Comparative synteny cloning of zebrafish you-too: mutations in the Hedgehog target gli2 affect ventral forebrain patterning - PubMed (original) (raw)
Comparative synteny cloning of zebrafish you-too: mutations in the Hedgehog target gli2 affect ventral forebrain patterning
R O Karlstrom et al. Genes Dev. 1999.
Abstract
Zebrafish you-too (yot) mutations interfere with Hedgehog (Hh) signaling during embryogenesis. Using a comparative synteny approach, we isolated yot as a zinc finger transcription factor homologous to the Hh target gli2. Two alleles of yot contain nonsense mutations resulting in carboxy-terminally truncated proteins. In addition to causing defects in midline development, muscle differentiation, and retinal axon guidance, yot mutations disrupt anterior pituitary and ventral forebrain differentiation. yot mutations also cause ectopic lens formation in the ventral diencephalon. These findings reveal that truncated zebrafish Gli2 proteins interfere with Hh signaling necessary for differentiation and axon guidance in the ventral forebrain.
Figures
Figure 1
(A) Genetic map of linkage group 9 (LG9) showing position of yot/gli2 in relation to previously mapped genes (Postlethwait et al. 1998) and SSLP markers (Knapik et al. 1998). yotty17 was mapped to LG9 using centromere linkage analysis with centromeric marker z5080 (boxed) (see B and Materials and Methods), followed by mapping in a standard sexual intercross with marker z3124 (6 recombinants in 44 meioses). To determine the position of yot more precisely, we scored a marker that cosegregates with yot (BAC 100D2, see below) along with SSLP markers z3124, z5080, and z5564 on a haploid mapping panel (M.A. Gates, A.F. Schier, W.S. Talbot, in prep.). yot maps to the same linkage group as the zebrafish hoxda cluster (Amores et al. 1998), dlx2, engrailed1 (eng1), evx2, desmin (des), dermo1, and activinbb (actbb). The orthologs of these genes are located on human chromosome 2, where gli2 is also located. Human cytological map and locations were derived from Genome DataBase (
). Zebrafish actbb corresponds to human inhibinbb (INHBB). (B) Mapping to LG9 using SSLP marker z5080. z5080 primers amplified an ∼120-bp fragment from yotty17 DNA but an ∼220-bp band from wild-type siblings, indicating linkage to the marker. No recombinants were seen in 16 individuals. (C) SSCP analysis links BAC 100D2 DNA to yot. PCR amplification of DNA from individual mutant embryos produced a faster migrating band (lines), whereas DNA from wild-type siblings produced a slower migrating band (homozygotes, arrow) or both bands (heterozygotes). No recombinants were seen in 230 mutant embryos, indicating tight linkage.
Figure 2
Sequence of zebrafish gli2 and identification of point mutations in yot alleles. (A) The deduced amino acid sequence of zebrafish gli2 (you-too) aligned with mouse gli2. The five zinc finger regions are indicated by lines and Roman numerals. The positions of stop codons in yotty119 and yotty17 are indicated by boxes and asterisks. Amino acid polymorphisms seen in wild-type strains are shown in circles over the zebrafish gli2 sequence. A putative VP16-like activation domain at the carboxyl terminus is boxed. (B) Sequencing gels showing point mutations in the two yot alleles. In yotty119 C3148 is mutated to U, changing Arg-929 to a stop codon. In yotty17 C3445 is mutated to U, changing Glu-1032 to a stop codon. (C) Schematic representation of the zebrafish gli2 protein sequence showing the positions of the stop codons (arrowheads) in the two mutant alleles. Alignment is shown with mouse Gli2, human GLI3, and fly Ci. Hatched regions have >70% sequence identity between the three Gli proteins. Overall amino acid sequence identities are zebrafish gli2 to mouse Gli2, 56%; zebrafish gli2 to mouse Gli1, 29%; zebrafish gli2 to zebrafish Gli1, 35%; zebrafish gli1 to mouse Gli1, 45%; zebrafish gli1 to mouse Gli2, 35% (R.O. Karlstrom, A. Kawakami, W.S. Talbot, A.F. Schier, unpubl.). Sites of stop codons resulting from nonsense mutations in Gli3 that lead to PAP-A and PHS are indicated by arrows. Site of cleavage that results in a repressor form of Ci is shown by an arrow. The carboxy-terminal region of Ci implicated in CBP binding is indicated by brackets. Zinc finger regions (Zn) are indicated by dark boxes.
Figure 3
Zebrafish gli2 RNA expression (purple). Embryos in E, G, and H were double labeled with shh (brown). Anterior is to the left in all panels except A. Dots in F_–_I show positions of anterior and postoptic commissures. (A) 80% epiboly. (Top) Dorsal view, animal pole up, arrows point to the vegetal margin of the gastrula. (Bottom) Optical cross section, dorsal up, arrowhead marks midline. (Arrows) Lateral mesoderm. (B) Tailbud stage, dorsal view. (Arrow) Anterior neural plate; (arrowhead) tailbud. (C) Six-somite stage, dorsal view. (Arrows) Optic vesicles; (arrowhead) developing somites. (D_–_I) Lateral views of the brain, medial focal plane, eyes have been removed. (D) Six-somite stage. (Arrowheads) Most dorsal telencephalon and ventral diencephalon do not express gli2; (P) mesendodermal tissue of the polster. (Arrows) Border between polster and forebrain tissue. (E) 10-somite stage. (Arrow) Optic recess. (F) 24 hr; (G) 30 hr; (H) 40 hr. Expression is reduced in cells adjacent to _shh_-expressing cells in the forebrain (arrowhead). (I) 48 hr. Line, rostral portion of the anterior pituitary anlage. (di) Diencephalon; (hy) hypothalamus; (MDB) mid-diencephalon boundary, (pit) anterior pituitary; (tel) telencephalon; (dots) anterior and postoptic commissures.
Figure 4
yot affects axon guidance in the ventral forebrain. (A_–_D) Ventral view, anterior up. (A,B) Anti-acetylated tubulin labeling. (A) In 36-hr wild-type embryos, commissural axons have crossed the forebrain to form the anterior (arrowhead) and postoptic (arrow) commissures. (B) In yot mutant embryos, the postoptic commissure fails to form (arrow) and the anterior commissure appears reduced (arrowhead). (C,D) ZN-8 antibody labeling. (C) In 48-hr embryos, retinal axons have crossed the midline to form the optic chiasm (arrow). (D) In yot mutant embryos, axons project ipsilaterally after leaving the eye and no chiasm forms. Lens-like structures form in yotty119 mutant embryos in the ventral diencephalon (arrow).
Figure 5
yot affects anterior pituitary formation and expression of Hh target genes in the brain. Expression of nk2.2 (A,B), six3 (C,D), ptc-1 (E,F), pax6 (G,H,K,L), and shh (I,J) in purple. Embryos in C, D, K, and L were double labeled with nk2.2 (C,D) or shh (K,L) (brown). Anterior is to the left in all panels; eyes have been removed. (A_–_D) 36 hr. (A,B) (Arrowheads) Anterior pituitary anlage; (square brackets) regions of the tegmentum; (arrows) ventral hindbrain. (C,D) In yot mutants, six3 expression (purple) is absent from the middle of its expression domain, which includes the rostral part of the anterior pituitary anlage (arrowheads). (E,F) 30 hr. (Arrow) Postoptic area. (G_–_L) 36 hr. Expansion of pax6 expression at mid-diencephalic boundary (thick arrows) and dorsal-to-_shh_–expressing cells (thin arrows). (K,L) (Dots) Anterior and postoptic commissures.
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