Developmental RT-PCR profile of wnt9a mRNAs. Amplification of β-actin (587 bp) was run in parallel to standardize input cDNA. RT-PCR amplification of a region of the wnt9a ORF (696 bp) reveals increasing expression levels after zygotic gene expression begins at 3 hpf peaking around 2 dpf then leveling off. Expression is maintained in mature zebrafish, indicated by the juvenile (1.5 months). Q-PCR analysis reveals that the highest level of expression is approximately 200x greater than the expression at 6 hpf which is normalized to 1. Negative control is no RT.

WISH analysis of zebrafish wnt9a expression. Discrete neural expression begins at 26 hpf (Panel A) and is maintained throughout development. Panel A′ is sense control, 26 hpf. By 37 hpf neural expression is maintained (B, arrow) and expression begins in the region of the gut (B, arrow head). Sections reveal that the cranial expression is localized to discrete tracts of cells of the PNS. Panel B′ is a section of the tissue indicated by the arrow in (B). Wnt9a is expressed in glial cell populations in the developing cranial ganglia. Expression is also present in glial cells in the optic chiasm (OC) (Panel B″). By 48 hpf the neuroglia can be seen as distinct punctate cells (dorsal view, Panel C, arrows), and in the developing pectoral fins (Panel C′, arrowheads). By 60 hpf expression is maintained in the head and is increased in the gut (Panel D, arrowheads indicate regions represented in D′–D″′). Sections reveal strong wnt9a expression in the glial cells occupying the optic tract, and chiasm (Panel D′), cranial ganglia posterior to the eye (Panel D″) and gut (Panel D″′). CNS and pronephric duct expression are maintained through 4 dpf, with expression found in the swim bladder as well (Panel E). Abbreviations: BA, branchial arch; E, eye; fb, fin bud; hb, hindbrain; OC, optic chiasm; SB, swim bladder; T, telencephalon.

Patterns of expression of Wnt9a and Wnt9b in developing pectoral fins. Whole mount in situ hybridization reveals wnt9a expression in the mesenchyme of the developing fin bud at 48 hpf (Panel A and A′) while wnt9b is ectodermally expressed at the same stage (Panel B and B′). Abbreviations: A, anterior fin; ec, ectoderm; M, mesenchyme; P, posterior fin.