The InteractiveFly: Drosophila as a Model for Human Diseases

Pitt-Hopkins syndrome
Reviews | PubMed | Wikipedia | More in IF | Recent Updates

Drosophila genes associated with
Pitt-Hopkins syndrome
Related terms

Overview of the disease

Pitt-Hopkins syndrome (PTHS) is a rare human disorder characterized by severe developmental delay, autistic behaviours, absence of speech, distinct facial features, epilepsy, constipation and hyperventilation. PTHS is caused by haploinsufficiency of the Transcription factor 4 (TCF4, located at 18q21.1). Large chromosomal deletions, partial gene deletions, frame shift, nonsense, splice site or missense mutations in the TCF4 gene have been found in PTHS patients. These mutations are usually sporadic, but in some cases children have inherited the mutant allele from a mosaic parent. In vitro, PTHS-associated missense mutations result in hypomorphic, non-functional or dominant-negative TCF4 alleles. It is unclear whether mutations causing PTHS impair development of the nervous system or functioning of the adult central nervous system (CNS), or both. Class I bHLH proteins, also called the E-proteins, comprise the mammalian TCF3/E2A, TCF4, TCF12/HEB and the Drosophila Daughterless (Da) (Tamberg, 2015 and references therein).

Go to top

Relevant studies of Pitt-Hopkins syndrome

Tamberg, L., Sepp, M., Timmusk, T. and Palgi, M. (2015). Introducing Pitt-Hopkins syndrome-associated mutations of TCF4 to Drosophila daughterless. Biol Open 4: 1762-1771. PubMed ID: 26621827

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. This study shows 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. da transgenic fruit fly strains with corresponding missense mutations R578H, R580W, R582P and A614V found in TCF4 of PTHS patients were generated and the impact of these mutations was studied in vivo. Overexpression of wild type Da as well as human TCF4 in progenitor tissues induces ectopic sensory bristles and the rough eye phenotype. By contrast, overexpression of DaR580W and DaR582P that disrupt DNA binding reduces the number of bristles and induces the rough eye phenotype with partial lack of pigmentation, indicating that these act dominant negatively. Compared to the wild type, DaR578H and DaA614V are less potent in induction of ectopic bristles and the rough eye phenotype, respectively, suggesting that these are hypomorphic. All studied PTHS-associated mutations introduced into Da lead to similar effects in vivo as the same mutations in TCF4 in vitro. Consequently, these Drosophila models of PTHS are applicable for further studies aiming to unravel the molecular mechanisms of this disorder (Tamberg, 2015).


  • Daughterless is the only orthologue of human E-proteins in Drosophila.
  • Mutated Daughterless proteins have variable transactivation capabilities in HEK293 cells in comparison to wild type protein.
  • TCF4 and Da, but not DaR578H, DaR580W, DaR580L or DaR582P, activate E-box controlled reporter gene expression in wing disc and induce ectopic thoracic bristles in Drosophila.
  • Da, DaD515G, DaR578H, DaA614V, TCF4-A and TCF4-B are capable of rescuing da null embryonic neuronal phenotype.
  • Overexpression of Da, DaPTHS, TCF4-A, or TCF4-B by GMR12B08-GAL4 results in the rough eye phenotype.
  • Overexpression of Da, TCF4-B and DaPTHS in young adult flies results in significantly altered survivorship.

This study shows that Da, the only E-protein in Drosophila with highly conserved bHLH domain, functions as human TCF4 orthologue. As the overall identity of a protein sequence between Drosophila and mammals is usually around 40% between homologues and 80-90% within conserved functional domains, Da can be considered the orthologue for all three human E-proteins. In all experiments conducted in this study, TCF4 was found to act very similarly as Da, proving the possibility of modelling PTHS in the fruit fly. The two human TCF4 isoforms, TCF4-A and TCF4-B, were able to activate E-box dependent lacZ expression in Drosophila, and more importantly, to induce ectopic bristle formation in the adult thorax, to rescue embryonic nervous system development in da null embryos, and to induce the rough eye phenotype when overexpressed in the nervous system identically to Da. Altogether these results show that TCF4 has comparable activity in the fruit fly as Da (Tamberg, 2015).

To further study the PTHS-associated mutations in Drosophila, four mutations found in TCF4 (R580W, R578H, R582P and A614V) and two control mutations (D515G and R580L) were introduced into Da. The mutants were analysed by luciferase assay in a mammalian cell line in order to compare the results of Da directly to results obtained with human TCF4. Subsequently PTHS-associated Da mutants were studied in vivo in E-box lacZ reporter assay, and in both rescue and overexpression experiments (Tamberg, 2015).

PTHS-associated arginine mutations R580W, R580L, and R582P abolish Da transactivation capability in luciferase reporter assays in HEK293 cells. DaR580W, DaR580L and DaR582P behave similarly to each other in both overexpression and rescue experiments in the fruit fly. The rescue by daG32-GAL4 driver of da null embryonic nervous system phenotype fails when using Da proteins with these arginine mutations. When Da carrying one of above mentioned mutations was overexpressed in flies under the control of the nervous system specific driver GMR12B08-GAL4, the strongest eye phenotype was observed. These flies have rough and partially unpigmented eyes with fused ommatidia consistent with Da having an important role in Drosophila eye development. In addition, overexpression of these arginine mutants under pnr-GAL4 causes malformation of the thorax. Altogether, these results indicate that mutations R580W, R580L and R582P abolish the Da transactivation capability resulting in dominant-negative effects. This is in line with the previous data about the corresponding mutations in TCF4 having dominant-negative effects in vitro (Tamberg, 2015).

R578H was found to differ from the other three arginine mutations (R580W, R580L and R582P) in in vivo experiments. Although DaR578H was unable to activate reporter gene expression in luciferase assay carried out in mammalian cell line HEK293 and in lacZ assay in vivo, it causes rough eye phenotype similar to Dawt when overexpressed by GMR12B08-GAL4. Furthermore, DaR578H rescues da null embryonic neuronal phenotype when expressed using daG32-GAL4. Also DaR578H shows weak induction of ectopic bristles. Taken together these results indicate that transactivation capability of DaR578H probably depends on its dimerisation partners, which could be lacking in mammalian cell line and weakly presented in the wing disc notum. Similarly, it has been previously shown that while TCF4 carrying the R578H mutation is unable to bind to E-box in vitro as a homodimer or in complex with either ASCL1 or NEUROD2, it does not act in dominant negative manner in reporter assays in mammalian cells (Tamberg, 2015).

The A614V mutation positioned in the second helix of the bHLH domain shows the mildest effects. DaA614V was able to activate E-box-specific transcription in vitro and in vivo. Expressing DaA614V using daG32-GAL4 rescues da null embryonic neuronal phenotype. Overexpression using GMR12B08-GAL4 results in the rough eye phenotype only when both of the transgenes are homozygous, indicating that this mutation causes hypomorphic effects. This is consistent with a recent study which shows that the A614V mutation leads to lower levels of TCF4 because of reduced protein stability (Tamberg, 2015).

The control mutation generated in this study, D515G, does not reduce Da transactivation capability in vitro and behaves similarly to Dawt in vivo. This shows that D515 positioned outside of the conserved bHLH is not required for Da transcriptional activity. The other control mutation generated in this study, R580L, where the same arginine is mutated as in DaR580W, leads to dominant-negative effects in vivo similarly to R580W. At least in the case of R580, the mutation specificity, whether it was mutated into tryptophan or leucine, was found to have no affect (Tamberg, 2015).

In rescue experiments with tested driver strains (69B-GAL4, tub-GAL4, ubi-GAL4, GMR12B08-GAL4, daG32-GAL4) all Da transgenes fail to rescue da null embryonic lethality. Apparently the successful rescue of da null lethality closely mimics the endogenous Da expression. daG32-GAL4, comprising of 3.2 kb of da gene covering the promoter, the first intron, and the upstream noncoding region, is widely used as a ubiquitous driver line. Most probably the expression of this driver line is far too strong compared to the native expression of da gene as Da has been shown to positively autoregulate its own expression via a transcriptional feedback loop. If daG32-GAL4 expression is regulated by Da itself, then Da overexpression might drive even stronger GAL4 expression, resulting in a positive feedback loop. Furthermore, it has been hypothesised that daG32-GAL4 lacks putative regulatory repressor elements since using a 15 kb genomic da transgene that has an additional 12 kb of downstream sequence rescues da null embryonic lethality (Tamberg, 2015).

Little is known about the role of E proteins in adult nervous system. This study shows that exact temporal and spatial expression of Da/TCF4 remains vitally important during adulthood of fruit flies. It was shown that overexpression of Da/TCF4 in adults leads to lethality within 2-3 days. Surprisingly, TCF4 isoforms A and B lead to strikingly different outcomes when overexpressed in adult fruit flies. While the long isoform TCF4-B behaves identically to Da, TCF4-A affects the survival only slightly compared to the control group. This could be related to the lack of interaction capability of much shorter N terminus of isoform A in fruit fly or different regulation of subcellular location and dimerisation of the alternative TCF4 isoforms. Analysis of survival divides the PTHS related mutations into severe (R580W, R580L and R582P) and milder (R578H and A614V) according to survivorship. The severe mutants lead to lethality within 3-4 days and the milder ones in 10-11 days. Further experiments with cell type or tissue-specific drivers would help to understand the role of E-proteins during adulthood in more details (Tamberg, 2015).

The fact that overexpression of wild type as well as dominant negative forms of Da causes comparable reduction in survival and induction of the rough eye phenotype raises the possibility that overexpression of wt protein is also eliciting dominant negative effects as suggested earlier. One explanation for this phenomenon could be that excess homodimers outcompete transcriptionally more potent heterodimers at various promoter sites. Intriguingly, recent studies suggest that in addition to TCF4 haploinsufficiency, increased TCF4 dose is also a risk factor for disturbed cognitive development as a TCF4 duplication has been described in a patient with developmental delay and a partial duplication in a patient with major depressive disorder. Nevertheless, in case of induction of ectopic bristles, opposite effects were observed for Dawt and dominant negative Da mutants, indicating that in addition to its dominant negative effects, excess wt protein also has specific effects during development (Tamberg, 2015).

In patients with PTHS just one copy of TCF4 is mutated or deleted. Seemingly the most relevant way to model PTHS in animal models would be to use the appropriate heterozygotes of the orthologous protein. However, in Drosophila there is a sole E-protein Da corresponding to all three mammalian E-proteins. In a way the heterozygous Da null mutation corresponds to the heterozygous deletion of all three E-proteins in mammals. Accordingly, Da as the only binding partner of class II bHLH proteins has a large variety of roles outside nervous system. As TCF4 is highly expressed in the nervous system, the mutated alleles were expressed specifically in the nervous system in a wild type background. Overexpression of DaPTHS under the nervous system specific GMR12B08-GAL4 leads to viable flies and stocks with each mutation generated in this study were created. An alternative tactic to model PTHS and to mimic dosage loss by TCF4 deletions would be to slightly downregulate Da expression nervous system specifically by RNAi. Additional studies are needed to generate and compare different PTHS models and to perform behavioural tests that would give valuable information about cognition and social behaviour of the PTHS model flies (Tamberg, 2015).

Go to top

Zweier, C., de Jong, E.K., Zweier, M., Orrico, A., Ousager, L.B., Collins, A.L., Bijlsma, E.K., Oortveld, M.A., Ekici, A.B., Reis, A., Schenck, A. and Rauch, A. (2009). CNTNAP2 and NRXN1 are mutated in autosomal-recessive Pitt-Hopkins-like mental retardation and determine the level of a common synaptic protein in Drosophila. Am J Hum Genet 85: 655-666.  PubMed ID: 19896112

Heterozygous copy-number variants and SNPs of CNTNAP2 and NRXN1, two distantly related members of the neurexin superfamily, have been repeatedly associated with a wide spectrum of neuropsychiatric disorders, such as developmental language disorders, autism spectrum disorders, epilepsy, and schizophrenia. This study identifies homozygous and compound-heterozygous deletions and mutations via molecular karyotyping and mutational screening in CNTNAP2 and NRXN1 in four patients with severe mental retardation (MR) and variable features, such as autistic behavior, epilepsy, and breathing anomalies, phenotypically overlapping with Pitt-Hopkins syndrome. With a frequency of at least 1% in the cohort of 179 patients, recessive defects in CNTNAP2 appear to significantly contribute to severe MR. Whereas the established synaptic role of NRXN1 suggests that synaptic defects contribute to the associated neuropsychiatric disorders and to severe MR as reported in this study, evidence for a synaptic role of the CNTNAP2-encoded protein CASPR2 has so far been lacking. Using Drosophila as a model, it was shown that, as known for fly Nrx-I, the CASPR2 ortholog Nrx-IV might also localize to synapses. Overexpression of either protein can reorganize synaptic morphology and induce increased density of active zones, the synaptic domains of neurotransmitter release. Moreover, both Nrx-I and Nrx-IV determine the level of the presynaptic active-zone protein bruchpilot, indicating a possible common molecular mechanism in Nrx-I and Nrx-IV mutant conditions. The study proposes that an analogous shared synaptic mechanism contributes to the similar clinical phenotypes resulting from defects in human NRXN1 and CNTNAP2 (Zweier, 2009).


  • Identification of recessive deletions and mutations in CNTNAP2 and NRXN1.
  • Analysis of CNTNAP2 and NRXN1 orthologs Nrx-IV and Nrx-I in Drosophila.
  • Drosophila Nrx-IV and Nrx-1 both determine the level of the synaptic protein Bruchpilot.
  • Drosophila Nrx-IV is present at synapses and can, like Nrx-I, reorganize them.

Go to top


Go to top

More in IF

Go to top

Recent Updates

Go to top

Back to Drosophila as a Model for Human Diseases

Date revised: 6 Jan 2016

Home page: The Interactive Fly © 2015 Thomas Brody, Ph.D.

The Interactive Fly resides on the web server of the Society for Developmental Biology