The Independent Probabilistic Firing of Transcription Factors: A Paradigm for Clonal Variability in the Zebrafish Retina.

Early retinal progenitor cells (RPCs) in vertebrates produce lineages that vary greatly both in terms of cell number and fate composition, yet how this variability is achieved remains unknown. One possibility is that these RPCs are individually distinct and that each gives rise to a unique lineage. Another is that stochastic mechanisms play upon the determinative machinery of equipotent early RPCs to drive clonal variability. Here we show that a simple model, based on the independent firing of key fate-influencing transcription factors, can quantitatively account for the intrinsic clonal variance in the zebrafish retina and predict the distributions of neuronal cell types in clones where one or more of these fates are made unavailable.BDS and SR acknowledge the support of the Wellcome Trust (098357/Z/12/Z). WAH and HB also acknowledge the support of the Wellcome Trust (100329/Z/12/Z). HB was also supported by the Swedish Research Council (2011-7054).This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.devcel.2015.08.01

Developmental and adult characterization of secretagogin expressing amacrine cells in zebrafish retina

Calcium binding proteins show stereotypical expression patterns within diverse neuron types across the central nervous system. Here, we provide a characterization of developmental and adult secretagogin-immunolabelled neurons in the zebrafish retina with an emphasis on co-expression of multiple calcium binding proteins. Secretagogin is a recently identified and cloned member of the F-hand family of calcium binding proteins, which labels distinct neuron populations in the retinas of mammalian vertebrates. Both the adult distribution of secretagogin labeled retinal neurons as well as the developmental expression indicative of the stage of neurogenesis during which this calcium binding protein is expressed was quantified. Secretagogin expression was confined to an amacrine interneuron population in the inner nuclear layer, with monostratified neurites in the center of the inner plexiform layer and a relatively regular soma distribution (regularity index > 2.5 across central-peripheral areas). However, only a subpopulation (~60%) co-labeled with gamma-aminobutyric acid as their neurotransmitter, suggesting that possibly two amacrine subtypes are secretagogin immunoreactive. Quantitative co-labeling analysis with other known amacrine subtype markers including the three main calcium binding proteins parvalbumin, calbindin and calretinin identifies secretagogin immunoreactive neurons as a distinct neuron population. The highest density of secretagogin cells of ~1800 cells / mm2 remained relatively evenly along the horizontal meridian, whilst the density dropped of to 125 cells / mm2 towards the dorsal and ventral periphery. Thus, secretagogin represents a new amacrine label within the zebrafish retina. The developmental expression suggests a possible role in late stage differentiation. This characterization forms the basis of functional studies assessing how the expression of distinct calcium binding proteins might be regulated to compensate for the loss of one of the others

Secretagogin expression in embryonic and adult zebrafish retina.

<p>Micrographs of vertical sections through zebrafish retina immunohistochemically labeled for secretagogin (SCGN–green) with nuclei counterstained by DAPI (blue). (A–E) Sections through retinas at 1–5 days post fertilization (dpf) show earliest secretagogin positive cells detected at 3 dpf (C) and maintained at subsequent days. (F) Collage through retinal section in 6 month old zebrafish. Secretagogin expression in the amacrine layer in the inner half of the inner nuclear layer (INL) remains strong throughout adulthood. (F’) Higher magnification inset of boxed region in F shows secretagogin labeled with stained processes showing monostratified band in the center of the inner plexiform layer (IPL). OS: outer segments; ONL: outer nuclear layer; OPL: outer plexiform layer; GCL: ganglion cell layer. Scale bar (E) for A-E is 50 μm, scale bar (F) is 100 μm, scale bar (F’) is 20 μm.</p

The chemokine receptor <it>cxcr5</it> regulates the regenerative neurogenesis response in the adult zebrafish brain

Abstract Background Unlike mammals, zebrafish exhibits extensive neural regeneration after injury in adult stages of its lifetime due to the neurogenic activity of the radial glial cells. However, the genes involved in the regenerative neurogenesis response of the zebrafish brain are largely unknown. Thus, understanding the underlying principles of this regeneration capacity of the zebrafish brain is an interesting research realm that may offer vast clinical ramifications. Results In this paper, we characterized the expression pattern of cxcr5 and analyzed the function of this gene during adult neurogenesis and regeneration of the zebrafish telencephalon. We found that cxcr5 was upregulated transiently in the RGCs and neurons, and the expression in the immune cells such as leukocytes was negligible during both adult neurogenesis and regeneration. We observed that the transgenic misexpression of cxcr5 in the ventricular cells using dominant negative and full-length variants of the gene resulted in altered proliferation and neurogenesis response of the RGCs. When we knocked down cxcr5 using antisense morpholinos and cerebroventricular microinjection, we observed outcomes similar to the overexpression of the dominant negative cxcr5 variant. Conclusions Thus, based on our results, we propose that cxcr5 imposes a proliferative permissiveness to the radial glial cells and is required for differentiation of the RGCs to neurons, highlighting novel roles of cxcr5 in the nervous system of vertebrates. We therefore suggest that cxcr5 is an important cue for ventricular cell proliferation and regenerative neurogenesis in the adult zebrafish telencephalon. Further studies on the role of cxcr5 in mediating neuronal replenishment have the potential to produce clinical ramifications in efforts for regenerative therapeutic applications for human neurological disorders or acute injuries.</p

Secretagogin positive cells from a regular mosaic with highest density along the horizontal midline.

<p>(A, B) Micrograph collage and schematic showing secretagogin immunostaining in the inner nuclear layer of a flat mounted whole adult zebrafish retina. (C, D) Density of secretagogin labeled cells across the nasal-temporal (F) or dorsal-ventral (G) axes indicate high even density along the horizontal meridian, with the density along the dorsal-ventral axis peaking in central retina and decreasing towards the periphery (n = 20–21 ROIs for each of n = 3 adult eyes). Density was counted in ROIs (200 μm x 200 μm) every 250 μm until the edge of the retina. (E–G) Individual examples showing nearest neighbor analysis of region of interest (200 μm x 200 μm) indicated by boxed regions in B. Secretagogin labeled cells at any eccentricity are distributed regularly. ROIs were located at 250 μm, 750 μm and 1250 μm distance from the optic nerve center. Scale bar (A) is 200 μm.</p

Comparative expression of secretagogin and other calcium binding proteins within the zebrafish retina.

<p>(A–C) Micrographs showing cross-sections through zebrafish retina at 5 days postfertilization. Higher magnification of boxed regions in each row show secretagogin expression in green and other calcium binding protein expression in red: Parvalbumin (PV–A), Calbindin (CB–B), Calretinin (CR–C). (D–F) Pie charts show quantification of singe and double labeling (asterisks). Secretagogin labeled cells are mutually exclusive from parvalbumin (D) expressing cells, but overlap partially with calbindin (E) and represent a subpopulation of calretinin (F) expressing cells. Scale bar (C) for A–C is 50 μm, scale bar (C”‘) for A’–C”‘ is 20 μm.</p

Exposure to an environmentally relevant concentration of 17α-ethinylestradiol disrupts craniofacial development of juvenile zebrafish

Endocrine disrupting chemicals (EDCs) can interact with native hormone receptors to interfere with and disrupt hormone signalling that is necessary for a broad range of developmental pathways. EDCs are pervasive in our environment, in particular in our waterways, making aquatic wildlife especially vulnerable to their effects. Many of these EDCs are able to bind to and activate oestrogen receptors, causing aberrant oestrogen signalling. Craniofacial development is an oestrogen-sensitive process, with oestrogen receptors expressed in chondrocytes during critical periods of development. Previous studies have demonstrated a negative effect of high concentrations of oestrogen on early craniofacial patterning in the aquatic model organism, the zebrafish (Danio rerio). In order to determine the impacts of exposure to an oestrogenic EDC, we exposed zebrafish larvae and juveniles to either a high concentration to replicate previous studies, or a low, environmentally relevant concentration of the oestrogenic contaminant, 17α-ethinylestradiol. The prolonged / chronic exposure regimen was used to replicate that seen by many animals in natural waterways. We observed changes to craniofacial morphology in all treatments, and most strikingly in the larvae-juveniles exposed to a low concentration of EE2. In the present study, we have demonstrated that the developmental stage at which exposure occurs can greatly impact phenotypic outcomes, and these results allow us to understand the widespread impact of oestrogenic endocrine disruptors. Given the conservation of key craniofacial development pathways across vertebrates, our model can further be applied in defining the risks of EDCs on mammalian organisms