Wnt signaling and tbx16 form a bistable switch to commit bipotential progenitors to mesoderm

Abstract

Anterior to posterior growth of the vertebrate body is fueled by a posteriorly located population of bipotential neuro-mesodermal progenitor cells. These progenitors have a limited rate of proliferation and their maintenance is crucial for completion of the anterior-posterior axis. How they leave the progenitor state and commit to differentiation is largely unknown, in part because widespread modulation of factors essential for this process causes organism-wide effects. Using a novel assay, we show that zebrafish Tbx16 (Spadetail) is capable of advancing mesodermal differentiation cell-autonomously. Tbx16 locks cells into the mesodermal state by not only activating downstream mesodermal genes, but also by repressing bipotential progenitor genes, in part through a direct repression of sox2. We demonstrate that tbx16 is activated as cells move from an intermediate Wnt environment to a high Wnt environment, and show that Wnt signaling activates the tbx16 promoter. Importantly, high-level Wnt signaling is able to accelerate mesodermal differentiation cell-autonomously, just as we observe with Tbx16. Finally, because our assay for mesodermal commitment is quantitative we are able to show that the acceleration of mesodermal differentiation is surprisingly incomplete, implicating a potential separation of cell movement and differentiation during this process. Together, our data suggest a model in which high levels of Wnt signaling induce a transition to mesoderm by directly activating tbx16, which in turn acts to irreversibly flip a bistable switch, leading to maintenance of the mesodermal fate and repression of the bipotential progenitor state, even as cells leave the initial high-Wnt environment.

Keywords: Bipotential; Neuromesodermal; Somitogenesis; Spadetail; Tbx16; Wnt.

Fig. 1.

tbx16 expression causes cells to exit the tailbud. (A) Outline of the assay used to test and quantify the ability of ectopic tbx16 to accelerate exit from the progenitor zone. (B,C) Overlays of transplanted cells labeled with fluorescent dextran (green) on a bright-field image from wild-type (B) or HS:tbx16 (C) donor zebrafish embryos at 36 hpf. (D) Quantification of the most posterior somite occupied by a fluorescently labeled cell with fiber-like morphology in embryos heat shocked at the 10 ss. Data are significantly different (Mann-Whitney U-test, P<0.05).

Fig. 2.

tbx16 expression represses genes involved in maintaining the progenitor zone. (A) Summary of changes in gene expression after the induction of ectopic tbx16 (based on the data in supplementary material Fig. S3 and Table S1). Arrows indicate the number of hours post-heat shock (HS) and the colors represent expansion (green), constriction (red) or no change (yellow) to expression, with intermediate shades marking the degree of difference. PM, progenitor markers; EDM, early differentiation markers; LDM, late differentiation markers. α-Tbx16 indicates Tbx16 assessed by immunostaining. (B-G) In situ hybridization shows changes to gene expression in the tailbud of HS:tbx16 (C,E,G) and control (B,D,F) embryos at 6 h after heat shock. The arrow (C) points to expansion of pcdh8 and the red boxes (E,G) highlight the reduction of sox2 and ntl in the tailbud.

Fig. 3.

tbx16-EnR expression represses sox2 and activates ntl. In situ hybridization shows changes to gene expression with HS:tbx16-EnR (B,D,F,H,J) or in control (A,C,E,G,I) 4 h after heat shock. The red boxes (B,D,F,H) highlight the reduction of tbx6l, pcdh8, her1 and sox2 in the tailbud and the arrow (J) points to an expansion of ntl.

Fig. 4.

tbx16 is Wnt responsive. (A-C) Immunofluorescence shows cells in the tailbud with nuclear β-catenin (nβ-catenin; A, orange; cytoplasmic and membrane-bound β-catenin have been eliminated from the image; see Materials and Methods) or Sox2 protein (B, orange). DAPI labels nuclei (blue). The highest levels of nβ-catenin and Sox2 are white and the lowest levels are purple. (C) A composite image shows that there is little overlap between the Sox2 domain (red) and the nuclear β-catenin domain (green). (D-G) Fluorescent whole-mount in situ hybridization shows tbx16 (red) and ntl (green) with HS:caβ-catenin (F,G) and in control (D,E) at 4 h post-heat shock. Single slices (D,F) and maximum intensity projections (E,G) are shown. In all images, anterior is to the left and posterior is to the right.

Fig. 5.

Wnt drives tbx16 reporter expression and tbx16 represses Wnt. (A) The zebrafish tbx16 locus, showing exons 1 and 2 (red) and a conserved region of the tbx16 promoter (green), which contains two predicted TCF binding sites (see supplementary material Fig. S5). The blue line marks the region used for the tbx16-1.2 construct. (B-E) Whole-mount in situ hybridization shows reporter expression from the tbx16-1.2 transgene with HS:caβ-catenin (D) and in control (B). Another reporter line with mutations in both of the predicted TCF sites (tbx16-1.2ΔTCF) showed no expression with (E) or without (C) HS:caβ-catenin. (F-I) In situ hybridization shows wild-type ntl expression (F; n=22 embryos) and the changes in ntl expression after heat shock induction of HS:caβ-catenin (G; n=16 embryos), HS:tbx16 (H; n=11 embryos), and both transgenes (I; n=9 embryos). The arrows (G,I) highlight the expansion of ntl and the red box (H) highlights the reduction of ntl. (J-L) In situ hybridization shows wild-type wnt3a expression (J) and the loss of wnt3a expression after heat shock induction of HS:tbx16 (K) or HS:tbx16-EnR (L). Red boxes (K,L) highlight the reduction of wnt3a in the tailbud.

Fig. 6.

caβ-catenin expression causes early exit of cells from the progenitor zone. (A,B) Overlays of transplanted cells labeled with fluorescent dextran (green) onto a bright-field image from control (A) or HS:caβ-catenin (B) donor embryos into wild-type hosts. (C) Quantification of the most posterior somite occupied by a fluorescently labeled cell in host embryos. Data are significantly different (Mann-Whitney U-test, P<0.05).

Fig. 7.

High levels of Wnt and activation of Tbx16 irreversibly commit progenitors to a mesodermal fate. The status of the regulatory network in different cell types in the tailbud. Levels of Wnt exposure are indicated by shades of blue. Progenitor cells are exposed to a moderate level of Wnt (medium blue), which sustains ntl expression but is insufficient to activate tbx16. If cells leave the progenitor zone and are exposed to a low-Wnt environment (light blue), they become neural. Cells exposed to high levels of Wnt (dark blue) as they leave the progenitor zone activate tbx16 and transition to a mesodermal fate. Cells expressing tbx16 are irreversibly committed to the mesodermal fate even as levels of Wnt eventually drop to a level comparable to that of neural cells, since Tbx16 activates mesodermal genes and turns off the progenitor genes ntl and sox2 through its repressive function. The red arrow highlights the movement of cells from the tailbud toward the domain of early mesodermal differentiation, which we propose requires an additional factor.