Optochemical dissection of T-box gene-dependent medial floor plate development

Abstract

In addition to their cell-autonomous roles in mesoderm development, the zebrafish T-box transcription factors no tail a (ntla) and spadetail (spt/tbx16) are required for medial floor plate (MFP) formation. Posterior MFP cells are completely absent in zebrafish embryos lacking both Ntla and Spt function, and genetic mosaic analyses have shown that the two T-box genes promote MFP development in a non-cell-autonomous manner. On the basis of these observations, it has been proposed that Ntla/Spt-dependent mesoderm-derived signals are required for the induction of posterior but not anterior MFP cells. To investigate the mechanisms by which Ntla and Spt regulate MFP development, we have used photoactivatable caged morpholinos (cMOs) to silence these T-box genes with spatiotemporal control. We find that posterior MFP formation requires Ntla or Spt activity during early gastrulation, specifically in lateral margin-derived cells that converge toward the midline during epiboly and somitogenesis. Nodal signaling-dependent MFP specification is maintained in the absence of Ntla and Spt function; however, midline cells in ntla;spt morphants exhibit aberrant morphogenetic movements, resulting in their anterior mislocalization. Our findings indicate that Ntla and Spt do not differentially regulate MFP induction along the anterior-posterior axis; rather, the T-box genes act redundantly within margin-derived cells to promote the posterior extension of MFP progenitors.

Figure 1. MFP defects are evident in zebrafish gastrulae lacking Ntla and Spt function

Expression of the MFP marker shhb in wildtype embryos and those without Ntla and/or Spt function at (a) 6 hpf, (b), 9 hpf, (c) 10 hpf, and (d) 13 hpf. Brackets demarcate posterior regions lacking shhb-expressing MFP progenitors, and arrowheads label ectopic shhb-positive cells in the germ ring (9 hpf), tailbud (10 hpf), and anterior midline (13 hpf). Embryo orientations: dorsal view, anterior up or posterior view, dorsal up. Scale bar: 200 μm.

Figure 2. Optochemical control of Spt function

Morphological phenotypes observed in zebrafish embryos injected with the spt cMO, either alone (a–b) or in combination with a ntla MO (c–d). Non-irradiated embryos exhibited wildtype (a) and ntla mutant (c) patterning, whereas those subjected to global, 360-nm irradiation at 3 hpf recapitulated spt (b) and ntla;spt (d) mutant defects. 24-hpf embryos are shown in lateral view, anterior left. Scale bar: 200 μm.

Figure 3. Spatiotemporal analysis of Ntla/Spt-dependent MFP development

(a) Classification of MFP patterning phenotypes, as determined by shhb expression. 24-hpf embryos are shown in lateral view, anterior left or dorsal view, anterior left. (b) Phenotypic distributions for embryos injected with the indicated oligonucleotides and either cultured in the dark or globally irradiated at the designated time points. (c) Regiospecific irradiation of the germ ring in 6-hpf embryos, as illustrated by cFD photoactivation within the lateral left margin. Graphical depictions and overlaid brightfield and fluorescence micrographs of an irradiated embryo are shown. Dorsal (D), ventral (R), and lateral (L) regions of the germ ring are labeled, and arrowheads denote the shield. Embryo orientations: lateral view, dorsal right or animal pole view, dorsal down. (d) Phenotypic distributions for embryos injected with the indicated oligonucleotides and either cultured in the dark or irradiated in the designated manner at 6 hpf. Scale bars: 200 μm.

Figure 4. Convergence of lateral margin-derived cells to the midline

Morphogenetic movement of cells originating from the right lateral margin, as determined by injecting Tg(−2.4shha-ABC:GFP) embryos with cRD and regiospecifically irradiating the germ ring at 6 hpf. Relative positions of the red-fluorescent, lateral margin-derived cells and the GFP-positive midline at 13 hpf are shown for control embryos (a) and those without Ntla and/or Spt function (b–d). Embryo orientations: dorsal view, anterior up. Scale bar: 200 μm.

Figure 5. Nodal signaling is maintained in zebrafish gastrulae lacking Ntla and Spt function

Expression of the Nodal signaling components (ndr2 and tdgf1; a–b) and transcriptional targets (pitx2a and lft2; c–d) in wildtype embryos and those without Ntla and/or Spt function. Embryos at 75% epiboly are shown in dorsal view, anterior up or lateral view, anterior up. Scale bar: 200 μm.

Figure 6. MFP cell number is conserved in the absence of Ntla and Spt function

(a) Relative shhb transcript levels in 22-hpf wildtype embryos and those without Ntla and/or Spt function, normalized with respect to eef1a1l1 expression. (b) Representative flow cytometry scatter plots identifying GFP-positive MFP cells in Tg(−2.4shha-ABC:GFP) embryos injected with a ntla MO or a mixture of ntla and spt MOs. (c) Quantification of GFP-positive MFP cells. Data are the average of triplicate samples ± s.e.m., and the asterisk indicates P < 0.01.

Figure 7. A model for T-box gene-dependent MFP patterning

Cells derived from the lateral margins (red) converge to flank the midline (green) in zebrafish embryos, promoting the extension of MFP progenitors along anterior-posterior axis. Loss of Ntla and Spt function in these cells alters their interactions with midline populations, leading to the anterior mislocalization of MFP progenitors. Dorsal (D), ventral (V), and lateral (L) regions of the margin and selected embryonic structures are labeled.