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Fig. 1. pia encodes the achaete-scute homologue Ascl1a. (A) (Top) Genetic map of a region of linkage group 4 (LG4), showing positions of the pia^t25215 mutation, the ascl1a gene and some markers used for mapping. Genetic distances of markers from the top of LG4 according to (http://zfin.org/cgi-bin/mapper_select.cgi) are indicated. (Bottom) Schematic representation of Ascl1a protein: the basic helix-loop-helix domain (blue and green) and the pia^t25215 mutation (red) are indicated. (B) Anti-Myc western blot, showing that translation of chimeric ascl1a-myc mRNA is efficiently blocked in the presence of ascl1a MO (upper panel). The same blot was probed with an anti-pan-cadherin antibody for loading control (lower panel). (C-H) Whole-mount in situ hybridization detecting expression of prl (26 hpf; frontal view, dorsal up; C-E) and pomc (48 hpf; dorsal view, anterior towards the left; F-H) in wild-type (wt; C,F), pia^t25215 mutant (pia; D,G) and ascl1a morphant embryos (ascl1aMO; E,H). (F-H) pomc-positive cells of the adenohypophysis are indicated with arrowheads, pomc-positive cells of the arcuate nucleus in the hypothalamus are indicated with arrows. (I-K) In situ hybridization at 48 hpf for the pituitary marker prl (p) and the epiphysis marker opsin (o). Injected BAC DNA usually does not distribute uniformly, but leads to chimeric embryos with only a subset of cells containing the injected DNA. Accordingly, the rescued pia embryo in K displays strong prl expression (arrow), but still lacks opsin expression in epiphysis, another phenotypic trait caused by loss of Ascl1a function (Cau and Wilson, 2003). The genotype of the embryo was further confirmed via PCR (see Materials and methods). (L-N) elavl3 in situ hybridization at 11 hpf, dorsal views, anterior towards the left, demonstrating that wild-type Ascl1a fused to the VP16 transactivation domain can induce primary neurons in inter-proneural domains of the neural plate (M), whereas a fusion between VP16 and the truncated Ascl1a protein encoded by the pia^t25215 allele is ineffective (N). hpf, hours post fertilization.

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