Direct and indirect roles of Fgf3 and Fgf10 in innervation and vascularisation of the vertebrate hypothalamic neurohypophysis.

The neurohypophysis is a crucial component of the hypothalamo-pituitary axis, serving as the site of release of hypothalamic neurohormones into a plexus of hypophyseal capillaries. The growth of hypothalamic axons and capillaries to the forming neurohypophysis in embryogenesis is therefore crucial to future adult homeostasis. Using ex vivo analyses in chick and in vivo analyses in mutant and transgenic zebrafish, we show that Fgf10 and Fgf3 secreted from the forming neurohypophysis exert direct guidance effects on hypothalamic neurosecretory axons. Simultaneously, they promote hypophyseal vascularisation, exerting early direct effects on endothelial cells that are subsequently complemented by indirect effects. Together, our studies suggest a model for the integrated neurohemal wiring of the hypothalamo-neurohypophyseal axis.

Immunocytochemical developmental patterns of the thoracolumbar sympathetic chain in the chick and a comparison with its adrenal counterpart.

The immunocytochemical development of the thoracolumbar sympathetic ganglion and its adrenal counterpart was studied in the chick from days 3.5 to 12 of incubation, using antibodies to 17 separate antigens, including antibodies to pan-neuroendocrine markers, catecholamine-synthesizing and proprotein-processing enzymes, and neuropeptides. Some of the antigens studied (Go protein-alpha subunit, thyrosine hydroxylase, and galanin) were strongly expressed from the first days of development, whereas others (chromogranin-A, chromogranin-B, 7B2 protein, and somatostatin) showed a diverse immunoreactive expression at different stages. Three different patterns were found in the development of both adrenal medulla and thoracolumbar sympathetic ganglion. In the first (chromogranin-A and B, Go protein-alpha subunit, tyrosine hydroxylase, HNK-1, and galanin), virtually all medullary and thoracolumbar sympathetic ganglion cells were strongly immunostained from day 4 onward. Except for HNK-1, chromogranin-A and B, there was a steady increase in immunoreactive cells for all the remaining antigens up to day 12. In the second (7B2 protein, proprotein convertase 2, and secretogranin II), full antigenic expression was reached in medullary and thoracolumbar sympathetic ganglion cells by day 10. In the third pattern (proprotein convertase 3, somatostatin, dopamine-beta-hydroxylase, neuron-specific enolase, vasoactive intestinal polypeptide, and met-enkephalin), differences in immunoreactivity were observed between the medullary and thoracolumbar sympathetic ganglion cells.

Apical accumulation of MARCKS in neural plate cells during neurulation in the chick embryo.

BACKGROUND: The neural tube is formed by morphogenetic movements largely dependent on cytoskeletal dynamics. Actin and many of its associated proteins have been proposed as important mediators of neurulation. For instance, mice deficient in MARCKS, an actin cross-linking membrane-associated protein that is regulated by PKC and other kinases, present severe developmental defects, including failure of cranial neural tube closure. RESULTS: To determine the distribution of MARCKS, and its possible relationships with actin during neurulation, chick embryos were transversely sectioned and double labeled with an anti-MARCKS polyclonal antibody and phalloidin. In the neural plate, MARCKS was found ubiquitously distributed at the periphery of the cells, being conspicuously accumulated in the apical cell region, in close proximity to the apical actin meshwork. This asymmetric distribution was particularly noticeable during the bending process. After the closure of the neural tube, the apically accumulated MARCKS disappeared, and this cell region became analogous to the other peripheral cell zones in its MARCKS content. Actin did not display analogous variations, remaining highly concentrated at the cell subapical territory. The transient apical accumulation of MARCKS was found throughout the neural tube axis. The analysis of another epithelial bending movement, during the formation of the lens vesicle, revealed an identical phenomenon. CONCLUSIONS: MARCKS is transiently accumulated at the apical region of neural plate and lens placode cells during processes of bending. This asymmetric subcellular distribution of MARCKS starts before the onset of neural plate bending. These results suggest possible upstream regulatory actions of MARCKS on some functions of the actin subapical meshwork.

Mapping the origin of the avian enteric nervous system with a retroviral marker.

The enteric nervous system is largely formed from the vagal neural crest which arises from the neuroaxis between somites 1-7. In order to evaluate the contribution of different regions of the vagal crest to the enteric nervous system, we marked crest cells by injecting somites 1-10 with a replication-defective spleen necrosis virus vector which contains the marker gene lacZ. After incubation in X-gal, lacZ-positive blue cells were found in the wall of the gut in three locations. Most were found at the peripheral edge of the developing circular muscle and within the developing submucosa, sites characteristic of developing ganglia. LacZ-positive cells in these ganglionic sites were always surrounded by HNK-1 immunostained cells, confirming their neural crest origin. LacZ-positive cells were also seen in a third location, the circular muscle layer of the esophagus and crop, and were separated from the HNK-1 positive ganglionic elements. These cells in the circular muscle are probably muscle cells derived from labeled mesodermal cells of the somite. Injection of somites 3, 4, 5, and 6 resulted in the largest percentage of preparations with lacZ-positive crest-derived cells and in the largest number of positive cells in the gut. After injection of these somites, lacZ-positive crest-derived cells were found in all regions of the gut from the proventriculus to the rectum. Very few positive crest-derived cells were found in the esophagus.(ABSTRACT TRUNCATED AT 250 WORDS)

Second messenger regulation of occlusion of the spinal neurocoel in the chick embryo.

We know that, once rostral neurulation is completed in the neuroaxis of the chick embryo, the caudal neurocoel becomes occluded and the brain rapidly expands. However, very little is known about the mechanisms maintaining occlusion. Studies had shown that occluded neurocoels reopened in embryos treated with chelators of cations, but the reasons remained unclear and the cations unidentified. To begin defining the role of cations, this study explored the effect of Ca2+, calmodulin, and cAMP on maintaining the occluded neurocoel. Chick embryos during the natural phase of neurocoel occlusion (stage 12) were cultured in vitro with drugs known to modulate Ca2+ transport, to inhibit calmodulin activity, or to elevate cAMP levels. To test if occlusion is a Ca(2+)-dependent process, embryos were treated with verapamil and ionophore A23187. To test if occlusion requires calmodulin, embryos were treated with antipsychotic agents. To test if occlusion is cAMP dependent, embryos were treated with methylisobutylxanthine (MIX), forskolin (FOR), or dibutyl cyclic adenosine (DbC). Following each treatment, occlusion of the neurocoel was tested by injecting dye into the midbrain. All treatments resulted in a predominant number of precocious reopenings of the occluded neurocoels. MIX-treated, naked neural tubes had a four-fold increase in cAMP, whereas FOR- and DbC-treated neural tubes showed ten- and 14-fold increases, respectively. The presence of calmodulin in the cells of the neural tube was confirmed by fluorescent tagging and 3H-chlorpromazine labelling. The combined results of this study show that occlusion of the spinal neurocoel depends on exogenous Ca2+, requires calmodulin, and is cAMP sensitive.

Effect of wound healing and tissue transplantation on the navigation of axons in organ-cultured embryonic chick eyes.

Wound closure and repair of embryonic neuroepithelium were studied in organ-cultured embryonic retinae. Eyes from 3 to 4-day-old embryos were cultured after removing pieces of retinal tissue. During the subsequent 24 hours of incubation, the 150 to 200 microns wide holes in the retina closed completely. Histological studies showed that the wound closure was not accomplished by cell migration or cell proliferation, but by an approximation of the wound edges mediated by extracellular matrix fibrils of the vitreous body. The wound contraction facilitated the integration of transplants into the retinal neuroepithelium with a perfect alignment of the implants with the host at the vitreal surface. Within 24 hours, a continuous inner limiting membrane between transplant and host retina was established. The effect of wound healing and tissue transplantation on the navigation of optic axons in the retina was investigated. The wound contraction in the retina caused the optic axons near the lesion site to grow to the wound center, where the axons traversed the retina and formed a neuroma at the ventricular side, resembling the organization of axons at the optic disc. In the transplantation paradigm, axons from the host retina migrated into the transplant and vice versa. However, due to the wound contraction around the transplant, most axons grew into the interface between the transplant and host tissue.