In vitro transcription and translation of tbx22 splice variants. Western blot analysis revealed distinct biotinylated Tbx22-1 and Tbx22-2 in vitro translation products corresponding to the predicted 50.3- and 45.2-kD Tbx22 isoforms, respectively. No putative 48.3-kD Tbx22-1 translation product was observed, consistent with the location of the Tbx22-1 start codon at bp 147. Plasmid concentrations used in the in vitro transcription reactions, and the amount of translation product loaded in each well, are as indicated. A no-template negative control is also shown. M, Molecular weight protein standard markers.

Developmental RT-PCR analysis of tbx22-1 and tbx22-2 mRNAs. A: The developmental expression of zebrafish tbx22-1 and tbx22-2 RT-PCR products, 402 and 515 bp, respectively, size fractionated in a non-denaturing TAE agarose gel. B: tbx22-1 and tbx22-2 RT-PCR products size fractionated in a denaturing, alkaline agarose gel. The non-denatured, heteroduplex, high molecular weight product present in A is eliminated in the alkaline denaturing gel. C: β-actin control RT-PCR products.

WISH analysis of zebrafish tbx22 expression in 38-42-hpf pharyngeal arch structures. B, H, I, O, P display ventral views, and all other panels present sagittal views. A: At 28 hpf, tbx22 is expressed in paraxial mesodermal tissues in a segmental pattern suggestive of vertebral pre-patterning (arrow). B, C: In 38-hpf embryos, tbx22 is first detectable as discrete, bilateral domains at either corner of the forming mouth (arrows). D: At 40 hpf, tbx22 is expressed in similar bilateral domains (arrow). E-G: Consecutive sagittal sections illustrate tbx22 expression underlying the bilaminar epithelium of the forming stomodeum, which bifurcates into dorsal and ventral elements (arrows). At 42 hpf, faint tbx22 expression is observed in tissues surrounding the mouth (H, arrows), and at each corner of the mouth opening (I, arrows), as shown in consecutive serial sections (J-N, arrows). O, P: At 50 hpf, bapx1 and tbx22 exhibit similar expression patterns in the developing jaw joint (arrows). e, eye; ec, ectoderm; m, mouth; s, stomodeum.