Abstract
BackgroundAberrant activation of the Hedgehog (Hh) signaling pathway in different organisms has shown the importance of this family of morphogens during development. Genetic screens in zebrafish have assigned specific roles for Hh in proliferation, differentiation and patterning, but mainly as a result of a loss of its activity. We attempted to fully activate the Hh pathway by removing both receptors for the Hh proteins, called Patched1 and 2, which are functioning as negative regulators in this pathway.ResultsHere we describe a splice-donor mutation in Ptc1, called ptc1hu1602, which in a homozygous state results in a subtle eye and somite phenotype. Since we recently positionally cloned a ptc2 mutant, a ptc1;ptc2 double mutant was generated, showing severely increased levels of ptc1, gli1 and nkx2.2a, confirming an aberrant activation of Hh signaling. As a consequence, a number of phenotypes were observed that have not been reported previously using Shh mRNA overexpression. Somites of ptc1;ptc2 double mutants do not express anteroposterior polarity markers, however initial segmentation of the somites itself is not affected. This is the first evidence that segmentation and anterior/posterior (A/P) patterning of the somites are genetically uncoupled processes. Furthermore, a novel negative function of Hh signaling is observed in the induction of the fin field, acting well before any of the previously reported function of Shh in fin formation and in a way that is different from the proposed early role of Gli3 in limb/fin bud patterning.ConclusionThe generation and characterization of the ptc1;ptc2 double mutant assigned novel and unexpected functions to the Hh signaling pathway. Additionally, these mutants will provide a useful system to further investigate the consequences of constitutively activated Hh signaling during vertebrate development.
Highlights
Aberrant activation of the Hedgehog (Hh) signaling pathway in different organisms has shown the importance of this family of morphogens during development
Identification and recovery of a splice-donor mutation in the ptc1 gene The dre, uki, and lep mutant class has shown that multiple regulators secure the activity of the Hh signaling pathway. This was illustrated by the fact that the concurrent loss of function of the negative regulators Suppressor of Fused (Sufu), Hedgehog interacting protein (Hip) and Ptc2 did not result in the typical Hh overexpression phenotype, http://www.biomedcentral.com/1471-213X/8/15 mainly concerning the chevron shape of the somites [14]
An ENU-induced mutation library of approximately 12,000 F1 fish was screened for mutations in this gene using the TILLING method [36]
Summary
Aberrant activation of the Hedgehog (Hh) signaling pathway in different organisms has shown the importance of this family of morphogens during development. We showed that a class of mutants consisting of dre, uki and lep, encode the negative regulators of Hh signaling, Sufu, Hip, and Ptc, respectively [14]. These mutants show an increased level of proliferation in different tissues but do not show the usual patterning defects described for Hh overexpression experiments. Optic cup vs stalk differentiation defects [17], dorsoventral patterning of the brain [18] and neural tube (for a review: [19]), are described to be controlled by Hh signaling None of these phenotypes could be observed in the dre, uki or lep mutant. Triple mutants for Sufu, Hip and Ptc did not show an enhanced phenotype, suggesting that additional regulators are still functioning to inhibit the Hedgehog pathway, most likely Ptc1 [14]
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