F. Harrisson

Neurochemical Coding of Enteric Neurons in Adult and Embryonic Zebrafish (Danio rerio)

Although the morphology and development of the zebrafish enteric nervous system have been extensively studied, the precise neurochemical coding of enteric neurons and their proportional enteric distribution are currently not known. By using immunohistochemistry, we determined the proportional expression and coexpression of neurochemical markers in the embryonic and adult zebrafish intestine. Tyrosine hydroxylase (TH), vasoactive intestinal peptide (VIP), and pituitary adenylate cyclase‐activating peptide (PACAP) were observed only in nerve fibers, whereas other markers were also detected in neuronal cell bodies. Calretinin and calbindin had similar distributions. In embryos, all markers, except for choline acetyltransferase (ChAT) and TH, were present from 72 hours postfertilization. Nitrergic neurons, evenly distributed and remaining constant in time, constituted the major neuronal subpopulation. The neuronal proportions of the other markers increased during development and were characterized by regional differences. In the adult, all markers examined were expressed in the enteric nervous system. A large percentage of enteric neurons displayed calbindin and calretinin, and serotonin was the only marker showing significant distribution differences in the three intestinal regions. Colocalization studies showed that serotonin was not coexpressed with any of the other markers. At least five neuronal subpopulations were determined: a serotonergic, a nitrergic noncholinergic, two cholinergic nonnitrergic subpopulations along with one subpopulation expressing both ChAT and neuronal nitric oxide synthase. Analysis of nerve fibers revealed that nitrergic neurons coexpress VIP and PACAP, and that nitrergic neurons innervate the tunica muscularis, whereas serotonergic and cholinergic nonnitrergic neurons innervate the lamina propria and the tunica muscularis. J. Comp. Neurol. 518:4419–4438, 2010.

Expression of different regional patterns of fibronectin immunoreactivity during mesoblast formation in the chick blastoderm

The appearance and distribution of the extracellular material glycoprotein, fibronectin, was investigated in gastrulating chick embryos using affinity-purified anti-human plasma fibronectin antibodies. Preservation of tissue structure and immunoreactivity was carried out by ethanol/acetic acid fixation or by formaldehyde/glutaraldehyde fixation. Using the former fixation method, fibronectin immunoreactivity was detected (1) at the ventral surface of the upper layer or epiblast, mainly anterior and lateral to Hensens node, in regions where middle-layer or mesoblast cells are not yet present, and (2) sparsely in extracellular spaces of the deep layer. Using the latter fixation method, fibronectin immunoreactivity was, moreover, found at the entire ventral surface of the upper layer, i.e., also at the epithelial-mesenchymal interface, where a basement membrane was previously described. At the light microscope level, we could not detect significant immunoreactivity in the middle layer. Treatment of sections of ethanol-fixed blastoderms with testicular hyaluronidase before immunostaining for fibronectin partially demasked the antigenic sites of this glycoprotein at the epithelial-mesenchymal interface. The present report indicates that the different regional patterns of fibronectin immunoreactivity in the basement membrane of the upper layer are spatially and temporally correlated with migration and positioning of mesoblast cells. These regional patterns are probably due to differences in the composition of fibronectin-associated material such as chondroitin sulfate A and/or C proteoglycans, and/or hyaluronate, before and after mesoblast expansion, rather than to differences in the distribution of fibronectin itself. In this respect. In this respect, it is noteworthy that the chemical composition of the basement membrane of an epithelium changes as mesenchyme cells migrate over it. The results also favor the idea that fibronectin is a structural component of the whole basement membrane which is used as a substrate for migration of mesenchymal cells.

Distribution of apoptosis-related proteins in the quail ovary during folliculogenesis: BCL-2, BAX and CPP32.

It is suggested that follicular apoptosis is driven by the status of the BCL-2: BAX rheostat, and that CPP32 is a key effector of granulosa cell death. In the present study, we have immunohistochemically localized two BCL-2 family members, BCL-2 and BAX, and one caspase, CPP32, in the quail ovary during folliculogenesis. BCL-2 was predominantly found in the granulosa cells of developing follicles. BAX was detected in some follicular cells of atretic follicles, and in the nucleus of some prelampbrush oocytes. Expression of CPP32 was detected in leukocytes and in follicular cells of atretic follicles. Immunostaining was also found in interstitial cells, in surface epithelial and vascular endothelial cells, and in some thecal cells of post-ovulatory follicles. In the granulosa cells of non-growing and small prehierarchal follicles, a weak immunostaining was observed. We can conclude that in the avian ovary, BAX and CPP32 are involved in atresia. The present results support the BCL-2: BAX rheostat hypothesis.

Immunohistochemical localization of S-100 protein, glial fibrillary acidic protein, and neuron-specific enolase in the pars distalis of quail, rat, and human hypophyses

SummaryIn the present study, we have localized immunohistochemically S-100 protein, glial fibrillary acidic (GFA) protein, and neuron-specific enolase (NSE) by the unlabelled antibody peroxidase-antiperoxidase technique. Special attention was paid to the influence of fixation and of pretreatment of sections with proteolytic enzymes. It appeared that the final immunostaining of a given antigen largely depends on the fixative and on the species used. Moreover, pepsin pretreatment proved to be necessary to unmask S-100 protein in quail and GFA protein in rat. S-100 protein (rat, human) and GFA protein (human) immunoreactivities were detected in the folliculo-stellate (FS) cells. In quail, S-100 protein was also found in cells, which were not arranged around a follicular lumen and, in rat, the endothelial cells were immunostained for GFA protein. Clusters of granular cells were weakly immunostained for NSE in all species. An exclusive relationship between FS cells and S-100 protein could not be ascertained from this study.

Transfer of extracellular matrix components between germ layers in chimaeric chicken-quail blastoderms

SummaryA chemical basis for the transmission of signals during gastrulation has been investigated by using chimaeric embryos resulting from the combination of 3H-glucosamine-labelled and unlabelled hypoblast with epiblast taken from chicken and quail embryos at stage 3 of Vakaet (1970). The ability to distinguish chicken from quail cells on the basis of their different nuclear distribution of heterochromatin after Feulgen staining made it possible to determine the origin of the cells in the chimaerae. Tritiated quail hypoblast (after incubation of the embryo in the presence of 3H-glucosamine) was transplanted onto unlabelled chicken blastoderm deprived of its hypoblast. After culture of the chimaera for 5 h, the autoradiographic pattern shows silver grains not only over the graft, but also at the ventral surface of the epiblast of the host. Transfer of label may occur to mesoblast cells, but not between chicken and quail hypoblast cells. Chase experiments exclude the possibility that unprocessed, tritiated glucosamine is transferred. Chemical fixation of the host before transplantation of a labelled quail hypoblast also allows visualization of a transfer of macromolecules from hypoblast to the basement membrane of the epiblast, suggesting that an intervention of the epiblast cells in this process is not necessary. The morphology of the chimaeric embryos, as studied by scanning electron microscopy, suggests a direct deposition of these macromolecules by filopodia of the dorsal surface of the hypoblast. The possibility of diffusion of free macromolecules has been considered and can reasonably be discarded on the basis of several observations. The reverse experiment, in which unlabelled quail hypoblast and possibly some mesoblast have been combined with a tritiated host deprived of its hypoblast, also shows the transfer of label from the host to the cellular surface of the graft. A two-way exchange of glucosamine-containing molecules thus occurs in the blastoderm. It is hypothesized that: (1) low molecular weight compounds, macromolecular material, and/or catabolic products, are exchanged between the different germ layers during gastrulation; (2) the components of the extracellular matrix turn over and are continuously changing; (3) this transfer is a possible mechanism of transmission for developmental or inductive signals during embryonic development. The present results also demonstrate the participation of underlying tissue in the biosynthesis of basement membrane components of an epithelium.

The relationship between the folliculo-stellate network and the thyrotropic cells of the avian adenohypophysis

SummaryThe folliculo-stellate network of the avian adenohypophysis consists of stellate cells surrounding colloid-containing follicular cavities into which cilia and microvilli project. Other identifying criteria are agranularity, junctional complexes at the apical pole, presence of cytoplasmic processes ramifying between adjacent secretory cells, and close appositions of plasma membranes linking folliculo-stellate cells and presumptive thyrotropic cells.Transmission electron microscopy reveals that TRH and L-DOPA induce simultaneous ultrastructural changes in the folliculo-stellate network and in the thyrotropic cells. TRH transforms at cell of the cephalic lobe into a highly hypertrophic cell in which enlargement of cisterns of rough endoplasmic reticulum containing secretory granules, development of a large Golgi complex, presence of newly synthesized secretory granules, and granulation of the cytoplasm are the main features. In the meantime, the follicular cavities become dilated by large amounts of homogeneous colloid. The administration of L-DOPA also leads to the development of dilated cisterns in presumptive thyrotropic cells of the cephalic lobe. Intracisternal granules, immature secretory granules, and large Golgi complexes, however, are not observed. Degranulation of the cytoplasm is obvious. The follicular cavities of both cephalic and caudal lobes are enlarged and filled with colloid in which granular elements are noted.The ultrastructural changes observed in thyrotropic cells and in the folliculo-stellate network reflect functional changes induced by the experimental manipulation. These changes may be related, directly or indirectly, or completely independent.

Fibronectin and its relation to the basal lamina and to the cell surface in the chicken blastoderm.

SummaryThe ultrastructural distribution of fibronectin immunoreactivity was investigated in the chicken embryo during late gastrulation. Sites of binding of anti-fibronectin antibodies were ascribed to the basal lamina and associated structures, and to the cell surface. The fibronectin-rich basal lamina was resolved into (1) a lamina densa, which appears as a continuous, dense sheet, (2) a lamina lucida, consisting of anchoring cords between lamina densa and epithelial cells, and (3) a lamina intima, closely juxtaposed to the cell surface. Cell-surface labelling was also observed in mesoblast cells, and along the dorsal side of the deep-layer cells. The ventral side of the latter cells was poorly stained in the endophyllic crescent, except in coated pits, and more regularly stained at the level of definitive endoblast. Some structures associated with the basal lamina reacted intensely with anti-fibronectin antibodies. These are (1) the interstitial bodies, which are aggregates of extracellular material, and (2) a kind of fibril or tubule, embedded in a fibronectin matrix and mainly found in the endophyllic crescent. Some intracellular labelling was found in most deep-layer cells, in few epiblast cells, never in mesoblast cells. These results extend previous studies on the localization of fibronectin, and correlate its presence and surface topology with its postulated role in migration of mesoblast cells on the basal lamina which, chemically, constitutes an appropriate substrate.

Microspectrographic analysis of trypan blue-induced fluorescence in oocytes of the Japanese quail

SummaryIt was shown that the vital dye trypan blue injected subcutaneously is adsorbed on exogenous yolk and stored in oocytes of Japanese quails. The binding sites of the dye could be visualized by fluorescence microscopy. The spectral distribution of the trypan blue-induced fluorescence emitted by yolk granules was analyzed microspectrographically. The analysis revealed that yolk granules exhibit a deep red fluorescence radiation with a maximum intensity at 670 nm, when blue or green excitation light is used. This fluorescence was exclusively induced by the presence of trypan blue, and not by contaminants of the dye. The fluorescence intensity did not decrease during processing of the tissue throughout the different solvents routinely used in light microscopy, especially after fixation in Heidenhains fluid, nor did it suffer from pronounced fading during irradiation of the tissue. Model experiments showed that the value of the fluorescence emission maximum was concentration-dependent, and that amounts as little as 5×10−3 mg trypan blue per ml solution containing an excess of yolk as a substrate for the dye, could clearly be detected and measured.It is suggested that a highly diluted solution of trypan blue can be used without teratogenic effects, as a tracer for exogenous yolk uptake and migration into oocytes, and that fluorescence microscopy is a reliable method for its further localization. A detailed account of the procedure is reported.

Ultrastructural study of the adenohypophysis of the male Chinese quail

SummaryThe ultrastructural analysis of the adenohypophysis in the male Chinese quail reveals seven different types of granular cells, and agranular folliculo-stellate cells. The cell types are assumed to be endocrine cells and are classified as: Type I cells (presumptive LH-gonadotrophs), with dilated endoplasmic reticulum, perinuclear spaces, and granules of 150–260 nm; Type II cells (presumptive FSH-gonadotrophs), with regularly-shaped cytoplasmic cisterns and small granules (80–150 nm); Type III cells (presumptive thyrotrophs), very close in appearance to the type II cells of normal birds; Type IV cells (presumptive prolactin cells), with very large secretory granules (up to 400 nm), Type V cells (presumptive corticotrophs), with abundant and electrondense granules (160–300 nm); Type VI cells, with irregularly-shaped granules; Type VII cells (presumptive somatotrophs), with abundant granules (130–220 nm) and less cytoplasmic structures.Cytological characteristics of the nucleus, and more particularly the presence of a Feulgen-positive nucleolus with a very particular ultrastructure are here reported. It is proposed that heterospecific associations of Chinese quail cells with chick cells can be used in embryological work for the study of cellular interactions.

Masking of antigenic sites of fibronectin by glycosaminoglycans in ethanol-fixed embryonic tissue

SummaryWe studied the interaction between glycosaminoglycans (GAGs) and fibronectin in the basement membrane of the epiblast in the chicken blastoderm using testicular-hyaluronidase digestion of GAGs either on fixed tissue sections or in vivo after microinjection of the enzyme preparation prior to immunostaining for fibronectin. In the choice of fixatives, special attention was paid to their preservation of GAGs. The controls included alcian-blue staining of serial sections to test the efficiency of the digestion, and incubations in the presence of protease inhibitors to abolish contaminating proteolytic activity in the commercial hyaluronidase preparations. The results indicate that fixation in solutions which preserve GAGs, i.e. ethanolic solutions or aqueous solutions containing cetylpyridinium chloride, allows the immunocytochemical demonstration of fibronectin in the basement membrane of the epiblast at the level of the endophyllic crescent, but masks this glycoprotein at the epithelial-mesenchymal interface. As shown by both approaches, this masking of immunoreactivity is reversible. Moreover, the in vivo clearance of GAGs before fixation shows that the masking at the epithelial-mesenchymal interface is not an experimental peculiarity due to the use of a particular technique, but is the consequence of an interaction between GAGs and fibronectin in that particular area of the basement membrane that is used by mesoblast cells as a substrate for migration. The observation that fibronectin may be masked by GAGs in ethanol-fixed tissue — a commonly used fixation method—may require the re-evaluation of some negative results mentioned in the literature.