DEV02485 Supplementary Material

Files in this Data Supplement:

• Supplemental Table - Adobe PDF

• Supplemental Figure 1 -

  Fig. S1. Efficacy and sepcificity of lhx5 morpholinos. (A-D) lhx5 morpholino knockdown alters pax6a and pax2a expression patterns. Embryos were dissected and flat mounted in glycerol after in situ hybridization. Dorsal views, rostral to the top. (E-G) lhx5 translation-blocking morpholino reduces lhx5-GFP mRNA translation. GFP fluorescence in the cell nucleus is reduced when 2 ng (F) or 4 ng (G) of the translational morpholino is injected into zebrafish embryos. (H) lhx5-splicing morpholino reduces wild-type lhx5 transcripts. Arrow indicates wild-type lhx5 or odc1 transcripts. Band above the wild-type lhx5 transcript is caused by splicing with a cryptic GT located in lhx5 intron 3 downstream from the canonical GT site at the exon 3/intron 3 junction; it results in a frame shift. Amplifications of odc1 transcripts serve as controls.

• Supplemental Figure 2 -

  Fig. S2. Lhx5, Sfrp1a and Sfrp5 regulate the size of the forebrain. Quantitative measurement of forebrain sizes. (A,B) An uninjected control embryo (A) and an lhx5 mRNA-injected embryo (B) are shown to illustrate how quantitative measurements are carried out. Injected embryos and uninjected control embryos were fixed and processed for in situ hybridization with ptc1 probes. Images of labeled embryos were analyzed with Axiovision software (Carl Zeiss). Two parameters were measured for each image. Green double arrow (head thickness) crosses the apex of forebrain and is approximately perpendicular to the tangent plane at the apex. Forebrain length was defined as the distance between the anterior limit and the diencephalic bending point of ptc1 labeling (red stars). The ptc1 bending point is located at the zona limitans intrathalamica that separates the anterior diencephalon and the posterior diencephalon. (C,D) Measurements were normalized by dividing by the values of uninjected controls and multiplying by 100. ** denotes significant differences between uninjected control and affected injected embryos (t-test, P<0.01). For each measurement, the sample size was n=10.

• Supplemental Figure 3 -

  Fig. S3. sfrp1a promoter fragments drive GFP expression in forebrain regions. Panels show GFP fluorescence from live embryos at the Prim-5 stage (24 hours post-fertilization).