Tcf4 encodescortical differentiation during development

Tcf4 has been linked to autism, schizophrenia, and Pitt-Hopkins Syndrome (PTHS) in humans, however, the mechanisms behind its role in disease development is still elusive. In the present study, we provide evidence that Tcf4 has a critical function in the differentiation of cortical regions during development. We show that Tcf4 is present throughout the developing brain at the peak of neurogenesis. Deletion of Tcf4 results in mis-specification of the cortical layers, malformation of the corpus callosum and hypoplasia of the hippocampus. RNA-sequencing on E14.5 cortex material shows that Tcf4 functions as a transcriptional activator and loss of Tcf4 results in downregulation of genes linked to the emergence of other neurodevelopmental disorders. Taken together, we show that neurogenesis and differentiation are severely affected in Tcf4 mutants, phenocopying morphological brain defects detected in PTHS patients. The presented data identifies new leads to understand the mechanism of human brain defects and will assist in genetic counseling programs.


Introduction
Correct cortical neurogenesis depends on a complex genetic program executed through correct spatio-temporal expression of transcription factors. A specific group of transcription factors present in the developing cortex, is the basic helix-loop-helix (bHLH) protein family (Powell and Jarman, 2008). The E-box protein sub-family of bHLH proteins consists out of three members; Tcf3 (E2A), Tcf12 (HEB), and Tcf4 (E2-2), which functions are dependent on specific bHLH binding partners through homo-or heterodimerization (Massari and Murre, 2000;Murre et al., 1989;Powell and Jarman, 2008). Mutations in the bHLH-domain, comprising the DNA-binding interface, inhibits such dimerization processes and proper DNAbinding, which directly interferes with their function in gene regulatory events (Sweatt, 2013). E-box proteins have mainly been studied in relation to their role in immune system development. They are found to be critical for the transition from CD4 + CD8 + double-positive T-cells to CD4 + or CD8 + single-positive T-cells (Wojciechowski et al., 2007) and loss of either Tcf12 or Tcf3 leads to an early depletion of T-cell progenitors and a decrease in mature T-cells (Wojciechowski et al., 2007). Tcf4 on the other hand, has only a minor role during early thymocyte development and it has been suggested that compensatory mechanism through Tcf3 and/or Tcf12 exists (Wikström et al., 2008). In the brain, E-box proteins are thought to regulate neurogenesis and neuronal differentiation based on their spatio-temporal presence and binding partners (Powell and Jarman, 2008). In humans, haplo-insufficiency of Tcf4 has been found to be determinative for Pitt-Hopkins Syndrome (PTHS) (Brockschmidt et al., 2007;Marangi et al., 2011;Marangi et al., 2012;Sweatt, 2013), a rare mental disorder, hallmarked by severe intellectual disability, typical facial gestalt, and additional features like breathing problems (Blake et al., 2010;Hasi et al., 2011;Peippo and Ignatius, 2012). Imaging of PTHS patients brains shows major neurodevelopmental defects, ranging from a smaller corpus callosum, underdeveloped hippocampi, and defective cortical development, to microcephaly, enlarged ventricles, and bulging caudate nuclei (Blake et al., 2010;Ghosh et al., 2012;Hasi et al., 2011;Peippo and Ignatius, 2012). Finally, Tcf4 has been implicated in other developmental brain disorders, like schizophrenia and autism (Brzózka and Rossner, 2013;Cousijn et al., 2014;Wirgenes et al., 2012). Until now the mechanism behind the observed neurodevelopmental defects as observed in humans is not known. Previous studies on a Tcf4 mouse mutant describe a loss of the pontine nucleus (Flora et al., 2007). Furthermore , Fischer et al., 2014 reported that Tcf4 promotes differentiation of neural stem cells to neurons in the adult forebrain and may thus be involved in postnatal neurogenesis by orchestrating neural stem cell lineage progression. In mammalian cell-lines Tcf4 has been found to interact with Mash1 and NeuroD2, important regulators of neuronal differentiation (Persson et al., 2000). Studies by Jung et al. (2018) and Grubišić et al. (2015) showed that the phenotype detected in the brain and in the gut of patients with PTHS could also (partially) be detected in heterozygous mutants of TCF4. Taken together, the available data point to a possible function of Tcf4 in neuronal development and links the developmental defects detected in heterozygous mutants to the phenotype in PTHS patients. In this study we aimed to gain more insight in and further specify the function of Tcf4 in the developing cortex. We demonstrate the specific presence of Tcf4 in specific cortical layers during development. Ablation of Tcf4 (Zhuang et al., 1996) induces major defects in cortical development, as cortical layering and specification of layer-specific neurons is severely affected. Furthermore, CUX1 positive neurons, a signature for upper layer neurons, are absent in Tcf4 mutants, whereas CTIP2 positive neurons (lower layer neurons) are decreased and

In situ hybridization
In situ hybridization with digoxigen (DIG)-labeled probes was performed as described previously (Smidt et al., 2004). Fresh frozen sections were fixed in 4% PFA for 30 min and acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine for 10 min. Probe hybridization was carried out at 68°C O/N with a probe concentration of 0.4 ng/μl in a hybridization solution containing 50% deionized formamide, 5x SSC, 5x Denhardt's solution, 250 μg/mL tRNA Baker's yeast, and 500 μg/mL sonificated salmon sperm DNA. The following day slides were washed in 0.2x SSC for 2 h at 68°C followed by blocking with 10% heat-inactivated fetal calf serum (HIFCS) in buffer 1 (100 mM Tris Hcl, pH 7.4 and 150 mM NaCl) for 1 h at RT. DIG-labeled probes were detected by incubating with an alkalinephosphatase-labeled anti-DIG antibody (Roche, Mannheim, 1:5000), using NBT-BCIP as a substrate. Slides were washed 2x5 min in T 10 E 5 , dehydrated with ethanol and embedded in Entellan. DIG in situ hybridization was performed with a 918 bp Tcf4 fragment bp 1101-2018 of mouse cDNA.

Tcf4 is present in cortical neurons at different stages of development.
In order to determine the role of Tcf4 in cortical development we first assessed its spatiotemporal expression pattern (Figure 1A). At E14.5 Tcf4 transcript is detected throughout the developing brain, although it is most abundantly expressed in the cortex. At E16.5 the expression of Tcf4 is still present throughout the cortex, however in the ventricular -and subventricular zone and outer parts of the cortical plate transcript levels appear to be higher, suggesting a layer specific intensity of the Tcf4 transcript. To confirm these data and to determine in which cortical layers TCF4 protein is present, we examined the cortex during development. As TCF4 protein was not detected in the cortex until E14.5 and becomes more apparent at later stages (Supplemental Figure 1), we focused on the expression pattern of TCF4 and layer-specific marks at E16.5 when most neurons of the developing cortex are specified (Molyneaux et al., 2007). At E16.5, similar to Tcf4 transcript, TCF4 is found in specific layers of the developing cortex. It co-localizes with CTIP2 ( Figure 1B-1 white arrowheads), a marker for layer V and VI, and TBR1 ( Figure 1B-2 white arrowheads), a marker for layer VI. Although Tcf4 transcript was detected in the ventricular-and subventricular zone at this stage, TCF4 protein is only marginally present in the VZ and SVZ of the cortex, identified by TBR2. Finally, some TBR2-positive cells, bordering the intermediate zone of the cortex, co-localize with TCF4 ( Figure 1B-3 white arrowheads).

Loss of Tcf4 results in defective cortical layering.
As shown above, Tcf4 is present in the murine developing cortex at the peek of neurogenesis. In order to determine the consequence of heterozygous or full deletion of Tcf4 on development of the cortex, we first examined the general cortical structure by means of DAPI-staining. At E17.5 the WT cortex shows a clear distinction in different layers. Based on neuronal density we were able to divide the cortex into a cortical plate (CP), Layer VI, intermediate zone (IZ), and the ventricular zone/subventricular zone ((S)VZ). Interestingly, the clear distinction between the IZ and layer VI was not present in Tcf4 heterozygous and full mutant embryos (Figure 2A white asterisk). Furthermore, the overall cortical thickness (CT) is reduced with ~14% in full mutants compared to WT (n=4; p<0.05, one-tailed), but is not significantly different between heterozygous and mutant or WT embryos ( Figure 2B). Besides the decrease in CT, a decrease in cortical plate thickness (CP) was detected in the full mutant of ~29% compared to WT (n=4; p<0.001, two-tailed) and ~20% compared to heterozygous embryos (n=4; p<0.01, two-tailed) ( Figure 2C). This change is reflected in the ratio of the CP compared to the CT, which is ~6% smaller in full mutants compared to WT (n=4; p<0.01, two-tailed) and ~4% smaller compared to heterozygous animals (n=4; p<0.05, two-tailed) (Figure 2D). At P0 we were able to distinguish several cortical layers based on cell-density; CP, Layer V-VI, sub-plate (SP), IZ, and VZ and SVZ. Similar as observed at E17.5, at P0 the layers of the cortex are less distinctive in both the heterozygous and mutant animals ( Figure 2E white asterisks), although the CT area has been recovered ( Figure 2F). The thickness of the CP is ~19% smaller in mutants compared to WT (n=3; p<0.01, two-tailed), and ~27% smaller compared to heterozygous animals (n=3; p<0.001, two-tailed) ( Figure 2G). Notably, the CP of the heterozygous animals shows an upward trend in thickness, as this is ~9% increased compared to WT animals (n=3; p=0.08, two-tailed) at P0. The ratio between the CP and the CT is more severely affected at P0 ( Figure 2H). The ratio of the CP to the CT is ~54% in WT and ~58% in the heterozygous animals, which is decreased to ~44% in mutant animals compared to WT (n=3; p=<0.01, two-tailed) and heterozygous (n=3; p<0.01, two-tailed) animals. Although TCF4 is not clearly present in the VZ and SVZ of the developing cortex, the increase in CT of the mutant cortex between E17.5 and P0 could be due to an elevated proliferation at E17.5 ( Figure 2I). Ki67 labeling, marking proliferating cells, showed that there is no significant difference in proliferation in the cortex of mutant or heterozygous embryos at E17.5 compared to WT (n=3; two-tailed) ( Figure 2J). Together, these data indicate that the correct developmental process of the murine cortex is affected upon loss of one or two alleles of Tcf4.

Loss of Tcf4 results in mis-specification of CTIP2-and SATB2-expressing neurons at E17.5.
Above we have shown that loss of one or two alleles of Tcf4 results in a difference in neuronal distribution throughout the cortex, suggesting a defective cortical layering at E17.5 and P0. To determine whether expression of layer-specific markers in the cortex is affected accordingly, we performed immunohistochemistry for SATB2 and CTIP2 at E17.5 in Tcf4 mutants (-/-and +/-) compared to WT. These two proteins mark two different neuronal populations, as SATB2 + neurons are callosal projection neurons (Alcamo et al., 2008;Leone et al., 2015;Srinivasan et al., 2012;Srivatsa et al., 2014) and CTIP2 + neurons project to other cortical areas in the same hemisphere (Leid et al., 2004;Srinivasan et al., 2012;Srivatsa et al., 2014). SATB2 is present throughout the differentiated layers of the cortex and not in the VZ and SVZ in Tcf4 WT, heterozygous, and mutant cortices at E17.5 ( Figure 3A). CTIP2, a marker for neurons in layer V and VI of the cortex, is also present in Tcf4 heterozygous and mutant cortices ( Figure 3B). However, quantification of CTIP2 + neurons at E17.5 shows that in full mutants there is a significant decrease of ~20% and ~17% in the amount of CTIP2 + cells compared to WT (n=4; p<0.05, one-tailed) and heterozygous (n=4; p<0.01, one-tailed) animals, respectively ( Figure  3D).

Deletion of Tcf4 results in a mis-specification of CTIP2-and SATB2-expressing neurons and loss of the Cux1 + neuronal population at P0.
Above we have shown that loss of one or two alleles of Tcf4 results in a defective segregation of CTIP2 and SATB2 neurons in the cerebral cortex. To determine whether this phenotype is persistent after birth, we performed similar experiments on P0 animals. Expression of SATB2 is still relatively normal at P0 in the heterozygous and mutant cortex compared to the WT ( Figure 4A). The amount of CTIP2-expressing cells on the other hand still displays a significant decrease (Figure 4B), although this decrease was lowered to ~12% between the mutant and the WT (n=3; p<0.05, one-tailed) and no significant difference was detected anymore between heterozygous and full mutants ( Figure 4D). Interestingly, the shift in distribution to lower parts of the cortex was still increased at P0 between WT and mutant animals, and a similar shift was detected for heterozygous compared to WT animals ( Figure  4E). Bin 4 (WT ~21.6%, heterozygous ~13.9%, mutant ~14.7%; n=3; p<0.001, two-tailed) and 5 (WT ~22%, heterozygous ~18%, mutant ~15.3%; n=3; p<0.001, two-tailed) showed a decrease in CTIP2 + neurons for both mutant and heterozygous compared to WT animals, whereas bin 7 (WT ~11%, heterozygous ~18%, mutant ~21.5%; n=3; p<0.001, p<0.01 respectively, two-tailed), bin 8 (WT ~2.6%, heterozygous ~7.8%, mutant ~9.3%; n=3; p<0.05, two-tailed), and bin 9 (WT ~0.7%, heterozygous ~2.9%, mutant ~1.8%; n=3; p<0.05, n.s. respectively, two-tailed) showed an increase in the amount of CTIP2-expressing cells compared to WT animals ( Figure 4E). Quantification of the population CTIP2 + /SATB2 + neurons showed that, confirming the E17.5 data, the amount of double-positive cells was ~8% of the CTIP2 + population in the WT cortex ( Figure 4C-1 and F). The amount of CTIP2 + /SATB2 + neurons showed an upward trend to ~12.7% in heterozygous animals ( Figure 4C-2) compared to WT animals (n=3; p=0.08, onetailed). Finally, the amount of double-positive neurons in the mutant cortex (Figure 4C-3) increased to ~39% of the total CTIP2-expressing population, which was significant compared to both WT (n=3; p<0.001, one-tailed) and heterozygous (n=3; p<0.01, one-tailed) animals ( Figure 4F). As the mis-specification of the cortical layers was more severely affected at P0 and the CP displayed a strong decrease in thickness, we set out to determine the presence of CUX1, a marker for the CP and future layer I-IV (Cubelos et al., 2008). CUX1 cells can be detected in the upper layers of the WT and Tcf4 heterozygous cortex, but is completely absent in the upper layers of the full Tcf4 mutant cortex (Figure 4G CP). Interestingly, CUX1 is still present in the VZ and SVZ in all genotypes (Figure 4G VZ). Taken together, these data further substantiate that cortical differentiation and layer specification is affected upon loss of Tcf4 and that this effect aggravates during development, leading to an absence of CUX1 + neurons in upper layers of the cortex in full Tcf4 mutants.
3.5 Loss of Tcf4 leads to defective development of the corpus callosum and hippocampus at E17.5. As shown above, deletion of Tcf4 has a measurable effect on the neuronal differentiation within the developing cerebral cortex. To determine whether this effect is represented by axonal projections and development of cortex-derived structures, we performed GAP43 immunohistochemistry in combination with DAPI to visualize the axonal tracts in the cortex, Page 9 of 31   272  273  274  275  276  277  278  279  280  281  282  283  284  285  286  287  288  289  290  291  292  293  294  295  296  297  298  299  300  301  302  303  304  305  306  307  308  309  310  311  312  313  314  315  316  317  318  319 hippocampus and the corpus callosum at E17.5 (Benowitz and Routtenberg, 1997;Grasselli and Strata, 2013;Strittmatter et al., 1995). GAP43 expression in the cortex can be divided at this stage in three compartments, an upper (CP), middle (layer VI), and lower (IZ) part, based on neuronal and axonal density. Quantification of the thickness of the compartments compared to the total thickness, shows a small but significant change in thickness of the middle compartment between the mutant-and both the WT (n=4; p<0.01, two-tailed) and heterozygous (n=4; p<0.01, two-tailed) animals of ~6% ( Figure 5A). As described above, we have detected a significant higher proportion of SATB2-CTIP2 double-positive neurons in full Tcf4 mutants. Since SATB2-expressing cells are known to project through the corpus callosum (Alcamo et al., 2008;Leone et al., 2015;Srinivasan et al., 2012;Srivatsa et al., 2014), we set out to determine whether this potential mis-specification influences the development of the corpus callosum ( Figure 5B). In both WT (Figure 5B-1) and heterozygous animals (Figure 5B-2) the corpus callosum seems unaffected (n=3), with the callosal bundle visualized through GAP43 IMHC and the callosal wedge ( Figure 5B white arrowheads) by DAPI staining. Importantly, in the full Tcf4 mutant (Figure 5B-3) the callosal bundle is completely absent (n=3; p<0.001, one-tailed), although the callosal wedge ( Figure 5B white arrowheads) seems unaffected. Examination of the hippocampus in the full Tcf4 mutant showed an absence of the dentate gyrus ( Figure 5C-1 to 3 for magnifications), next to a clear incomplete development in heterozygous animals. Together, these data indicate that not only the development of the cerebral cortex is disrupted by the loss of Tcf4, also the development of cortex-related structures, like the corpus callosum and hippocampus, is affected at this stage of development.

Corpus callosum and hippocampal defects persists towards birth in Tcf4 mutants.
As shown above, the corpus callosum and hippocampus show an aberrant development upon loss of Tcf4. To determine whether this effect persists towards birth, we tracked the development of these structures by GAP43 IMHC at P0, similar as shown for E17.5 above (Figure 6). Whereas at E17.5 distribution of GAP43 only showed a significant change in the middle compartment, at P0 GAP43 distribution in the cerebral cortex displays mild but significant changes in all subsections analyzed ( Figure 6A). The upper (CP) compartment of GAP43 is increased in both the heterozygous with ~3% (n=3 p<0.05, two-tailed) and mutant with ~9% (n=3 p<0.001, two-tailed) compared to WT. The difference of ~6% between the heterozygous and mutant animals is similarly significant (n=3 p<0.01, two-tailed). Interestingly, whereas at E17.5 the middle (V-VI) compartment was increased in thickness in the mutant, at P0 this compartment shows a decrease of ~6% between the WT and full mutants (n=3 p<0.05, two-tailed). In the lower (SP-IZ) compartment only a significant difference was detected between the WT and heterozygous animals of ~2.5% (n=3 p<0.05, two-tailed). The difference in GAP43 between E17.5 and P0 indicates that axonal tract formation in the cortical layers is affected even at late stages of development. The development of the corpus callosum is affected in full mutants (Figure 6B-3) compared to both WT (Figure 6B-1) (n=3 p<0.001, one-tailed) and heterozygous (Figure 6B-2) (n=3 p<0.001, one-tailed) animals at P0, confirming the initial defects observed at E17.5 including the apparent absence of a defective callosal wedge (Figure 6B-1 to 3 white arrowheads). Confirming the data observed at E17.5, the development of the hippocampus, and specifically the dentate gyrus, is affected in full Tcf4 mutants, although at this stage some initial cortical folding could be detected (Figure 6C-1 to 3). Taken together, the initial aberrations in cortical development, as identified at E17.5, persisted towards birth.

Tcf4 acts as a transcriptional activator during cortical development and regulates genes involved in neuronal differentiation and maturation.
In order to provide a better insight in the molecular mechanism of TCF4 action during cortical development we aimed to determine the early and therefore possible direct effects of Tcf4 ablation through RNA-sequencing of E14.5 dissected cortices of WT and mutant embryos (n=3; 2 pooled embryos per biological replicate). These analyzes showed that Tcf4 mainly acts as a transcriptional activator since the we observed 131 downregulated genes and 6 upregulated. Analysis of the top 25 upregulated (Figure 7A) and the top 5 downregulated genes (Figure 7B), based on fold-change of the transcript compared to WT samples indicates that Tcf4 may regulate genes that are involved in the regulation of neuronal differentiation en neuronal migration (e.g. NeuroD1, Mash1, Nos1, and Id2) (Casarosa et al., 1999;Kim, 2013;Park et al., 2013;Zhang et al., 2015) and regulates its own expression ( Figure 7C). Interestingly, these developmental processes that are typically affected in patients with ID. Confirming the data previously shown and described above, GO-term analysis (PANTHER Over-representation Test, Figure 8A) shows that Tcf4 is involved in the regulation of (neuronal) differentiation, cell signaling, synaptic plasticity, and development of the telencephalon and hippocampus. Further analysis of the genes regulated by Tcf4 shows that 26 of the 137 genes regulated by Tcf4 (19%) have previously been shown to be mutated in cases of ID ( Figure 8B) (Alders et al., 2014;Backx et al., 2010;Brockschmidt et al., 2007;Cocchella et al., 2010;Ehmke et al., 2017;Gerber et al., 2016;Gillentine et al., 2017;Guo et al., 2016;Labonne et al., 2016;Magoulas and El-Hattab, 2012;Merla et al., 2002;Metsu et al., 2014;Mikhail et al., 2011;Montesinos, 2014;Moore et al., 2016;Mulatinho et al., 2012;Myers et al., 2012;Nesbitt et al., 2015;Poot et al., 2010;Schoonjans et al., 2016;Schuurs-Hoeijmakers et al., 2013;Srivatsa et al., 2014;Tassano et al., 2015;Thevenon et al., 2014;Ţuţulan-Cunită et al., 2012). Characteristics of these cases range from moderate to severe ID, autistic phenotypes, brain malformations, speech impairments, and epilepsy. These traits can similarly be detected in patients with PTHS (Blake et al., 2010;Hasi et al., 2011;Peippo and Ignatius, 2012). Taken together, the transcriptome analyzes shows that Tcf4 acts mainly as a transcriptional activator of the neurogenic profile and the genetic program clearly relates to neurodevelopmental disorders as PTHS and ID.

Discussion
Here, we have shown that Tcf4 is specifically expressed in different layers of the murine cortex during development and that it has a central role in cortical differentiation and maturation. Loss of Tcf4 results in a disorganized cortex, in which the clear distinction between the different layers is merely affected, and postmitotic developing neurons are misspecified. Furthermore, cortex-related structures, like the corpus callosum and hippocampus show clear developmental defects. Transcriptome analyzes through RNA-sequencing suggested that Tcf4 acts mainly as a transcriptional activator involved in neuronal differentiation and maturation, and that Tcf4 directly or indirectly regulates genes that are known to be mutated in cases of ID. In humans, mutations within the bHLH domain in one copy of the Tcf4 gene results in PTHS (Brockschmidt et al., 2007;Marangi et al., 2011;Marangi et al., 2012;Pontual et al., 2009;Sweatt, 2013), a rare mental disorder that is characterized by severe mental retardation, breathing abnormalities, and distinctive facial features (Blake et al., 2010;Hasi et al., 2011;Peippo and Ignatius, 2012). Brain-defects seen in PTHS patients include a smaller corpus callosum, bulging caudate nuclei, underdeveloped hippocampi, enlarged ventricles, and in some cases microcephaly (Blake et al., 2010;Ghosh et al., 2012;Hasi et al., 2011;Peippo and Ignatius, 2012). Many of these traits we were able to trace back in Tcf4 mouse mutants, like the defects in the corpus callosum and hippocampi, initial underdevelopment of the cortex, and in some embryos we detected bulging caudate nuclei (Supplemental Figure 2A) or enlarged ventricles (Supplemental Figure 2B). In accordance to humans, loss of one copy of the Tcf4 allele resulted in developmental defects, although we did detect a dose dependency, where the full Tcf4 mutants displays the most consistent phenotypic characteristics as observed in human PTHS patients. The smaller hippocampi, initial underdevelopment of the cortex, and agenesis of the corpus callosum detected in this study match the data from Jung et al. (2018). However, when analyzing these mutants it is important to keep in mind that the study of Jung et al. focuses on the heterozygous mice of a different mouse model, which lacks exon 4 of the Tcf4 gene, than used in this study, in which the bHLH domain of the Tcf4 gene is replaced by a Neo-casette (Zhuang et al., 1996). As stated above patients with PTHS mainly show missense mutations or deletions in the basic region of the bHLH domain, resulting in a defective gene and possible protein in the human brain, indicating that the model used in this study could be used as a proper tool to mimic the effects of the main defects detected in PTHS patients. Furthermore, we described that Tcf4 is involved in the regulation of genes that are known to be mutated in cases of ID, together with traits like epilepsy, loss of the corpus callosum, and speech impairment also detected in patients with PTHS. For example patients with a mutation in NeuroD1 show ID and speech impairments (Cocchella et al., 2010), whereas mutations in and Zeb2, related to the Mowat Wilson syndrome, is characterized by facial features, mental retardation, and an absent corpus callosum (Moore et al., 2016). Interestingly, although Ascl1 is not directly related to ID, mutation of this gene is related to congenital central hypoventilation syndrome, a syndrome characterized by breathing abnormalities, which is also a known characteristic of PTHS patients (de Pontual et al., 2003). Besides its role in PTHS and other syndromes related to PTHS and ID, the Tcf4 mouse mutant could be used in to better understand the onset and progression of autism and schizophrenia, both linked to Tcf4 mutations (Brzózka and Rossner, 2013;Cousijn et al., 2014;de Munnik et al., 2014), with regard to its role in correct cortical development and synaptic plasticity. For example, in our RNA-sequencing data we detected a ~60% down-regulation of the Lpl gene, which is located on the 8p21.3 locus part of the 8p21 locus, a known schizophrenia susceptibility locus (Blouin et al., 1998;Fallin et al., 2011), and shows a ~42% and ~32% downregulation of Kcnj3 and Grm5 respectively, which are both associated to schizophrenia (Matosin et al., 2015;Yamada et al., 2012). Taken together, our data show that Tcf4 is of major importance in the murine brain for proper development of the cortex. Furthermore, loss of Tcf4 leads to similar effects on the brain as observed in PTHS patients, making the Tcf4 mutant mouse-line a good model to study molecular mechanisms of brain development and specifically in relation to PTHS patients. Our data provides a clear overview on the effects of Tcf4 on brain development, next to previous roles described in development of the pons (Flora et al., 2007), and gives new insights in possible treatment of specific traits like PTHS and genes that could be involved in the development of PTHS-like syndromes and ID. Finally, our data set of genes regulated by Tcf4 provide new leads for clinical genetics and or clinical diagnostics in, until now, genetically undefined neurodevelopmental disorders in humans.