Planarian Hedgehog/Patched establishes anterior–posterior polarity by regulating Wnt signaling

Abstract

Despite long-standing interest, the molecular mechanisms underlying the establishment of anterior-posterior (AP) polarity remain among the unsolved mysteries in metazoans. In the planarians (a family of flatworms), canonical Wnt/␤-catenin signaling is required for posterior specification, as it is in many animals. However, the molecular mechanisms regulating the posterior-specific induction of Wnt genes according to the AP polarity have remained unclear. Here, we demonstrate that Hedgehog (Hh) signaling is responsible for the establishment of AP polarity via its regulation of the transcription of Wnt family genes during planarian regeneration. We found that RNAi gene knockdown of Dugesia japonica patched (Djptc) caused ectopic tail formation in the anterior blastema of body fragments, resulting in bipolar-tails regeneration. In contrast, RNAi of hedgehog (Djhh) and gli (Djgli) caused bipolar-heads regeneration. We show that Patched-mediated Hh signaling was crucial for posterior specification, which is established by regulating the transcription of Wnt genes via downstream Gli activity. Moreover, differentiated cells were responsible for the posterior specification of undifferentiated stem cells through Wnt/␤-catenin signaling. Surprisingly, Djhh was expressed in neural cells all along the ventral nerve cords (along the AP axis), but not in the posterior blastema of body fragments, where the expression of Wnt genes was induced for posteriorization. We therefore propose that Hh signals direct head or tail regeneration according to the AP polarity, which is established by Hh signaling activity along the body's preexisting nervous system. body polarity ͉ regeneration ͉ anteroposterior axis ͉ neuron ͉ stem P lanarians are one of the most useful animals for investigation of the molecular and cellular mechanisms by which the anteriorposterior (AP) polarity of the body is established during regeneration. It is well known that planarians regenerate a head or a tail from the anterior and posterior ends, respectively, of the amputated stump, with maintenance of the original AP polarity. More than a century ago, T.H. Morgan reported one of the earliest descriptions of ''polarity'' in planarian regeneration (1), based on his findings that very thin cross-sectional fragments made by cutting along the AP axis regenerated bipolar two-headed planarians (2), termed ''Janus-heads'' (an allusion to the Roman god Janus; 3, 4). Since then, much effort has been focused on this fascinating maintenance of the original polarity, and its disruption, such as the induction of Janus-heads formation by treatment with colchicine or colcemid (5, 6), although the molecular mechanisms still remain unknown.Recently, canonical Wnt/␤-catenin signaling was shown to be required for posterior specification in planarians. Wnt/␤-catenin signaling is evolutionarily well conserved and plays a role in establishing the AP axis during development in several animal species. In the planarian Schmidtea mediterranea, gene knockdown by RNA interference (RNAi) of ...

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

“…3A). Staining of the regenerants for expression of Dj frizzled-T (DjfzT), a posterior-specific Wnt receptor gene (Yazawa et al, 2009), showed that there was a dramatic reduction of the tissue expressing DjfzT in both Djislet(RNAi) and Djwnt1/P-1(RNAi) (Fig. 3B).…”

“…Double staining for detection of mRNA expression was performed essentially as described (Yazawa et al, 2009). For the detection of DIG-or fluorescein-labeled RNA probes, samples were incubated with specific antibodies conjugated with alkaline phosphatase or horseradish peroxidase (1:2000; Roche Diagnostics).…”

“…, 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.…”

The planarian P2X homolog in the regulation of asexual reproduction

“…Recent findings have shown that various growth factors are involved in specifying the body polarity of planarians, such as the antero-posterior and dorsal-ventral axes. Hh (Hedgehog) and Wnt family members induce the differentiation of posterior cells from neoblasts (Gurley et al, 2008;Yazawa et al, 2009). BMP (Bone morphogenetic protein) is involved in dorsal cell specification of neoblasts (Molina et al, 2007).…”

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian <em>Dugesia japonica</em>

“…The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs 8,9,15 . Both approaches can produce visually striking phenotypes at high penetrance-for example, regeneration of bipolar animals [16][17][18][19][20][21] . To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.…”