Calcitonin receptor-like receptor guides arterial differentiation in zebrafish

Abstract

The calcitonin receptor-like receptor (crlr) is a major endothelial cell receptor for adrenomedullin, a peptide vasodilator involved in cardiovascular development, homeostasis, and disease. Here, we used the zebrafish (Danio rerio) model to characterize the role of crlr in vascular development. Crlr is expressed within somites from the 4-to the 13-somite stage and by arterial progenitors and axial vessels during zebrafish development. Loss of crlr results in profound alterations in vascular development and angiogenesis, including atrophic trunk dorsal aorta and interruption of anterior aortic bifurcation, delay in intersomitic vessel development, and lack of blood circulation. Remarkably, crlr morphants are characterized by the loss of arterial endothelial cell identity in dorsal aorta, as shown by the lack of expression of the arterial markers ephrin-B2a, DeltaC, and notch5. Down-regulation of crlr affects vascular endothelial growth factor (vegf) expression, whereas vegf overexpression is sufficient to rescue arterial differentiation in crlr morphants. Finally, genetic and biochemical evidences indicate that somitic crlr expression is under the control of sonic hedgehog. These data demonstrate that crlr plays a nonredundant role in arterial differentiation, representing a novel element of the sonic hedgehog- vegf IntroductionThe calcitonin receptor-like receptor (crlr) is a promiscuous 7-transmembrane G protein-coupled receptor (GPCR) that binds various ligands by forming complexes with different receptor activity-modifying proteins (RAMPs). 1 The interaction of crlr protein with RAMP-2 or RAMP-3 leads to the formation of heterodimeric complexes representing the functional receptors for adrenomedullin, a peptide vasodilator involved in cadiovascular stresses and tumor angiogenesis. 2 crlr is highly expressed in endothelium, 3 and crlr-null mice die in utero at midgestation with hydrops fetalis and developmental abnormalities in cardiovascular tissues. 4 Remarkably, adrenomedullin-null mice show a similar cardiovascular phenotype that provides compelling genetic and in vivo evidence that crlr is the primary receptor through which adrenomedullin acts during embryonic development. 4 In vertebrates, the large midline artery and vein are the first blood vessels to develop via a vasculogenic process that involves migration, differentiation, and assembly of the vascular endothelial growth factor (vegf) receptor-2 (kdr) ϩ angioblasts from the lateral posterior mesoderm. 5 Molecular differences exist between arterial and venous endothelial cells before the onset of circulation and the activation of complex genetic pathways specifies artery and vein identity, leading to the expression of specific arterial and venous markers (eg, ephrin-B2a and ephB4, respectively). 6 Genetic alterations of arterial/venous specification are hypothesized to be responsible for congenital hereditary arteriopathies. Indeed, mutations of notch pathway components involved in arterial specification are associated with congenital defects of the...

Sphingosine-1-Phosphate Receptor-1 Controls Venous Endothelial Barrier Integrity in Zebrafish

“…It must be pointed out that zS1P 1 knockdown did not affect arterial/venous differentiation of endothelial cells in zebrafish. Indeed, zS1P 1 morphants did not show any change in the levels of expression of shh, vegf, kdr, and fli-1, as well as of the arterial markers ephrinB2 30 and crlr 34 and of the venous markers flt4 35 and dab2, 36 thus confirming previous observations in S1P 1 −/− mice. 5 Nevertheless, the expression of the Eph receptor EphB4a was significantly downregulated in zS1P 1 -MO-injected embryos.…”

“…As shown in Figure 8, zS1P 1 downregulation does not affect the expression of the specific arterial markers ephrinB2 30 and crlr 34 in the DA of zS1P 1 morphants. Also, zS1P 1 -MO injection does not alter the expression of the venous markers flt4 35 and dab2 36 in the PCV.…”

RNA polymerase III component Rpc9 regulates hematopoietic stem and progenitor cell maintenance in zebrafish

“…However, in that report, only Rpc9, not other CGRP receptor components, was expressed in granulocytes (Harzenetter et al, 2002), implying that Rpc9 may function beyond the CGRP receptor complex. Crlr, another component of this CGRP receptor complex, was reported to be required for zebrafish arterial patterning (Nicoli et al, 2008;Wilkinson et al, 2012). However, we observed no obvious alteration of expression of arterial markers in rpc9 −/− embryos ( Fig.…”

Arterial–Venous Specification During Development

“…125 Somite expression of calcitonin receptor-like receptor (crlr), a known receptor for adrenomedullin, may be under the regulation of shh, and control arterial differentiation upstream of VEGF. 126 Potential effectors of arterial/venous specification such as these will undoubtedly be located as the biology of artery-vein specification continues to be studied.…”