Several transcripts that exhibited an expression change trig

Several transcripts that exhibited an expression change triggered by T3 are promising candidates for being responsible for the observed increased cell extension growth of ENS cells in vitro. The calcium dependent cell adhesion molecule cadherin 12 (Cdh12, N-cadherin, 5.6 fold upregulated), the gene with the highest T3-triggered upregulation, is an integral membrane protein that strongly induces neurite outgrowth in retinal ganglion cells (Riehl et al., 1996) by interacting with the receptor-type tyrosine–protein phosphatase T (PTPRT1; +2.8 fold upregulated) (Burden-Gulley and Brady-Kalnay, 1999). N-cadherin is not essential for proper ENS development, but its knockout in mice delays the colonization of the gut with enteric neural crest cells and further. A combined knockout of N-cadherin and β1-integrins resulted in severe aganglionosis, mainly of the proximal hindgut (Broders-Bondon et al., 2012). Upregulation of transcription factor Kruppel-like factor 9 (KLF9, BTEB, +3.6) and the corepressor hairless (Hr, +3.8) results in increased neurite outgrowth and arborization in developing mouse topirimate (Denver et al., 1999). Both genes also play a role in the autoinduction of the Trβ1 receptor (Bagamasbad et al., 2008; Thompson, 1996). In our experiments, the Trβ receptor was 2.5 fold upregulated by T3. Tissue transglutaminase (TGM2, +2.2) was also picked as a possible candidate because a 10 fold increase of TGM2 expression concomitant with neurite outgrowth was observed when serum was withdrawn from neuroblastoma cells (Maccioni and Seeds, 1986). Solute carrier family 12 (sodium/potassium/chloride transporters), member 2 (SLC12A2, +1.6) plays a fundamental role in NGF-induced neurite outgrowth in pheochromocytoma PC12D cells (Nakajima et al., 2007). Neurotrimin (Ntm, −1.6) regulates the development of neuronal projections via attractive and repulsive mechanisms that are cell type specific (Gil et al., 1998). The small proline-rich protein 1A (Sprr1a, +1.5) promotes axonal outgrowth in embryonic and adult dorsal root ganglia neurons (Bonilla et al., 2002). The hepatocyte growth factor receptor c-Met (Met, −1.7) is needed for sensory nerve development and Met/HGF signaling seems to enhance axonal growth (Maina et al., 1997). However, some of our identified T3 regulated genes have been described with an antagonistic regulation in other reports about cell extension outgrowth: Neuronal pentraxin-2 (NPTX2, NARP, −1.6), a pentraxin family member that promotes neurite outgrowth in cortical explants, is downregulated in differentiating T3-induced ENS progenitor cells and therefore might have a different effect or might not be involved at all in our observed extension outgrowths (Tsui et al., 1996). One interesting issue was the interaction of T3 with the expression of guidance molecules. During ENS development, migration processes of neural crest cells are regulated by various guidance cues that are part of several signaling pathways, e.g. GDNF/GFRα1/Ret (Natarajan et al., 2002), Sema3A/neuropilin-1 (Shepherd and Raper, 1999), netrins/DCC/neogenin (Jiang et al., 2003), neurturin/GFRα2/Ret (Rossi et al., 1999), and the slit/robo pathway (Young et al., 2004). Attractive and repellent guidance molecules are necessary for the establishment of the neural patterns of the ENS. Other genes are mainly involved in proliferation and or maturation of neural crest cells (NCCs) in the developing gut, e.g. genes of the endothelin/endothelin receptor pathway and transcription factors like Phox2a/b and Sox10 (Newgreen and Young, 2002). Mutations in some of these genes were identified to trigger different kinds of gut aganglionoses with different characteristics like in HSCR, Waardenburg syndrome or Haddad syndrome (Amiel et al., 2008). Analysis of our microarray experiments exhibited some interesting regulations of members of these pathways, e.g. a change in endothelin 3, netrin-G1, semaphorins and slit-1 expression. In our in vitro experiments, endothelin 3 was upregulated 3.2 fold. In embryonic mice, lack of endothelin 3 delays the migration of neural crest cells leading to aganglionosis of the distal bowel (Woodward et al., 2003; Baynash et al., 1994). In humans, it is involved in Morbus Hirschsprung, the most common developmental disease of the ENS. Mutations in endothelin 3 are responsible for 5% of the Hirschsprung disease cases (Newgreen and Young, 2002). 5–10% of cases are based on mutations in its receptor endothelin receptor beta (EDNRB). In contrast to endothelin 3, the endothelin receptor was not regulated by T3 in our experiments.