Fig. S4

Tbx2b acts in parallel with Nr2e3 during parapineal specification. (A) Dorsal views of antibody labeling of parapineal neurons (krt4:eGFP+) as well as axons and dendrites (acetylated tubulin+) in the epithalamus at 5 dpf. Parapineal neurons in tbx2b^c144 or tbx2b^c144/nr2e3 morphants do not migrate correctly and remain within the pineal organ region (dashed circles). Scale bar: 30 µm. Loss of Nr2e3 function partially suppressed the tbx2b^c144 parapineal specification phenotype, but did not suppress the migration defect phenotype (B,C). (B) Quantification of the number of parapineal neurons in non-injected controls (NICs) or nr2e3 splice morphants that either have both WT copies of tbx2b (WT) or two mutated copies (tbx2b^c144-/-); mean±s.e.m. and number of samples (n) are shown. ****p<0.0001, *p<0.05, one-way ANOVA with Tukey’s post hoc analysis, comparisons with WT;NIC (*), comparisons with tbx2b^c144;NIC (#), and a comparison with nr2e3 MO^splice (+) are shown. (C) Quantification of the percentages of larvae that have normal, bilateral, mixed (some cells migrate and others do not), or medial (cells remain near the midline) migration phenotypes and number of samples (n) are shown. Parapineal migration phenotypes were determined based on the position of parapineal neurons relative to acetylated tubulin as well as the projection direction of parapineal neurons. When two parapineal organs were observed, parapineal migration phenotypes were assessed for each parapineal organ and classified as mixed (when only one of two parapineal organs migrated correctly) or bilateral (when one of the parapineal organs migrate to the left and the other to the right of the pineal organ). Loss of Tbx2b function leads to an increased percentage of larvae with parapineal migration defects.