Planarian Hh Signaling Regulates Regeneration Polarity and Links Hh Pathway Evolution to Cilia

Abstract

The Hedgehog (Hh) signaling pathway plays multiple essential roles during metazoan development, homeostasis, and disease. Although core protein components are highly conserved, the variations in Hh signal transduction mechanisms exhibited by existing model systems (Drosophila, fish, and mammals) are difficult to understand. We characterize the Hh pathway in planarians. Hh signaling is essential for establishing the Anterior/Posterior axis during regeneration by modulating wnt expression. Moreover, RNAi methods to reduce signal transduction proteins Cos2/Kif27/Kif7, Fused, or Iguana do not result in detectable Hh signaling defects; however, these proteins are essential for planarian ciliogenesis. Our study expands the understanding of Hh signaling in the animal kingdom and suggests an ancestral mechanistic link between Hh signaling and the function of cilia.The Hh signaling pathway plays numerous evolutionarily conserved roles in the regulation of cell growth and patterning during the embryonic and postembryonic development of animals as diverse as frutiflies and humans. The misregulation of this pathway has equally profound consequences resulting in defects such as holoprosencephaly (cyclopia) and tumorigenesis. Secreted Hh protein alters gene transcription by binding the cell-surface receptor Patched (Ptc), preventing repression of the 7 membrane spanning receptor Smoothened (Smo) by Ptc. This activates Gli transcription factors and inactivates their inhibitor Suppressor of Fused (SuFu). Despite conservation of these core components and their mode of function (1,2), Hh signal transduction mechanisms appear to have diversified throughout evolution (3). Drosophila Hh signaling is cilia-independent and requires the kinesin Costal2 (4) (Kif7/27 in vertebrates) and the kinase Fused (5). The mouse Hh pathway requires primary cilia (6,7) and Kif7 (8-10), but not Fused (11,12). Zebrafish utilize cilia, Kif7, Fused, and Iguana/Dzip1 (Igu) (13)(14)(15)(16)(17)(18)(19). C. elegans has lost a functional Hh pathway altogether (20). Since planarians belong to a group of animals that evolved independently from flies, fish, and mammals (Sup. Fig. 1) an analysis of planarian Hh signaling could reveal how the mechanistic differences in a highly conserved signaling pathway arose.Systematic sequence homology searching of the S. mediterranea genome identified single homologs for planarian Hh (Smed-hh), Patched (Smed-ptc), Smoothened (Smed-smo) and Supressor of Fused (Smed-sufu), but three Gli homologs (37) (Sup. Fig. 2,3). Of the Gli homologs, only Smed-gli-1 exhibited an obvious role in Hh signaling (see below). We cloned (see SOM) and analyzed the expression of these planarian Hh components by in-situ hybridization (Fig. 1A-C,Sup. Fig. 4). ptc expression was reduced by RNAi of pathway activators (hh, smo, gli-1) and elevated by RNAi of pathway inhibitors (ptc, sufu) (Fig. 1B), suggesting that as in other animals (21-23),

A LIM-homeobox gene is required for differentiation of Wnt-expressing cells at the posterior end of the planarian body

“…S9 in the supplementary material) and this decrease was not influenced by anteriorization. In addition, Dj-cateninB(RNAi) had little effect on the expression of Djwnt1/P-1 at day 1, as previously described (Petersen and Reddien, 2009;Rink et al, 2009). This result showed that, at least in the first phase, Djwnt1/P-1 is expressed independently of the -catenin pathway, in contrast to the other Wnt and posterior genes.…”

“…, however, shows a tail formation defect and inappropriate midline patterning after posterior amputation (Gurley et al, 2010). Furthermore, it has been shown that Hedgehog (Hh) signaling is upstream of Wnt/-catenin signaling and is involved in the establishment of anterior-posterior (AP) polarity (Rink et al, 2009;Yazawa et al, 2009). These studies clearly indicate that signaling molecules plays a crucial role, not only in regeneration, but also in cell differentiation.…”

teashirt is required for head-versus-tail regeneration polarity in planarians

“…nou darake (ndk), ndl-4, sFRP-1, notum and prep were selected as anterior PCGs (Cebrià et al, 2002;Gurley et al, 2008;Reddien, 2008, 2011;Rink et al, 2009;Felix and Aboobaker, 2010); wnt2 and ndl-3 were selected as prepharyngeal PCGs (Petersen and Reddien, 2008;Rink et al, 2009); and wntless, wntP-2, frizzled-4 ( fz-4), axinB and wnt1 were selected as posterior PCGs (Gurley et al, 2008;Petersen and Reddien, 2008;Adell et al, 2009;Iglesias et al, 2011). We used a highly parallel microfluidic qRT-PCR platform (Fluidigm) to assess gene expression in wild-type, control, β-catenin-1 and teashirt RNAi animals (supplementary material Fig.…”

Congenital Hydrocephalus in Genetically Engineered Mice

“…81 In planaria worms, a reduction in signal transduction proteins coded by cos2/kif27/kif7, fused, or iguana does not result in detectable Hh signaling defects but appears to be essential for planarian ciliogenesis. 81 Interestingly, the protein KIF27 was recently shown to physically interact with Fused (STK36), a critical protein in the construction of the central pair apparatus of motile 9þ2 cilia in mice. 106 Taken together, these earlier reports, plus our findings of hydrocephalus and other lesions that are characteristic of ciliary dyskinesia, support an interactive role for both Kif27 and Stk36 in ciliogenesis.…”