Isolation and expression analysis of three zebrafish angiopoietin genes.

The Tie1 and Tie2 receptor tyrosine kinases and the Tie2 ligands, the angiopoietins, play critical roles in vertebrate vascular embryogenesis, helping to mediate the interaction between endothelial cells and the pericytes or vascular smooth muscle cells that envelop and support them. We have obtained full-length cDNA sequences for zebrafish orthologs of angiopoietin-1 (ang1), angiopoietin-2 (ang2), and angiopoietin-like-3 (angptl3). Ang1 is expressed in head ventral mesenchyme, in the ventromedial region of somites, in mesenchyme surrounding trunk axial vessels, and in the hypochord, a transient embryonic structure of endodermal origin that has been implicated in dorsal aorta assembly in both zebrafish and Xenopus. Ang2 is expressed in head and anterior trunk ventral mesenchyme and the developing pronephric glomeruli. Angptl3 is expressed in the yolk syncytial layer.

2,3,7,8-tetrachlorodibenzo-p-dioxin inhibits luminal cell differentiation and androgen responsiveness of the ventral prostate without inhibiting prostatic 5alpha-dihydrotestosterone formation or testicular androgen production in rat offspring.

In utero and lactational exposure to a single maternal dose of 1 microg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)/kg causes some overt toxicity and impairs prostate growth in male offspring. As similar effects on the ventral prostate can be caused by decreased testosterone production during perinatal development, we determined whether intratesticular testosterone content, testicular responsiveness to gonadotropin stimulation, or plasma testosterone concentrations were reduced in fetal and newborn rats. Because these endpoints were not affected, the ability of TCDD exposure to inhibit synthesis of the proximal androgen in prostate development, 5alpha-dihydrotestosterone (DHT), from the circulating precursor testosterone and 5alpha-androstane-3alpha,17ss-diol (3alpha-Diol), was studied on postnatal days (PNDs) 14, 21, and 32. The ability of the ventral prostate to form DHT from 3alpha-Diol was slightly impaired on PND 14, but this transient effect was not statistically significant, and recovery was evident by PND 21. Subsequent experiments used organ culture to study the effects of in vivo TCDD exposure on androgen metabolism, androgen responsiveness, androgen receptor expression, and luminal epithelial cell differentiation after in vitro exposure to graded androgen concentrations. In utero and lactational TCDD exposure had no effect on DHT formation in organ culture, but transiently reduced the androgen -induced expression of prostatic-binding protein subunit C3, decreased ventral prostate epithelial cell androgen receptor expression, and inhibited the formation of androgen responsive luminal epithelial cells. These results suggest that TCDD exposure impairs prostate growth and androgen responsiveness by inhibiting prostatic epithelial cell differentiation.

Universal GFP reporter for the study of vascular development.

We report the generation and characterization of transgenic mouse and zebrafish expressing green fluorescent protein (GFP) specifically in vascular endothelial cells in a relatively uniform fashion. These reporter lines exhibit fluorescent vessels in developing embryos and throughout adulthood, allowing visualization of the general vascular patterns with single cell resolution. Furthermore, we show the ability to purify endothelial cells from whole embryos and adult organs by a single step fluorescence activated cell sorting. We expect that these transgenic reporters will be useful tools for imaging vascular morphogenesis, global gene expression profile analysis of endothelial cells, and high throughput screening for vascular mutations.

Building the vertebrate vasculature: research is going swimmingly.

The vertebrate vasculature develops in remarkably similar fashion in all vertebrates. A cohort of unspecified mesodermal cells differentiates into primitive endothelial cells, which migrate to and occupy positions within the stereotypical blueprint of the primitive vasculature. Once in position, these cells coalesce and form cords, which lumenize and become ensheathed by supporting pericytes and smooth muscle cells. This primitive vascular network is extensively remodeled in some places, and expanded by sprouting in others. Various studies using the mouse, quail/chick, and frog have uncovered a number of signals that guide these complex processes but many gaps still exist in our understanding of the mechanisms by which the embryonic vasculature is built. Because many questions will require in vivo studies to be properly addressed, the zebrafish, with its unique accessibility to analysis by combined embryological, molecular, and genetic methods, should prove invaluable in identifying new molecules involved in blood vessel development and integrating pathways that influence embryonic blood vessel formation.

Responsiveness of the adult male rat reproductive tract to 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure: Ah receptor and ARNT expression, CYP1A1 induction, and Ah receptor down-regulation.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) either in adulthood or during late fetal and early postnatal development causes a variety of adverse effects on the male rat reproductive system. It was therefore of interest to identify male rat reproductive organs and cell types within these organs that might be direct targets of TCDD exposure. Because TCDD toxicity could possibly be the result of alterations in gene transcription mediated by the TCDD/aryl hydrocarbon receptor (AhR)/AhR nuclear translocator (ARNT) complex, the presence of the AhR and ARNT in the various organs of the adult male reproductive tract was examined using Western blotting. Both proteins were detectable in all organs examined (testis, epididymis, vas deferens, ventral prostate, dorsolateral [combined dorsal and lateral] prostate, and seminal vesicle). Although technical difficulties precluded the immunohistochemical evaluation of AhR distribution in these organs, ARNT was localized in all organs in a variety of cell types, including germ cells, epithelial cells, fibroblasts, smooth muscle cells, and endothelial cells. Subcellular localization varied across organs and across cell types within these organs. In order to determine whether TCDD exposure could alter gene expression in these organs, animals were dosed with TCDD (25 micrograms/kg po) or vehicle and euthanized at 24 h, and cytochrome P4501A1 (CYP1A1) expression was evaluated. By Western blotting, only the ventral and dorsolateral prostates exhibited significant induction of CYP1A1. Immunohistochemistry confirmed this induction and localized CYP1A1 expression to epithelial cells of the ventral and lateral lobes of the prostate. Immunohistochemistry also revealed CYP1A1 induction in select epithelial cells in the epididymis and seminal vesicle, as well as endothelial cells in the vas deferens and seminal vesicle. No induction was observed in the testis. Finally, AhR and ARNT expression in TCDD-exposed and control animals was evaluated by Western blotting. Results revealed no effect of TCDD exposure on ARNT protein expression, while AhR expression was decreased to 5-51% of control in all organs examined. In summary, both AhR and ARNT were expressed in all organs of the adult male rat reproductive tract examined, and epithelial and/or endothelial cells within each of these organs (with the exception of the testis) were responsive to TCDD exposure in terms of CYP1A1 induction. In addition, all tissues exhibited marked reductions in AhR protein content after TCDD exposure that did not correlate with the magnitude of the CYP1A1 response.

In utero and lactational exposure of the male rat to 2,3,7,8-tetrachlorodibenzo-p-dioxin impairs prostate development. 1. Effects on gene expression.

In utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure decreases rat prostate weight without decreasing circulating androgen concentrations. Because one mechanism by which TCDD is thought to cause toxicity is by aryl hydrocarbon receptor (AhR)-mediated alterations in gene transcription, the goals of this study were to determine whether the developing prostate expresses the AhR and its dimerization partner, the AhR nuclear translocator (ARNT); to determine whether in utero and lactational TCDD exposure is capable of directly activating gene transcription in the developing prostate; and to identify prostatic mRNAs that exhibit altered abundance in response to in utero and lactational TCDD exposure. Pregnant Holtzman rats were administered TCDD (1.0 microgram/kg po) or vehicle on Gestation Day (GD) 15, and male offspring were euthanized between Postnatal Days (PNDs) 1 and 63. Using reverse transcriptase-polymerase chain reaction (RT-PCR), mRNAs encoding the AhR and ARNT were detected in both ventral and dorsolateral prostates from control animals throughout postnatal development. ARNT protein was expressed in the majority of stromal nuclei early in development, whereas ARNT expression in the prostate epithelium was initially cytoplasmic but became nuclear as development progressed. GD 15 TCDD exposure increased cytochrome P4501A1 (CYP1A1) mRNA and protein in whole prostates between PNDs 7 and 21. In these TCDD-exposed animals, CYP1A1 protein was localized to the epithelium. In order to define other genes in the developing prostate that might be regulated by TCDD at the level of mRNA, RNA samples from PND 21 whole prostates from control and TCDD-exposed animals were compared using mRNA differential display. Although no growth-regulatory candidates were identified using this screening technique, a ventral prostate-specific, androgen-regulated mRNA (20-kDa protein) was identified that seemed to be downregulated by TCDD exposure. Northern blot analysis confirmed this decrease at PND 21 and further showed that the downregulation was transient. Similar results were obtained for four additional androgen-regulated prostatic mRNAs (prostatic binding protein [PBP], Royal Winnipeg Ballet [RWB], probasin, and dorsal protein-1 [DP-1]), all of which are markers of a differentiated ductal epithelium. In contrast, TCDD exposure of adult male rats (25 micrograms TCDD/kg, 24 h) greatly induced CYP1A1 mRNA without affecting the abundance of prostate-specific, androgen-regulated mRNAs. These results suggest that the transient decreases in androgen-regulated prostatic mRNA abundance observed in response to in utero and lactational TCDD exposure were probably not the result of direct action of the activated AhR on these genes but instead were reflective of a TCDD-induced delay in prostate development.

In utero and lactational exposure of the male rat to 2,3,7,8-tetrachlorodibenzo-p-dioxin impairs prostate development. 2. Effects on growth and cytodifferentiation.

In the male Holtzman rat, in utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure decreases prostate weight without inhibiting testicular androgen production or decreasing circulating androgen concentrations. Therefore, the present study sought to characterize effects of TCDD exposure on prostate development, from very early outgrowth from the urogenital sinus (Gestation Day [GD] 20) until rapid growth and differentiation are essentially complete (Postnatal Day [PND] 32). Pregnant Holtzman rats were administered a single dose of TCDD (1.0 microgram/kg po) or vehicle on GD 15 and offspring were exposed via placental transfer (GD 20 euthanasia) or placental and subsequent lactational transfer until euthanasia (if before PND 21) or weaning. Results show that the prostatic epithelial budding process was impaired by in utero TCDD exposure, as evidence by significant decreases in the number of buds emerging from dorsal, lateral, and ventral aspects of the GD 20 urogenital sinus. Ventral prostate cell proliferation index was significantly decreased on PND 1 but was similar to or higher than control at later times, whereas apoptosis was an extremely rare event in ventral prostates from both control and TCDD-exposed animals. Delays were noted in the differentiation of pericordal smooth muscle cells and luminal epithelial cells. In addition, ventral prostates from approximately 40% of TCDD-exposed animals examined on PNDs 21 and 32 exhibited alterations in the histological arrangement of cell types that could not be explained by a developmental delay. Compared to controls, these ventral prostates exhibited a disorganized, hyperplastic epithelium containing fewer luminal epithelial cells and an increased density or continuous layer of basal epithelial cells, as well as thicker periductal smooth muscle sheaths. In addition, in ventral prostates from TCDD-exposed animals, the intensity of androgen receptor staining was relatively low in the central and distal epithelium, and the number of androgen receptor-positive cells was relatively high in the periductal stroma. These data suggest that in utero and lactational TCDD exposure interferes with prostate development by decreasing very early epithelial growth, delaying cytodifferentiation, and, in the most severely affected animals, producing alterations in epithelial and stromal cell histological arrangement and the spatial distribution of androgen receptor expression that may be of permanent consequence.

In utero and lactational exposure of the male rat to 2,3,7,8-tetrachlorodibenzo-p-dioxin: impaired prostate growth and development without inhibited androgen production.

To determine whether in utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure decreases male rat accessory sex organ weights during postnatal development secondary to decreases in testicular androgen production or changes in peripheral androgen metabolism, pregnant Holtzman rats were administered a single dose of TCDD (1.0 microgram/kg, po) or vehicle on Gestation Day 15 and offspring were exposed via placental and subsequent lactational transfer until weaning on Postnatal Day (PND) 21. Between PNDs 21 and 63, circulating androgen concentrations and intratesticular androgen content tended to be decreased by in utero and lactational TCDD exposure, but in most cases decreases were not statistically significant. In vitro human chorionic gonadotropin-stimulated testosterone production by decapsulated testes from TCDD-exposed animals was not different from control, although 5 alpha-androstane-3 alpha,17 beta-diol production was decreased on PNDs 32 and 49 and increased on PND 63. Taken together, these results imply that in utero and lactational TCDD exposure can cause subtle decreases in testicular androgen production. However, the biological relevance of these reductions is equivocal because they do not correlate temporally with one another or with decreases in androgen-dependent male accessory sex organ weights. Of the male accessory sex organs, ventral prostate (VP) and dorsolateral prostate (DLP) were the most severely affected. Between PNDs 21 and 63, relative VP and DLP weights were decreased to 65-84% and 57-80% of control, respectively, and the magnitude of observed decreases was greatest at early times. In contrast, relative weights of the seminal vesicle and coagulating gland ranged from 80 to 104% of control, and the magnitude of observed decreases was greatest at later times. The sensitivity of the prostate to TCDD could not be explained by tissue-specific decreases in dihydrotestosterone (DHT) concentrations. Although VP DHT concentration was decreased to 63% of control on PND 21, DHT concentration was not decreased in the VP between PNDs 32 and 63 or in the DLP at any time. We conclude that in utero and lactational TCDD exposure selectively impairs rat prostate growth and development without inhibiting testicular androgen production or consistently decreasing prostate DHT concentration.