Aberrant Global and Jagged-Mediated Notch Signaling Disrupts Segregation Between wt1-Expressing and Steroidogenic Tissues in Zebrafish.

Although the zebrafish interrenal tissue has been used as a model for steroidogenesis and genesis of the adrenal gland, its specification and morphogenesis remains largely unclear. In the present study, we explored how the Wilms tumor 1 (WT1)-expressing cells are segregated from the SF-1-expressing steroidogenic cells in the zebrafish model. The interrenal tissue precursors expressing ff1b, the equivalent of mammalian SF-1, were derived from wt1-expressing pronephric primordia in the zebrafish embryo. Through histochemistry and in situ hybridization, we demonstrated that the size of functionally differentiated interrenal tissue was substantially increased on global inhibition of the Notch signaling pathway and was accompanied by a disrupted segregation between the wt1- and ff1b-expressing cells. As the Notch pathway was conditionally activated during interrenal specification, differentiation, but not ff1b expression, of interrenal tissue was drastically compromised. In embryos deficient for Notch ligands jagged 1b and 2b, transgenic reporter activity of wt1b promoter was detected within the steroidogenic interrenal tissue. In conclusion, our results indicate that Jagged-Notch signaling is required (1) for segregation between wt1-expressing cells and differentiated steroidogenic tissue; and (2) to modulate the extent of functional differentiation in the steroidogenic interrenal tissue.

Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish.

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying differentiated steroidogenic tissues through chromogenic histochemical staining of 3-ß-Hydroxysteroid dehydrogenase /Δ5-4 isomerase (3ß-Hsd) activity-positive cells is critical for monitoring the morphology and differentiation of adrenocortical and interrenal tissues in mammals and teleosts, respectively. In the zebrafish model, the optical transparency and tissue permeability of the developing embryos and larvae allow for whole-mount staining of 3ß-Hsd activity. This staining protocol, as performed on transgenic fluorescent reporter lines marking the developing pronephric and endothelial cells, enables the detection of the steroidogenic interrenal tissue in addition to the kidney and neighboring vasculature. In combination with vibratome sectioning, immunohistochemistry, and confocal microscopy, we can visualize and assay the vascular microenvironment of interrenal steroidogenic tissues. The 3ß-Hsd activity assay is essential for studying the cell biology of the zebrafish interrenal gland because to date, no suitable antibody is available for labeling zebrafish steroidogenic cells. Furthermore, this assay is rapid and simple, thus providing a powerful tool for mutant screens targeting adrenal (interrenal) genetic disorders as well as for determining disruption effects of chemicals on steroidogenesis in pharmaceutical or toxicological studies.

Analysis of steroidogenic pathway key transcripts in interrenal cells isolated by laser microdissection (LMD) in stressed rainbow trout.

An assessment of the key transcripts expression of the steroidogenesis-related genes in rainbow trout subjected to either acute or chronic stress was performed in both interrenal cells and whole head kidney tissue. The analysis of interrenal cells was possible thanks to the use, for the first time in this specific type of cells, of the technique of laser microdissection (LMD) which allows to isolate specific cells and process them independently of other surrounding cells in the tissue. The results indicated that both acute and chronic stressors induced a significant up-regulation of the steroidogenesis-related genes with a higher but expected degree in the isolated cells. In addition, under acute stress a delay between cortisol levels and transcript expression was found. Under chronic stress a clear relation between plasma cortisol levels, mRNA transcription and interrenal tissue area was observed, since all parameters were concomitantly increased at day 5 after stress. Moreover results indicated that the LMD technique allowed ascertaining with more precision and accuracy whether and when the steroidogenesis-related genes were significantly expressed, disregarding the noise produced by other cells present in the head kidney. Results also showed a typical physiological response in plasma parameters and a positive relationship between plasma cortisol data and transcript abundance in isolated cells. The present results may help to better understand the mechanisms behind the interrenal response to stress challenges in fish.

The hemodynamically-regulated vascular microenvironment promotes migration of the steroidogenic tissue during its interaction with chromaffin cells in the zebrafish embryo.

BACKGROUND: While 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 FINDINGS: In 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 ß-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 SIGNIFICANCE: These 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.

Anteroposterior variation of the cell types in the interrenal gland of the male toad Rhinella arenarum (Amphibia, Anura).

The interrenal gland of anurans synthesizes the steroids aldosterone and corticosterone, but it is unknown whether these hormones are synthesized by the same cell type. In this work, we aim to elucidate whether there are different steroidogenic cell types and whether they have specific regionalization in the interrenal gland of the male toad Rhinella arenarum. We characterized all cell types using histological, immuhistochemical, and histochemical methods as well as transmission electron microscopy. Furthermore, we evaluated the organization of the cell types in the gland and anteroposterior variations in the synthesis of the steroids. We found evidence of five cell types: two morphologically different steroidogenic cells, type 1: polyhedral cells tightly attached to each other that have spherical euchromatic nuclei and type 2: retracted cells loosely attached to each other that have oval heterochromatic nuclei. Cell type 2 is mainly observed in the inner zone of the gland. In addition, we observed two types of chromaffin cells, called type 3 and 4 cells, randomly distributed throughout the interrenal gland, as well as type 5 cells, recognized as summer cells. Morphometric analyses of the cell types in the anterior and posterior zones of the interrenal showed that the ratio "area of type 2 cells/total interrenal area" is significantly lower in the posterior zone. In vitro incubations showed that the posterior portion of the gland produces significantly higher amounts of both corticosterone and aldosterone. Overall, our results suggest that the type 2 cells are less active to synthesize both aldosterone and corticosterone, compared to type 1 cells. Unlike most previous reports on the interrenal gland of anurans, in R. arenarum there is a zonation of the steroidogenic cell types, which implies that the organ is not anteroposterior or dorsoventrally homogeneous.

Survey of the adrenal homolog in teleosts.

The adrenal homolog of teleosts is not a compact organ as the adrenal glands of most vertebrates but is composed by aminergic chromaffin and interrenal steroidogenic cells located mostly inside the head kidney that, in this taxon, generally has a hematopoietic function. The two tissues can be mixed, adjacent, or completely separated and line the endothelium of the venous vessels or are located in close proximity. The chromaffin cells in some species are also present in the posterior kidney. Histological and ultrastructural work revealed cytological peculiarities of both types of cells as compared to those of other vertebrate species. In particular, the interrenal ones can show some variations in ultrastructure depending on sex, time of the year, and relation to stress events. A periodic renewal of the whole gland tissue is also sustained by some studies. Research regarding development is scanty as compared to mammals and most studies go back to the early years of the past century. The adrenal homolog of teleosts is under hormonal and neuronal control. Moreover, local paracrine interactions may play an important role in modulating a system involved in stress response and osmoregulation. Most previous studies involved a few species with the object of intensive rearing for commercial purposes; in fact cortisol, the main hormone secreted by the interrenal cells, can also influence reproduction and growth. This review summarizes data from morphocytological work and refers to other excellent reviews regarding physiology. Some of the results are compared to data available from other fishes and vertebrate classes with the aim of including them in an evolutionary and environmental framework.

Irreversible binding of o,p'-DDD in interrenal cells of Atlantic cod (Gadus morhua).

Precision-cut tissue slices of the anterior kidney from Atlantic cod (Gadus morhua) were prepared with a Krumdieck tissue slicer and exposed to 2-(2-chlorophenyl)-2-(4-chloro-(14C)phenyl)-1,1-dichlorethane (o,p(')-[14C]DDD) in vitro. Microautoradiography revealed irreversible o,p(')-DDD-derived binding confined to the glucocorticoid producing interrenal cells (adrenocortical analogues). This cell-selective binding was confirmed by means of autoradiography at different levels of resolution on Atlantic cod administered o,p(')-[14C]DDD intragastrically. The results provide evidence for a site-specific metabolic activation and irreversible binding of o,p(')-DDD in the interrenal cells, which, in turn, may modify glucocorticoid homeostasis.

Structural and ultrastructural characteristics of interrenal gland and chromaffin cell of matrinxã, Brycon cephalus Gunther 1869 (Teleostei-Characidae).

This work presents the structure and ultrastructure of the interrenal gland and chromaffin cells, as well as the morphology of the head kidney of Brycon cephalus. The head kidney is composed of fused bilateral lobes located anterior to the swim bladder and ventrolateral to the spinal column. The parenchyma revealed lympho-haematopoietic tissue, melano-macrophage centres, interrenal gland and chromaffin cells. The interrenal gland consisted of cords or strands of cells grouped around the posterior cardinal vein and their branches. Chromaffin cells are found in small groups, closely associated with the interrenal gland and/or under the endothelium of the posterior cardinal vein. So far, the ultrastructural analysis has revealed only one interrenal cell type which contained abundant smooth endoplasmic reticulum and numerous mitochondria with tubulo-vesicular cristae, characteristic of steroid-producing cells. Two types of chromaffin cells were observed. The first type was characterized by the presence of vesicles with round, strongly electron-dense granules, which were eccentrically located. Such cells were interpreted as noradrenaline cells. Meanwhile, cells which contained smaller vesicles and electron-lucent granules, with a small halo separating the granule from the vesicular limiting membrane, were identified as adrenaline cells.

LHRH induced changes in the interrenal gland of the frog Rana cyanophlyctis (Schn.).

Administration (i.m.) of synthetic mammalian LHRH (40 ng/frog, alternate day for 30 days) to adult male intact frogs R. cyanophlyctis resulted in activation of the interrenal cells as shown by increases in nuclear diameters of the interrenal cells, activity (histochemical demonstration) of delta 5-3 beta-hydroxysteroid dehydrogenase and glucose-6-phosphate dehydrogenase and concomitant decrease in sudanophilic lipid droplets in the interrenal gland when compared to those of controls. These changes were not observed in the interrenal gland of LHRH treated hypophysectomized frogs. The results indicate that LHRH stimulates secretory activity of the interrenal cells and action may not be directly on the adrenal gland.

Ontogeny of the cortisol stress response in larval rainbow trout.

The ontogeny of the interrenal stress response in rainbow trout was characterized by measuring resting and acute-stress-induced changes in whole-body cortisol levels in embryos and larvae at different early developmental stages. In Experiment 1, resting cortisol levels averaged 6.0 ng/g in newly fertilized eggs, fell to less than 0.3 ng/g by the time of hatching at Week 4 (incubation at 10 degrees), and increased to 1.4 ng/g by Week 5. Cortisol levels did not change in response to acute stress in 3-, 4-, or 5-week-old fish. In Experiment 2, resting cortisol averaged 1.4 ng/g in newly fertilized eggs, fell to less than 0.03 ng/g by Week 2, and then steadily increased between Weeks 3 and 6 to a peak of 4.8 ng/g before falling to 1.2 ng/g by Week 7. Cortisol levels did not change in response to acute stress in 3-, 4-, or 5-week-old fish. Six-week-old fish showed a 2.3-fold increase in cortisol levels at 1 hr poststress, indicating that the hypothalamic-pituitary-interrenal axis first develops responsiveness to stress 2 weeks after hatching and 1 week before the onset of exogenous feeding. The stress hyporesponsive period after hatching in rainbow trout may be homologous to the 2-week stress hyporesponsive period after birth in rodents, the function of which may be to maintain low, constant corticosteroid levels during a critical developmental period when these steroids can have permanent effects on neural organization. As suggested for mammals, this period may be a time when rainbow trout are particularly vulnerable to environmental effects on their subsequent development.

[The effect of vasotocin, mesotocin and adrenaline on the interrenal gland in the frog Rana temporaria under in-vitro conditions]. / Vliianie vazototsina, mezototsina i adrenalina na interrenalovuiu zhelezu liagushki Rana temporaria v usloviiakh in vitro.

Morphometric studies indicate that vasotocin increases the activity of the adrenal gland in frogs, whereas mesotocin and epinephrine do not essentially affect it. Combination of epinephrine with nonapeptides in cultural medium showed that the former may abolish the effect of vasotocin on the adrenal gland.

[The effect of injections of arginine vasotocin and mesotocin on the function of the interrenal gland in the frog Rana temporaria 2 months after the removal of the anterior lobe of the hypophysis]. / Vliianie in''ektsii arginin-vazototsina i mezototsina na sostoianie interrenalovoi zhelezy liagushki Rana temporaria spustia dva mesiatsa posle udaleniia perednei doli gipofiza.

Morphometric and biochemical studies have been made on the activity of interrenal gland in the frog Rana temporaria after removal of the anterior lobe of pituitary and injections of arginine vasotocin and mesotocin. It was demonstrated that the activity of this gland after the operation decreases increasing after arginine vasotocin injections.

The chromaffin cells of urodele amphibians.

Different conditions in the arrangement of the adrenal gland are observed in urodeles. The gland consists of islets scattered on the ventral surface of the kidneys, the amount, size and position of the islets varying consistently within different families and even within genera. The infraordinal variation also extends to the fine structure of the gland, as observed in 14 species belonging to 6 different families. The ultrastructural characteristics of chromaffin cells and their relationships with interrenal cells appear to be related to the phyletic position. In primitive urodeles (Sirenidae, Proteidae) the chromaffin cells are isolated or in small groups, mostly separated from interrenal cells and often in contact with renal cells. In neourodeles (Amphiumidae, Ambystomidae, Salamandridae, Plethodontidae) the chromaffin cells appear generally grouped and intermingled with steroidogenic cells. Some cytological characteristics of chromaffin cells, such as nerve supply and the shape and electron density of chromaffin granules exhibit a variability related to phyletic position.

[Morphofunctional research on the interrenal gland of the frog Rana temporaria following arginine vasotocin administration]. / Morfofunktsional'noe issledovanie interrenalovoi zhelezy liagushki Rana temporaria posle vvedeniia arginin-vazototsina.

Studies have been made of the effect of injections of hypothalamic nonapeptide neurohormone, arginine vasotocin, on functional condition of the interrenal gland in mature frogs. In unoperated, sham-operated and in animals 10 days after hypophysectomy, single and especially three subsequent injections of arginine vasotocin (5 x 10(-9) M per 1 kg of the body weight) result in evident activation of glandular cells of the interrenal gland which is manifested in the increase of the volume of their nuclei and cytoplasmic area, as well as in the dilatation of the blood vessels. Activation of the interrenal gland in hypophysectomized frogs, which lack endogenous ACTH, indicate the direct para-adenohypophyseal influences of nonapeptide hypothalamic hormones on the activity of glandular cells in the peripheral endocrine glands, in particular, the interrenal gland of the grass frog.

Interrenal activity during metamorphosis of the tiger salamander, Ambystoma tigrinum.

Plasma corticosterone concentrations were low in premetamorphic tiger salamander larvae (Norman Stage I; M. F. Norman (1985) Anat. Rec. 211, 102-109). Corticosterone levels were significantly elevated at midmetamorphosis (Norman Stage IV) but decreased at the end of metamorphosis (Norman Stage VII). Corticosterone levels remained low 2 weeks after metamorphosis. Interrenal 3 beta-hydroxysteroid dehydrogenase activity was low in premetamorphic larvae (Norman Stage I) but was significantly elevated by midmetamorphosis (Norman Stage IV) and remained elevated at the end of metamorphosis (Norman Stage VII). There were no significant changes in interrenal cell nuclear size during metamorphosis. There was a significant decrease in body weight as well as a significant increase in hematocrit accompanying metamorphosis. The increase in plasma corticosterone concentration seen during metamorphosis of the tiger salamander is accompanied by an increase in interrenal steroidogenesis.

Contrasting effects of ACTH and cyanoketone on delta 5-3 beta-hydroxysteroid dehydrogenase activity in interrenal glands of tadpoles of Rana catesbeiana in vitro.

The present communication describes an investigation of stimulation and inhibition of delta 5-3 beta-hydroxysteroid dehydrogenase in interrenal glands of tadpoles of Rana catesbeiana. Frozen sections of interrenal glands, together with kidneys, were prepared histochemically for assay of delta 5-3 beta-HSD activity. Concentrations of 0.01, 0.1, 1, and 10 IU/ml of ACTH or of 0.01, 0.1, 1, and 10 micrograms/ml of cyanoketone were added to the incubation media. The reaction products of the histochemically prepared slides, in terms of absorbance, were scanned at a defined area with a computerized microscope spectrophotometer. The results indicate that ACTH causes a significant dose-response stimulation of delta 5-3 beta-HSD activity in tadpole interrenals; cyanoketone, on the other hand, causes significant dose-dependent inhibition.

Effects of metopirone on the pars distalis, the interrenal gland and the testis of the frog, Rana cyanophlyctis (Schn.).

Administration (i.m.) of metopirone (2.5 mg/frog/day for 10 days) to adult male frog, R. cyanophlyctis, resulted in marked degranulation and hypertrophy of B3 cells and a moderate degranulation of B2 cells of the pars distalis. Concomitantly, there was hypertrophy of the interrenal cells and regression of the Leydig cells, however spermatogenesis was not affected. The results suggest a functional correlation between pituitary B cells and the interrenals in R. cyanophlyctis.

Cytophysiological studies on the effects of amphenone 'B' and dexamethasone on the pars distalis and interrenal gland of Rana cyanophlyctis (Schn.).

Amphenone 'B' treatment resulted in the hypertrophy and degranulation of the B3 cells of the pars distalis and stimulation of interrenal cells, whereas administration of dexamethasone caused regression of B3 and interrenal cells. Following 15-day cessation of amphenone or dexamethasone treatment, there was no recovery in the B3 or the interrenal cells. The results suggest that B3 cells are the probable source of ACTH and there exists a relationship between the pituitary and interrenal gland of the frog, R. cyanophlyctis.

[Chloride cells of the gills and the interrenal gland of Dasyatis pastinaca (L.) (Elasmobranchii) skates during adaptation to distilled sea water]. / Khloridnye kletki zhabr i interrenalovaia zheleza skatov Dasyatis pastinaca (L.) (Elasmobranchii) pri adaptatsii k opresnennoi morskoi vode.

During adaptation of the skate Dasyatis pastinaca (L) to distilled sea water (7.1%) resulted in decreasing concentration of sodium, urea in blood serum, reduction in the chloride cells volume, their nuclei and increase of the apical part of the cell free from mitochondria (and occupied by the endoplasmic reticulum) are observed. Owing to uniformity of the morphometric changes of the chloride cells revealed in two types, during adaptation of the skate to the distilled sea water, as well as adaptation to its increased salinity, it is possible to conclude that in the gill of selachians there are chloride cells of one type with various functional activity. Adaptation of the skate to the distilled sea water is connected with removal of lipid inclusions out of the cells of the interrenal gland.