Transcription factors lhx1/5-1 and pitx are required for the maintenance and regeneration of serotonergic neurons in planarians

Abstract

SUMMARYIn contrast to most adult organisms, freshwater planarians can regenerate any injured body part, including their entire nervous system. This allows for the analysis of genes required for both the maintenance and regeneration of specific neural subtypes. In addition, the loss of specific neural subtypes may uncover previously unknown behavioral roles for that neural population in the context of the adult animal. Here we show that two homeodomain transcription factor homologs, Smed-lhx1/5-1 and Smed-pitx, are required for the maintenance and regeneration of serotonergic neurons in planarians. When either lhx1/5-1 or pitx was knocked down by RNA interference, the expression of multiple canonical markers for serotonergic neurons was lost. Surprisingly, the loss of serotonergic function uncovered a role for these neurons in the coordination of motile cilia on the ventral epidermis of planarians that are required for their nonmuscular gliding locomotion. Finally, we show that in addition to its requirement in serotonergic neurons, Smed-pitx is required for proper midline patterning during regeneration, when it is required for the expression of the midline-organizing molecules Smed-slit in the anterior and Smed-wnt1 in the posterior.

Genome-wide analysis of the bHLH gene family in planarians identifies factors required for adult neurogenesis and neuronal regeneration

“…Analyses of the planarian photoreceptor, excretory and serotonergic cells have shown that tissue-specific transcription factors are detected in the stem cells in intact (Lapan and Reddien, 2012) and regenerating tissues (Lapan and Reddien, 2011;Scimone et al, 2011;Currie and Pearson, 2013); these studies have identified the first sets of precursor cells in planarians outside of the germ cells (Newmark et al, 2008) and have generated a working model in which planarians possess diverse lineage-committed progenitors that contribute to the maintenance and regeneration of tissues (Reddien, 2013;Rink, 2013). In contrast to the well-defined excretory system and photoreceptors, the nervous system represents a formidable challenge.…”

“…Finally, in flies and crustaceans, sim functions as a master regulator of midline cells by regulating the specification of midline progenitors (Nambu et al, 1991;Vargas-Vila et al, 2010), whereas in vertebrates, sim controls the differentiation (Michaud et al, 1998;Eaton and Glasgow, 2006) and migration (via plexinC1) (Xu and Fan, 2007) (Lapan and Reddien, 2012), protonephridia (Scimone et al, 2011), serotonergic (Currie and Pearson, 2013) and novel CNS (bHLH) progenitors, respectively. *Progenitors that are only observed during regeneration.…”

“…The bHLH superfamily consists of six monophyletic groups (Groups A-F), which are also characterized by the presence or absence of various additional protein domains (Simionato et al, 2007). Due to the large number of putative paralogs in Groups A and B, the predicted protein sequences were aligned using T-Coffee (Notredame et al, 2000) and subjected to Bayesian analyses as described previously (Currie and Pearson, 2013;Zhu and Pearson, 2013); Group C-F genes were categorized based on clear top BLASTP hits against the Swiss-Prot database (UniProt) and the presence of class-specific protein domains (supplementary material Table S1). bHLH sequences were obtained from a cDNA collection (Zayas et al, 2005) or cloned from cDNA into pJC53.2 (Collins et al, 2010) or pPR244 (Reddien et al, 2005a) using gene specific primers or 3′ RACE, respectively.…”

“…For γ-irradiation treatments, animals were exposed to 100 Gy in a JL Shepherd Mark I Cesium-137 irradiator and fixed 6 days after treatment. To visualize bHLH transcripts by multiple fluorescent in situ hybridization (FISH), we used horseradish peroxidase substrates as described previously (Pearson et al, 2009) or the alkaline phosphatase (AP) substrate Fast Blue (Lauter et al, 2011;Currie and Pearson, 2013). For Fast Blue staining, animals were developed in 0.25 mg/ml Fast Blue BB (Sigma F3378) and NAMP (Sigma 855) in AP staining buffer (0.1 M Tris-HCl pH 8.2, containing 50 mM MgCl 2 , 100 mM NaCl, 0.1% Tween 20) (Hauptmann, 2001;Lauter et al, 2011).…”

Cerberus–Nodal–Lefty–Pitx signaling cascade controls left – right asymmetry in amphioxus

“…First, in ecdysozoan lineages, Nodal appears to have been lost (7, 9), whereas Pitx is generally symmetrically expressed (8) and, where studied, does not function in LR asymmetry (10, 11). Second, some lophotrochozoans do not show asymmetric Pitx expression (8,12,13). Third, functional studies on protostome Nodal signaling are currently restricted to molluscs (5), so it is unclear whether the regulatory connection between Nodal and Pitx is conserved even in those species that show asymmetric expression.…”