Introducing Pitt-Hopkins syndrome-associated mutations ofTCF4toDrosophila daughterless

Abstract

Pitt-Hopkins syndrome (PTHS) is caused by haploinsufficiency of Transcription factor 4 (TCF4), one of the three human class I basic helix-loop-helix transcription factors called E-proteins. Drosophila has a single E-protein, Daughterless (Da), homologous to all three mammalian counterparts. Here we show that human TCF4 can rescue Da deficiency during fruit fly nervous system development. Overexpression of Da or TCF4 specifically in adult flies significantly decreases their survival rates, indicating that these factors are crucial even after development has been completed. We generated da transgenic fruit fly strains with corresponding missense mutations R578H, R580W, R582P and A614V found in TCF4 of PTHS patients and studied the impact of these mutations in vivo. Overexpression of wild type Da as well as human TCF4 in progenitor tissues induced ectopic sensory bristles and the rough eye phenotype. By contrast, overexpression of DaR580W and DaR582P that disrupt DNA binding reduced the number of bristles and induced the rough eye phenotype with partial lack of pigmentation, indicating that these act dominant negatively. Compared to the wild type, DaR578H and DaA614V were less potent in induction of ectopic bristles and the rough eye phenotype, respectively, suggesting that these are hypomorphic. All studied PTHS-associated mutations that we introduced into Da led to similar effects in vivo as the same mutations in TCF4 in vitro. Consequently, our Drosophila models of PTHS are applicable for further studies aiming to unravel the molecular mechanisms of this disorder.

Type I bHLH Proteins Daughterless and Tcf4 Restrict Neurite Branching and Synapse Formation by Repressing Neurexin in Postmitotic Neurons

“…Da, and its mammalian ortholog, Tcf4, share share conserved functions which were recently highlighted by the finding that expression of human TCF4 can rescue loss of function da mutant phenotypes in Drosophila (Tamberg et al, 2015). To explore the postmitotic neuronal function of Tcf4 in mammals, we first tested for specificity of Tcf4 antibody and efficiency of Tcf4 siRNA knockdown in mouse NSCs/NPCs using Western blot and RT-PCR analysis (Figure S5).…”

Diagnosis and management in Pitt‐Hopkins syndrome: First international consensus statement

“…TCF4 is necessary for neurodevelopment and it plays an important role in cognition and behavior . Individuals with PTHS often have disrupted sensorimotor gating, indicating disturbed filtering of external and internal stimuli.…”

“…Loss‐of‐function mutations in Tcf4 lead to the near‐complete lack of language acquisition. Language acquisition is virtually absent in most PTHS individuals . Up to 55% of individuals express only single words before 10 years of age, and indeed many have little to no expressive language throughout their lifespan ( R34 ).…”

“…Deficits in social and communication interaction skills combined with patterns of repetitive behavior, as well as impairments in adaptive skills are prominent in individuals with PTHS . Few studies have implemented direct in‐person assessments of autism characteristics, although various studies have reported on language development, insistence on sameness and on behavioral patterns of repetitive behaviors and stereotypies such as hand clapping and flapping, head banging, body rocking, or finger mannerisms …”

Extramacrochaetae promotes branch and bouton number via the sequestration of daughterless in the cytoplasm of neurons

“…Haploinsufficiency of TCF4 has been associated with Pitt-Hopkins syndrome (Amiel et al, 2007;Tamberg, Sepp, Timmusk, & Palgi, 2015), while common small nucleotide polymorphisms in TCF4 have been associated with schizophrenia (The Schizophrenia Psychiatric Genome-Wide Association Study (GWAS) Consortium, 2011). Defects in human ID protein function are associated with a number of disorders in other cell types including eye disease (Fan et al, 2018;Guo et al, 2015), hydronephrosis (Aoki et al, 2004), and bone disease (Fiori, Billings, de la Pena, Kaplan, & Shore, 2006).…”