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.

Ultrafast phasor-based hyperspectral snapshot microscopy for biomedical imaging

“…In order to demonstrate the advantages of our method in a complex biological sample, we applied ultrafast phasor-based hyperspectral snapshot microscopy in zebrafish embryos, a prime model to study developmental processes that is also widely used to model human diseases. We took advantage of the Spectrum of Fates approach (SoFa), in which a transgenic line was generated to study zebrafish retinal development by driving the expression of differently tuned fluorescent proteins (FPs) from the promoters of a set of transcription factors 22 . In SoFa embryos, each FP is expressed in one or multiple cell types resulting in a different fluorophore ratio associated with each of the major cell types.…”

“…This difference in FP ratios creates a unique spectral fingerprint for each cell type. We imaged 72 hours post fertilization (hpf) embryos of SoFa1 zebrafish 22 expressing Crx:gapCFP, Ptf1a:cytGFP, and Atoh7:gapRFP, together with SYTOX Orange (SO) nucleic acid stain added after embryo fixation. Importantly, the emission spectra of CFP/EGFP and RFP/SO completely overlap and could not be separated by conventional bandpass filters ( Supplementary Fig.…”

Feedback from each retinal neuron population drives expression of subsequent fate determinant genes without influencing the cell cycle exit timing

“…Because gene expression starts in the anterior‐ventral patch and spreads three dimensionally as a wave both toward posterior regions and deeper eye regions, transplanted cells, even under the control condition, show vastly different timing of gene expression (>12 hours for a single label; see Fig. ; Almeida et al, ). This is reflected by the increase in the standard deviation of these data even within the Atoh7 control expression.…”

A non-cell autonomous actin redistribution enables isotropic retinal growth

“…37 Some of the molecular factors governing cell and tissue geometry have been 38 extensively studied. Both microtubule reorganization and cortical actomyosin 39 rearrangements can influence shapes of individual epithelial cells, as well as the 40 shape of whole tissues [3][4][5][6][7][8][9][10]. When studying these factors however, most 41 investigations so far focused on anisotropic tissue shape changes occurring during 42 development, while isotropic growth and shape maintenance is not as well 43…”

“…understood. In addition, most studies explored tissue growth and shape in two 44 dimensions [5,9,[11][12][13]. Thus, a thorough evaluation of how tissue growth is 45 coordinated by cell and tissue parameters in 3D is currently lacking.…”