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• W2032967567 abstract "The hypothalamo-neurohypophyseal system (HNS) is the neurovascular structure through which the hypothalamic neuropeptides oxytocin and arginine-vasopressin exit the brain into the bloodstream, where they go on to affect peripheral physiology. Here, we investigate the molecular cues that regulate the neurovascular contact between hypothalamic axons and neurohypophyseal capillaries of the zebrafish. We developed a transgenic system in which both hypothalamic axons and neurohypophyseal vasculature can be analyzed in vivo. We identified the cellular organization of the zebrafish HNS as well as the dynamic processes that contribute to formation of the HNS neurovascular interface. We show that formation of this interface is regulated during development by local release of oxytocin, which affects endothelial morphogenesis. This cell communication process is essential for the establishment of a tight axovasal interface between the neurons and blood vessels of the HNS. We present a unique example of axons affecting endothelial morphogenesis through secretion of a neuropeptide. The hypothalamo-neurohypophyseal system (HNS) is the neurovascular structure through which the hypothalamic neuropeptides oxytocin and arginine-vasopressin exit the brain into the bloodstream, where they go on to affect peripheral physiology. Here, we investigate the molecular cues that regulate the neurovascular contact between hypothalamic axons and neurohypophyseal capillaries of the zebrafish. We developed a transgenic system in which both hypothalamic axons and neurohypophyseal vasculature can be analyzed in vivo. We identified the cellular organization of the zebrafish HNS as well as the dynamic processes that contribute to formation of the HNS neurovascular interface. We show that formation of this interface is regulated during development by local release of oxytocin, which affects endothelial morphogenesis. This cell communication process is essential for the establishment of a tight axovasal interface between the neurons and blood vessels of the HNS. We present a unique example of axons affecting endothelial morphogenesis through secretion of a neuropeptide. Live imaging of neurovascular interfaces reveals mechanisms driving morphogenesis Hypothalamic neurons are necessary for pituitary vascular organization Oxytocin signaling regulates the neurovascular interface in the pituitary The neuroendocrine system is composed of neurosecretory brain cells that transfer hormones into the bloodstream to influence the function of target cells throughout the body. The hypothalamo-neurohypophyseal system (HNS) is a major neuroendocrine conduit through which the brain controls peripheral physiology (Burbach et al., 2001Burbach J.P. Luckman S.M. Murphy D. Gainer H. Gene regulation in the magnocellular hypothalamo-neurohypophysial system.Physiol. Rev. 2001; 81: 1197-1267Crossref PubMed Scopus (271) Google Scholar). The anatomy and activities of the HNS are conserved in all vertebrates. Ramon Cajal was the first to provide a description of the nerve fibers that connect the hypothalamus with the posterior pituitary (Cajal, 1911Cajal S.R. Histologie du Systeme Neurveux de L'Homme et de Vertebres. A. Maloine, Paris1911Google Scholar). It has since been established that these hypothalamic neurons themselves secrete neurohormones directly into the blood circulation, a finding that arose from experiments in both fish and mammalian models (Bargmann, 1949Bargmann W. J. Mol. Med. 1949; 27: 617-622Google Scholar, Harris, 1948bHarris G.W. Neural control of the pituitary gland.Physiol. Rev. 1948; 28: 139-179PubMed Google Scholar, Scharrer, 1928Scharrer E. [The light sensitivity of blind minnows (Investigations About the diencephalon of the fish I)]. Journal of Comparative Physiology A.Neuroethology. 1928; 7: 1-38Google Scholar). The hypothalamic neuropeptides arginine-vasopressin (AVP) and oxytocin (OXT) are synthesized in massive magnocellular neurons in the hypothalamus, transported along axons all the way down to the neurohypophysis, where they are secreted (Brownstein et al., 1980Brownstein M.J. Russell J.T. Gainer H. Synthesis, transport, and release of posterior pituitary hormones.Science. 1980; 207: 373-378Crossref PubMed Scopus (615) Google Scholar). Within the neurohypophysis, AVP and OXT are released from axons of the supraopticohypophyseal tract into fenestrated capillaries, thus leaving the brain and entering the general circulation without disrupting the blood-brain barrier (Burbach et al., 2001Burbach J.P. Luckman S.M. Murphy D. Gainer H. Gene regulation in the magnocellular hypothalamo-neurohypophysial system.Physiol. Rev. 2001; 81: 1197-1267Crossref PubMed Scopus (271) Google Scholar). In the general circulation, secreted AVP regulates water homeostasis by increasing water permeability of the collecting duct of the kidney, and oxytocin regulates labor and milk let down by causing the respective contraction of the smooth muscle of the uterus and of the myoepithelial cells of breast ducts (for review, see Burbach et al., 2001Burbach J.P. Luckman S.M. Murphy D. Gainer H. Gene regulation in the magnocellular hypothalamo-neurohypophysial system.Physiol. Rev. 2001; 81: 1197-1267Crossref PubMed Scopus (271) Google Scholar, Gimpl and Fahrenholz, 2001Gimpl G. Fahrenholz F. The oxytocin receptor system: structure, function, and regulation.Physiol. Rev. 2001; 81: 629-683Crossref PubMed Scopus (2106) Google Scholar, Verbalis, 2007Verbalis J.G. How does the brain sense osmolality?.J. Am. Soc. Nephrol. 2007; 18: 3056-3059Crossref PubMed Scopus (44) Google Scholar). These physiological activities are conserved: in teleost fish, the AVP-like neuropeptide (Avpl) (a.k.a. arginine-vasotocin) regulates water balance by affecting filtration in the kidney (Amer and Brown, 1995Amer S. Brown J.A. Glomerular actions of arginine vasotocin in the in situ perfused trout kidney.Am. J. Physiol. 1995; 269: R775-R780PubMed Google Scholar, Macfarlane and Maetz, 1974Macfarlane N.A. Maetz J. Effects of hypophysectomy on sodium and water exchanges in the euryhaline flounder, Platichthys flesus (L).Gen. Comp. Endocrinol. 1974; 22: 77-89Crossref PubMed Scopus (6) Google Scholar, Peter and Fryer, 1983Peter R.E. Fryer J.N. Endocrine Functions of the Hypothalamus of Actinopterygians. Volume 2. University of Michigan Press, Ann Arbor, MI1983Google Scholar) and oxytocin-like neuropeptide (Oxtl) (a.k.a. isotocin) regulates contraction of smooth muscles in the ovary and oviduct during parturition or oviposition of live bearing and egg laying fish (La Pointe, 1977La Pointe J.L. Comparative physiology of neurohypophysial hormone action on vertebrate oviduct uterus.Am. Zool. 1977; 17: 763-773Google Scholar, Peter and Fryer, 1983Peter R.E. Fryer J.N. Endocrine Functions of the Hypothalamus of Actinopterygians. Volume 2. University of Michigan Press, Ann Arbor, MI1983Google Scholar). Avpl and Oxtl also regulate blood pressure in the ventral aorta (Chan, 1977Chan D.K.O. Comparative physiology of the vasomotor effects of neurohypophysial peptides in the vertebrates.Am. Zool. 1977; 17: 751-761Google Scholar, Kulczykowska, 1998Kulczykowska E. Effects of arginine vasotocin, isotocin and melatonin on blood pressure in the conscious atlantic cod (Gadus morhua): hormonal interactions?.Exp. Physiol. 1998; 83: 809-820PubMed Google Scholar, Le Mevel et al., 1993Le Mevel J.C. Pamantung T.F. Mabin D. Vaudry H. Effects of central and peripheral administration of arginine vasotocin and related neuropeptides on blood pressure and heart rate in the conscious trout.Brain Res. 1993; 610: 82-89Crossref PubMed Scopus (53) Google Scholar, Peter and Fryer, 1983Peter R.E. Fryer J.N. Endocrine Functions of the Hypothalamus of Actinopterygians. Volume 2. University of Michigan Press, Ann Arbor, MI1983Google Scholar). The HNS is therefore a central point of interface between the hormonal, neuronal, and vascular systems common to all vertebrate species. The neurohypophysis is an elaborate three-dimensional structure, which substantially complicates the interpretation of cellular interactions and dynamics based solely on tissue sections. Although the anatomical structures of the neurohypophyseal axons and blood vessels have been the focus of intense study for over a century (Bargmann, 1949Bargmann W. J. Mol. Med. 1949; 27: 617-622Google Scholar, Fink and Smith, 1971Fink G. Smith G.C. Ultrastructural features of the developing hypothalamo-hypophysial axis in the rat. A correlative study.Z. Zellforsch. Mikrosk. Anat. 1971; 119: 208-226Crossref PubMed Scopus (102) Google Scholar, Harris, 1948aHarris G.W. Further evidence regarding the endocrine status of the neurohypophysis.J. Physiol. 1948; 107: 436-448Crossref PubMed Scopus (1) Google Scholar, Scharrer, 1928Scharrer E. [The light sensitivity of blind minnows (Investigations About the diencephalon of the fish I)]. Journal of Comparative Physiology A.Neuroethology. 1928; 7: 1-38Google Scholar), little progress has been made in uncovering the molecular and cellular processes that underlie formation of the interface between hypothalamic axons and hypophyseal vasculature. Here, we present a unique transgenic approach in which both zebrafish oxytocinergic axonal termini and vascular endothelia cells within the neurohypophysis are labeled. We use this system to analyze the morphogenesis of the neurohypophyseal axonal trajectories and neuroendocrine vasculature in live zebrafish embryos and demonstrate that neurohypophyseal angiogenesis is regulated by the developing hypothalamo-neurohypophyseal nerve termini. Our results suggest that local secretion of oxytocin into the neurohypophysis is an intrinsic developmental event essential for the formation of the hypophyseal neurovascular connection. The optically transparent zebrafish embryo offers a unique tool to study the ontogenesis of the HNS in vivo without the need for surgical intervention, although the formation and structure of the zebrafish neurohypophysis is, to date, still poorly characterized. We recently identified the genomic regulatory region of the zebrafish oxtl gene and have generated a transgenic reporter line, oxtl:EGFP, in which zebrafish oxytocinergic cell bodies in the hypothalamus are labeled (Blechman et al., 2011Blechman J. Amir-Zimberstein L. Gutnick A. Ben-Dor S. Levkowitz G. The metabolic regulator PGC-1α directly controls the expression of the hypothalamic neuropeptide oxytocin.J. Neurosci. 2011; (in press)PubMed Google Scholar). We first examined whether this line can be used to track axonal projections from the hypothalamus to the neurohypophysis. Three-dimensional reconstitution of optically-sectioned 9-day-old oxtl:EGFP larvae revealed extensive EGFP-positive axonal trajectories of oxytocinergic neurons throughout the nervous system (Figures 1A and 1B; see Movie S1 available online). In particular, we observed a prominent oxtl:EGFP+ tract originating from the two hemispheres of the neurosecretory preoptic areas (NPO) converging into the midline and terminating at the ventral part of the hypothalamus at the presumed location of the pituitary (Figure 1B). Immunostaining of the oxtl:EGFP with an antioxytocin antibody confirmed that these ventral hypothalamic axonal termini colocalize with oxytocinergic-positive vesicles of the presumed neurohypophysis of embryonic and adult brains (Figures 1C and 1D and Figure S1). To further validate this observation we crossed the oxtl:EGFP line with another transgenic line in which the adenohypophyseal pituitary cell-type, prolactin (Prl) is labeled (Liu et al., 2003Liu N.A. Huang H. Yang Z. Herzog W. Hammerschmidt M. Lin S. Melmed S. Pituitary corticotroph ontogeny and regulation in transgenic zebrafish.Mol. Endocrinol. 2003; 17: 959-966Crossref PubMed Scopus (93) Google Scholar, Liu et al., 2006Liu N.A. Liu Q. Wawrowsky K. Yang Z. Lin S. Melmed S. Prolactin receptor signaling mediates the osmotic response of embryonic zebrafish lactotrophs.Mol. Endocrinol. 2006; 20: 871-880Crossref PubMed Scopus (53) Google Scholar). Two-color imaging of this double-transgenic line revealed that the ventral hypothalamic oxytocinergic trajectories terminate near the Prl-positive adenohypophyseal cells (Figure 2A ). This transgenic system provided us with a new tool to study the morphogenesis of the zebrafish preopticohypophyseal axonal tract analogous to the supraopticohypophyseal tract in mammals.Figure 2Axovasal Interactions in the Zebrafish PituitaryShow full caption(A–D) Three-dimensional reconstructions of the hypophysis of 9-day-old zebrafish embryos carrying pairs of transgenic fluorescent markers of different hypophyseal components.(A) The oxtl:EGFP transgene (green) marks oxytocinergic axons terminating at the neurohypophysis, just dorsal and posterior to the adenohypophyseal prolactin-producing cells marked with prl:RFP (red).(B) Double transgenic animals expressing oxtl:EGFP (green) together with the vascular endothelial marker vegfr2:Cherry (red) reveal a distinct, previously unidentified structure of the zebrafish hypophyseal vasculature.(C and D) Hypophyseal vasculature, visualized by either vegfr2:EGFP or vegfr2:Cherry, together with the adenohypophyseal markers prl:RFP and pomc:EGFP. The ring-like vascular structure resided dorsal to the adenohypophysis. (B′) and (D′) show optical Z-slices that demonstrate the position of the NH in an indentation of the adenohypophysis.(E) Schematic map of ventral head vasculature of the zebrafish larvae, including the location of the hypophyseal vasculature. The asterisk marks the location of the neurohypophysis.AH, adenohypophysis; CrDI, cranial division of the internal carotid artery; NH, neurohypophysis; OA, optic artery; PLA, palatocerebral artery; PLV, palatocerebral vein; pomc, proopiomelanocortin; prl, prolactin; vegfr, vascular endothelial growth factor receptor. Scale bar, 20 μm. See also Movie S2. Visualization of Axons and Blood Vessels of the HNS (Related to Figure 2), Movie S3. Neurovascular Interaction in the Zebrafish Neurohypophysis (Related to Figure 2), Movie S4. Hypophyseal Blood Vessels and Adenohypophyseal Cells (Related to Figure 2).View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A–D) Three-dimensional reconstructions of the hypophysis of 9-day-old zebrafish embryos carrying pairs of transgenic fluorescent markers of different hypophyseal components. (A) The oxtl:EGFP transgene (green) marks oxytocinergic axons terminating at the neurohypophysis, just dorsal and posterior to the adenohypophyseal prolactin-producing cells marked with prl:RFP (red). (B) Double transgenic animals expressing oxtl:EGFP (green) together with the vascular endothelial marker vegfr2:Cherry (red) reveal a distinct, previously unidentified structure of the zebrafish hypophyseal vasculature. (C and D) Hypophyseal vasculature, visualized by either vegfr2:EGFP or vegfr2:Cherry, together with the adenohypophyseal markers prl:RFP and pomc:EGFP. The ring-like vascular structure resided dorsal to the adenohypophysis. (B′) and (D′) show optical Z-slices that demonstrate the position of the NH in an indentation of the adenohypophysis. (E) Schematic map of ventral head vasculature of the zebrafish larvae, including the location of the hypophyseal vasculature. The asterisk marks the location of the neurohypophysis. AH, adenohypophysis; CrDI, cranial division of the internal carotid artery; NH, neurohypophysis; OA, optic artery; PLA, palatocerebral artery; PLV, palatocerebral vein; pomc, proopiomelanocortin; prl, prolactin; vegfr, vascular endothelial growth factor receptor. Scale bar, 20 μm. See also Movie S2. Visualization of Axons and Blood Vessels of the HNS (Related to Figure 2), Movie S3. Neurovascular Interaction in the Zebrafish Neurohypophysis (Related to Figure 2), Movie S4. Hypophyseal Blood Vessels and Adenohypophyseal Cells (Related to Figure 2). We next visualized the neurovascular interface within the neurohypophysis by crossing our HNS specific line with a vascular endothelial reporter expressing mCherry in vegfr2-positive cells (Chi et al., 2008Chi N.C. Shaw R.M. De Val S. Kang G. Jan L.Y. Black B.L. Stainier D.Y. Foxn4 directly regulates tbx2b expression and atrioventricular canal formation.Genes Dev. 2008; 22: 734-739Crossref PubMed Scopus (228) Google Scholar, Jin et al., 2005Jin S.W. Beis D. Mitchell T. Chen J.N. Stainier D.Y. Cellular and molecular analyses of vascular tube and lumen formation in zebrafish.Development. 2005; 132: 5199-5209Crossref PubMed Scopus (530) Google Scholar). Previous work, based on microangiography, has provided an extensive map of the vascular network in the zebrafish head (Isogai et al., 2001Isogai S. Horiguchi M. Weinstein B.M. The vascular anatomy of the developing zebrafish: an atlas of embryonic and early larval development.Dev. Biol. 2001; 230: 278-301Crossref PubMed Scopus (603) Google Scholar). Using our axovasal double transgenic line, we were able to identify GFP+ oxytocinergic projections converging posteriorly and medially into the neurohypophysis, arborizing extensively and interfacing with a formation of blood vessels previously annotated as palatocerebral veins (Figures 2B and 2E and Movie S2. Visualization of Axons and Blood Vessels of the HNS (Related to Figure 2), Movie S3. Neurovascular Interaction in the Zebrafish Neurohypophysis (Related to Figure 2)). The hypothalamo-neurohypophyseal (HN) axonal termini project onto the posterior part of this loop-shaped vascular structure. To ascertain the position of this structure in relation to the adenohypophysis, we generated double transgenic lines with labeled blood vessels (vegfr2:EGFP or vegfr2:mCherry) and adenohypophyseal cell types (pomc:GFP or prl:RFP). Three-dimensional image reconstruction of the ventral diencephalon in these embryos revealed that these blood vessels engulf part of the pituitary area abutting the adenohypophysis (Figures 2C and 2D and Movie S4). The NH is positioned in a pocket-like indentation formed by the ventrally located adenohypophysis (Figures 2B′ and 2D′). The vessels forming the hypophyseal vascular structure has been previously annotated as median palatocerebral vein (MPLV) and palatocerebral vein (PLV) (Isogai et al., 2001Isogai S. Horiguchi M. Weinstein B.M. The vascular anatomy of the developing zebrafish: an atlas of embryonic and early larval development.Dev. Biol. 2001; 230: 278-301Crossref PubMed Scopus (603) Google Scholar). As we now identify these vessels as the zebrafish hypophyseal vessels, we set out to reassess their arterial and venous identity. We performed live imaging of hypophyseal blood flow using time-lapse microscopy of a triple transgenic line with labeled erythrocytes (gata1:dsRed), blood vessels (vegfr2:EGFP) and adenohypophyseal cells for positional reference (prl:RFP). By following individual dsRed+ erythrocytes, we determined the direction of blood flow into and out of the hypophysis to the lateral venous sinuses (Figure 3A and Movie S5). This allowed us to deduce the identity of the hypophyseal artery and veins (Figure 3B and Figure S2). Taken together, our analyses identify the organization of the zebrafish neurovascular HNS structure in relation to the other pituitary cell populations (Figure 3C). We set out to delineate the morphogenic events leading to the formation of axovasal contact between hypothalamic axons and neurohypophyseal blood capillaries. Although the expression of oxytocin is apparent from the Prim-15 stage (30–36 hr; Unger and Glasgow, 2003Unger J.L. Glasgow E. Expression of isotocin-neurophysin mRNA in developing zebrafish.Gene Expr. Patterns. 2003; 3: 105-108Crossref PubMed Scopus (44) Google Scholar), we found that our oxtl:EGFP did not express sufficient levels of the EGFP protein to detect axonal expression at this embryonic stage (data not shown). To resolve this, we sought an earlier transgenic reporter that would allow detection of the initial stages of hypophyseal innervation. We previously reported that the homeodomain-containing protein Orthopedia (Otp) is expressed in hypothalamic neuronal progenitors, including the magnocellular oxytocinergic neurons of the NPO area (Blechman et al., 2007Blechman J. Borodovsky N. Eisenberg M. Nabel-Rosen H. Grimm J. Levkowitz G. Specification of hypothalamic neurons by dual regulation of the homeodomain protein Orthopedia.Development. 2007; 134: 4417-4426Crossref PubMed Scopus (78) Google Scholar, Machluf et al., 2011Machluf Y. Gutnick A. Levkowitz G. Development of the zebrafish hypothalamus.Ann. N Y Acad. Sci. 2011; 1220: 93-105Crossref PubMed Scopus (60) Google Scholar). Moreover, we recently identified a genomic cis-regulatory region driving expression of the zebrafish otpb gene in the NPO (Fujimoto et al., 2011Fujimoto E. Stevenson T.J. Chien C.B. Bonkowsky J.L. Identification of a dopaminergic enhancer indicates complexity in vertebrate dopamine neuron phenotype specification.Dev. Biol. 2011; 352: 393-404Crossref PubMed Scopus (32) Google Scholar). We made use of this cis-regulatory element to generate a transgenic otpb:EGFP-caax line driving expression of a membrane-tagged EGFP in Otp+ cell bodies and axons, including hypothalamo-neurohypophyseal (HN) projections (Figures 4A–4C ). We analyzed neurovascular formation at 36, 54, and 72 hr using an otpb:caax-EGFP;fli:dsRed double transgenic line. This analysis shows that HN axonal projections innervate the neurohypophysis before the vessels are formed and a tight neurovascular interface is established between 2 and 3 days of development (Figures 4D–4F). A dynamic analysis of hypophyseal vascularization/angiogenesis has never been reported. We used time-lapse microscopy to track vascular endothelial cells in the ventral diencephalon of zebrafish embryos carrying the vegfr2:Cherry vascular reporter transgene in which a membrane-localized monomeric Cherry is expressed in endothelial precursors and mature vessels (Chi et al., 2008Chi N.C. Shaw R.M. De Val S. Kang G. Jan L.Y. Black B.L. Stainier D.Y. Foxn4 directly regulates tbx2b expression and atrioventricular canal formation.Genes Dev. 2008; 22: 734-739Crossref PubMed Scopus (228) Google Scholar). The embryos were imaged from 2 days postfertilization (dpf), when the hypophyseal vascular structure is yet absent, through 3 dpf, at which time the vascular loop around the neurohypophysis is fully established. Three-dimensional reconstitution followed by cell tracking of such time-lapse movies (n = 4) showed that formation of the hypophyseal artery begins prior to that of the veins; the artery is initiated at the hypophysis itself and extends anteriorly until it connects with the palatocerebral arteries at day 2.5 (Figure 4G). As the primary lateral vein sinuses extend forward through the diencephalon, they sprout medially and wrap around the posterior edge of the neurohypophysis to form the hypophyseal veins. These fuse bilaterally with one another and with the hypophyseal arteries to form a ring around the neurohypophysis and connect it to the general circulation at approximately 3 days. (Figure 4G and Movie S6). High-resolution 3D rendering of an embryo fixed at this time point clearly shows that the hypophyseal veins connect bilaterally to the primary vein sinuses (Figure S2). The above analyses of neurohypophyseal axons and blood vessels highlight key cellular events of HNS morphogenesis: (1) HN axons appears at the site of the prospective neurohypophysis as early as 24–36 hr after fertilization. (2) Endothelial cells forming the hypophyseal artery and veins appear at 2 days, approximately 24 hr after the axons start to innervate the neurohypophysis. (3) It takes another 24 hr for the endothelial cells of the artery and veins of the hypophysis to sprout and fuse to form the aforementioned loop-like structure, creating a tight interface with the neurohypophyseal axons. During its morphogenesis, the neurohypophysis becomes a point of intersection between axons and vessels. We next examined the interdependence of blood vessels for axon patterning in the neurohypophysis. The zebrafish mutant cloche (clo) has an overall deficiency of endothelial and hematopoietic lineages, including a complete lack of head vasculature (Stainier et al., 1995Stainier D.Y. Weinstein B.M. Detrich 3rd, H.W. Zon L.I. Fishman M.C. Cloche, an early acting zebrafish gene, is required by both the endothelial and hematopoietic lineages.Development. 1995; 121: 3141-3150Crossref PubMed Google Scholar). We examined whether the neurohypophyseal axonal projections are affected in cloche mutants by comparing oxytocinergic-positive neurohypophyseal axonal termini in mutant and wild-type siblings. This analysis revealed no significant differences in oxytocinergic immune-reactive projections indicating that the presence of endothelial cells/vessels is not required for HN innervation (Figures 5A and 5B ; n = 13/15). This result is consistent with the observation that axons begin to innervate the NH before the blood vessel network is established (Figures 4D–4F). We next examined whether the presence of HN neuronal tracts is important for morphogenesis of the neuroendocrine vasculature. We undertook a conditional HN cell ablation strategy using tissue-specific expression of the nitroreductase (NTR), an enzyme that converts the prodrug metronidazole into a cytotoxic agent (Curado et al., 2008Curado S. Stainier D.Y. Anderson R.M. Nitroreductase-mediated cell/tissue ablation in zebrafish: a spatially and temporally controlled ablation method with applications in developmental and regeneration studies.Nat. Protoc. 2008; 3: 948-954Crossref PubMed Scopus (207) Google Scholar, Davison et al., 2007Davison J.M. Akitake C.M. Goll M.G. Rhee J.M. Gosse N. Baier H. Halpern M.E. Leach S.D. Parsons M.J. Transactivation from Gal4-VP16 transgenic insertions for tissue-specific cell labeling and ablation in zebrafish.Dev. Biol. 2007; 304: 811-824Crossref PubMed Scopus (241) Google Scholar). We chose to target Otp+ neurons as this cell population includes all NPO neurons that innervate the neurohypophysis (Figure 4 and Machluf et al., 2011Machluf Y. Gutnick A. Levkowitz G. Development of the zebrafish hypothalamus.Ann. N Y Acad. Sci. 2011; 1220: 93-105Crossref PubMed Scopus (60) Google Scholar). To this end, we generated a transgenic line, otpb:Gal4;UAS:NTR-Cherry, in which the NTR-Cherry fusion protein is expressed in Otp+ neurons using a tissue-specific Gal4 transgene to drive an NTR-Cherry that was placed under multiple Gal4 upstream activation sequences (Davison et al., 2007Davison J.M. Akitake C.M. Goll M.G. Rhee J.M. Gosse N. Baier H. Halpern M.E. Leach S.D. Parsons M.J. Transactivation from Gal4-VP16 transgenic insertions for tissue-specific cell labeling and ablation in zebrafish.Dev. Biol. 2007; 304: 811-824Crossref PubMed Scopus (241) Google Scholar). We crossed the otpb:Gal4;UAS:NTR-Cherry fish with the vascular-specific vegfr2:EGFP line to generate triple transgenic embryos in which vascular development can be monitored in the presence or absence of Otp+ HN cells. The metronidazole drug was applied to embryos at 24–72 hr, from the stage at which the first HN neuronal tracts are detected and until the intact hypophyseal vascular structure is formed and contacts the axons. The efficiency of HN cell ablation was assessed by monitoring NTR-Cherry+ (Figures 5D and 5J) and oxtl+ neurons (Figures S3A and S3B) in control and metronidazole-treated triple transgenic (otpb:Gal4;UAS:NTR-Cherry;vegfr2:EGFP) embryos. To verify the deleterious effects of Otp+ cell ablation on HN neurosecretion, we measured the levels of the Oxytocin protein in the neurohypophysis by anti-oxytocin immunostaining and determined that Otp+ cell ablation causes a marked decrease in neurohypophyseal oxytocin levels (Figures 5F, 5I, and 5L and Figure S3C). We then went on to analyze the head vasculature in metronidazole-treated vegfr2:EGFP embryos carrying the otpb:Gal4 and UAS:NTR-Cherry transgenes, as compared to siblings not carrying them. As expected, application of the metronidazole prodrug to embryos not carrying the UAS:NTR-Cherry transgene had no significant effect on hypophyseal or other blood vessels, indicating that in the absence of NTR-Cherry, metronidazole treatment is not toxic and does not affect vasculature (Figures 5G–5I, n = 15/15). In contrast, Otp+ cell ablation in metronidazole-treated triple transgenic (vegfr2:EGFP;otpb:Gal4;UAS:NTR-Cherry) embryos was accompanied by a marked impairment of hypophyseal vasculature, indicating that impaired HN neurosecretion is associated with hypophyseal vascular abnormality (Figures 5J–5L, n = 12/15). While the hypophyseal vasculature of normal embryos tightly surrounds the neurohypophysis forming an elliptical loop (Figures 5E and 5H), ablation of Otp+ neurons caused abnormalities characterized by: failure to close the neurohypophyseal vascular loop, incomplete connection of the hypophyseal artery to the PLA and overall thin hypophyseal vascular structures (Figure 5K). We used two measurable parameters to quantify changes to this morphology (Figure 5C): (1) “loop roundness index”– samples with ablated axons display a more round loop as opposed to the ellipsoid shaped loop in the control samples, and (2) “loop area”– axonal ablated embryos exhibit a significant increase in the loop area. In short, ablation of Otp+ neurons led a significantly rounder loop surrounding a larger area (n = 15, ∗p < 0.05). In all cases, all neighboring head vessels remained unaffected. These results suggest that a tissue interaction between HN neurons projecting to the neurohypophysis and endothelial cells contributes to the formation of" @default.
• W2032967567 created "2016-06-24" @default.
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• W2032967567 date "2011-10-01" @default.
• W2032967567 modified "2023-10-18" @default.
• W2032967567 title "The Hypothalamic Neuropeptide Oxytocin Is Required for Formation of the Neurovascular Interface of the Pituitary" @default.
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