|ZFIN ID: ZDB-IMAGE-170921-36|
The tortugab644 allele is a 1.46 Mb deficiency that includes the pnrc2 gene. Haploid-based mapping revealed that the tortuga lesion lies 0.30 cM to the left (4/1434 recombinants) and 0.37 cM to the right (4/1083 recombinants) of the SSLP markers z13511 and z9511, respectively (A). The extent of the deletion was refined by PCR-based screening of regions within and around the deletion interval from pooled genomic DNA samples of 10 wild-type (WT) and 10 tortuga mutant embryos (B); regions that fail to amplify in mutants are deleted in the torb644 allele (A, B). Among BACs spanning the deletion interval, only BAC AL844887 restores her1 expression when injected into tor mutants (A; Fig S1A-D). In wild-type embryos, her1 is expressed in a striped pattern (n=54/54) (C). In contrast, embryos injected with 6 ng of pnrc2 splice-blocking morpholino (sbMO) have a tortuga-like her1 expression defect (n=37/39) (D). When pnrc2 mRNA is co-injected with 4 ng pnrc2 sbMO, her1 expression is partially restored in a dose-dependent manner (n=3/19 WT her1 expression, 150 pg pnrc2 mRNA; n=7/17 WT her1 expression, 600 pg pnrc2 mRNA) (E; Table 1). Alignment of vertebrate Pnrc2 amino acid sequences reveals a conserved 10 amino acid N-terminus (purple) and conserved C-terminal SRC-Homology 3 (SH3) (red) and Nuclear Receptor (NR box) domains (green) (F). Clustal alignments were performed in consultation with published alignments for vertebrate Pnrc2 (Valkov et al., 2016).
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