Components of the Wnt signaling pathway are expressed in the developing inner ear. To explore their role in ear patterning, we used retroviral gene transfer to force the expression of an activated form of beta-catenin that should constitutively activate targets of the canonical Wnt signaling pathway. At embryonic day 9 (E9) and beyond, morphological defects were apparent in the otic capsule and the membranous labyrinth, including ectopic and fused sensory patches. Most notably, the basilar papilla, an auditory organ, contained infected sensory patches with a vestibular phenotype. Vestibular identity was based on: (1) stereociliary bundle morphology; (2) spacing of hair cells and supporting cells; (3) the presence of otoliths; (4) immunolabeling indicative of vestibular supporting cells; and (5) expression of Msx1, a marker of certain vestibular sensory organs. Retrovirus-mediated misexpression of Wnt3a also gave rise to ectopic vestibular patches in the cochlear duct. In situ hybridization revealed that genes for three Frizzled receptors, c-Fz1, c-Fz7, and c-Fz10, are expressed in and adjacent to sensory primordia, while Wnt4 is expressed in adjacent, nonsensory regions of the cochlear duct. We hypothesize that Wnt/beta-catenin signaling specifies otic epithelium as macular and helps to define and maintain sensory/nonsensory boundaries in the cochlear duct.

The signaling molecule retinoic acid (RA) regulates rod and cone photoreceptor fate, differentiation, and survival. Here we elucidate the role of RA in differential regulation of the tandemly-duplicated long wavelength-sensitive (LWS) cone opsin genes. Zebrafish embryos were treated with RA from 48 hours post-fertilization (hpf) to 75 hpf, and RNA was isolated from eyes for microarray analysis. ~170 genes showed significantly altered expression, including several transcription factors and components of cellular signaling pathways. Of interest, the LWS1 opsin gene was strongly upregulated by RA. LWS1 is the upstream member of the tandemly duplicated LWS opsin array and is normally not expressed embryonically. Embryos treated with RA 48 hpf to 100 hpf or beyond showed significant reductions in LWS2-expressing cones in favor of LWS1-expressing cones. The LWS reporter line, LWS-PAC(H) provided evidence that individual LWS cones switched from LWS2 to LWS1 expression in response to RA. The RA signaling reporter line, RARE:YFP indicated that increased RA signaling in cones was associated with this opsin switch, and experimental reduction of RA signaling in larvae at the normal time of onset of LWS1 expression significantly inhibited LWS1 expression. A role for endogenous RA signaling in regulating differential expression of the LWS genes in postmitotic cones was further supported by the presence of an RA signaling domain in ventral retina of juvenile zebrafish that coincided with a ventral zone of LWS1 expression. This is the first evidence that an extracellular signal may regulate differential expression of opsin genes in a tandemly duplicated array.

BackgroundRetinoic acid (RA) is important for vertebrate eye morphogenesis and is a regulator of photoreceptor development in the retina. In the zebrafish, RA treatment of postmitotic photoreceptor precursors has been shown to promote the differentiation of rods and red-sensitive cones while inhibiting the differentiation of blue- and UV-sensitive cones. The roles played by RA and its receptors in modifying photoreceptor fate remain to be determined.ResultsTreatment of zebrafish embryos with RA, beginning at the time of retinal progenitor cell proliferation and prior to photoreceptor terminal mitosis, resulted in a significant alteration of rod and cone mosaic patterns, suggesting an increase in the production of rods at the expense of red cones. Quantitative pattern analyses documented increased density of rod photoreceptors and reduced local spacing between rod cells, suggesting rods were appearing in locations normally occupied by cone photoreceptors. Cone densities were correspondingly reduced and cone photoreceptor mosaics displayed expanded and less regular spacing. These results were consistent with replacement of approximately 25% of positions normally occupied by red-sensitive cones, with additional rods. Analysis of embryos from a RA-signaling reporter line determined that multiple retinal cell types, including mitotic cells and differentiating rods and cones, are capable of directly responding to RA. The RA receptors RXRγ and RARαb are expressed in patterns consistent with mediating the effects of RA on photoreceptors. Selective knockdown of RARαb expression resulted in a reduction in endogenous RA signaling in the retina. Knockdown of RARαb also caused a reduced production of rods that was not restored by simultaneous treatments with RA.ConclusionsThese data suggest that developing retinal cells have a dynamic sensitivity to RA during retinal neurogenesis. In zebrafish RA may influence the rod vs. cone cell fate decision. The RARαb receptor mediates the effects of endogenous, as well as exogenous RA, on rod development.

Head and neck cancer (HNC) therapy often leads to caries development. Our goal was to characterize the oral microbiome of HNC patients who underwent radiation therapy (RT) at baseline (T0), and 6 (T6) and 18 (T18) months post-RT, and to determine if there was a relationship with increased caries. HOMINGS was used to determine the relative abundance (RA) of >600 bacterial species in oral samples of 31 HNC patients. The DMFS score was used to define patient groups with tooth decay increase (DMFS[+]) or no increase (DMFS[-]).A change in microbiome beta-diversity was observed at T6 and T18. The Streptococcus mutans RA increased at T6 in both DMFS[+] and DMFS[-] groups. The RA of Prevotella melaninogenica, the species often associated with caries in young children, decreased at T6 in the DMFS[-] group. The RA of the healthassociated species, Abiotrophia defective, decreased in the DMFS[+] group. The oral microbiome underwent significant changes in radiation-treated HNC patients, whether they developed caries or not. Caries rates were not associated with a difference in salivary flow reduction between DMFS [+] andDMFS[-] groups. Patients who develop caries might be more susceptible to certain species associated with oral disease or have fewer potentially protective oral species.

Background Functions for the early embryonic vasculature in regulating development of central nervous system tissues, such as the retina, have been suggested by in vitro studies and by in vivo manipulations that caused additional ocular vessels to develop. Here we use an avascular zebrafish embryo, cloche−/− (clo−/−), to begin to identify necessary developmental functions of the ocular vasculature in regulating development and patterning of the neural retina, in vivo. These studies are possible in zebrafish embryos, which do not yet rely upon the vasculature for tissue oxygenation. Results clo−/− embryos lacked early ocular vasculature and were microphthalmic, with reduced retinal cell proliferation and cell survival. Retinas of clo mutants were disorganized, with irregular synaptic layers, mispatterned expression domains of retinal transcription factors, morphologically abnormal Müller glia, reduced differentiation of specific retinal cell types, and sporadically distributed cone photoreceptors. Blockade of p53-mediated cell death did not completely rescue this phenotype and revealed ectopic cones in the inner nuclear layer. clo−/− embryos did not upregulate a molecular marker for hypoxia. Conclusions The disorganized retinal phenotype of clo−/− embryos is consistent with a neural and glial developmental patterning role for the early ocular vasculature that is independent of its eventual function in gas exchange.