• tcf4
• tcf4 variants
• in vivo models
• functional characterization
• zebrafish
• Xenopus laevis
• craniofacial alterations
• craniofacial development
• CRISPR/Cas9

The Transcription Factor 4 (TCF4) is a member of the basic-Helix-Loop-Helix (bHLH) transcription factor family involved in neurodevelopment, lymphoid development and epithelial-mesenchymal transition (Forrest et al., 2014). Deletions, nonsense and missense mutations in TCF4 are known to be responsible for the Pitt-Hopkins syndrome (PTHS), a severe neurodevelopmental disorder associated with a facial gestalt, mental retardation and autonomic nervous system dysfunction (Zweier et al. 2007; Amiel et al. 2007, Brockschmidt et al. 2007).

The project in which I take part, has the aim to functionally characterize three de novo TCF4 variants by using two vivo models, namely, Danio rerio (zebrafish) and Xenopus laevis. Interestingly, these variants are associated to a new pathological phenotype presents in three children with craniofacial alterations different from that of PTHS’s patients and without intellectual disability, a hallmark of this syndrome. We are collaborating with Prof. Vitobello’s group (University of Dijon, France), which has identified these variants by exome sequencing analysis. Specifically, the described TCF4 variants are different point-mutations that cause missense substitutions in exon 18, which encodes the bHLH protein domain, crucial for dimerization and DNA-binding (Zweier et al., 2007; Ross et al., 2003). Considering the importance of the bHLH domain and the phenotypic differences with PTHS, it has been hypothesised that these mutations in TCF4 may cause a gain of function mechanism which affects craniofacial development.

Before starting with experiments, it was verified that TCF4 sequence (especially that in exon 18), was conserved in orthologues of both zebrafish and Xenopus. After this essential confirmation, I performed in situ hybridization on whole mount embryos of both animal models to verify whether tcf4 expression profile was comparable to that described in human embryos. To that end, tcf4 cDNA from zebrafish and Xenopus embryos were firstly cloned into an expression plasmid (pGEM-T Easy vector) and in vitro transcribed to obtain the antisense RNA digoxigenin-labelled probes. In situ hybridization was performed on 24, 48 and 72 hpf (hours post fertilization) zebrafish larvae as well as on Xenopus embryos from18 to 38 stage of development.

The following step was the overexpression of the human wild type TCF4 (wt TCF4) to assess whether the gain of function of TCF4 could alter the craniofacial morphogenesis. Initially, different cloning steps were required to subclone wt TCF4 into a suitable plasmid for in vitro transcription of 5’-capped mRNA, which was then microinjected into one-cell Danio rerio embryos and into one blastomere of Xenopus embryos. At later developmental stages, embryos were stained with Alcian blue in order to visualise the craniofacial cartilages. Intriguingly, morphological alterations were observed in the ethmoid plate, homolog to the mammalian hard palate, both in zebrafish and Xenopus embryos. The same overexpression experiments are currently ongoing using TCF4 variants found in patients, starting with the one associated to the most severe phenotype in patients (T1781→ A; Met594→ Lys).

Lastly, our last goal would be using CRISPR/Cas9 technique to generate a Danio rerio line carrying the exact patient’s mutation. In the experimental schedule, I first used PCR technique with specific primers (flanking the CRISPR/Cas9 target site), to amplify the region of zebrafish tcf4 in which the mutation will be inserted. The obtained amplicon of genomic tcf4 was sequenced by Sanger sequencing to confirm the sequence of our wildtype zebrafish did not present polymorphisms. Moreover, I took part in the design of two Cas9 RNA guides, checking for possible off-targets, which will be further limited by using Cas9 nickase through an approach of “obliged heterodimers”. Finally, I identified the ssDNA sequences that will be injected along with the Cas9/gRNAs to serve as DNA templates for homologous recombination repair.