DEV02046 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Rat and mouse lov-related genes are expressed in medial habenular nuclei. (A,B) In situ hybridization of rat Kctd8, the putative orthologue of zebrafish dex, in a transverse cryosection (20 mm) of adult rat brain.  Expression is restricted to the medial habenular nuclei. (C,D) In situ hybridization of mouse Kctd12/Pfetin, a gene with homology to lov and ron, in a transverse section of a postnatal day-6 mouse brain.  High levels of expression are found in the medial habenular nuclei and in the hippocampus (granule cells of the dentate gyrus and pyramidal cells of CA1-CA3). Scale bars: 150 mm.

• Supplemental Figure 2 -

  Fig. S2. Specificity of Leftover and Right on antisera. (A) Western blot showing detection of bacterially expressed Lov or Ron protein (1 mg) by anti-Lov (lanes 1, 2) and anti-Ron (lanes 3, 4) rabbit polyclonal antiserum, and the lack of cross-reactivity. (B-J) Embryos injected with 3 ng of lov or ron morpholino (complementary to the 5¢ UTR proximal to the start ATG) were harvested at 3.5 days, at which time Lov and Ron proteins were detected by immunofluorescence of habenular projections to the interpeduncular nucleus.  Upon injection of Lov MO, Lov (E) but not Ron immunofluorescence (F) was significantly reduced relative to  uninjected control larvae (B,C).  Conversely, ron MO injections resulted in reduced  Ron immunofluorescence (I) at the IPN, but did not affect  Lov labeling (J). All confocal images are lateral views of the dorsal (d) and ventral (v) IPN, as indicated in B.

• Supplemental Figure 3 -

  Fig. S3. Southpaw (Spaw) regulates the direction of epithalamic asymmetry. Spaw MO-depleted larvae show either a  WT (sinistral; A,C,E,G,H) or  reversed (dextral; B,D,F,I,J) pattern of epithalamic laterality.  (A,B) Dorsal view of fluorescently labeled pineal complex in foxD3:GFP transgenic larvae at 2 days. Position of the parapineal is indicated (white arrowhead). (C,D) Dense neuropil in the habenular nuclei is revealed by confocal imaging of anti-acetylated tubulin immunofluorescence in a 4-day larva. (E,F) Asymmetric expression pattern of the lov gene at 4 days. (G-J) Whole-mount in situ hybridization to detect lov habenular expression (open arrowhead) and insulin transcripts in the pancreas (black arrowhead) reveals the four major classes of larvae obtained following spaw MO injection. The direction of habenular asymmetry and L-R positioning of the pancreas are randomized, but in an independent manner. All images are dorsal views, anterior to the top. Scale bar: 8 mm (A,B); 20 mm (C,D).