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Fig. 5. Embryonic expression of tbx-8 and tbx-9 reporters. The earliest detectable tbx-8::GFP and tbx-9::GFP expression is seen in the nuclei of the gut cell precursors Ea and Ep at the onset of gastrulation, around 100 minutes following first cleavage (A-B). Ea and Ep are moving dorsally, into the interior. (A) TBX-8::GFP (Ea, arrowhead, Ep, arrow), (B) TBX-9::GFP (Ea, arrowhead, Ep, arrow). tbx-8 and tbx-9 are subsequently expressed in muscle and hypodermal cells. (C) Expression of tbx-8::GFP in dorsal muscle cells (arrows) and lateral hypodermal cells (arrowheads) at the 1.5-fold stage. To confirm these cells types, co-staining was performed with muscle and hypodermal specific markers. (D) TBX-9 stained green with a GFP antibody. The outlines of muscles cells are stained red with the muscle antibody NE8/4C6.3 and dorsal muscle cells expressing tbx-9 are indicated (arrows). Similar results were obtained for TBX-8 (data not shown). (E-I) Hypodermal expression of tbx-8::GFP and tbx-9::GFP. TBX-8::GFP (E) and TBX-9::GFP (F) can be seen in the nuclei of dorsal hypodermal cells undergoing intercalation (around 300 minutes following first cleavage). The expression is particularly strong in cells that are just starting to intercalate (E,F, arrows). Outlines of cells undergoing the early stages of intercalation are shown with a black dotted line. Weaker expression can also be seen in lateral hypodermal cells (E,F, arrowheads). (G-I) Embryo co-stained with antibodies to show TBX-9::GFP in green and LIN-26 (expressed in all hypodermal cells) in red. (G) Green channel only showing cells expressing TBX-9::GFP stained with a GFP antibody. (H) Red channel only showing cells expressing LIN-26. (I) Merged image. Yellow nuclei strongly expressing both LIN-26 and TBX-9 can be seen in dorsal hypodermal cells that are beginning to intercalate (white solid arrow). Once cells are intercalated at the anterior TBX-9 expression is weaker and nuclei appear orange-yellow (dashed arrow). Cells at the posterior that have yet to intercalate express TBX-9 very weakly in this embryo and the nuclei appear orange-red (dotted arrow). Weak TBX-9 expression can also be seen in lateral hypodermal cells, in which the nuclei appear orange (arrowhead). Ventral hypodermal cells (not visible in this focal plane), which express LIN-26 but not TBX-9, stained red (not shown). Underlying muscle cell nuclei, which express TBX-9 but not LIN-26, appear green (blue arrow). Similar results were obtained with a tbx-8::GFP reporter. Posterior is to the right in all panels. Scale bar, 10 µm.

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