Endothelial Restoration of Receptor Activity-Modifying Protein 2 Is Sufficient to Rescue Lethality, but Survivors Develop Dilated Cardiomyopathy.

RAMPs (receptor activity-modifying proteins) serve as oligomeric modulators for numerous G-protein-coupled receptors, yet elucidating the physiological relevance of these interactions remains complex. Ramp2 null mice are embryonic lethal, with cardiovascular developmental defects similar to those observed in mice null for canonical adrenomedullin/calcitonin receptor-like receptor signaling. We aimed to genetically rescue the Ramp2(-/-) lethality in order to further delineate the spatiotemporal requirements for RAMP2 function during development and thereby enable the elucidation of an expanded repertoire of RAMP2 functions with family B G-protein-coupled receptors in adult homeostasis. Endothelial-specific expression of Ramp2 under the VE-cadherin promoter resulted in the partial rescue of Ramp2(-/-) mice, demonstrating that endothelial expression of Ramp2 is necessary and sufficient for survival. The surviving Ramp2(-/-) Tg animals lived to adulthood and developed spontaneous hypotension and dilated cardiomyopathy, which was not observed in adult mice lacking calcitonin receptor-like receptor. Yet, the hearts of Ramp2(-/-) Tg animals displayed dysregulation of family B G-protein-coupled receptors, including parathyroid hormone and glucagon receptors, as well as their downstream signaling pathways. These data suggest a functional requirement for RAMP2 in the modulation of additional G-protein-coupled receptor pathways in vivo, which is critical for sustained cardiovascular homeostasis. The cardiovascular importance of RAMP2 extends beyond the endothelium and canonical adrenomedullin/calcitonin receptor-like receptor signaling, in which future studies could elucidate novel and pharmacologically tractable pathways for treating cardiovascular diseases

Tricellulin deficiency affects tight junction architecture and cochlear hair cells

The two compositionally distinct extracellular cochlear fluids, endolymph and perilymph, are separated by tight junctions that outline the scala media and reticular lamina. Mutations in TRIC (also known as MARVELD2), which encodes a tricellular tight junction protein known as tricellulin, lead to nonsyndromic hearing loss (DFNB49). We generated a knockin mouse that carries a mutation orthologous to the TRIC coding mutation linked to DFNB49 hearing loss in humans. Tricellulin was absent from the tricellular junctions in the inner ear epithelia of the mutant animals, which developed rapidly progressing hearing loss accompanied by loss of mechanosensory cochlear hair cells, while the endocochlear potential and paracellular permeability of a biotin-based tracer in the stria vascularis were unaltered. Freeze-fracture electron microscopy revealed disruption of the strands of intramembrane particles connecting bicellular and tricellular junctions in the inner ear epithelia of tricellulin-deficient mice. These ultrastructural changes may selectively affect the paracellular permeability of ions or small molecules, resulting in a toxic microenvironment for cochlear hair cells. Consistent with this hypothesis, hair cell loss was rescued in tricellulin-deficient mice when generation of normal endolymph was inhibited by a concomitant deletion of the transcription factor, Pou3f4. Finally, comprehensive phenotypic screening showed a broader pathological phenotype in the mutant mice, which highlights the non-redundant roles played by tricellulin

EGFL7 is required for junctional reorganization but not for the hierarchical assembly of tight junctions.

<p>(<b>A-H</b>). Representative confocal images of transverse sections of the dorsal aorta (DA) and posterior cardinal veins (PCV) stained with Claudin-5 (red), GFP to mark cell membranes (MEM-GFP; green), and DAPI to mark nuclei (blue). Phase contrast images for the PCVs represent the <i>Erg</i> in situ signal. All images taken at 63x magnification with scale bars indicating 20 μm. Dotted white and red lines in all panels delineate the endothelial cells comprising each vessel. As anatomical references, the hypochord in (A-D) and the kidney in (E-H) are indicated by solid white lines. (<b>A-B</b>). Claudin-5 expression is absent from endothelial cells of the DA in late tailbud stage (stage 33/34) control and EGFL7-depleted embryos. (<b>C</b>). Claudin-5 localizes to tight junctions between endothelial cells once the DA lumen has formed in early tadpole stage (stage 35/36) control embryos. (<b>D</b>). Claudin-5 expression is apparent in tight junctions between EGFL7-depleted endothelial cells of the DA despite failed lumen formation at early tadpole stage (stage 35/36). (<b>E-F</b>). Claudin-5 expression is absent from endothelial cells of the PCV in late tailbud stage (stage 33/34) control and EGFL7-depleted embryos. (<b>G</b>). Claudin-5 localizes to distinct tight junctions between endothelial cells around the periphery of the PCV lumen in early tadpole stage (stage 35/36) control embryos. (<b>H</b>). Claudin-5 localizes to tight junctions along endothelial cell contacts within the PCV of early tadpole stage (stage 35/36) EGFL7-depleted embryos. 3–4 embryos from each condition/stage were assessed from at least three independent injection batches at the same position along the anterior-posterior axis of the embryo.</p

EGFL7 is required for lumen formation.

<p>(<b>A-D</b>). In situ hybridization with endothelial-specific <i>Erg</i> shows a requirement for EGFL7 in lumen formation of the dorsal aorta (DA) and posterior cardinal veins (PCV). (<b>A</b>). Endothelial cells coalesce at the appropriate positions of the DA and PCVs in late tailbud stage (stage 33/34) control embryos. (<b>B</b>). DA and PCV lumens become evident by early tadpole stage (stage 35/36) in control embryos. (<b>C</b>). Endothelial cells appear as aggregates in late tailbud stage (stage 33/34) EGFL7-depleted vessels similar to controls. (<b>D</b>). Lumens fail to form in early tadpole stage (stage 35/36) EGFL7-depleted embryos. 3–4 embryos from each condition/stage were assessed from at least three independent injection batches at the same position along the anterior-posterior axis of the embryo. Top image of each panel was taken at 20x magnification. Black boxes correspond to enlarged images of each vessel displayed below. Scale bars represent 20 μm. (<b>E</b>). Frequency of lumenized PCVs and DAs in early tadpole stage (stage 35/36) embryos. Lumens are detected in 81% of control embryos vs. 21% of EGFL7-depleted embryos. n = 209 control, n = 216 MO, three independent experiments. Student's t-test was used to calculate the p-value and bars represent ± SEM. (<b>F</b>). Measurement of the luminal area within the PCV in early tadpole stage (stage 35/36) embryos. The size of control lumens = 218 μm. Of EGFL7-depleted vessels that were lumenized, lumens were significantly smaller,75 μm. n = 70 control, n = 29 MO. A Mann-Whitney test was used to determine significance and bars represent ± SEM.</p

Cellular morphology of endothelial cells undergoing lumen formation.

<p>(<b>A-H</b>). Representative confocal images of transverse sections of the dorsal aorta (DA) and posterior cardinal veins (PCV) stained with GFP to mark cell membranes (MEM-GFP; green) and DAPI to mark nuclei (blue). Phase contrast images for the PCVs represent the <i>Erg</i> in situ signal. All images taken at 63x magnification with scale bars indicating 20 μm. Dotted white and red lines in all panels delineate individual endothelial cells. As anatomical references, the hypochord in (A-D) and the kidney in (E-H) are indicated by solid white lines. (<b>A</b>). Endothelial cells of the DA are oblong-shaped at late tailbud stage (stage 33/34) control embryos. (<b>B</b>). Endothelial cells of late tailbud stage (stage 33/34) EGFL7-depleted DAs display a similar morphology to controls. (<b>C</b>). Endothelial cells elongate during DA lumen formation in early tadpole stage (stage 35/36) control embryos. (<b>D</b>). Endothelial cells of the DA remain oblong-shaped and do not elongate to accommodate the vascular lumen in early tadpole stage (stage 35/36) EGFL7-depleted embryos. (<b>E</b>). Endothelial cells of the PCVs have a polygonal cobblestone-like shape in late tailbud stage (stage 33/34) control embryos. (<b>F</b>). Endothelial cells of late tailbud stage (stage 33/34) EGFL7-depleted PCVs are similarly shaped as controls. (<b>G</b>). Endothelial cells elongate, becoming narrower as PCV lumens form by early tadpole stage (stage 35/36) control embryos. (<b>H</b>). Endothelial cells of the PCVs retain their polygonal morphology in early tadpole stage (stage 35/36) EGFL7-depleted embryos. 3–4 embryos from each condition/stage were assessed from at least three independent injection batches at the same position along the anterior-posterior axis of the embryo.</p

EGFL7 is required for proper reorganization of tight junctions.

<p>(<b>A-H</b>). Representative confocal images of transverse sections of the dorsal aorta (DA) and posterior cardinal veins (PCV) stained with ZO-1 (red), GFP to mark cell membranes (MEM-GFP; green), and DAPI to mark nuclei (blue). Phase contrast images for the PCVs represent the <i>Erg</i> in situ signal. All images taken at 63x magnification with scale bars indicating 20 μm. Dotted white and red lines in all panels delineate the endothelial cells comprising each vessel. As anatomical references, the hypochord in (A-D) and the kidney in (E-H) are indicated by solid white lines. (<b>A</b>). ZO-1 localizes to tight junctions between endothelial cells of the DA in late tailbud stage (stage 33/34) control embryos. (<b>B</b>). ZO-1 tight junctions between endothelial cells of the DA in late tailbud stage (stage 33/34) EGFL7-depleted embryos appear similar to controls. (<b>C</b>). Tight junctions are redistributed to distinct puncta between endothelial cells at the periphery of the lumen in early tadpole stage (stage 35/36) control embryos. (<b>D</b>). Tight junctions are retained between endothelial cells of the DA in early tadpole stage (stage 35/36) EGFL7-depleted embryos. (<b>E</b>). Tight junctions assemble along the cord center between endothelial cells of the PCV in late tailbud stage (stage 33/34) control embryos. (<b>F</b>). Similar to controls, tight junctions assemble between adjacent endothelial cells of the PCV in late tailbud stage (stage 33/34) EGFL7-depleted embryos. (<b>G</b>). Tight junctions are redistributed to the periphery of the lumenized PCV and appear as distinct points of cell-cell contact in early tadpole stage (stage 35/36) control embryos. (<b>H</b>). Tight junctions are retained along the cord center of the PCV in early tadpole stage (stage 35/36) EGFL7-depleted embryos. 3–4 embryos from each condition/stage were assessed from at least 3–4 independent injection batches at the same position along the anterior-posterior axis of the embryo.</p

Apical polarity is not established in <i>Xenopus</i> vessels.

<p>(<b>A-H</b>). Representative confocal images of transverse sections of the dorsal aorta (DA) and posterior cardinal veins (PCV) stained with atypical PKCζ (aPKCζ; red), GFP to mark cell membranes (MEM-GFP; green), and DAPI to mark nuclei (blue; A,B,E,F) or Phalloidin to mark F-actin (green) and DAPI (blue; C,D,G,H). Phase contrast images for the PCVs in (E) and (F) represent the <i>Erg</i> in situ signal. All images taken at 63x magnification with scale bars indicating 20 μm. Dotted white and red lines in all panels delineate the endothelial cells comprising each vessel. As anatomical references, the hypochord in (A-D) and the kidney in (E-H) are indicated by solid white lines. Asterisks denote the vessel lumen. (<b>A-B</b>). aPKCζ enrichment is absent from the cell-cell contact in late tailbud stage (stage 33/34) control embryos and the apical membrane of the DA of early tadpole stage (stage 35/36) control embryos. 3–4 embryos from each condition/stage were assessed from at least three independent injection batches at the same position along the anterior-posterior axis of the embryo. (<b>C-D</b>). F-actin fails to localize to the cell-cell contact or apical surface of the dorsal aorta at late tailbud and early tadpole stages. (<b>E-F</b>). aPKCζ can readily be detected on the apical surface of the kidney (arrow) but not at the cell-cell contact or apical membrane of the PCV at late tailbud (stage 33/34) or early tadpole stages (stage 35/36). (<b>G-H</b>). F-actin is evenly distributed in venous endothelial cells. 3–4 embryos from each condition/stage were assessed from at least two independent injection batches at the same position along the anterior-posterior axis of the embryo.</p

Vascular lumen formation proceeds independently of circulation in <i>Xenopus</i>.

<p>(<b>A-B</b>). In situ hybridization with endothelial-specific <i>Erg</i> followed by sectioning was performed to determine the time course of vascular lumen formation of the dorsal aorta (DA) and posterior cardinal veins (PCV) in <i>Xenopus</i>. (<b>A</b>). Endothelial cells are coalesced together at the correct positions of the DA and PCVs in late tailbud stage (stage 33/34) control embryos. (<b>B</b>). Lumenized DA and PCVs become apparent by early tadpole stage (stage 35/36) in control embryos. 3–4 embryos from each condition/stage were assessed from at least three independent injection batches at the same position along the anterior-posterior axis of the embryo. (<b>C-D</b>). In situ hybridization with <i>Erg</i> followed by sectioning. (<b>C</b>). Transverse section of an early tadpole stage (stage 35/36) wildtype embryo depicting lumenized DA and PCVs. (<b>D</b>). Following the removal of the anterior and heart of early tailbud embryos, explants were cultured until early tadpole stage (stage 35/36) and processed as indicated above. PCV and DA lumens readily form in the absence of a functioning heart or circulatory system. 3–4 embryos from each condition were assessed from two independent experiments. Top image of each panel was taken at 20x magnification. Black boxes correspond to enlarged images of each vessel displayed below. Scale bars represent 20 μm.</p

Basement membrane proteins are deposited on both the basal and apical surfaces of early vessels in <i>Xenopus</i>.

<p><b>(A-G)</b> Representative confocal images of transverse sections of the dorsal aorta (DA) and posterior cardinal veins (PCV) stained with laminin or fibronectin (red). In A,B,D,E,F staining with GFP to mark cell membranes (MEM-GFP) and DAPI to mark nuclei (blue) is also shown. In C and G, Flk-1:GFP transgenic animals were used to identify endothelial cells (green). All images taken at 63x magnification with scale bars indicating 20 μm. Dotted white lines in all panels delineate the endothelial cells comprising each vessel. As anatomical references, the hypochord in (A-B) and the kidney in (D-F) are indicated by solid white lines. Dotted lines in the magnified images represent vessels. Asterisks denote the vessel lumen. (<b>A</b>). The DA of early tadpole stage (stage 35/36) control embryos display deposition of fibronectin on both apical and basal surfaces (arrowheads point to apical staining). (<b>B</b>). DA of early tadpole stage (stage 35/36) control embryo exhibits the laminin staining on the basal and the apical surfaces of the vessel (arrowheads point to apical staining). (<b>C</b>). By late tadpole stage (stage 46), proper polarity is established in the dorsal aorta with laminin localizing to the basal surface of endothelial cells (arrowheads point to basal staining). (<b>D</b>) PCV of early tadpole stage (stage 35/36) control embryo exhibits apically and basally deposited fibronectin (arrowheads point to apical staining). (<b>E-F</b>). The kidney displays appropriate basal expression of laminin (arrow), however, the PCV of early tadpole stage (stage 35/36) control embryos display aggregates of laminin staining on the apical surface (arrowheads). (<b>G</b>) Laminin becomes distributed on the basal surface of the posterior cardinal vein in late tadpole stage (stage 46) embryos (arrowheads point to basal staining). 3–4 embryos from each condition/stage were assessed from at least three independent injection batches at the same position along the anterior-posterior axis of the embryo.</p