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.
Highlights
The boundary between the midbrain and the hindbrain is essential for establishing a clear demarcation between the midbrain and the anterior hindbrain
To test if the CRISPR/Cas9-mediated strategy can be used for knocking-in reporters into the zebrafish genome, we chose otx2 as a candidate gene because, first the caudal limit of the Otx2 marks the midbrain-hindbrain boundary (MHB), and second, no transgenic otx2 reporter lines that recapitulate its endogenous expression are currently available (Kurokawa et al, 2006)
The position of the cross section of the midbrain with optic tectum, hypothalamus, and pituitary shown in panel (B) is indicated. (B) Cross section of the midbrain labeled with otx2:venus, the pan neuronal marker HuC/D and the nuclear counterstain DAPI. (C) Higher magnification image of the region indicated by white box in panel (B) showing that numerous neurons express otx2:venus. (D–F) Higher magnification images of the regions indicated by white boxes in (C). otx2:venus is co-expressed in HuC/D expressing neurons in the various tectal sub layers that is characteristic of a teleost midbrain
Summary
The boundary between the midbrain (mesencephalon, mes) and the hindbrain (metencephalon, met) is essential for establishing a clear demarcation between the midbrain and the anterior hindbrain This midbrain-hindbrain boundary (MHB), known as the isthmic organizer or midbrain-hindbrain organizer, acts as a local signaling center (Wurst and Bally-Cuif, 2001; Raible and Brand, 2004; Rhinn et al, 2006; Dworkin and Jane, 2013). This interface is established by mutual transcriptional repression and initiates formation of the prospective MHB Morphogens such as Wnt, Fgf, and transcription factors like Engrailed1/2, and Pax2/5/8 induce the formation of the MHB, and their subsequent interplay is critical for maintenance of the MHB (Rhinn and Brand, 2001; Wurst and Bally-Cuif, 2001; Raible and Brand, 2004; Rhinn et al, 2006; Dworkin and Jane, 2013). The above-mentioned factors (Otx, Gbx, Wnt, Fgf, Pax, and Eng) form the core of the MHB signaling machinery and a disruption of any of these factors interferes with the formation and function of the MHB
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