CRISPR/Cas9-Mediated Zebrafish Knock-in as a Novel Strategy to Study Midbrain-Hindbrain Boundary Development

Abstract

The midbrain-hindbrain boundary (MHB) acts as an organizer and controls the fate of neighboring cells to develop into either mesencephalic (midbrain) or metencephalic (hindbrain) cells by secreting signaling molecules like Wnt1 and Fgf8. The zebrafish is an excellent vertebrate model for studying MHB development due to the ease of gene manipulation and the possibility of following cellular dynamics and morphogenetic processes using live imaging. Currently, only very few reporter and/or Cre-driver lines are available to study gene expression at the MHB, hampering the understanding of MHB development, and traditional transgenic technologies using promoter/enhancer fragments or bacterial artificial chromosome (BAC)-mediated transgenesis often do not faithfully recapitulate endogenous expression patterns. In contrast, CRISPR/Cas9-mediated genome editing technology now provides a great opportunity to efficiently knock-in or knock-out genes. We have generated four CRISPR/Cas9-based knock-in fluorescent reporter lines for two crucial genes involved in MHB development, namely otx2 and pax2a. The coding sequences of the reporters were knocked-in upstream of the corresponding ATG and are, thus, under the control of the endogenous promoter/enhancer elements. Interestingly, this strategy does not disturb endogenous gene expression. Using the fast maturing fluorescent protein reporter, Venus, enabled us to follow MHB development using cell tracking and live imaging. In addition, we show that these reporter lines label various neuronal and glial cell types in the adult zebrafish brain, making them highly suitable for investigating embryonic and adult midbrain, hindbrain, and MHB development.

Cell-fate plasticity, adhesion and cell sorting complementarily establish a sharp midbrain-hindbrain boundary

“…The coding sequences for the reporters were knocked-in upstream of the corresponding ATG and, thus, under the control of the endogenous promoter/enhancer elements ( Fig 1A, schematic representation). Importantly, as shown previously, reporter expression faithfully reproduces the endogenous gene expression patterns (Kesavan et al, 2017;Kesavan et al, 2018).…”

“…The knock-in lines for various genes expressed at MHB were generated and maintained as previously described (Kesavan et al, 2017;Kesavan et al, 2018).…”

“…Embryos at specific developmental stages were derived by crossing wild type fish (Ab strain). They were fixed in 4% PFA, stored in 100% methanol at -20°C, and whole mount in situ hybridization was performed as described elsewhere (Kesavan et al, 2017, Reifers et al, 1998. Briefly, using a RNA labeling and detection kit (Roche), digoxigenin (DIG)-labeled probes were synthesized from linear DNA, and the hybridized probes were detected using anti-digoxigenin antibody.…”

“…Therefore, we have addressed these questions using the developing zebrafish as a model. Specially, we visualized the establishment of the MHB in real-time by generating various novel fluorescent reporter and Cre driver lines for otx2b, wnt1, gbx1, gbx2, and fgf8a using CRISPR/Cas9-mediated knock-in strategies (Kesavan et al, 2017;Kesavan et al, 2018). Using a combination of time-lapse imaging and Cre/lox-mediated lineage tracing, we show that lineage restriction at the MHB occurs by the end of gastrulation.…”

Polyacrylamide Bead Sensors for in vivo Quantification of Cell-Scale Stress in Zebrafish Development

“…To obtain membrane-tagged eGFP embryos, adults from the transgenic strain Tg(Bactin:HRas-EGFP) (ZDB-ALT-061107-2) 41 were outcrossed with those from wild type AB strain. Furthermore, a transgenic line which expressed a nucleus-targeted venus fluorescent protein at the otx2 locus (knock-in) was established using the CRISPR knock-in strategy as previously described 42 ; this line was used to identify the midbrain hindbrain boundary.…”