A temperature-sensitive mutation in thenodal-related genecyclopsreveals that the floor plate is induced during gastrulation in zebrafish

Abstract

The floor plate, a specialized group of cells in the ventral midline of the neural tube of vertebrates, plays crucial roles in patterning the central nervous system. Recent work from zebrafish, chick, chick-quail chimeras and mice to investigate the development of the floor plate have led to several models of floor-plate induction. One model suggests that the floor plate is formed by inductive signalling from the notochord to the overlying neural tube. The induction is thought to be mediated by notochord-derived Sonic hedgehog (Shh), a secreted protein, and requires direct cellular contact between the notochord and the neural tube. Another model proposes a role for the organizer in generating midline precursor cells that produce floor plate cells independent of notochord specification, and proposes that floor plate specification occurs early, during gastrulation. We describe a temperature-sensitive mutation that affects the zebrafish Nodal-related secreted signalling factor, Cyclops, and use it to address the issue of when the floor plate is induced in zebrafish. Zebrafish cyclops regulates the expression of shh in the ventral neural tube. Although null mutations in cyclops result in the lack of the medial floor plate, embryos homozygous for the temperature-sensitive mutation have floor plate cells at the permissive temperature and lack floor plate cells at the restrictive temperature. We use this mutant allele in temperature shift-up and shift-down experiments to answer a central question pertaining to the timing of vertebrate floor plate induction. Abrogation of Cyc/Nodal signalling in the temperature-sensitive mutant embryos at various stages indicates that the floor plate in zebrafish is induced early in development, during gastrulation. In addition, continuous Cyclops signalling is required through gastrulation for a complete ventral neural tube throughout the length of the neuraxis. Finally, by modulation of Nodal signalling levels in mutants and in ectopic overexpression experiments, we show that, similar to the requirements for prechordal plate mesendoderm fates, uninterrupted and high levels of Cyclops signalling are required for induction and specification of a complete ventral neural tube.

The pro-domain of the zebrafish Nodal-related protein Cyclops regulates its signaling activities

“…Although Cyc and Sqt share 68% identity and 78% sequence similarity in their mature domains, their pro-domains are more divergent (Erter et al, 1998;Rebagliati et al, 1998a). In particular, the Cyc pro-domain has several proline-rich regions that are not shared by any of the other vertebrate Nodal-related factors (Hudson and Yasuo, 2005;Jones et al, 1995;Morokuma et al, 2002;Rebagliati et al, 1998a;Sampath et al, 1998;Tian et al, 2003;Toyama et al, 1995;Yu et al, 2002;Zhou et al, 1993). Although it is not clear how this region evolved, the presence of the proline-rich repeats is suggestive of insertion by (Constam and Robertson, 1999).…”

“…Capped mRNA was synthesized from linearized plasmids using the mMessage mMachine kit (Ambion), and 0.5 pg, 1.25 pg, 5 pg or 25 pg aliquots of capped mRNA were injected into zebrafish embryos at the one-cell stage. The embryos were fixed in 4% paraformaldehyde (PFA) at 50% epiboly and processed for in situ hybridization to detect gsc or ntl expression (Tian et al, 2003). For detecting the range of signaling, 5 or 10 pg aliquots of in vitro synthesized capped mRNA encoding Sqt, Cyc, CycΔ2, Cyc pro :Sqt mat , Sqt pro :Cyc mat , Sqt Cyc2 :Sqt were injected together with the lineage tracers fluorescein-dextran and biotinylated dextran (Molecular Probes) into a single cell of 64-to 128-cell zebrafish wild-type embryos.…”

“…Analysis of mutations that affect cyc and sqt has shown that these genes have overlapping functions in the embryo during mesoderm induction (Erter et al, 1998;Feldman et al, 1998;Long et al, 2003;Rebagliati et al, 1998a;Rebagliati et al, 1998b;Sampath et al, 1998). Loss of cyc function results in severe deficiencies in the ventral neural tube and in cyclopia, whereas sqt function is required for formation of the mesendoderm and dorsal structures (Erter et al, 1998;Feldman et al, 2000;Feldman et al, 1998;Gore et al, 2005;Hagos et al, 2007;Hatta et al, 1991;Rebagliati et al, 1998b;Sampath et al, 1998;Sirotkin et al, 2000;Tian et al, 2003). Spw regulates left-right asymmetry of the visceral organs during somitogenesis (Long et al, 2003).…”

The Integrator subunits function in hematopoiesis by modulating Smad/BMP signaling

“…Whole-mount in situ hybridization was performed as described (Tian et al, 2003). The following plasmids were linearized and antisense probes were synthesized by in vitro transcription: pBSints5 (BamHI, T3 RNA Polymerase), pGEMT-Easyscl (NcoI, SP6 RNA Polymerase), pBSgata1 (XbaI, T7 RNA Polymerase), pBSflk1 (SmaI, T7), pKSpax2a (BamHI, T7) (Detrich et al, 1995;Gering et al, 1998;Liao et al, 1997;Majumdar et al, 2000), pBShgg1 (XbaI, T7) (Thisse et al, 1994), pBSgsc (EcoRI, T7) (Thisse et al, 1994), pBSntl (XhoI, T7) (Schulte-Merker et al, 1994), pBSspt (EcoRI, T7) (Griffin et al, 1998), pBSsox17 (EcoRI, T7) (Alexander and Stainier, 1999), pBSmyoD (BamHI, T7) (Weinberg et al, 1996) and pBSdlx3 (SalI, T7) (Akimenko et al, 1994).…”

Medial floor plate formation in zebrafish consists of two phases and requires trunk-derived Midkine-a

“…In zebrafish, mutant analyses and lineage tracing have shown that MFP cells originate from a pool of midline precursor cells in the gastrula organizer, the embryonic shield (Appel et al 1999;Le Douarin and Halpern 2000). Accordingly, the induction of the MFP in zebrafish occurs during gastrulation by shield-derived factors (Tian et al 2003). Initially induced MFP precursors persist in the tailbud and contribute to the MFP during later embryonic development.…”