Fig. S2

Gfra3 function is required for opercular muscle development. (A) Radiation hybrid panel mapping of zebrafish gfra3 (red) reveals that it segregates with other genes lying between z1812 and z5435 in agreement with SSLP mapping data. Filled boxes represent a positive result from the PCR on pooled hybridoma cells containing defined regions of the zebrafish genome. (B) Quantification of muscle fibers in gfra3 morphants reveals a statistically significant reduction of opercular muscles similar to stm^hy024 mutants (n=10) but also reveals that muscles recover between 80 hpf to 144 hpf in gfra3 morphants. Error bars represent s.e.m. (C) RT-PCR amplification of a 380 bp fragment of gfra3 using primers designed to exons 2 and 4 from animals injected by gfra3 morpholinos (m) reveals a reduction of spliced gfra3 RNA compared to uninjected controls (c) at 24, 48 and 72 hpf. (D) TUNEL labelling of cells undergoing programmed cell death in the pharyngeal arches revealed no significant differences (P<0.01) between stm^hy024 and wild type (WT) at 60, 76 and 120 hpf. Error bars represent s.e.m. (E-J) Very few muscle precursors of the lap and do expressing En2 (green) are labelled by anti-phosphohistone3 (PH3, red), a marker of proliferating cells in WT (E,G,I) or stm^hy024 mutants (F,H,J) at 47, 49 and 51 hpf relative to DAPI (blue). There appears to be no change to the number of proliferating En2+ stained nuclei (arrowheads) in stm^hy024 mutants, although there are fewer En2+ cells overall by 51 hpf. (K-N) gfra3 expression is reduced in the arches (1,2) of stm^hy024 (L) at 25 hpf relative to WT (K) and, likewise, is reduced in gfra3 morphants (N) at 25 hpf relative to uninjected control animals (M).