Cloning of zebrafish nkx6.2 and a comprehensive analysis of the conserved transcriptional response to Hedgehog/Gli signaling in the zebrafish neural tube

Abstract

Sonic Hedgehog (Shh) signaling helps pattern the vertebrate neural tube, in part by regulating the dorsal/ventral expression of a number of homeodomain containing transcription factors. These Hh responsive genes have been divided into two classes, with Class II genes being activated by Hh signaling and Class I genes being repressed by Hh signaling. While the transcriptional response to varying Hh levels is well defined in chick and mouse, it is only partially described in zebrafish, despite the fact that zebrafish has emerged as a powerful genetic system for the study of neural patterning. To better characterize the Hh response in the zebrafish neural tube, we cloned the zebrafish Class II Hh target genes nkx2.9 and nkx6.2. We then analyzed the expression of a number of Class I and Class II Hh responsive genes in wild type, Hh mutant, and Hh over-expressing zebrafish embryos. We show that expression of Class I and Class II genes is highly conserved in the vertebrate neural tube. Further, ventral-most Class II gene expression was completely lost in all Hh pathway mutants analyzed, indicating high levels of Hh signaling are blocked in all of these mutants. In contrast, more dorsally expressed genes were variably affected in different Hh pathway mutants, indicating mid-levels of Hh signaling are differentially affected. This comprehensive expression study provides an important tool for the characterization of Hh signaling in zebrafish and provides a sensitive assay for determining the degree to which newly identified zebrafish mutants affect Hh signaling. dbx1a; dbx2; hlx1; hlxb9; irx1a; irx3a; irx5; nkx2.2a; nkx2.9; nkx6.1; nkx6.2; pax3; pax6a; pax7; chameleon; detour; slow muscle omitted; you-too; cyclopamine; floorplate; motor neurons Keywords Results and DiscussionSecreted proteins of the Hedgehog (Hh) family are well known for their role in patterning the vertebrate and invertebrate embryo (Ingham and McMahon, 2001). During development, Hh functions in a concentration dependent manner to influence cell differentiation (Ericson et al., 1997a). Hh can also act as a survival factor (Miao et al., 1997) or a mitogen (Dahmane et al., 2001) to help shape the embryo. The morphogen activity of Hh has been well studied in the neural tube of chick and mouse (Ericson et al., 1995;Wijgerde et al., 2002), where Hh secreted from the notochord induces floorplate and ventral neural fates based on the concentration of Hh seen by a neural progenitor cell (Echelard et al., 1993). Cells respond to Hh through a * Corresponding author (e-mail: Karlstrom@bio.umass.edu) Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers t...

Regulation and function of Dbx genes in the zebrafish spinal cord

“…Therefore, Hedgehog signaling is dispensable for the induction of these genes but it also prevents their expression in the ventral spinal cord. Our results differ from a previous study, which shows a more subtle ventral expansion of dbx1a in the smoothened mutant zebrafish (Guner and Karlstrom, 2007). This difference is likely due to the presence of maternally supplied Smoothened protein in the mutant, allowing for Hedgehog signaling to partially repress dbx1a from the most ventral extent of the spinal cord.…”

“…2A-AЉ). These data differ from those presented previously, which show that pax3 expression was unaffected in smoothened mutants (Guner and Karlstrom, 2007). When cyclopamine was added at 18 hpf, we did not observe a shift in msxc (Fig.…”

“…In fact, sequence comparison between the three genes indicates dbx2 is the most divergent (Seo et al, 1999). Recent reports in zebrafish have shown that many of the spinal progenitor genes have conserved spatial relationships (Cheesman et al, 2004;Lewis et al, 2005;Guner and Karlstrom, 2007). We have demonstrated that the relationship of Dbx expression to other progenitor genes is evolutionarily conserved in zebrafish, which we find intriguing given the size difference of the zebrafish spinal cord compared with amniotes.…”

“…3C,CЈ ;Cheesman et al, 2004) and nkx6.2 ( Fig. 3E,EЈ; Guner and Karlstrom, 2007) were abolished in embryos treated with cyclopamine at 50% epiboly, whereas dimethyl sulfoxide (DMSO) -treated embryos displayed normal expression of these markers. Interestingly, when we applied cyclopamine at 18 hpf and fixed at 24 hpf, low levels of nkx6.1 (Fig.…”

Roles of Hedgehog pathway components and retinoic acid signalling in specifying zebrafish ventral spinal cord neurons

“…In zebrafish, several laboratories have investigated the effects of severely reducing Hh signalling on the expression of spinal cord ventral progenitor domain markers (e.g. Guner and Karlstrom, 2007;Park et al, 2002;Park et al, 2004;Schauerte et al, 1998) and the formation of MNs (Chen et al, 2001;Lewis and Eisen, 2001;Park et al, 2004;Varga et al, 2001) and V3 domain cells (Schäfer et al, 2007), but none of these studies has investigated whether loss of Hh signalling affects V2, V1 or V0v cells.…”