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Oxytocin and Its Receptor Are Necessary for Pituitary Vascularization
(A–D) High-resolution confocal images of the head vasculature of 3-day-old embryos following targeted knockdown of the gene encoding for oxytocin (oxtl-MO) or oxytocin receptor (oxtlr-MO) using antisense morpholino oligonucliotides, leads to severe impairment of neurohypophyseal vasculature, but does not impede development of neighboring vessels. A schematic representation of the corresponding phenotype with arterial (red) and venous (blue) color-coding is shown in (A2)–(C2).
(D and E) By crossing nuclear- and membrane-tagged fluorescent vascular reporter lines, we are able to count the number of vascular endothelial cells in each vessel. A dot-plot histogram showing the x,y positions of EGFP+;Cherry+ endothelial cells nuclei of the hypophyseal vessels as well as the adjacent internal carotid arteries (CrDI, between the PICA and the PLA) is shown in (D2) and (E2). Individual cells belonging to the hypophyseal vessels (red) and CrDI (gray) are color coded. The oxtlr-MO-injected embryos (E) display a markedly reduced number of endothelial cells in the hypophyseal vessel but no change in the nearby internal carotid artery.
(F) Bar histogram showing EGFP+;Cherry+ cell counts in the internal carotid arteries and hypophyseal vessels in control and following oxtlr knockdown. *p < 0.01, n = 14.
(G) RT-PCR analysis of vegfr2:Cherry-positive and -negative FACS sorted endothelial cells demonstrating vascular expression of the oxytocin receptor (oxtlr).
CaDI, caudal division of internal carotid; CrDI, cranial division of the internal carotid artery; MO, morpholino antisense oligonucleotide; OA, optic artery; PICA, primitive internal carotid artery; PLA, palatocerebral artery; PLV, palatocerebral vein. The asterisk (*) demarcates the location of the neurohypophysis. See also Figure S4.
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