Transcriptional regulation of TLX2 and impaired intestinal innervation: possible role of the PHOX2A and PHOX2B genes

Abstract

TLX2 (also known as HOX11L1, Ncx and Enx) is a transcription factor playing a crucial role in the development of the enteric nervous system, as confirmed by mice models exhibiting intestinal hyperganglionosis and pseudo-obstruction. However, congenital defects of TLX2 have been excluded as a major cause of intestinal motility disorders in patients affected with intestinal neuronal dysplasia (IND) or pseudo-obstruction. After demonstrating the direct regulation of TLX2 expression by the homeoprotein PHOX2B, in the present work, we have focused on its paralogue PHOX2A. By co-transfections, electrophoretic mobility shift assays and chromatin immunoprecipitation, we have demonstrated that PHOX2A, like PHOX2B, is involved in the cascade leading to TLX2 transactivation and presumably in the intestinal neuronal differentiation. Based on the hypothesis that missed activation of the TLX2 gene induces the development of enteric nervous system defects, PHOX2A and PHOX2B have been regarded as novel candidate genes involved in IND and pseudo-obstruction and consequently analyzed for mutations in a specific set of 26 patients. We have identified one still unreported PHOX2A variant; however, absence of any functional effect on TLX2 transactivation suggests that regulators or effectors other than the PHOX2 genes must act in the same pathway, likely playing a non redundant and direct role in the pathogenesis of such enteric disorders.

Defining the transcriptomic landscape of the developing enteric nervous system and its cellular environment

“…Phox2a is also similar to Phox2b in its expression in the enteric neurons, cranial glia and other differentiating autonomic neurons [64, 147–149] but its expression appears to be more restricted to a subset of enteric neural crest [150]. Both Phox2b and Phox2a have been shown to transcriptional regulate Tlx2 [91]. Loss of Phox2b gives rise to aganglionic gastrointestinal tract [54] while in the absence of Phox2a some enteric neurons are present in the oesophogeal region [151].…”

“…Interestingly, Tlx2 (Hox11l) has been shown to be a target for Phox2b [152] and Phox2a [90, 91]. Tlx2 deficiency leads to megacolon and myenteric neuronal hyperplasia in mice [90–92], which may explain its association with intestinal motility disorders like Intestinal neuronal dysplasia (IND).…”

Causative and common PHOX2B variants define a broad phenotypic spectrum

“…Among PHOX2B common variants, a cohort of 26 Italian patients affected with intestinal disorders such as intestinal neuronal dysplasia and/or chronic intestinal pseudo‐obstruction has already been reported to show a cumulative frequency of PHOX2B p.Ala254 = and c.754_774del21, this latter corresponding to 7Ala contraction (−7Ala), higher than in the European population, thus suggesting that also conditions similar to Hirschsprung disease are associated with apparently benign PHOX2B variants and confirming the role of the pan‐neural PHOX2B protein in intestinal development.…”

Functional characterization of a minimal sequence essential for the expression of human TLX2 gene

“…On the other hand, our previous studies of the 5'-flanking regions of the other two family members could disclose a minimal promoter sequence without cell-specificity for TLX3 (8) and a neural-specific PHOX2B/PHOX2A binding site with enhancer property for TLX2 (9,10). This latter transcriptional pathway accounts for the observation that TLX2 is mainly expressed in tissues derived from neural crest cells at the end of their migration.…”