Vacik T , Stubbs JL , Lemke G .

EY017478 NEI NIH HHS , R01 EY017478 NEI NIH HHS

	Figure 6. DnTcf7l2 is essential for Xenopus forebrain development. (A) A 59 RACE experiment in X. laevis reveals the existence of an alternative XTcf7l2 first exon in intron 5, which contains an ATG start codon in-frame with Ser 180 in XTcf7l2 exon 6. The positions of the translation-blocking (MO-ATG) and splicing- blocking (MO-SPL) morpholinos are indicated. Arrows represent the primers used for qRTCR. (B) In situ hybridization analysis of dnTcf7l2 expression at stages 13, 15, and 28 of X. laevis development. (A) Anterior; (P) posterior; (D) dorsal; (V) ventral; (bp) blastopore. (C) Knockdown of XdnTcf7l2 with MO-ATG or MO-SPL leads to embryos with severely truncated anterior head regions at stage 40. Phenotype ranges from moderate (gray bars), showing small head and small eyes, to strong (black bars), where no head and no eyes are present.
	Figure S8. Expression of Spemann organizer and anterior neural markers during embryogenesis of Xenopus morphants lacking XdnTcf7l2. In situ hybridization was carried out for: the organizer marker chordin (Chd) at st. 10.5 (1st column, vegetal view), the organizer+anterior neuroectodermal marker Otx2 (Otx2) at st. 12.5 (2nd column, anterior up); the anterior neuroectodermal marker Otx2 at st. 20 (3rd column, anterior on); and the midbrain/hindbrain boundary marker engailed 2 (En2) at st. 20 (4th column, anterior on). These hybridizations were carried out in embryos injected with morpholinos in one animal blastomere (left, injected in all panels) of 2-cell stage embryos, with the uninjected side (right in all panels) as a control. (Injected side marked with lacZ.) Morpholinos used were: control (top row); ATG morpholino (middle row), and splicing morpholino (bottom row). Percent tally of results from repeated analyses in multiple embryos (n = 18-46, indicated by white numbers), with respect to level of marker expression on the injected side (same, reduced, or absent), is given in the histograms below each column. Anterior neural development is unaffected by either morpholino prior to st. 12.5, the time at which XdnTcf7l2 mRNA first appears.
	Figure S9. Misexpression of XdnTcf7l2 in Xenopus laevis embryos results in multiple developmental anomalies. (A) As in the mouse, Tcf7l2 and dnTcf7l2 in Xenopus exist in multiple isoforms due to alternative splicing of exons 13-16. We identified the indicated XdnTcf7l2 isoforms, designated 1-4, as most abundant in stage 13 and 24 embryos, with their relative abundance at each stage indicated as a percentage. Color coding of XdnTcf7l2 protein domains within exons is as for Figure 3. X indicates the presence of a stop codon. Isoforms 1-3 contain the short SFLSS motif of Pukrop et al. (2001) in exon 9 (indicated by , while isoform 4 does not (indicated by . (B) We injected mRNAs encoding the indicated isoforms, either singly or in combination (1+2 and 3+4), into either the two dorsal blastomeres (orsal injections or one ventral blastomere (entral injections of 4-cell-stage embryos, with nuclear -galactosidase mRNA co-injected as a marker (or control). Embryos were scored for the indicated patterning phenotypes at st. 30-32. N indicates number of embryos scored. (C) Selected examples of patterning defects observed. Although these studies indicate that XdnTcf7l2 has potent developmental effects in vivo, there are two important caveats to interpreting these effects in a biological context. First, we are over-expressing exogenous XdnTcf7l2 much earlier in development than its normal first appearance at st. 13, and therefore may be affecting developmental events in which XdnTcf7l2 does not participate. And second, it is possible that this early expression of XdnTcf7l2 interferes with (inhibits) the activity of other XTcf proteins that may act earlier in development than does XTcf7l2.

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