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. 2008 Feb 19:8:15.
doi: 10.1186/1471-213X-8-15.

Genetic analysis of the two zebrafish patched homologues identifies novel roles for the hedgehog signaling pathway

Marco J Koudijs  1 , Marjo J den Broeder, Evelyn Groot, Fredericus Jm van Eeden
Affiliations

Genetic analysis of the two zebrafish patched homologues identifies novel roles for the hedgehog signaling pathway

Marco J Koudijs et al. BMC Dev Biol. .

Abstract

Background: Aberrant 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.

Results: Here 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.

Conclusion: The 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.

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Figures

Figure 1
Figure 1
Identification and characterization of a splice-donor mutation in the zebrafish ptc1 gene. A splice donor mutation was identified in the first base pair of intron 10, changing the consensus sequence GT to AT, probably affecting splicing (A). The intron after exon 10, shown in lower case in yellow, contains 81 bp, and the splice donor position mutated in ptc1hu1602 is indicated in red (B). RT-PCR analysis confirms that splicing is affected as a result of the mutation, which extends the transcript with 81 bp compared to wild type (C). Schematic representation of the Ptc1 protein, showing the 12 transmembrane domains (black dots), and the extension of the second extracellular loop with 27 AA in red. The blw mutation is positioned directly after the eighth transmembrane domain of the protein (D).
Figure 2
Figure 2
Phenotypic consequences of zebrafish ptc1 mutants. Homozygous ptc1hu1602 mutants show a subtle somite phenotype at 32 hpf, where the average angle of the somite becomes more obtuse (A,B). At 72 hpf, ptc1hu1602 mutants exhibit an eye phenotype where the pigmented epithelium is extended into the diencephalon. The similar phenotype described for the blw mutant is weaker compared to the ptc1hu1602 mutant (C-E). The expression level of ptc1, a general readout for Hh activity, shows a mild increase in the blw mutant compared to wild type (F-H). The ptc1hu1602 mutant shows a severely increased level of ptc1, where wild types, heterozygotes and mutants can be distinguished based on ptc1 levels (I-K). Additional to the difference in the strength of the eye phenotype, the activation of the pathway is significantly higher in ptc1hu1602 mutants compared to blw mutants. An increased expression level of gli1 confirms an activation of the Hh pathway in the ptc1hu1602 mutant (L-N).
Figure 3
Figure 3
Concurrent inactivation of ptc1 and ptc2 results in severe developmental defects. At 18 hpf, a somite phenotype becomes apparent in the ptc1;ptc2 double mutants, where the chevron shaped form of the somites becomes straight (A,B), which is a typical consequence of increased activity of the Hh pathway. At 24 hpf, ptc1;ptc2 double mutants do not develop a lens but the primitive eye field is still present (C,D). At 48 hpf the eyes are completely absent. Additionally, reduced pigmentation, an absence of the nose, and an underdeveloped ear can be observed at 48 hpf (E,F). Expression levels of nkx2.2a confirms that the pathway becomes more activated upon losing wild type alleles of ptc1 or ptc2, with the highest expression in the ptc1;ptc2 mutant, mainly in the anterior brain structures (G-J).
Figure 4
Figure 4
Ptc1;ptc2 double mutants show mediolateral and anteroposterior somite patterning defects. Patterning of adaxial cells and slow muscle cell precursors is disturbed in ptc1;ptc2 mutants. The region of myod positive adaxial cells and eng1 expressing slow muscle precursors are expanded at 19 hpf (A-D). At 19 hpf, prdm1 expression is highly induced in the ptc1;ptc2 mutant, suggesting that the myotome is mainly developing slow muscle type precursors (E,F). Anteroposterior patterning of the somites is lost in segmented somites, since the posterior somite marker uncx4.1 (G,H) and anterior somite marker fgf8 (I,J) are strongly reduced or not detectable at 19 hpf. myf5 expression in 11 somite stage wildtype (K,L) and ptc1;ptc2 double mutant embryos (M,N). L and N are higher magnification of relevant areas of K and M, respectively. In wild type, myf5 is expressed at higher levels in the posterior of the somites during their formation, in more posterior (younger) segments this appears to include the adaxial cells (*). In more anterior (more mature) somites more anterior adaxial cells appear to show higher levels of labeling (arrowheads). In ptc1;ptc2 double mutant embryos (N) a "salt and pepper" type staining suggests that anterior and posterior cells are intermingled. Additionally, dlc necessary for proper segmentation is present in presomitic mesoderm but failed to be expressed in the posterior part of segmented somites (O,P), suggesting that somite formation and A/P patterning of formed somites are genetically uncoupled processes.
Figure 5
Figure 5
Hh signaling has an early negative role in the induction of the fin field. Expression analysis of fgf24 and prdm1 in a wild type embryo shows that these genes are restricted to the fin field at 26 hpf (A,C). ptc1;ptc2 mutants do not express these markers confirming that aberrant activation inhibits fin bud formation (B,D). To determine at which level Hh is inhibiting fin formation, the expression of tbx5, one of the earliest markers expressed in the finbud, was analyzed. At 20 hpf (E,F) tbx5 expression is lost in the presumptive finbud region (scale bar 100 μm). At 40 hpf (G,H), the fin bud has established and tbx5 expression is restricted to the pectoral fin in a wild type situation. However, in the ptc1;ptc2 double mutant a scattered low expression can be observed, showing that the pectoral fin bud is not formed. At the 10 ss, however, the initial expression domain of tbx5, encompassing heart and fin primordia is established. hand2, acting upstream of tbx5 is not expressed in the future pectoral fin area (I-L: white arrow), suggesting a very early negative role for Hh signaling in fin bud induction.
Figure 6
Figure 6
Cyclopamine treatment determines time window where Hh signaling inhibits fin induction. Treatment with 10 μM cyclopamine between 50% epiboly and the indicated developmental stages, identified the critical time window for Hh where it actively inhibits fin induction. From a dorsal (A,D, I-L) and lateral (E-H, M-P) view recruitment of tbx5 positive cells in the fin field can be slightly rescued when cyclopamine is administered between 50%- and 100% epiboly (J,N). However, tbx5 is more highly expressed when cyclopamine is administered between 50% epiboly and 5- and 10 somite stage (K,L,O,P). The constitutive activation of Hh signaling after removing cyclopamine, inhibits the outgrowth of the fin bud, clearly visible from a lateral view (M-P). Inhibiting Hh signaling in ptc1;ptc2 double mutants from the 18 ss till 40 hpf, rescues a restricted expression of tbx5, which is not, observed when cyclopamine is administered at 24 ss or 24 hpf (Q-T). These data show that Hh signaling inhibits fin induction during late gastrulation and the segmentation stage.
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References

    1. McMahon AP, Ingham PW, Tabin CJ. Developmental roles and clinical significance of hedgehog signaling. Curr Top Dev Biol. 2003;53:1–114. - PubMed
    1. Lum L, Beachy PA. The Hedgehog response network: sensors, switches, and routers. Science. 2004;304:1755–1759. doi: 10.1126/science.1098020. - DOI - PubMed
    1. Huangfu D, Anderson KV. Signaling from Smo to Ci/Gli: conservation and divergence of Hedgehog pathways from Drosophila to vertebrates. Development. 2006;133:3–14. doi: 10.1242/dev.02169. - DOI - PubMed
    1. Schauerte HE, van Eeden FJ, Fricke C, Odenthal J, Strahle U, Haffter P. Sonic hedgehog is not required for the induction of medial floor plate cells in the zebrafish. Development. 1998;125:2983–2993. - PubMed
    1. Barresi MJ, Stickney HL, Devoto SH. The zebrafish slow-muscle-omitted gene product is required for Hedgehog signal transduction and the development of slow muscle identity. Development. 2000;127:2189–2199. - PubMed

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