The Zebrafish Mutants dre, uki, and lep Encode Negative Regulators of the Hedgehog Signaling Pathway

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. In a screen aimed at finding genes that control proliferation in the zebrafish embryo, three mutants were identified. Mutants showed an increase in size of several structures including the brain, the retina, and the fins. Surprisingly, although size was increased in the embryos, adults remained small. Cloning of these genes revealed that increased Hedgehog signaling was at the basis of the phenotype, because all three genes encoded known repressors of the Hedgehog signaling pathway: Ptc2, Su(Fu), and Hip. Hedgehog is known to play a role in proliferation. For instance, ectopic Hedgehog signaling can lead to several tumors including basal cell carcinoma and medulloblastoma. However, the phenotypes were still a surprise, because earlier experiments in zebrafish embryos suggested that activation should lead to patterning rather than proliferation defects. Current models of the pathway predict that these genes act independently to inhibit the signal but curiously, redundancy amongst these genes was not found, because triple mutants looked like the single mutants. The conclusion is that weak activation of Hedgehog signaling can already lead to stimulation of growth in the absence of patterning defects, and that the Hedgehog signal is probably kept in check by the last inhibitor: Ptc1. A mutant for the ptc1 gene has recently been created and will put the model to the test.