The Hemodynamically-Regulated Vascular Microenvironment Promotes Migration of the Steroidogenic Tissue during Its Interaction with Chromaffin Cells in the Zebrafish Embryo

Abstract

BackgroundWhile the endothelium-organ interaction is critical for regulating cellular behaviors during development and disease, the role of blood flow in these processes is only partially understood. The dorsal aorta performs paracrine functions for the timely migration and differentiation of the sympatho-adrenal system. However, it is unclear how the adrenal cortex and medulla achieve and maintain specific integration and whether hemodynamic forces play a role.Methodology and Principal FindingsIn this study, the possible modulation of steroidogenic and chromaffin cell integration by blood flow was investigated in the teleostean counterpart of the adrenal gland, the interrenal gland, in the zebrafish (Danio rerio). Steroidogenic tissue migration and angiogenesis were suppressed by genetic or pharmacologic inhibition of blood flow, and enhanced by acceleration of blood flow upon norepinephrine treatment. Repressed steroidogenic tissue migration and angiogenesis due to flow deficiency were recoverable following restoration of flow. The regulation of interrenal morphogenesis by blood flow was found to be mediated through the vascular microenvironment and the Fibronectin-phosphorylated Focal Adhesion Kinase (Fn-pFak) signaling. Moreover, the knockdown of krüppel-like factor 2a (klf2a) or matrix metalloproteinase 2 (mmp2), two genes regulated by the hemodynamic force, phenocopied the defects in migration, angiogenesis, the vascular microenvironment, and pFak signaling of the steroidogenic tissue observed in flow-deficient embryos, indicating a direct requirement of mechanotransduction in these processes. Interestingly, epithelial-type steroidogenic cells assumed a mesenchymal-like character and downregulated β-Catenin at cell-cell junctions during interaction with chromaffin cells, which was reversed by inhibiting blood flow or Fn-pFak signaling. Blood flow obstruction also affected the migration of chromaffin cells, but not through mechanosensitive or Fn-pFak dependent mechanisms.Conclusions and SignificanceThese results demonstrate that hemodynamically regulated Fn-pFak signaling promotes the migration of steroidogenic cells, ensuring their interaction with chromaffin cells along both sides of the midline during interrenal gland development.

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Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish

“…NOTE: For analyzing the vascular microenvironment of the steroidogenic tissue, the 3β-Hsd activity-stained embryos with a transgenic endothelium-specific fluorescent reporter are subjected to transverse vibratome sectioning and subsequent IHC analysis 12,18 .…”

“…The differentiation of the interrenal tissue is not compromised even in the complete absence of blood and vasculature. Therefore, how endothelium-derived signals shape the developing interrenal organ can be determined 12,13 . Overall, this histochemical assay has been successfully used for studying specification, differentiation, NOTE: In our experience, repeated freezing and thawing of the aliquots of all the aforementioned solutions do not affect the intensity of 3β-Hsd activity staining.…”