Spectrum of Fates: a new approach to the study of the developing zebrafish retina

Abstract

The ability to image cells live and in situ as they proliferate and differentiate has proved to be an invaluable asset to biologists investigating developmental processes. Here, we describe a Spectrum of Fates approach that allows the identification of all the major neuronal subtypes in the zebrafish retina simultaneously. Spectrum of Fates is based on the combinatorial expression of differently coloured fluorescent proteins driven by the promoters of transcription factors that are expressed in overlapping subsets of retinal neurons. Here, we show how a Spectrum of Fates approach can be used to assess various aspects of neural development, such as developmental waves of differentiation, neuropil development, lineage tracing and hierarchies of fates in the developing zebrafish retina.

Slit2 is necessary for optic axon organization in the zebrafish ventral midline

“…Embryos were raised at temperatures ranging from 28.5 to 32 °C and staged in hours post-fertilization (hpf) according to Kimmel and collaborators (Kimmel et al, 1995). We used wild-type (Tab5), SoFa1 triple transgenic (atoh7:gap-RFP/ptf1a:cytGFP/crx:gap-CFP; Almeida et al, 2014) and the CRISPRgenerated mutant line NM_131753.1:g.30_39del (hereafter denoted as slit2-/-). All the manipulations were carried out following the approved local regulations (CEUA-Institut Pasteur de Montevideo, and CNEA).…”

Independent modes of ganglion cell translocation ensure correct lamination of the zebrafish retina

“…Transgenic lines used line structures labeled reference Tg(bactin:GFP-DCX) all microtubules this study aPKC in all membranes this study Tg(bactin:ras-GFP) all membranes gift from C.P. Heisenberg lab Tg(ath5:gap-GFP) membranes of RGCs and photoreceptors (Zolessi et al, 2006) Tg(ath5:gap-RFP) membranes of RGCs and photoreceptors (Zolessi et al, 2006) Tg(crx:gap-CFP) membranes of photoreceptors and bipolar cells (Almeida et al, 2014) Tg(ptf1a:DsRed) amacrine and horizontal cells (Jusuf et al, 2012) Tg(ptf1a:Gal4-VP16, UAS:gap-YFP) membranes of amacrine and horizontal cells (Pisharath and Parsons, 2009) (Williams et al, 2010) Tg(SoFa2) all retinal cells (Almeida et al, 2014) Tg(vsx1:GFP) bipolar cells (Kimura et al, 2008) DNA constructs ath5:mKate2-aPKC-CAAX The coding sequence of mKate2-aPKC-CAAX was assembled by classical cloning in the pCS2+ backbone and used as a template for creating the mKate2-aPKC-CAAX middle entry clone by PCR using Phusion polymerase (NEB) with primers: F: 5′-ggggacaagtttgtacaaaaaagcaggctggATGGTGAGCGAGCTGATTAAGG-3′ R: 5′-ggggaccactttgtacaagaaagctgggtcTTAGGAGAGCACACACTTG-3′ The Ath5 promoter 5′ entry clone (Kwan et al, 2007) was combined with mKate2-aPKC-CAAX middle entry clone and pTol2+pA R4-R2 backbone (Villefranc et al, 2007).…”

“…Heat shock was performed between 30 hpf and 32 hpf so that aPKC-CAAX was present when RGC emergence was at its peak. Live imaging revealed that in this condition RGCs indeed lost their basal process after apical division and for more than 6 hours did not lose their apical process To assess whether this impairment of RGC translocation had long-lasting effects on retinal lamination, we performed long-term imaging of Tg(hsp70:mKate2-aPKC-CAAX) fish crossed with the SoFa2 line (Almeida et al, 2014), which labels membranes of all retinal cells using a combination of three fluorescent proteins. Fish were imaged in the LSFM every 12 hours between 36 hpf and 84 hpf to follow retinal lamination ( Fig.…”

Dynamic contrast with reversibly photoswitchable fluorescent labels for imaging living cells

“…Further increasing this number requires spectral unmixing but at signicant cost in terms of photon budget and computation time. [15][16][17][18][19][20][21][22] Such constraints currently limit the discriminative power of emerging genetic engineering strategies, [23][24][25] which can already be implemented to label a larger number of biomolecules or cells in order to interrogate signaling pathways, 26 cycling states, 27 genotypes, 28,29 neuronal projections, 23,30,31 clones, [26][27][28][29][30][31][32][33] cell types, 34 or microbiomes. 35 Since uorescence should remain a much favored observable for imaging live cells, 36 the spectral dimension has to be complemented by another dimension for further discriminating uorophores (see Fig.…”