Background: Adult zebrafish spontaneously regenerate their retinas after damage. Although a number of genes and signaling pathways involved in regeneration have been identified, the exact mechanisms regulating various aspects of regeneration are unclear. microRNAs were examined for their potential roles in regulating zebrafish retinal regeneration. Results: To investigate the requirement of miRNAs during zebrafish retinal regeneration, we knocked down the expression of Dicer in retinas prior to light-induced damage. Reduced Dicer expression significantly decreased the number of proliferating Müller glia-derived neuronal progenitor cells during regeneration. To identify individual miRNAs with roles in neuronal progenitor cell proliferation, we collected retinas at different stages of light damage and performed small RNA high-throughput sequencing. We identified subsets of miRNAs that were differentially expressed during active regeneration but returned to basal levels once regeneration was completed. We then knocked down five different miRNAs that increased in expression and assessed the effects on retina regeneration. Reduction of miR-142b and miR-146a expression significantly reduced INL proliferation at 51 hours of light treatment, while knockdown of miR-7a, miR-27c and miR-31 expression significantly reduced INL proliferation at 72 hours of constant light. Conclusions: miRNAs exhibit dynamic expression profiles during retinal regeneration and are necessary for neuronal progenitor cell proliferation.

Damage of the zebrafish retina triggers a spontaneous regeneration response that is initiated by Müller Glia (MG) dedifferentiation and asymmetric cell division to produce multipotent progenitor cells. Subsequent expansion of the progenitor pool by proliferation is critical for retina regeneration. Pax6b expression in the progenitor cells is necessary for their proliferation, but exact regulation of its expression is unclear. Here, we show that miR-203 is downregulated during regeneration in proliferating progenitor cells. Elevated miR-203 levels inhibit progenitor cell expansion without affecting MG dedifferentiation or progenitor cell generation. Using GFP-reporter assays and gain and loss of function experiments in the retina, we show that miR-203 expression must be suppressed to allow pax6b expression and subsequent progenitor cell proliferation.

Skeletal muscle satellite cells are a muscle stem cell population that mediate posthatch muscle growth and repair. Satellite cells respond differentially to environmental stimuli based upon their fiber‐type of origin. The objective of this study was to determine how temperatures below and above the in vitro control of 38°C affected the proliferation and differentiation of satellite cells isolated from the chicken anaerobic pectoralis major (p. major) or mixed fiber biceps femoris (b.femoris) muscles. The satellite cells isolated from the p. major muscle were more sensitive to both cold and hot temperatures compared to the b.femoris satellite cells during both proliferation and differentiation. The expressions of myogenic regulatory transcription factors were also different between satellite cells from different fiber types. MyoD expression, which partially regulates proliferation, was generally expressed at higher levels in p. major satellite cells compared to the b.femoris satellite cells from 33 to 43°C during proliferation and differentiation. Similarly, myogenin expression, which is required for differentiation, was also expressed at higher levels in p. major satellite cells in response to both cold and hot temperatures during proliferation and differentiation than b. femoris satellite cells. These data demonstrate that satellite cells from the anaerobic p. major muscle are more sensitive than satellite cells from the aerobic b. femoris muscle to both hot and cold thermal stress during myogenic proliferation and differentiation.

Satellite cells are multipotential stem cells that mediate postnatal muscle growth and respond differently to temperature based upon aerobic versus anaerobic fiber-type origin. The objective of this study was to determine how temperatures below and above the control, 38°C, affect the fate of satellite cells isolated from the anaerobic pectoralis major (p. major) or mixed fiber biceps femoris (b. femoris). At all sampling times, p. major and b. femoris cells accumulated less lipid when incubated at low temperatures and more lipid at elevated temperatures compared to the control. Satellite cells isolated from the p. major were more sensitive to temperature as they accumulated more lipid at elevated temperatures compared to b. femoris cells. Expression of adipogenic genes, CCAAT/enhancer-binding protein β (C/EBPβ) and proliferator-activated receptor gamma (PPARγ) were different within satellite cells isolated from the p. major or b. femoris. At 72 h of proliferation, C/EBPβ expression increased with increasing temperature in both cell types, while PPARγ expression decreased with increasing temperature in p. major satellite cells. At 48 h of differentiation, both C/EBPβ and PPARγ expression increased in the p. major and decreased in the b. femoris, with increasing temperature. Flow cytometry measured apoptotic markers for early apoptosis (Annexin-V-PE) or late apoptosis (7-AAD), showing less than 1% of apoptotic satellite cells throughout all experimental conditions, therefore, apoptosis was considered biologically not significant. The results support that anaerobic p. major satellite cells are more predisposed to adipogenic conversion than aerobic b. femoris cells when thermally challenged.

A zebrafish ortholog of human lengsin was identified by EST analysis of an adult lens cDNA library. During zebrafish development, lengsin transcription is first detected at 24 hours post-fertilization (hpf). Immunolocalization, using polyclonal antiserum generated against a Lengsin bacterial fusion protein, detects lens-specific protein in whole-mount embryos at 30 hpf. Lengsin expression in zebrafish follows the temporal expression of the αA-αB1-and βB1-crystallin proteins in the lens. At 72 hpf, Lengsin is localized to a subpopulation of differentiating secondary fiber cells, while no expression is detected in the lens epithelial cells or central lens fibers. In the adult lens, Lengsin is restricted to a narrow band of cortical fibers and co-localizes with actin at the lateral faces of these interdigitating cells. Stable transgenic lines, using a 3 kb lengsin genomic fragment to regulate EGFP expression, recapitulate the Lengsin temporal and spatial expression patterns. Lengsin function in zebrafish lens formation was examined by antisense morpholino-mediated translation and mRNA splice inhibition. At 72 hpf, the lengsin morphant lenses are reduced in size and exhibit separations within the cortex due to defects in secondary fiber morphogenesis. The location of the morphant lens defects correlates with the Lengsin protein localization at this age. These results demonstrate Lengsin is required for proper fiber cell differentiation by playing roles in either cell elongation or the establishment of cell interactions.