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. 2005 Aug;1(2):e19.
doi: 10.1371/journal.pgen.0010019. Epub 2005 Aug 19.

The zebrafish mutants dre, uki, and lep encode negative regulators of the hedgehog signaling pathway

Marco J Koudijs  1 , Marjo J den Broeder, Astrid Keijser, Erno Wienholds, Saskia Houwing, Ellen M H C van Rooijen, Robert Geisler, Fredericus J M van Eeden
Affiliations

The zebrafish mutants dre, uki, and lep encode negative regulators of the hedgehog signaling pathway

Marco J Koudijs et al. PLoS Genet. 2005 Aug.

Abstract

Proliferation is one of the basic processes that control embryogenesis. To identify factors involved in the regulation of proliferation, we performed a zebrafish genetic screen in which we used proliferating cell nuclear antigen (PCNA) expression as a readout. Two mutants, hu418B and hu540A, show increased PCNA expression. Morphologically both mutants resembled the dre (dreumes), uki (ukkie), and lep (leprechaun) mutant class and both are shown to be additional uki alleles. Surprisingly, although an increased size is detected of multiple structures in these mutant embryos, adults become dwarfs. We show that these mutations disrupt repressors of the Hedgehog (Hh) signaling pathway. The dre, uki, and lep loci encode Su(fu) (suppressor of fused), Hip (Hedgehog interacting protein), and Ptc2 (Patched2) proteins, respectively. This class of mutants is therefore unique compared to previously described Hh mutants from zebrafish genetic screens, which mainly show loss of Hh signaling. Furthermore, su(fu) and ptc2 mutants have not been described in vertebrate model systems before. Inhibiting Hh activity by cyclopamine rescues uki and lep mutants and confirms the overactivation of the Hh signaling pathway in these mutants. Triple uki/dre/lep mutants show neither an additive increase in PCNA expression nor enhanced embryonic phenotypes, suggesting that other negative regulators, possibly Ptc1, prevent further activation of the Hh signaling pathway. The effects of increased Hh signaling resulting from the genetic alterations in the uki, dre, and lep mutants differ from phenotypes described as a result of Hh overexpression and therefore provide additional insight into the role of Hh signaling during vertebrate development.

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Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. PCNA and Proliferation Patterns
(A) PCNA pattern as scored during the screen. In a dorsal view at 40 hpf, PCNA staining is observed in the medial and posterior part of the tectum, and in the cerebellum, the neural crest (arrowhead), and the pectoral fin (arrow). (B) In a sideview (42 hpf), a ring of positive cells is visible around the lens. (C) In a whole-mount BrdU labeling from day 3.5 to 4.5, similar regions are labeled indicating that PCNA RNA expression prefigures where BrdU will be incorporated. (D and E) Sibling and hu418B mutants, respectively, showing increased PCNA labeling in the CMZ, but most prominently in the tectum.
Figure 2
Figure 2. Phenotypes of ukihu418B Mutant Embryos
(A and B) Lateral view of a wild-type (wt) and ukihu418B mutant. (B) Showing an increased volume of the head. The size of the pupil is reduced in the ukihu18B mutant without affecting the size of the eye. (C–E) The size of the pupil is reduced in the uki hu18B mutant compared to wild-type, without affecting the size of the eye. Measurements revealed that the length and width of the pupil is significantly reduced in the uki418B mutant (n = 5, *p < 0,001). (F–H) Pectoral fins showing the increased size of an uki418B mutant in which the dorsal/ventral (D/V) size of the pectoral fin is increased by 50%, (n = 3, *0.01 < p < 0.02). (I) The anterior/posterior size is not significantly affected, but the fin area has increased by 65% (n = 3, p < 0.001). (J and K) ukihu418B mutants lack the dorsolateral septum in the ear (arrowhead). Scale bar is 100 μm.
Figure 3
Figure 3. Patterning of the Branchial Arches in a 5-Mo-Old dre Mutant
(A and B) The strict organization of the brachial arch into primary (p) and secondary (s) lamellae in a wild-type situation (100× magnification). Higher magnification shows stacks of single chondrocytes in the primary lamellae. (C and D) Sectioning of a dre mutant shows disturbed patterning, resulting in the absence of secondary lamellae and the presence of foci of chondrocyte-like cells in the primary lamellae (arrowsHE, hematoxylin and eosin stain; wt, wild-type. (E and F) Alcian Blue staining reveals the presence of differentiated chondrocytes in the wild-type (wt) primary lamellae, but not in the dre mutant, indicating that the differentiation of these chondrocytes is affected (200×). (G and H) Branchial arches of uki and lep mutants appear to be wild-type (wt).
Figure 4
Figure 4. Positional Cloning of the dre Mutant
(A) Schematic representation of assembled contig 11890 of the Zv2 genome assembly. SSLP markers z5395 and z25745 and newly identified SSLPs 11890.2A and 11890.2 were closely linked with the dre locus. Remaining recombinants of a complete panel of 765 mutant embryos are indicated. Four genes were predicted in the region of marker 11890.2A that encode Su(fu), TRC8, ubiquitin conjugating enzyme E2, and β-mannosidase precursor protein. (B) The dre mutation is a C to an A substitution, changing a threonine to a lysine. (C) Multiple alignment of Su(fu) homologs revealed that the induced mutation changes an amino acid in a highly conserved region of Su(fu).
Figure 5
Figure 5. MO Injection Experiments against Su(fu), Hip, and Ptc2
(A) Dorsal view of the eye showing the lens in the eye chamber. (B–D) Dorsal view of embryos injected with the indicated MOs, resulting in a phenocopy of dre, uki, and lep mutants. (E) A wild-type ear showing the presence of the dorsolateral septum (arrow), which is not present after injections with the indicated MOs (F–H, arrow). (I) Injections with control MOs against the initiation codon of Su(fu) results in chevron-shaped somites with an angle of 97°. (J) Injection of MOs against Su(fu) results in a more obtuse angle of the somite (126°).
Figure 6
Figure 6. Premature Stop Codons Were Identified in the uki and lep Mutant in Hip and Ptc2, Respectively
(A) Schematic representation of the genomic organization of the Hip gene. All three alleles of the uki mutation contain premature stop codons positioned in exon 5 and exon 7. (B) Representation of the protein structure of Ptc2 shows that the identified nonsense mutation is positioned after the sixth transmembrane domain, probably resulting in a malfunctioning protein. (C) ISH experiments show that Ptc1 expression is increased in uki and lep (arrow), confirming the aberrant activation of the Hh signaling pathway.
Figure 7
Figure 7. Phenotypic Analysis of dre/uki/lep Triple Mutants
(A) Wild-type (wt) embryo at 96 hpf. (B–E) The indicated double and triple mutants do not show severe enhancement of the phenotype. (F–I) dre/lep double mutants have an ear phenotype comparable with a single mutant. In the uki/lep and dre/uki/lep triple mutants, the epithelial projections (arrows) fail to grow out to fuse in the middle of the ear to form the ear lumen.
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