Tbx1 controls the morphogenesis of pharyngeal pouch epithelia through mesodermal Wnt11r and Fgf8a

The pharyngeal pouches are a segmental series of epithelial structures that organize the embryonic vertebrate face. In mice and zebrafish that carry mutations in homologs of the DiGeorge syndrome gene TBX1, a lack of pouches correlates with severe craniofacial defects, yet how Tbx1 controls pouch development remains unclear. Using mutant and transgenic rescue experiments in zebrafish, we show that Tbx1 functions in the mesoderm to promote the morphogenesis of pouch-forming endoderm through wnt11r and fgf8a expression. Consistently, compound losses of wnt11r and fgf8a phenocopy tbx1 mutant pouch defects, and mesoderm-specific restoration of Wnt11r and Fgf8a rescues tbx1 mutant pouches. Time-lapse imaging further reveals that Fgf8a acts as a Wnt11r-dependent guidance cue for migrating pouch cells. We therefore propose a two-step model in which Tbx1 coordinates the Wnt-dependent epithelial destabilization of pouch-forming cells with their collective migration towards Fgf8a-expressing mesodermal guideposts.

(J and K) Fluorescent in situ hybridization for sox10 (green) at 12 hpf. Dorsal views with anterior to the left show sox10 expression in two bilateral fields of neural crest cells in both wild types and tbx1 mutants. We also note that sox10 expression was consistently higher in tbx1 mutants.
(L-O) Fluorescent in situ hybridization for dlx2a (green) to detect neural-crest-derived ectomesenchyme. While dlx2a is expressed in three distinct neural crest streams in 11/11 wild-type siblings at 16.5 hpf, its expression is reduced in the first and second streams and nearly lost in the third stream in 3/5 tbx1 mutants. Compared to wild-type siblings at 18 hpf (n=21), the arch expression of dlx2a was greatly reduced in 4/4 tbx1 mutants. her5:GFP labeling of endoderm at 18 hpf (red) shows that pouches have yet to form at this stage. Scale bar represents 40 μM.
(R and S) Lysotracker Red staining (red) labels dying cells relative to sox10:GFP+ neural-crest-derived cells (green) in wild-type siblings (n=6) and tbx1 mutants (n=6). Arrows show dying cells in the posterior-most pharyngeal arches of a tbx1 mutant.
(T) Quantification of cell death in tbx1 mutants. Dying cells were counted in the entire head (from the posterior-most arch to the anterior limit of the embryo, including the brain, arches, and other tissues), as well as in sox10:GFP+ cells of the arches. A one-tailed Student's t test with unequal variance was utilized to quantify the number of dying cells, and p values are shown.
(O-Q) Alcama immunohistochemistry shows a series of five pouches at 34 hpf. Compared to wild types, we observed normal pouches in 49/49 nkx2.5:Wnt11r and 27/36 nkx2.5:Fgf8a-GFP embryos. In 9/36 nkx2.5:Fgf8a-GFP embryos, we observed delayed and/or disorganized formation of the last pouch. Sensory ganglia are indicated with red asterisks. Anterior is to the left and ventral is down. Scale bar represents 40 μm.
(N-Q) Fluorescent in situ hybridization for fgf8a or wnt11r mRNA (green) at 30 hpf. GFP immunohistochemistry labels the nkx2.5:GFP+ mesoderm in red. fgf8a expression is unaffected in 10/13 and slightly reduced in 3/13 wnt11r mutants. wnt4a expression is unaffected in 15/19 and slightly reduced in 4/19 fgf8a mutants. Scale bar represents 40 μM (F) Quantification of the contribution of cells to the pouches based on whether they were initially dusp6:dGFP-(left bar) or dusp6:dGFP+ (right bar). For initially dusp6:dGFP-cells, we then quantified the number of cells that stayed GFP-or turned on dusp6:dGFP (GFP+). For initially dusp6:dGFP+ cells, we quantified the number of cells that maintained dus-p6:dGFP (GFP+) or extinguished GFP (GFP-). For both, we also scored the contribution of each category to pouches. Both cells that turned on dusp6:dGFP (p<0.001) and maintained dusp6:dGFP (p=0.002) contributed to pouches at a high frequency than cells that remained or turned off dusp6:dGFP. y-axis represents % of cells of each category.

Development | Supplementary Material
(G-I) High magnification confocal sections show her5:mCherryCAAX labeling of endodermal cell membranes within the fourth pouch (p4) and presumptive fifth pouch (p5) at 28 hpf. The epithelium of nascent pouch p5 was multilayered in 8/8 wild-type siblings and 5/5 fgf8a mutants but only in 1/4 wnt11r mutants. Instead, pre-pouch cells in 3/4 wnt11r mutants retained a columnar epithelial morphology. Scale bars represent 20 μM. (C and D) Higher magnification and gain-enhanced sections focused on the posterior pouches seen in (A) and (B) show dusp6:dGFP fluorescence within alternating clusters of endodermal epithelial cells (arrowheads), as well as in clusters of adjacent mesodermal cells (asterisks). While pouch outpocketing is delayed in wnt11r mutants, segmental dusp6:dGFP fluorescence is still detectable within both the mesoderm and endoderm. Scale bar represents 20 μM.

Fig. S7, Criteria for scoring pouch rescue
Several examples of 34 hpf Alcama staining are shown that illustrate lack of pouch rescue (score = 0) or partial/full pouch rescue (score = 1). Pouches were scored as "partial rescue" when they were greater than 50% the length of a corresponding wild-type pouch at that position. These criteria were applied consistently across all experimental groups.
Movie S1. Wild-type and tbx1 -/pouch development In these 24-34 hpf time-lapse recordings, her5:mCherryCAAX labels pharyngeal endodermal cell membranes. In the wildtype example (A), sequential formation of the fourth through six pouches is observed. In the tbx1 -/embryo (B), her5:m-CherryCAAX+ endodermal epithelial cells are present at 24 hpf but fail to display outpocketing behavior during the same period. Lateral is down with anterior to the left.

Movie S2. Contribution of dusp6:dGFP cells to pouches
In this 24-36 hpf recording of wild-type pouch development, her5:mCherryCAAX labels endodermal epithelial cell membranes in red and dusp6:dGFP dynamically reports Fgf activity in green. Tracking of endodermal cells (colored dots) allows correlation of dusp6:dGFP expression with future contributions to pouches.

Movie S3. Pouch development in wild types and fgf8a -/mutants
Time-lapse recordings of pouch development from 24 to 34 hpf in embryos expressing her5:mCherryCAAX (red) and dusp6:dGFP (green). In the wild-type example (B), dusp6:GFP fluorescence emergences in largely coherent clusters of cells that contribute to pouches. In contrast, dusp6:dGFP fluorescence is patchy in fgf8a -/mutants and pouch formation appears delayed and disorganized. Bottom images show the cell tracking employed for our analyses of endodermal cell velocity and persistence.

Movie S4. Effect of Fgf8a-GFP misexpression on pouch development in wild types and wnt11r mutants
Time-lapse recordings from 25-32 hpf highlight continued development of the third pouch. One-cell-stage embryo injection of transgenes produces mosaic misexpression (green) within the mesoderm. her5:mCherryCAAX labels the developing pharyngeal endoderm in red. In A and B, two examples are shown of mosaic misexpression of nkx2.5:Fgf8a-GFP in wild types. Arrows highlight the transient diversion of third pouch cells by nkx2.5:Fgf8a-GFP+ clones. In C, mesodermal clones expressing a control nkx2.5:GFP transgene have no effect on surrounding third pouch cells (arrowheads) in wild types. In D, endodermal cells of wnt11r -/mutants display delayed outpocketing behavior and little response towards ectopic clones of nkx2.5:Fgf8a-GFP-expressing mesodermal cells. Single sections are shown for A, C, and E, and thin projections of five sections are shown in B for better appreciation of diverted third pouch cells.