kidney vasculature facilitates the excretion of wastes, the dissemination of hormones, and the regulation of blood chemistry. To carry out these diverse functions, the vasculature is regionalized within the kidney and along the nephron. However, when and how endothelial regionalization occurs remains unknown. Here, we examine the developing kidney vasculature to assess its 3-dimensional structure and transcriptional heterogeneity. First, we observe that endothelial cells (ECs) grow coordinately with the kidney bud as early as E10.5, and begin to show signs of specification by E13.5 when the first arteries can be identified. We then focus on how ECs pattern and remodel with respect to the developing nephron and collecting duct epithelia. ECs circumscribe nephron progenitor populations at the distal tips of the ureteric bud (UB) tree and form stereotyped cruciform structures around each tip. Beginning at the renal vesicle (RV) stage, ECs form a continuous plexus around developing nephrons. The endothelial plexus envelops and elaborates with the maturing nephron, becoming preferentially enriched along the early distal tubule. Lastly, we perform transcriptional and immunofluorescent screens to characterize spatiotemporal heterogeneity in the kidney vasculature and identify novel regionally enriched genes. A better understanding of development of the kidney vasculature will help instruct engineering of properly vascularized ex vivo kidneys and evaluate diseased kidneys.
Proper lumen morphogenesis during pancreas development is critical to endocrine and exocrine cell fate. Recent studies showed that a central network of lumens (termed core), but not the surrounding terminal branches (termed periphery), produces most islet endocrine cells. To date, it remains unclear how pancreatic lumens form and remodel and which aspects of lumen morphogenesis influence cell fate. Importantly, models testing the function of the central lumen network as an endocrine niche are lacking. Here, we identify mechanisms underlying lumen formation and remodeling and show that central lumen network morphogenesis impacts pancreatic endocrine mass. We show that loss of the scaffolding protein Afadin disrupts de novo lumenogenesis and lumen continuity in the tip epithelium. Codepletion of the actomyosin regulator RhoA and Afadin results in defects in the central lumens and arrests lumen remodeling. This arrest leads to prolonged perdurance of the central lumen network over developmental time and expansion of the endocrine progenitor population and, eventually, endocrine mass. Our study uncovers essential roles of Afadin and RhoA in pancreatic central lumen morphogenesis, which subsequently determines endocrine cell mass.
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