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ORIGINAL RESEARCHpublished: xx July 2020

doi: 10.3389/fnana.2020.00042

Edited by:Emmanuel Valjent,

Centre National de la RechercheScientifique (CNRS), France

Reviewed by:Tonis Timmusk,

Tallinn University of Technology,Estonia

José L. Ferran,University of Murcia, Spain

Q4 *Correspondence:Benjamin Philpot

bphilpot@med.unc.edu

Received: 20 April 2020Accepted: 22 June 2020Published: xx July 2020

Citation:Kim H, Berens NC,

Ochandarena NE and Philpot BD(2020) Region and Cell Type

Distribution of TCF4 in the PostnatalMouse Brain.

Front. Neuroanat. 14:42.doi: 10.3389/fnana.2020.00042

Region and Cell Type Distribution ofTCF4 in the Postnatal Mouse Brain

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Hyojin Kim1,2, Noah C. Berens3, Nicole E. Ochandarena4 and Benjamin D. Philpot1,2,5*

1 Department of Cell Biology & Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States,2 Neuroscience Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States, 3 Department of Biology,University of North Carolina at Chapel Hill, Chapel Hill, NC, United States, 4 MD-Ph.D. Program, University of North Carolinaat Chapel Hill, Chapel Hill, NC, United States, 5 Carolina Institute for Developmental Disabilities, University of North Carolinaat Chapel Hill, Chapel Hill, NC, United States

Transcription factor 4 is a class I basic helix-loop-helix transcription factor regulatinggene expression. Altered TCF4 gene expression has been linked to non-syndromicintellectual disability, schizophrenia, and a severe neurodevelopmental disorder knownas Pitt-Hopkins syndrome. An understanding of the cell types expressing TCF4 proteinin the mouse brain is needed to help identify potential pathophysiological mechanismsand targets for therapeutic delivery in TCF4-linked disorders. Here we developed a novelgreen fluorescent protein reporter mouse to visualize TCF4-expressing cells throughoutthe brain. Using this TCF4 reporter mouse, we observed prominent expression of TCF4in the pallial region and cerebellum of the postnatal brain. At the cellular level, bothglutamatergic and GABAergic neurons express TCF4 in the cortex and hippocampus,while only a subset of GABAergic interneurons express TCF4 in the striatum. Among glialcell groups, TCF4 is present in astrocytes and immature and mature oligodendrocytes.In the cerebellum, cells in the granule and molecular layer express TCF4. Our findingsgreatly extend our knowledge of the spatiotemporal and cell type-specific expressionpatterns of TCF4 in the brain, and hence, lay the groundwork to better understandTCF4-linked neurological disorders. Any effort to restore TCF4 functions through smallmolecule or genetic therapies should target these brain regions and cell groups to bestrecapitulate TCF4 expression patterns.

Keywords: transcription factor 4, Pitt-Hopkins syndrome, schizophrenia, autism spectrum disorder,neurodevelopmental disorder, intellectual disability

INTRODUCTION

Transcription factor 4 (TCF4, Gene ID 6925) is a basic helix-loop-helix (bHLH) transcriptionfactor, acting as both a repressor and activator of gene expression (Massari and Murre, 2000).The protein’s functional domains include a first activation domain, a nuclear localization signal,a second activation domain, and a bHLH domain. The bHLH domain consists of a basic regionthat directly mediates DNA binding and amphipathic helices that provide a dimerization interface.TCF4 can form homo- and hetero-dimers with cell type-specific bHLH proteins, which modulateits function (Murre et al., 1994). The human TCF4 gene can be transcribed from multiplepromoters, and the usage of alternative 5′ exons and splicing produces protein isoforms with18 different N′-termini and variable functional domains (Sepp et al., 2011). Genomic alterationsthat affect TCF4 function or levels increase the risk of neurodevelopmental or psychiatric

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disorders (Sepp et al., 2012; Bedeschi et al., 2017). For example,haploinsufficiency of TCF4 is the main pathogenic mechanismin Pitt-Hopkins syndrome (PTHS), which is characterized byintellectual disability, sensory processing deficits, anxiety, andspeech and motor delay (Amiel et al., 2007; Zweier et al.,2007). PTHS is associated with enlarged ventricles, cerebellaratrophy, and hippocampal and corpus callosum hypoplasia(Peippo et al., 2006; Amiel et al., 2007; Zweier et al., 2008;Goodspeed et al., 2018; Zollino et al., 2019), suggesting thatgross brain development is sensitive to dramatic changes inTCF4 expression and function. More subtle alterations in TCF4gene expression have been linked to non-syndromic intellectualdisability, schizophrenia, and bipolar diseases (Pickard et al.,2005; Kharbanda et al., 2016; Maduro et al., 2016; Forrest et al.,2018; Ma et al., 2018; Mary et al., 2018). These structural andbehavioral phenotypes emphasize the importance of TCF4 generegulation for normal brain function.

Mouse models carrying mutations or deletions of the bHLHregion of Tcf4 display many PTHS-like phenotypes, includingmemory and learning deficits, anxiety, hyperactivity, and sensorydysfunction. Perturbations of Tcf4 disrupt synaptic function inthe hippocampus and cortex, likely contributing to impairedlearning and memory (Kennedy et al., 2016; Rannals et al.,2016; Thaxton et al., 2018). At the cellular level, reduced TCF4protein levels impair dendritic development, neuronal migration,and cortical laminar organization (Chen et al., 2016; Li et al.,2019; Wang et al., 2020). In glial cells, TCF4 loss leads todelayed differentiation of oligodendrocyte progenitors (Fu et al.,2009). Thus, evidence from mouse studies implicates TCF4 in avariety of critical processes in brain development and function,including progenitor cell differentiation, neuronal migration andmorphogenesis, and synaptic plasticity.

Human TCF4 is expressed in the prosencephalon and theventricular zone of the central nervous system during fetaldevelopment, and its expression remains sustained in the adultforebrain (de Pontual et al., 2009). Similarly, mouse Tcf4 isprominently expressed in the isocortex and hippocampus duringdevelopment and in adulthood (Chen et al., 2016; Jung et al.,2018). While these studies highlight broad regions in whichTCF4 is particularly active, much less is known regarding thespecific identity of cell types in which TCF4 is expressed. TCF4expression has been reported in a subset of cortical neurons(Jung et al., 2018). However, it is not yet characterized whichcortical neurons express TCF4, and whether brain regions outsidethe cortex contain TCF4-expressing cells. Moreover, TCF4-expressing hippocampal cell groups are largely unknown despitethe prominent expression in the hippocampus.

Eventual pharmacological or genetic approaches to treatPTHS and other TCF4-linked disorders require knowledge ofTCF4 distribution at the resolution of discrete brain areasand specific cell lineages and types. This is particularly truefor gene therapy strategies that are attempting to addressTCF4 haploinsufficiency in PTHS by normalizing levels of geneexpression. In order to facilitate these therapeutic efforts andfurther contextualize roles for TCF4 in brain development, wedeveloped and validated a novel mouse model incorporating aCre-dependent TCF4 green fluorescent protein (GFP) reporter.

Using this line, we track TCF4-expressing brain regions andcell groups throughout postnatal development, with greaterreliability and resolution than could previously be achieved usingavailable antibodies (Jung et al., 2018).

MATERIALS AND METHODS

AnimalsWe generated Tcf4LGSL/+ mice through the University of NorthCarolina, Chapel Hill (UNC) Animal Models Core facility. Weutilized CRISPR/Cas9-mediated homologous recombination togenerate Tcf4-LoxP-GFP-Stop-LoxP (Tcf4LGSL) knock-in mice onthe C57BL/6J background. The Tcf4LGSL allele was generatedby inserting a cassette, comprised of a LoxP site, adenovirussplice acceptor, porcine teschovirus-1 2A (P2A) site, EGFPcoding sequence, 3 copies of SV40 polyadenylation sequence(Stop), FRT site, and another LoxP site (Figure 1A). Thiscassette was inserted into Tcf4 intron 17. The sequence of theguide RNA (gRNA) was 5′- GTCGTGCCTTACGTAGCTGGG-3.′ Mouse embryos were injected with a mixture of 400 nMCas9 protein, 50 ng/µl in vitro transcribed gRNA, and 20 ng/µlsupercoiled donor plasmid. The donor plasmid was constructedwith 1017 bp 5′ homology arm, the LoxP-GFP-Stop-LoxPcassette, and 884 bp 3′ homology arm. Potential founder animalswere screened for the presence of the insertion event by 5′ and 3′polymerase chain reaction (PCR) assays consisting of one primeroutside the targeting vector homology arms and one primerunique to the insertion event. The 5′ assay primers were Tcf4-5ScF1 (5′-GCACTTCAGGGATCGCTTA-3′) and AdSA-R2 (5′-GGGACAGGATAAGTATGACATCATC-3′), which produceda 1224 bp band. The 3′ assay primers were SV40pA-F2 (5′-GCTGATCCGGAACCCTTAAGC-3′) and Tcf4-3ScR1(5′-CCGCCCTAATTGTTCAAAGAG-3′), which produced a1109 bp band. Two chosen founders were checked for off-targetmutations at 10 predicted off-target sites. No mutations weredetected at the off-target sites screened in two founder animals.The Tcf4LGSL/+ knock-in mice were genotyped via PCR. Theprimer set of Tcf4-5ScF1 and Tcf4-3ScR1 or SV40pA-F2 and Tcf4-3ScR1 was, respectively, used to amplify the wildtype or LGSLknock-in allele.

The female Tcf4LGSL/+ mice were mated with heterozygousmales from one of three Cre-expressing lines: Nex-Cre(Goebbels et al., 2006), which Klaus-Armin Nave generouslyprovided, Actin-Cre (RRID:IMSR_JAX:019099), and Gad2-Cre(RRID:IMSR_JAX:010802). All mice were maintained on acongenic C57BL/6J background. All research procedures usingmice were approved by the Institutional Animal Care and UseCommittee at the UNC and conformed to National Institutes ofHealth guidelines.

Western BlottingEmbryonic day 16.5–18.5 brains were dissected in ice-coldphosphate-buffered saline (PBS, pH = 7.3) and then immediatelyfrozen with dry ice. Frozen brain samples were homogenizedin glass homogenizers with ice-cold RIPA buffer [50 mMTris–HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1%

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FIGURE 1 | ValidationQ5 of

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Tcf4-LGSL mice that faithfully report TCF4 expression. A Schematic of the strategy to generate C57BL/6J mice carrying theLoxP-P2A-GFP-STOP-LoxP cassette upstream of the basic helix-loop- helix region. Adenovirus splicing acceptor is shown by the blue box. B RepresentativeWestern blot for TCF4, GFP, and GAPDH loading control protein in embryonic brain lysates from Tcf4+/+ (WT), Tcf4LGSL/+ (Het), and Tcf4LGSL/LGSL (Homo) mice.The TCF4 antibody (recognizes mouse TCF4 aa 50–150) is designed to detect a long isoform of TCF4. We detected a TCF4 full length protein (TCF4-FL) band atapproximately 76 kDa that corresponds to the long isoform in WT lysates. A TCF4 truncated protein (TCF4-Trunc.) was detected at approximately 65 kDa in Hetlysates. A band for GFP or GAPDH protein was detected at approximately 26 or 35 kDa, respectively. C Quantification of Western blotting for TCF4-FL and for GFP.D–F Dual fluorescence ISH for Tcf4 (magenta) and GFP (green) from PFC of P80 WT and Tcf4LGSL/+, and CA1 of Tcf4LGSL/+ mice. Asterisk indicates a cellexpressing only Tcf4, and arrows indicate cells co-expressing Tcf4 and GFP. Insets are higher magnifications. Scale bars = 10 µm. G, H Quantification ofGFP-positive and -negative cells in Tcf4-expressing cells in the PFC and CA1 region (n = 3 mice). Data represent mean ± SEM.

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sodium dodecyl sulfate (SDS), and 0.5% Na deoxycholate]supplemented with 2 mM EDTA pH 8.0 and a protease inhibitorcocktail (Sigma, Saint Louis, MO). Tissue homogenates werecleared by centrifugation at 4◦C for 20 min. Protein sampleswere mixed with 4x protein loading buffer (Li-COR, Lincoln,NE) and 2-mercaptoethanol (Sigma) and incubated in 95◦Cfor 5–7 min. They were resolved by SDS-polyacrylamide gelelectrophoresis and transferred to nitrocellulose membranes.Membranes were blocked for 1 h at room temperaturein Odyssey blocking buffer (Li-COR) prior to incubationovernight at 4◦C with primary antibodies diluted 1:500with blocking buffer. Membranes were subsequently washedrepeatedly with PBS (0.1 M Phosphate, 1.5 M NaCl) containing0.1% Tween-20 (PBSTween) prior to incubation for 1 h atroom temperature with secondary antibodies prepared in thedilution of 1:5000 in blocking buffer. The following secondaryantibodies were used: donkey anti-mouse 800CW (Li-COR, 926-32212) or donkey anti-rabbit Alexa 680 (Invitrogen, A10043).Finally, blots were washed repeatedly in PBSTween followedby PBS alone prior to imaging with the Odyssey imagingsystem (Li-COR).

Tissue PreparationPostnatal mice were anesthetized with sodium pentobarbital(60 mg/kg i.p.) before transcranial perfusion with 25 mlof PBS immediately followed by phosphate-buffered 4%paraformaldehyde (pH 7.4). Brains were postfixed overnight at4◦C before 24-h incubations in PBS with 30% sucrose. Brainswere sectioned coronally or sagittally at 40 µm using a freezingsliding microtome (Thermo Scientific, Kalamazoo, MI). Sectionswere stored at −20◦C in a cryopreservative solution (45% PBS,30% ethylene glycol, and 25% glycerol by volume).

Histology and ImmunostainingFor chromogenic staining, sections were rinsed several timeswith PBS, and endogenous peroxidases were quenched byincubating for 5 min in 1.0% H2O2 in MeOH, followedby PBS rinsing. Sections were washed with PBS containing0.2% Triton X-100 (PBST) several times. Then sections wereblocked with 5% normal goat serum in PBST (NGST) for1 h at room temperature. Blocked sections were incubated inprimary antibodies diluted in NGST for 24 h at 4◦C. Afterincubation in primary antibodies, sections were rinsed severaltimes in PBST and incubated for 1 h at room temperaturein biotinylated goat anti-rabbit secondary antibodies (VectorBA-1000, Burlingame, CA) diluted 1:500 in NGST. Sectionswere then rinsed in PBST prior to tertiary amplificationfor 1 h with the ABC elite avidin-biotin-peroxidase system(Vector PK-7100). Further rinsing with PBST preceded a 3-min incubation at room temperature in 3,3′-diaminobenzidine(DAB) chromogenic substrate (0.02% DAB and 0.01% H2O2in PBST) to visualize immune complexes amplified by avidin-biotin-peroxidase.

For immunofluorescent staining, sections were rinsedseveral times with PBS and PBST before blocking withNGST or 5% bovine serum albumin (BSA) in PBST for1 h at room temperature. Sections were then incubated

with primary antibodies diluted in NGST or BSA at 4◦Covernight. The list of primary antibodies used is providedin Table 1. Sections were rinsed several times with PBSTand then incubated with secondary antibodies for 1 h atroom temperature. The following secondary antibodies fromInvitrogen (Carlsbad, CA) were used at a 1:1000 dilution:goat anti-mouse Alexa 568 (A11031); goat anti-mouse Alexa647 (A21240); goat anti-rabbit Alexa 568 (A11011); goat anti-chicken Alexa 488 (A11039); or donkey anti-goat Alexa 568(A11057). In all experiments, 4′,6-diamidino-2-phenylindole(DAPI; Invitrogen D1306) was added during the secondaryantibody incubation at a concentration of 700 ng/ml fornuclear counterstaining. Brain sections compared withinfigures were stained within the same experiment, underidentical conditions.

In situ HybridizationRNAscope Fluorescent Multiplex Assay, designed to visualizemultiple cellular RNA targets in fresh frozen tissues (Wang et al.,2012), was used to detect Tcf4 (Cat No. 423691), EGFP (CatNo. 400281-C2), vGat (Cat No. 319191-C3), and vGlut1 (CatNo. 416631-C2) in mouse brain (Advanced Cell Diagnostics,Newark, CA). The target region of the Tcf4 probe is 1120–2020 bp of mouse Tcf4 mRNA (NM_001083967.1). Brainswere extracted and frozen in dry ice. Sections were taken ata thickness of 16 µm. Staining procedure was completed tomanufacturer’s specifications.

Imaging and Figure ProductionImages of brain sections stained with DAB histochemistrywere obtained with a Nikon Ti2 Eclipse Color andWidefield Microscope (Nikon, Melville, NY). Images ofbrain sections stained by using fluorophore-conjugatedsecondary antibodies were obtained with Zeiss LSM 710Confocal Microscope, equipped with ZEN imaging software(Zeiss, Jena, Germany). Images compared within the samefigures were taken within the same imaging session usingidentical imaging parameters. Images within figure panelswent through identical modification for brightness andcontrast by using Fiji Image J software. Figures were preparedusing Adobe Illustrator software (Adobe Systems, San Jose,CA, United States).

Data AnalysisImages for in situ hybridization (ISH) colocalization analysiswere captured from consistent coronal section planes acrossdifferent mouse brains (PFC, STR: ∼ 1.10 mm; CA1, BLA,TH: ∼ −2.06 mm; VC: ∼ −2.70 mm from bregma). TheDAPI image from each brain region (265.69 × 265.69 µm) wasconverted to 8-bit in black and white, and its threshold wasadjusted using the Huang method built into Fiji software. Forthe image with Tcf4 or GFP staining, the ISODATA thresholdmethod was consistently applied. To identify mean Tcf4 or GFPfluorescence intensity level for each nucleus (DAPI), we usedCellProfiler software, which is a free open-source software thatallows one to measure and analyze cell images automatically(Kamentsky et al., 2011).

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TABLE 1 | Primary antibodies used.

Antigen Manufacturer Dilution

APC Millipore (Billerica, MA, United States), mouse monoclonal, clone CC-1, OP80 1:500

Calbindin Santa Cruz (Dallas, TX, United States), goat polyclonal, sc-7691 1:500

ChAT Millipore, goat polyclonal, AB144P 1:1,000

DARPP-32 Millipore, rabbit polyclonal, AB10518 1:1,000

GAPDH Millipore, mouse monoclonal, clone 6C5, MAB374 1:5,000

GFAP Dako (Glostrup, Denmark), rabbit polyclonal, Z0334 1:1,000

GFP Novus (Centennial, CO), rabbit polyclonal, NB600-308 1:1,000

GFP Aves Labs (Tigard, OR), chicken polyclonal, GFP-1020 1:10,000

IBA1 Wako (Osaka, Japan), rabbit polyclonal, 019-19741 1:500

NeuN Millipore, mouse monoclonal, clone A60, MAB377 1:1,000

Olig2 Millipore, rabbit polyclonal, AB9610 1:1,000

PV Swant (Marly, Switzerland), mouse monoclonal, PV235 1:1,000

SOM Peninsula Laboratories (San Carlos, CA), rabbit polyclonal, T-4103 1:1,000

TCF4 Abcam (Cambridge, United Kingdom), rabbit polyclonal, ab130014. Synthetic peptide corresponds to Mouse TCF4 aa 50–150. 1:500 or 1,000

VIP Immunostar (Hudson, WI), rabbit polyclonal, 20077 1:1,000

RESULTS

Validation of Tcf4-LGSL Mouse ModelTo investigate the spatiotemporal profile of TCF4-expressingcells, we engineered mice with a LoxP-GFP-STOP-LoxP (LGSL)cassette introduced into intron 17 of the Tcf4 allele (Figure 1A).An adenovirus splicing acceptor was included in the cassetteto avoid alternative splicing of intron 17 (Figure 1A). Thisdesign allowed us to examine TCF4-expressing cells withhigh confidence, as GFP can be detected by commercialantibodies. Moreover, the insertion of a 2A self-cleaving peptide(P2A) enables GFP molecules to freely diffuse throughout thecytoplasm, making it possible to track axonal projections fromTCF4-expressing neurons, though at the cost of not being able touse it to identify the subcellular localization of TCF4. The GFPand STOP cassette is flanked by LoxP sites, enabling their Cre-mediated deletion, and in turn, reinstating the capacity to expressfull-length, functional TCF4 from the locus.

As predicted from our design, brain lysates from Tcf4+/+

(WT) mice produced a single full-length TCF4 band by Westernblot, whereas lysates from Tcf4LGSL/+ (Het) mice produced boththe full-length and truncated TCF4 protein, and lysates fromTcf4LGSL/LGSL (Homo) mice produced only a truncated TCF4band (Figure 1B). GFP was present only in Tcf4LGSL/+ andTcf4LGSL/LGSL lysates (Figure 1B). The band intensity of full-length TCF4 was reduced by approximately half in lysates fromTcf4LGSL/+ compared to WT mice (Figure 1C: WT: 1.00 ± 0.02,n = 5; Het: 0.54 ± 0.03, n = 11; Homo: 0.00 ± 0.00, n = 3).GFP levels were higher in lysates from Tcf4LGSL/LGSL comparedto Tcf4LGSL/+ mice (Figure 1C: WT: 0.00 ± 0.00; n = 5,Het: 0.36 ± 0.01, n = 11; Homo: 1.00 ± 0.00, n = 3). Theseresults validated that the LGSL cassette produced GFP andtruncated TCF4 protein.

To verify that GFP faithfully reports TCF4 expression, weperformed dual ISH using probes specific to Tcf4 or GFPmRNA. GFP signals were detected in cells from adult Tcf4LGSL/+mice, but absent in cells from WT mice (Figure 1D), proving

the specificity of the GFP probe detection. Tcf4 signals wereobserved in both WT and Tcf4LGSL/+ mice (Figures 1E,F).Quantification of cells expressing both GFP and Tcf4 revealedan approximate 97% overlap (Figures 1G,H: prefrontal cortex(PFC): 97.56 ± 0.31 %; CA1: 97.58 ± 0.13 %), as only2.4 % of Tcf4-expressing cells lacked detectable GFP mRNA(Figures 1F,H: PFC: 2.44 ± 0.32 %; CA1, 2.42 ± 0.13 %).These results verify that the GFP expression in Tcf4LGSL/+ micefaithfully reports Tcf4 expression.

Comparison of GFP Reporter and TCF4AntibodiesOf commercially available TCF4 antibodies, only one has beenvalidated for immunostaining using homozygous Tcf4 knock-outtissues (Jung et al., 2018). We used this antibody to visualizeTCF4-expressing cells in WT brain. We observed weak proteinsignals in brain cell nuclei at postnatal day (P) 7 (Figure 2A).Under identical experimental conditions, we failed to detectappreciable TCF4 protein signals at P15 and P80 (Figures 2B,C).TCF4 expression may dwindle to undetectable levels, or ceasealtogether, over the course of brain maturation. To distinguishbetween these possibilities, we performed ISH for Tcf4 in age-matched WT brains. We observed comparable numbers of Tcf4-expressing cells between neonatal and adult brains (Figures 2D–F), indicating expression of Tcf4 transcript persisted in mostcells across postnatal development, albeit likely at reduced levels.Thus, failure to immunodetect TCF4 protein in adult brain is dueto the limited sensitivity of the TCF4 antibody, not the absence ofthe target protein.

To directly compare sensitivities for detecting TCF4 andGFP antibodies, we performed double immunohistochemistry inbrain sections of Tcf4LGSL/+ mice, from birth into adulthood.GFP and TCF4 labeling patterns were similar across postnataldevelopment, though GFP labeling was of visually greaterintensity than TCF4 labeling (Figures 2G–I). The disparity inlabeling intensity was also apparent at P10 and was even morepronounced by adulthood when TCF4 labeling outside of the

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FIGURE 2 | GFP reporter enhances sensitivity to detect TCF4 by immunohistochemistry. A–C Immunostaining and (D–F) ISH of TCF4/Tcf4 and DAPI from P7, P15,and P80 mouse cortex. Immunostaining shows decreased detection of TCF4 protein using TCF4 antibody, whereas ISH shows comparable number of Tcf4expressing cells during postnatal development. Scale bars = 20 µm. G–I Dual immunostaining of P0, P7, and P80 using TCF4 and GFP antibodies in coronalsections from Tcf4LGSL/+ mice. Image is taken from the same double-labeled section. Scale bar = 1 mm.

hippocampus was barely detectable (Figures 2H,I). We alsodetected GFP labeling within axonal projections (Figure 2G).These data highlight advantages of the GFP reporter — increasedsensitivity and the capacity to track the axonal projectionsof TCF4-expressing neurons—for mapping TCF4 expressionpatterns across all postnatal ages.

TCF4 Expression Patterns of the AdultMouse BrainTo examine adult patterns of TCF4 expression, we stained forGFP across the rostral to caudal extent in coronal sectionsfrom Tcf4LGSL/+ mice (Figures 3A–H). We observed themost prominent GFP labeling intensity in the pallial region,

which contains the olfactory bulb, cortex, and hippocampus(Figures 3B–G). Cells in the glomerular (gm), external plexiform(pl), and granule layers (gr) of the olfactory bulb (OLF) werestrongly labeled with GFP (Figure 3A). Throughout the entirecortex, intense GFP staining was seen in almost all areasand in every layer (Figures 3B–G,I). Expression was strongin the hippocampus, especially in the pyramidal cell layer ofAmmon’s horn (Figure 3J), and in the cerebellum, highlightedby concentrated GFP labeling in the molecular (mo) and granulecell (gl) layers (Figures 3H,M).

While the entire pallial region and cerebellum stainedintensely for GFP, subsets of other brain regions were lightlyand sparsely labeled for GFP. In the pallial derivatives, cellsin the basolateral amygdala nucleus (BLA) and claustrum

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FIGURE 3 | TCF4 expression patterns of adult mouse brain. A–H DAB immunostaining of GFP (for TCF4) in coronal brain sections of adult Tcf4LGSL/+ mice. I–NHigh magnification view of CRX, CA1, CP, TH, and CBX. TCF4-expressing cells are prominently found in CRX, CA1, and CBX. Scale bars = 1 mm and 200 µm forhigher magnification insets. The list of abbreviations used is provided in Table 2.

(CLA) were stained for GFP. In the subpallial derivatives, wedetected GFP-positive cells in the central amygdala nucleus(CEA) and medial amygdala nucleus (MEA) (Figures 3C–E).We also noted GFP labeling of cells in the caudoputamen (CP),nucleus accumbens (ACB), lateral septal nucleus (LS), medial

septal complex (MS), and nucleus of the diagonal band (NDB)(Figures 3B,C,K), although this labeling was much lighter, andthe stained cell density was much lower than what we observedin the pallial region. In the hypothalamus, we observed thehighest density of GFP-expressing cells in posterior hypothalamic

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TABLE 2 | Abbreviations.

ACB Nucleus accumbens

BLA Basolateral amygdalar nucleus

CA1 Cornu ammonis1

CA3 Cornu ammonis3

CBX Cerebellum

CC Corpus callosum

CEA Central amygdalar nucleus

CLA Claustrum

CP Caudate putamen

CRX Cortex

CS Superior central nucleus raphe

egl External granule layer of cerebellum

gl Granule layer of cerebellum

gm Glomerular layer of olfactory bulb

gr Granule layer of olfactory bulb

HY Hypothalamus

igl Inner granule layer of cerebellum

LSr Lateral septal nucleus, rostral (rostroventral) part

MB Midbrain

MEA Medial amygdalar nucleus

MH Medial habenula

ml Molecular layer of cerebellum

MM Medial mammillary nucleus

MS Medial septal nucleus

NDB Diagonal band nucleus

OLF Olfactory bulb

PAG Periaqueductal gray

PFC Prefrontal cortex

PG Pontine gray

PIR Piriform area

pl Plexiform layer of olfactory bulb

PRP Nucleus prepositus

SC Superior colliculus

SPV Spinal nucleus of the trigeminal

STR Striatum

TH Thalamus

VC Visual cortex

VNC Vestibular nuclei

ZI Zona incerta

nucleus (PH) (Figures 3D–E). In the diencephalic prosomeres,the medial habenula (MH) stood out for its strong GFPlabeling intensity (Figure 3D), contrasting sharply with otherthalamic nuclei that were generally devoid of detectable GFP(Figures 3D,E,L). In the prethalamic structure, we observed GFP-positive cells in zona incerta (ZI). In the midbrain, GFP labeledcells in periaqueductal gray (PAG) and superior colliculus (SC)(Figures 3E–G). In the hindbrain, we observed GFP-expressingcells in the superior central nucleus raphe (CS), pontine gray(PG), vestibular nuclei (VNC), nucleus prepositus (PRP), andspinal nucleus of the trigeminal (SPV) (Figures 3G,H).

The contrast in labeling intensity of GFP detected in the pallialregion along with cerebellum and the rest of the brain suggestsdifferences in TCF4-expressing cell densities. To compare theexpression across different brain regions, we fluorescently labeled

Tcf4 in adult WT tissues via ISH and quantified Tcf4-containingcells. We detected Tcf4 signals in all examined brain regions,including CA1, visual cortex (VC), BLA, PFC, CP, and TH(Figure 4). Consistent with our qualitative observations of GFPlabeling intensity (Figure 3), the percentage of cells expressingTcf4 transcript was dramatically higher in CA1, VC, BLA, andPFC compared to CP and TH (Figure 4).

TCF4 Expression Patterns of theNeonatal and Juvenile Mouse BrainWe investigated the spatial dynamics of TCF4 expressionduring postnatal brain development by examining GFP reporterexpression at P1, P10, P20, and P60. At P1, the pallial regionstood out with the strongest GFP staining. Other derivatives fromprosencephalon, mesencephalon, and rhombencephalon werealso stained for GFP. Cell densities were lower in these derivativesthan the pallial region. The lowest level of GFP expressionwas detected in the thalamus and inferior colliculus. Intenselylabeled axonal projections were unique to the P1 timepoint. Mostnotably, some GFP-stained axons were extended from the corticalneurons into discrete thalamic nuclei. Other GFP-stained corticalaxons were extended to invade the hypothalamus and pons(Figure 5A). We also detected the cerebral peduncle intenselystained for GFP. These labeling patterns demonstrate that, at anearly postnatal stage, corticothalamic and subcerebral projectionneurons expressed TCF4. Additionally, axons coursing throughthe corpus collosum, fimbria, internal capsule, fornix, andanterior commissure were labeled strongly for GFP (Figures 5A,2G). GFP expression remained high in the pallial regionand cerebellum at P10. We also detected GFP-expressingcells throughout the hypothalamus, midbrain, and hindbrain.Strikingly, GFP expression level was slightly increased in thethalamus at this age compared with P1 (Figure 5B). This slightincrease is potentially caused by axonal fibers spreading intothe midline nuclei. A similar pattern of corticothalamic fiberswas reported at this age in transgenic mice that drive GFP inearly cortical preplate and subplate neurons (Jacobs et al., 2007).At P20, GFP expression level was reduced in the thalamus,hypothalamus, midbrain, and hindbrain. The pallial region,cerebellum, and some hindbrain and hypothalamic nuclei wereintensely stained for GFP (Figure 5C). The expression patternobserved in P20 brain was conserved in P60 brain, althoughthe overall expression level of P60 brain was slightly decreasedcompared with P20 brain. Our data show that high levels ofGFP labeling were persistently detected in the pallial region andcerebellum in all ages (Figure 5). These data suggest that TCF4could be involved in early stages of neuronal development acrossthe entire brain, but as the brain matures, TCF4 function becomesincreasingly restricted to the pallial region and cerebellum.

Glutamatergic and GABAergic Cells,Astrocytes, and OligodendrocytesExpress TCF4 in the Prefrontal CortexWe used the GFP reporter line to characterize the cell type-specific expression of TCF4 in the PFC. GABAergic andglutamatergic neurons represent two major neuronal classes that

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FIGURE 4 | Quantification of Tcf4-expressing cells in multiple brain regions of adult WT brain. Representative ISH images of Tcf4 and DAPI and proportionality ofTcf4-positive (magenta) and -negative (white) cell populations in CA1, VC, BLA, PFC, STR, and TH. Tcf4 mRNA is present at high levels in the CA1, VC, BLA, andPFC. The total numbers in the pie chart center represent the quantified DAPI cells per brain region. Values represent the mean percentages. n = 3 mice. Scalebars = 20 µm.

we could more easily distinguish upon reciprocal Cre deletion,which succeeded in eliminating expression of the GFP reporterone class at a time. We generated LGSL::Gad2-Cre mice todelete GFP expression from GABAergic neurons (Taniguchiet al., 2011). We detected relatively light GFP staining inputative glutamatergic neurons throughout the cortical layers(Figure 6A). We also generated LGSL::Nex-Cre mice to deleteGFP selectively from forebrain glutamatergic neurons (Goebbelset al., 2006). We detected strong residual labeling in GABAergiccells (Figure 6B). To confirm that Tcf4 expression is ubiquitousin these neuronal classes, we performed double ISH in adultWT PFC for Tcf4 in combination with either vGlut1 or vGat,which encode the vesicular transporters for glutamate andGABA, respectively. We found almost all vGlut1- and vGat-expressing cells contained Tcf4 (Figures 6C–H). These findingssuggested that TCF4 may be ubiquitously expressed in corticalglutamatergic and GABAergic cell populations.

Nearly all cortical GABAergic interneurons belong to one ofthree groups defined by the expression of parvalbumin (PV),somatostatin (SOM), and the ionotropic serotonin receptor5HT3a (5HT3aR) (Rudy et al., 2011). Each group differsin its morphological and electrophysiological properties andplays unique roles in cortical circuit function (DeFelipe, 1993;Gonchar and Burkhalter, 1997; Markram et al., 2004). Todetermine whether TCF4 is expressed in specific GABAergic

interneuron subtypes, we performed coimmunostaining for GFPand representative subgroup-specific markers in the juvenileand adult LGSL::Nex-Cre mice. There are currently no suitableantibodies for staining 5HT3aR, so we chose vasoactive intestinalpeptide (VIP) as an alternative marker, which is expressed byapproximately half of all 5HT3aR-expressing neurons (Lee et al.,2010; Rudy et al., 2011). We found that nearly all SOM, PV, andVIP labeled interneurons were copositive with GFP in the PFC(Figures 6I–K) at P20 and P80, suggesting that TCF4-expressingGABAergic cells consist of SOM, PV, and VIP interneurons.

Over the course of our study, we observed that a subset ofGFP-stained cells did not stain positive for NeuN (data notshown), indicating that TCF4 may be expressed in glial cellpopulations. We costained for GFP and either the astrocytemarker glial fibrillary acid protein (GFAP), or the microgliamarker ionized calcium binding adaptor molecule 1 (IBA1),in LGSL::Nex-Cre mice. GFP/GFAP copositive astrocytes werepresent throughout the PFC of both juvenile and adult mice(Figure 6L). However, GFP-stained glia did not costain forIBA1 (Figure 6M). Due to the recently established rolefor TCF4 in regulating the maturation of oligodendrocyteprogenitors (Phan et al., 2020), we expected that TCF4 wouldbe expressed in oligodendrocyte lineage cells. Olig2 marks allstages of oligodendrocyte lineage, and APC (or CC1) marksthe maturational process (Bhat et al., 1996). The majority

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FIGURE 5 | TCF4 expression patterns of the neonatal and juvenile mousebrain. A–D DAB immunostaining of GFP (for TCF4) in sagittal brain sections ofTcf4LGSL/+ mice at P1, P10, P20, and P60. A similar staining pattern largelypersists throughout postnatal development. Scale bars = 1 mm.

of Olig2/APC positive cells, reflecting mature, myelinatingoligodendrocytes, stained for GFP in the PFC and corpuscallosum at P20 (Figure 6N). Similarly, a subset of immatureoligodendrocytes, labeled only by Olig2, stained for GFP(Figure 6N). Our results show that among major glial cellpopulations in the brain, astrocytes and both immature andmature oligodendrocytes express TCF4, while microglia appearto lack TCF4 expression.

Pyramidal Cells, GABAergicInterneurons, and Astrocytes ExpressTCF4 in the HippocampusTcf4 deficient mice exhibited deficits in the behavioral tasksthat require proper hippocampal functions. Additionally, aform of hippocampal synaptic plasticity was altered in these

mice (Kennedy et al., 2016; Thaxton et al., 2018). Therefore,we characterized TCF4-expressing cell types in this brainregion to reveal which cell types might contribute to thesephenotypes. First, we examined glutamatergic and GABAergiccell populations by staining for GFP in LGSL::Gad2-Creand LGSL::Nex-Cre mice. As expected from our ISH data(CA1, Figure 4), glutamatergic pyramidal cells of the CA1region exhibited strong GFP labeling (Figure 7A). Moreover,we detected strong residual labeling in GABAergic cellsacross the layers (Figure 7B). The hippocampal GABAergicinhibitory circuits consist of SOM-, PV-, VIP-, neuropeptide Y-,calretinin-, and cholecystokinin-expressing interneurons (Pelkeyet al., 2017). We tested whether some of these inhibitoryinterneurons expressed TCF4 by performing coimmunostainingin LGSL::Nex-Cre brain. We found that SOM-, PV- andVIP-positive neurons stained for GFP at P20 and P80(Figures 7C–E). GFP staining in LGSL::Nex-Cre mice revealedclearly identifiable star-shaped cells (rad. layer, Figure 7B).Our coimmunostaining result showed that GFAP-positiveastrocytes stained for GFP (Figure 7F). But, IBA-positivemicroglial cells were devoid of GFP (Figure 7G). Our resultsdemonstrated that TCF4-expressing hippocampal cell groupsconsist of astrocytes, pyramidal cells, and SOM-, PV-, and VIP-containing interneurons.

SOM and PV Interneurons andAstrocytes Express TCF4 in the StriatumThe vast majority of striatal neurons signal through GABAto inhibit their target cells (Koos and Tepper, 1999; Gittiset al., 2010). Because we observed that only ∼19% of striatalcells express Tcf4 (STR, Figure 4), we speculated that thesewould comprise specific subgroups of GABAergic neurons. Usingdouble ISH, we detected Tcf4 signals in a subset of vGat-expressing cells (Figures 8A–C). We subsequently employeda double immunostaining approach in juvenile and adultTcf4LGSL/+ mice to further define TCF4-expressing GABAergicpopulation. We found that the GFP-labeled cells were notcolocalized with medium spiny neurons (MSNs), marked byDARPP32 (Figure 8D), indicating that GABAergic MSNsdo not express TCF4. Cholinergic interneurons, marked bycholine acetyltransferase (ChAT), represent another major cellGABAergic class in the striatum in which GFP was notexpressed (Figure 8E). SOM and PV expression characterizesother GABAergic interneuron types in the striatum (Munoz-Manchado et al., 2018). We detected GFP in SOM- and PV-positive interneurons at P20, and this colocalization persisted inthe adult striatum (Figures 8F,G). Interestingly, a few SOM orPV positive cells did not stain for GFP, raising the possibility thatTCF4 expression could confer unique functional properties tosubsets of PV and SOM interneurons. We showed earlier in thisstudy that TCF4 was expressed in astrocytes, but not microglialcells, in the cortex and hippocampus (Figures 6L,M, 7F,G).Thus, we asked whether this expression pattern also applied tothe striatum. We detected GFP in GFAP-positive cells, but notin IBA1-positive cells (Figures 8F,G). Collectively, these datasuggest that TCF4 expression in the striatum is restricted to PV

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FIGURE 6 | Glutamatergic, GABAergic cells, astrocytes, and oligodendrocytes express TCF4 in the PFC. A, B DAB immunostaining of GFP (for TCF4) in coronalsections of P80 LGSL::Gad2-cre or LGSL::Nex-cre mice where GFP protein is deleted in inhibitory or excitatory neurons, respectively. Both glutamatergic andGABAergic cells express TCF4. Scale bars = 0.5 mm. C–E, F–H Dual ISH for vGult1 and Tcf4 and for vGat and Tcf4 in P80 WT brain tissue, confirming that Tcf4mRNA is present in vGlut1- and vGat-expressing cells. Scale bars = 20 µm. I–K Dual immunostaining of interneuron subtype-specific markers, SOM, PV, or VIP, andGFP (for TCF4) in P20 and P80 LGSL::Nex-cre mice. TCF4 is expressed in nearly all SOM-, PV-, and VIP-positive interneurons (arrows). Asterisk represents rareinterneuron that does not express GFP. Scale bars = 30 µm. L, M: Dual immunostaining of astrocyte marker, GFAP, or microglial marker, IBA1, and GFP (for TCF4) inP20 and P80 LGSL::Nex-cre mice. GFAP-labeled cells express GFP (arrows), but IBA1-labeled cells do not express GFP (asterisk). Scale bars = 30 or 10 µm forhigher magnification insets. N Triple immunostaining of APC, Olig2, and GFP (for TCF4) in the PFC (top panel) and corpus callosum (bottom panel) of P20LGSL::Nex-cre mice. TCF4 is expressed in mature (arrow) and immature (double arrow) oligodendrocytes. Scale bars = 20 µm.

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FIGURE 7 | Pyramidal cells, GABAergic interneurons, and astrocytes express TCF4 in the hippocampus. A, B DAB immunostaining of GFP (for TCF4) in the CA1 ofP80 LGSL::Gad2-cre or LGSL::Nex-cre mice. Both pyramidal layer cells and GABAergic cells express TCF4. Scale bars = 0.5 mm. C–E Dual immunostaining ofinterneuron subtype-specific markers, SOM, PV, or VIP, and GFP (for TCF4) in P20 and P80 LGSL::Nex-cre mice. TCF4 is expressed nearly all SOM-, PV-, orVIP-positive interneurons (arrows). F, G Dual immunostaining of GFAP or IBA1, and GFP (for TCF4) in P20 and P80 LGSL::Nex-cre mice. GFAP-labeled cells expressGFP (arrow), but IBA1-labeled cells do not express GFP (asterisks). Scale bars = 30 or 10 µm for higher magnification insets.

and SOM interneurons and astrocytes, but not to medium spiny,cholinergic neurons, and microglial cells.

TCF4 Is Enriched in the Molecular andGranule Cell Layer of the CerebellarCortexWe consistently observed strong GFP immunoreactivity in thecerebellum across postnatal development (Figures 3M, 5). Thus,we further characterized TCF4 distribution in this structure,focusing on the molecular, Purkinje cell, and granule cell layers.At P10, a timepoint of ongoing cerebellar histogenesis (Altman,1969), we found that GFP was enriched in the extracellular areaof the molecular layer and inner granule layer, but absent in the

external granule layer and Purkinje cell layer (Figure 9A). NeuNstaining clearly marked neurons with a multipolar morphology,presumably traversing the molecular layer toward the innergranule layer (Figure 9B). These cells were negative for GFP(Figures 9A–C), suggesting that migrating granule cells donot express TCF4. In the inner granule layer, where post-migratory granule cells undergo maturation, we infrequentlyfound NeuN-positive cells that costained with GFP (Figures 9A–C). By adulthood, however, nearly all NeuN-positive neuronsin the granule layer costained for GFP (Figures 9E–G), leadingus to surmise that cerebellar granule cells only upregulateTCF4 expression as they mature. Regardless of age, GABAergicPurkinje cell bodies, labeled by calbindin, lacked GFP staining(Figures 9D,H). Consistent with our GFP immunostaining

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FIGURE 8 | StriatalQ12 interneurons, but not medium spiny neurons, express TCF4. A Representative ISH images for Tcf4 and vGat from adult WT striatum, showingthat specific subtypes of interneurons express Tcf4 (arrows). Scale bar = 20 µm. D–G Dual immunostaining of DARPP32, ChAT, SOM, or PV and GFP (for TCF4) inP20 and P80 Tcf4LGSL/+ mice. The representative staining images reveal that SOM- and PV-positive subtype interneurons express TCF4 (arrow). Asterisks representonly GFP-positive neurons. Double arrows represent interneuron subtypes that do not express GFP. Scale bars = 20 µm. H, I Dual immunostaining of GFAP or IBA1,and GFP (for TCF4) in P20 and P80 Tcf4LGSL/+ mice. GFAP-labeled cells express GFP (arrow), but IBA1-labeled cells do not express GFP (asterisk). Scale bars = 30or 10 µm for higher magnification insets.

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results, ISH for Tcf4 in adult wildtype cerebellum confirmed thatmost granule cells expressed Tcf4, while GABAergic Purkinjecells did not (Figures 9I–K). We also detected Tcf4-expressingcells in most GABAergic interneurons of the molecular layer(Figures 9I–K).

DISCUSSION

It is imperative to understand the cellular distribution of TCF4during postnatal development in order to guide the delivery oftherapeutics for TCF4-linked disorders. Toward this goal, wedeveloped a mouse with a TCF4-GFP reporter that conferredgreater sensitivity for detecting TCF4 expression than existingantibody detection methods (Figure 2). We validated the TCF4-GFP reporter mouse model by using double in situ labelingto show that about 98% of Tcf4-containing cells express GFP,proving the mouse model as a faithful reporter for TCF4(Figures 1E–H). While the GFP reporter was designed to diffusefreely through the cytoplasm, and thus is not a marker of TCF4subcellular localization, the reporter offers the advantage thatit can label dendritic arborizations and axonal projections ofTCF4-expressing neurons (Figures 2G, 5A). To improve ourability to observe TCF4-expressing cell types, we conditionallydeleted the GFP reporter in a Cre-dependent manner. Thisallowed us to more easily observe the remaining GFP-positivecells with an improved signal to noise ratio (Figures 6A,B,7A,B). We used these approaches, coupled with double-labelingimmunohistochemistry and in situ hybridization, to characterizethe cell type-specific and spatiotemporal expression of TCF4 inthe postnatal mouse brain.

TCF4 Expression Patterns and TheirImplications in Pathology ofTCF4-Linked DisordersCommon genetic variants in and around TCF4 are associatedwith a range of neurodevelopmental and psychiatric disorders.Rare TCF4 single nucleotide variants have been described inschizophrenia patients whose symptoms include impairmentsof attention, memory, social cognition, and executive functions(Basmanav et al., 2015; Forrest et al., 2018). TCF4 mutationshave been found in large-scale genotyping studies in patientswith intellectual disability and autism spectrum disorder (ASD)(Kharbanda et al., 2016; Maduro et al., 2016). Haploinsufficiencyof TCF4 causes PTHS – a rare form of intellectual disabilityassociated with characteristic facial features and motor andspeech dysfunction (Goodspeed et al., 2018; Zollino et al.,2019). Collectively, these studies implicate TCF4 in a range ofbrain disorders that are commonly associated with cognitivedysfunction. The prefrontal cortex is linked with a rangeof cognition including cognitive control, lower-level sensoryprocessing, memory, and motor operations (Miller, 2000). Thehippocampus supports learning and memory functions in aspatiotemporal context (Dupret et al., 2010; Rubin et al., 2014).The prefrontal cortex and hippocampus are thus suspectedpathophysiological loci for TCF4-linked disorders. TCF4 isenriched in most cortical and hippocampal cells, including

excitatory and inhibitory neurons, as well as astrocytes, andoligodendrocytes, in the juvenile and adult mouse brain(Figures 5–7). These findings in TCF4-expressing cell groupssupport the idea that functions of the prefrontal cortex andhippocampus are particularly susceptible to subtle changes inTCF4 expression. TCF4 loss is associated with defects in corticalcell positioning, dendritic spines, and arborizations (Chen et al.,2016; Li et al., 2019). TCF4 haploinsufficiency results in reducedhippocampal volume and cortical thickness in mice (Jung et al.,2018). These structural phenotypes are likely linked to functionalconsequences, including abnormal neuronal excitability andsynaptic plasticity in the prefrontal cortex and hippocampus,which are consistently observed across multiple PTHS mousemodels (Kennedy et al., 2016; Rannals et al., 2016; Thaxton et al.,2018). These cell physiological defects in turn likely contribute tothe impairments in cognition and memory functions in patientswith TCF4-linked disorders.

Severe motor delay and stereotypic behavior are consistentphenotypes observed in patients with PTHS (Goodspeed et al.,2018; Zollino et al., 2019). However, the potential mechanismunderlying motor deficits and stereotypies remains unknown.The striatum is involved in translating cortical activity intoadaptive motor actions and controlled movement (Kreitzer andMalenka, 2008). At the circuit levels, some striatal interneuronsreceive direct cortical afferents. For example, activity of striatalPV interneurons, known to inhibit MSNs, are enhanced bycortical stimulation. Regardless of cortical projections, SOMinterneurons locally target MSNs and ChAT-positive neurons(Straub et al., 2016). TCF4 is expressed in PV and SOMinterneurons, but not in MSNs and ChAT-positive neurons(Figure 8), suggesting that TCF4 loss may alter striatal circuitfunctions through PV and SOM interneurons. Disruptionsin GABAergic circuits of the striatum have been found inneuropsychiatric disorders and autism (Maia and Frank, 2011;Rapanelli et al., 2017; Skene et al., 2018). Further experiments willbe required to determine whether GABAergic circuit dysfunctionoccurs with TCF4 loss, and if so, whether it is the direct cause ofmotor delay and stereotypic behaviors.

The cerebellum contributes to motor coordination, cognitiveprocessing and emotional control (Schmahmann and Caplan,2006). It is structurally and functionally abnormal in patientsdiagnosed with ASD and other neurodevelopmental disorders(Rogers et al., 2013). Cognitive functions are impaired inindividuals with developmental reductions in cerebellar volume.Also, the degree of volume reduction is correlated with the degreeof cognitive impairment (Steinlin, 2008; Bolduc et al., 2012).Patients with PTHS display reduced volume of the cerebellum(Peippo et al., 2006; Whalen et al., 2012), which may contributeto severity of cognitive and motor impairment. The adult humancerebellum expresses high levels of TCF4 (Jung et al., 2018).Similar to the human brain, TCF4 is prominently expressedin the mouse cerebellum during postnatal development andin adulthood (Figures 3H, 5). Our data thus suggest that thecerebellum is a candidate brain region that needs to be evaluatedto determine whether TCF4 regulates cerebellar structure, andperhaps function. We found that differentiated and migratinggranule cells repress TCF4 expression, while post-migratory

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FIGURE 9 | Cerebellar granule and molecular layer cells, but not Purkinje cells, express TCF4. A–H Triple immunostaining of GFP (for TCF4), NeuN, and Purkinje cellmarker, calbindin, in P20 and P80 Tcf4LGSL/+ mouse cerebellum. The representative images confirm that migrating NeuN-positive granule cells in the molecular layer(ml) lack TCF4 (double arrows), and post-migratory granule cells in the inner granule layer (igl) express TCF4 (arrows). Purkinje cells do not express TCF4.egl = External granule layer. I–K Representative ISH images for Tcf4 and DAPI in WT adult cerebellum, showing that Tcf4 mRNA is present in granule and molecularlayer (gl and ml) cell nuclei, but it is absent in Purkinje cell nuclei (dashed line). Scale bars = 30 µm.

mature granule cells upregulate TCF4 expression (Figures 9A–C,E–G). Our findings indicate that TCF4 is positioned tomodulate maturation of the granule cells after migration. Futurestudy will need to address whether TCF4 loss or dysfunctionalters cerebellar anatomy and local circuit function, and if so,whether changes in cerebellar circuit directly cause motor andcognitive deficits.

Neurons are produced in the proliferative ventricular zone(VZ) and the subventricular zone (SVZ) of the embryonictelencephalon during development of the cortex (Bystron et al.,2008). These neurons migrate along radial glia fibers throughthe intermediate zone to form six-layer laminar structures(Rakic, 1972; Rakic et al., 2009). Differentiation and synapseformation occur once neurons are properly positioned (Katzand Shatz, 1996; Bystron et al., 2008; Frank and Tsai, 2009).Alterations in any of these processes are involved in pathogenesisof neurodevelopmental disorders such as autism, intellectualdisability, and schizophrenia (Fan et al., 2013; Fang et al., 2014;Stoner et al., 2014). TCF4 is present in the VZ/SVZ of the dorsaltelencephalon at an early embryonic stage in both humans andmice (de Pontual et al., 2009; Jung et al., 2018). The mouse

cortex produces TCF4 protein at the highest level during earlyembryonic and neonatal development (Chen et al., 2016). Ourpostnatal immunostaining study shows that TCF4 is upregulatedin the mouse cortex at birth, but as mice age, it is downregulated(Figure 5). After birth and through the first 7 to 10 days ofpostnatal development, cells undergo migration, differentiation,and maturation processes. Therefore, TCF4 is well positioned toinfluence these critical steps of corticogenesis. TCF4 loss delaysneuronal migration, resulting in a thin cortical upper layer (Liet al., 2019). Beyond migration, dendritic and synaptic formationare abnormal in Tcf4 haploinsufficient mice (Li et al., 2019).These previous and current findings suggest that TCF4 may bean upstream gene of the molecular network regulating migrationand maturation processes.

Spatial specificity of axonal projections across different brainregions is important for normal brain development and function(Abelson et al., 2005; Matsuda and Cepko, 2007; Mortazavi et al.,2008), and TCF4 could be positioned to affect such projections.The TCF4 reporter mouse allowed us to visualize projectingaxons, as the GFP reporter was free to diffuse throughoutthe cytoplasmic compartment (Figure 1A). The GFP reporter

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revealed corticothalamic projections and what appeared to bethe corticospinal and corticobulbar tracts (Figure 5A). Becausecorticothalamic neurons are largely localized in layer 6, and thecorticospinal and corticobulbar tracts are largely localized tolayer 5 (Chen et al., 2005; Jacobs et al., 2007), our data suggestthat TCF4 may be expressed in both layer 5 and 6 projectionneurons, although additional experiments will be required todirectly confirm this. Several studies demonstrated that TCF4regulates the laminar pattern and structure of the cortex (Chenet al., 2016; Li et al., 2019), and our findings suggest thatTCF4 may also be critical to the development of corticofugalprojections. To test this possibility, the consequences of TCF4 losson axonal projections during embryonic development need to bethoroughly examined.

Insights Into Genetic NormalizationStrategies to Treat TCF4-RelatedDisordersTCF4 is a major transcription modulator that differentiallycontrols the expression of hundreds of genes (Forrest et al.,2013; Hill et al., 2017; Xia et al., 2018). Thus, it is whollyimpracticable to develop therapeutic tools that adjust thedosage of each impacted gene. Ideally, TCF4-linked disorderscan be treated by normalizing TCF4 gene expression levels.A slight upregulation of TCF4 rescues learning and memoryphenotypes in adult PTHS mouse model (Kennedy et al., 2016).Studies from similar neurodevelopmental disorders, includingRett and Angelman syndrome, show that reinstatement ofaffected gene expression can provide therapeutic benefits (Guyet al., 2007; Silva-Santos et al., 2015; Sinnett et al., 2017).These convergent lines of evidence support the idea thatTCF4-linked disorders can benefit from normalizing TCF4levels. Gene therapy using adeno-associated virus (AAV) hasbeen clinically tested as a potential therapeutic interventionfor genetic disorders (Deverman et al., 2018; Hudry andVandenberghe, 2019). In principle, disorders linked to the lossof TCF4 function should be amenable to correction followingtreatment with viral vectors coding for TCF4. Key experimentalparameters requiring AAV-mediated gene therapy strategiesinclude distribution of viral vector and the age at time oftreatment. TCF4 is distributed in nearly all neurons, astrocytes,and oligodendrocytes in the forebrain at all ages (Figures 6, 7). Incontrast, only selective cell types express TCF4 in the striatum,thalamus, midbrain, hindbrain, and cerebellum (Figures 3–5,8). Optimal design of viral vectors will thus require carefulchoice of promoter, capsid, and delivery method to promoteexpression in forebrain neurons over other brain regions.Moreover, microglial cells, medium spiny neurons, ChAT-positive striatal cells, and Purkinje cells lack TCF4 expression(Figures 6–9). Thus, a major challenge for successful therapyis avoiding upregulation of TCF4 in these cell types, as it isunclear how TCF4 expression in these cells will modify thetranscriptional machinery.

The other critical parameter that must be considered intreating TCF4-linked disorders is timing of TCF4 expression.Based on the expression profiling of TCF4 (Figure 5;

Jung et al., 2018), we predict that earlier interventions willhave a larger therapeutic impact on TCF4-linked disorders.After proliferation and maturation, which occur in the prenataland neonatal periods, there is no need to increase the numberof neurons in the brain, except for the hippocampal dentategyrus. Therefore, after the critical timepoint of neurogenesisand synaptogenesis, the brain undergoes limited plastic changes(Bystron et al., 2008; Budday et al., 2015). Late onset therapiesare unlikely to exert as dramatic a phenotypic improvementcompared to early intervention, yet partial improvement ofsome phenotypes in adults or prevention of disease progressionwould be significant achievements. Our novel TCF4 conditionalmouse model allows us to reinstate wildtype Tcf4 under itsown promoter and regulatory elements (Figure 1A). Usingthis powerful tool, future experiments must be performed todetermine the latest age by which normalizing TCF4 expressioncan improve or even rescue PTHS-associated phenotypes.

DATA AVAILABILITY STATEMENT

The Q8raw data supporting the conclusions of this article will bemade available by the authors, without undue reservation.

ETHICS STATEMENT

The Q9animal study was reviewed and approved by Colleen Fritsche,UNC at Chapel Hill.

AUTHOR CONTRIBUTIONS

HK Q10designed the experiments with guidance from BP, performedthe experiments and analyzed the data. NB and NO performedin situ hybridizations and provided histological assistance. HKand BP wrote the manuscript. All authors contributed scientificinsights and provided critical readings of the manuscript.

FUNDING

This work was supported by the Pitt Hopkins ResearchFoundation and NINDS grant R01NS114086 to BP Microscopywas performed at the Neuroscience Microscopy Core Facility,supported, in part, by funding from the NIH-NINDSNeuroscience Center Support Grant P30 NS045892 and theNIH-NICHD Intellectual and Developmental DisabilitiesResearch Center Support Grant U54 HD079124.

ACKNOWLEDGMENTS

We thank Matthew C. Judson, Mason T. Riley, and Alain C.Burette for critical readings of the manuscript, Dale Cowley atthe UNC Animal Models Core for designing and generating thenew mouse model, Bonnie Taylor-Blake for sharing some of theantibodies, and Klaus-Armin Nave for providing Nex-Cre mice.

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REFERENCESAbelson, J. F., Kwan, K. Y., O’Roak, B. J., Baek, D. Y., Stillman, A. A.,

Morgan, T. M., et al. (2005). Sequence variants in SLITRK1 are associatedwith Tourette’s syndrome. Science 310, 317–320. doi: 10.1126/science.1116502

Altman, J. (1969). Autoradiographic and histological studies of postnatalneurogenesis. IV. Cell proliferation and migration in the anteriorforebrain, with special reference to persisting neurogenesis in theolfactory bulb. J. Comp. Neurol. 137, 433–457. doi: 10.1002/cne.901370404

Amiel, J., Rio, M., de Pontual, L., Redon, R., Malan, V., Boddaert, N.,et al. (2007). Mutations in TCF4, encoding a class I basic helix-loop-helixtranscription factor, are responsible for Pitt-Hopkins syndrome, a severeepileptic encephalopathy associated with autonomic dysfunction. Am. J. Hum.Genet. 80, 988–993. doi: 10.1086/515582

Basmanav, F. B., Forstner, A. J., Fier, H., Herms, S., Meier, S., Degenhardt, F.,et al. (2015). Investigation of the role of TCF4 rare sequence variants inschizophrenia. Am. J. Med. Genet. B Neuropsychiatr. Genet. 168B, 354–362.doi: 10.1002/ajmg.b.32318

Bedeschi, M. F., Marangi, G., Calvello, M. R., Ricciardi, S., Leone, F. P. C., Baccarin,M., et al. (2017). Impairment of different protein domains causes variableclinical presentation within Pitt-Hopkins syndrome and suggests intragenicmolecular syndromology of TCF4. Eur. J. Med. Genet. 60, 565–571. doi: 10.1016/j.ejmg.2017.08.004

Bhat, R. V., Axt, K. J., Fosnaugh, J. S., Smith, K. J., Johnson, K. A., Hill, D. E., et al.(1996). Expression of the APC tumor suppressor protein in oligodendroglia.Glia 17, 169–174.

Bolduc, M. E., du Plessis, A. J., Sullivan, N., Guizard, N., Zhang, X., Robertson,R. L., et al. (2012). Regional cerebellar volumes predict functional outcome inchildren with cerebellar malformations. Cerebellum 11, 531–542. doi: 10.1007/s12311-011-0312-z

Budday, S., Steinmann, P., and Kuhl, E. (2015). Physical biology of humanbrain development. Front. Cell. Neurosci. 9:257. doi: 10.3389/fncel.2015.00257

Bystron, I., Blakemore, C., and Rakic, P. (2008). Development of the humancerebral cortex: Boulder Committee revisited. Nat. Rev. Neurosci. 9, 110–122.doi: 10.1038/nrn2252

Chen, B., Schaevitz, L. R., and McConnell, S. K. (2005). Fezl regulates thedifferentiation and axon targeting of layer 5 subcortical projection neurons incerebral cortex. Proc. Natl. Acad. Sci. U.S.A. 102, 17184–17189. doi: 10.1073/pnas.0508732102

Chen, T., Wu, Q., Zhang, Y., Lu, T., Yue, W., and Zhang, D. (2016). Tcf4 ControlsNeuronal Migration of the Cerebral Cortex through Regulation of Bmp7. Front.Mol. Neurosci. 9:94. doi: 10.3389/fnmol.2016.00094

de Pontual, L., Mathieu, Y., Golzio, C., Rio, M., Malan, V., Boddaert, N., et al.(2009). Mutational, functional, and expression studies of the TCF4 genein Pitt-Hopkins syndrome. Hum. Mutat. 30, 669–676. doi: 10.1002/humu.20935

DeFelipe, J. (1993). Neocortical neuronal diversity: chemical heterogeneityrevealed by colocalization studies of classic neurotransmitters, neuropeptides,calcium-binding proteins, and cell surface molecules. Cereb. Cortex 3, 273–289.doi: 10.1093/cercor/3.4.273

Deverman, B. E., Ravina, B. M., Bankiewicz, K. S., Paul, S. M., and Sah, D. W. Y.(2018). Gene therapy for neurological disorders: progress and prospects. Nat.Rev. Drug Discov. 17:767. doi: 10.1038/nrd.2018.158

Dupret, D., O’Neill, J., Pleydell-Bouverie, B., and Csicsvari, J. (2010). Thereorganization and reactivation of hippocampal maps predict spatialmemory performance. Nat. Neurosci. 13, 995–1002. doi: 10.1038/nn.2599

Fan, Y., Abrahamsen, G., Mills, R., Calderon, C. C., Tee, J. Y., Leyton, L., et al.(2013). Focal adhesion dynamics are altered in schizophrenia. Biol. Psychiatry74, 418–426. doi: 10.1016/j.biopsych.2013.01.020

Fang, W. Q., Chen, W. W., Jiang, L., Liu, K., Yung, W. H., Fu, A. K. Y., et al.(2014). Overproduction of upper-layer neurons in the neocortex leads toautism-like features in mice. Cell Rep. 9, 1635–1643. doi: 10.1016/j.celrep.2014.11.003

Forrest, M. P., Hill, M. J., Kavanagh, D. H., Tansey, K. E., Waite, A. J., andBlake, D. J. (2018). The psychiatric risk gene transcription factor 4 (TCF4)regulates neurodevelopmental pathways associated with schizophrenia, autism,and intellectual disability. Schizophr. Bull. 44, 1100–1110. doi: 10.1093/schbul/sbx164

Forrest, M. P., Waite, A. J., Martin-Rendon, E., and Blake, D. J. (2013). Knockdownof human TCF4 affects multiple signaling pathways involved in cell survival,epithelial to mesenchymal transition and neuronal differentiation. PLoS One8:e73169. doi: 10.1371/journal.pone.0073169

Frank, C. L., and Tsai, L. H. (2009). Alternative functions of core cell cycleregulators in neuronal migration, neuronal maturation, and synaptic plasticity.Neuron 62, 312–326. doi: 10.1016/j.neuron.2009.03.029

Fu, H., Cai, J., Clevers, H., Fast, E., Gray, S., Greenberg, R., et al. (2009). A genome-wide screen for spatially restricted expression patterns identifies transcriptionfactors that regulate glial development. J. Neurosci. 29, 11399–11408. doi: 10.1523/JNEUROSCI.0160-09.2009

Gittis, A. H., Nelson, A. B., Thwin, M. T., Palop, J. J., and Kreitzer, A. C. (2010).Distinct roles of GABAergic interneurons in the regulation of striatal outputpathways. J. Neurosci. 30, 2223–2234. doi: 10.1523/JNEUROSCI.4870-09.2010

Goebbels, S., Bormuth, I., Bode, U., Hermanson, O., Schwab, M. H., and Nave, K. A.(2006). Genetic targeting of principal neurons in neocortex and hippocampusof NEX-Cre mice. Genesis 44, 611–621. doi: 10.1002/dvg.20256

Gonchar, Y., and Burkhalter, A. (1997). Three distinct families of GABAergicneurons in rat visual cortex. Cereb. Cortex 7, 347–358. doi: 10.1093/cercor/7.4.347

Goodspeed, K., Newsom, C., Morris, M. A., Powell, C., Evans, P., and Golla,S. (2018). Pitt-Hopkins syndrome: a review of current literature, clinicalapproach, and 23-patient case series. J. Child Neurol. 33, 233–244. doi: 10.1177/0883073817750490

Guy, J., Gan, J., Selfridge, J., Cobb, S., and Bird, A. (2007). Reversal of neurologicaldefects in a mouse model of Rett syndrome. Science 315, 1143–1147. doi: 10.1126/science.1138389

Hill, M. J., Killick, R., Navarrete, K., Maruszak, A., McLaughlin, G. M., Williams,B. P., et al. (2017). Knockdown of the schizophrenia susceptibility gene TCF4alters gene expression and proliferation of progenitor cells from the developinghuman neocortex. J. Psychiatry Neurosci. 42, 181–188.

Hudry, E., and Vandenberghe, L. H. (2019). Therapeutic AAV gene transfer to thenervous system: a clinical reality. Neuron 102:263. doi: 10.1016/j.neuron.2019.03.020

Jacobs, E. C., Campagnoni, C., Kampf, K., Reyes, S. D., Kalra, V., Handley, V.,et al. (2007). Visualization of corticofugal projections during early corticaldevelopment in a tau-GFP-transgenic mouse. Eur. J. Neurosci. 25, 17–30. doi:10.1111/j.1460-9568.2006.05258.x

Jung, M., Haberle, B. M., Tschaikowsky, T., Wittmann, M. T., Balta, E. A., Stadler,V. C., et al. (2018). Analysis of the expression pattern of the schizophrenia-riskand intellectual disability gene TCF4 in the developing and adult brain suggestsa role in development and plasticity of cortical and hippocampal neurons. Mol.Autism 9:20. doi: 10.1186/s13229-018-0200-1

Kamentsky, L., Jones, T. R., Fraser, A., Bray, M. A., Logan, D. J., Madden, K. L.,et al. (2011). Improved structure, function and compatibility for CellProfiler:modular high-throughput image analysis software. Bioinformatics 27, 1179–1180. doi: 10.1093/bioinformatics/btr095

Katz, L. C., and Shatz, C. J. (1996). Synaptic activity and the construction of corticalcircuits. Science 274, 1133–1138. doi: 10.1126/science.274.5290.1133

Kennedy, A. J., Rahn, E. J., Paulukaitis, B. S., Savell, K. E., Kordasiewicz, H. B.,Wang, J., et al. (2016). Tcf4 regulates synaptic plasticity, DNA methylation, andmemory function. Cell Rep. 16, 2666–2685. doi: 10.1016/j.celrep.2016.08.004

Kharbanda, M., Kannike, K., Lampe, A., Berg, J., Timmusk, T., and Sepp, M.(2016). Partial deletion of TCF4 in three generation family with non-syndromicintellectual disability, without features of Pitt-Hopkins syndrome. Eur. J. Med.Genet. 59, 310–314. doi: 10.1016/j.ejmg.2016.04.003

Koos, T., and Tepper, J. M. (1999). Inhibitory control of neostriatal projectionneurons by GABAergic interneurons. Nat. Neurosci. 2, 467–472. doi: 10.1038/8138

Kreitzer, A. C., and Malenka, R. C. (2008). Striatal plasticity and basal gangliacircuit function. Neuron 60, 543–554. doi: 10.1016/j.neuron.2008.11.005

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2052

Kim et al. TCF4 Distribution

Lee, S., Hjerling-Leffler, J., Zagha, E., Fishell, G., and Rudy, B. (2010). The largestgroup of superficial neocortical GABAergic interneurons expresses ionotropicserotonin receptors. J. Neurosci. 30, 16796–16808. doi: 10.1523/JNEUROSCI.1869-10.2010

Li, H., Zhu, Y., Morozov, Y. M., Chen, X., Page, S. C., Rannals, M. D., et al.(2019). Disruption of TCF4 regulatory networks leads to abnormal corticaldevelopment and mental disabilities. Mol. Psychiatry 24, 1235–1246. doi: 10.1038/s41380-019-0353-0

Ma, C., Gu, C., Huo, Y., Li, X., and Luo, X. J. (2018). The integrated landscapeof causal genes and pathways in schizophrenia. Transl. Psychiatry 8:67. doi:10.1038/s41398-018-0114-x

Maduro, V., Pusey, B. N., Cherukuri, P. F., Atkins, P., du Souich, C., Rupps, R., et al.(2016). Complex translocation disrupting TCF4 and altering TCF4 isoformexpression segregates as mild autosomal dominant intellectual disability.Orphanet. J. Rare Dis. 11:62. doi: 10.1186/s13023-016-0439-6

Maia, T. V., and Frank, M. J. (2011). From reinforcement learning models topsychiatric and neurological disorders.Nat. Neurosci. 14, 154–162. doi: 10.1038/nn.2723

Markram, H., Toledo-Rodriguez, M., Wang, Y., Gupta, A., Silberberg, G., and Wu,C. (2004). Interneurons of the neocortical inhibitory system. Nat. Rev. Neurosci.5, 793–807. doi: 10.1038/nrn1519

Mary, L., Piton, A., Schaefer, E., Mattioli, F., Nourisson, E., Feger, C., et al. (2018).Disease-causing variants in TCF4 are a frequent cause of intellectual disability:lessons from large-scale sequencing approaches in diagnosis. Eur. J. Hum.Genet. 26, 996–1006. doi: 10.1038/s41431-018-0096-4

Massari, M. E., and Murre, C. (2000). Helix-loop-helix proteins: regulators oftranscription in eucaryotic organisms. Mol. Cell. Biol. 20, 429–440. doi: 10.1128/mcb.20.2.429-440.2000

Matsuda, T., and Cepko, C. L. (2007). Controlled expression of transgenesintroduced by in vivo electroporation. Proc. Natl. Acad. Sci. U.S.A. 104, 1027–1032. doi: 10.1073/pnas.0610155104

Miller, E. K. (2000). The prefrontal cortex and cognitive control. Nat. Rev. Neurosci.1, 59–65. doi: 10.1038/35036228

Mortazavi, A., Williams, B. A., McCue, K., Schaeffer, L., and Wold, B. (2008).Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat.Methods 5, 621–628. doi: 10.1038/nmeth.1226

Munoz-Manchado, A. B., Bengtsson Gonzales, C., Zeisel, A., Munguba, H.,Bekkouche, B., Skene, N. G., et al. (2018). Diversity of interneurons in the dorsalstriatum revealed by single-cell RNA Sequencing and PatchSeq. Cell Rep. 24,2179–2190.e7. doi: 10.1016/j.celrep.2018.07.053

Murre, C., Bain, G., van Dijk, M. A., Engel, I., Furnari, B. A., Massari, M. E., et al.(1994). Structure and function of helix-loop-helix proteins. Biochim. Biophys.Acta 1218, 129–135. doi: 10.1016/0167-4781(94)90001-9

Peippo, M. M., Simola, K. O., Valanne, L. K., Larsen, A. T., Kahkonen, M.,Auranen, M. P., et al. (2006). Pitt-Hopkins syndrome in two patients andfurther definition of the phenotype. Clin. Dysmorphol. 15, 47–54. doi: 10.1097/01.mcd.0000184973.14775.32

Pelkey, K. A., Chittajallu, R., Craig, M. T., Tricoire, L., Wester, J. C., and McBain,C. J. (2017). Hippocampal GABAergic Inhibitory Interneurons. Physiol. Rev. 97,1619–1747. doi: 10.1152/physrev.00007.2017

Phan, B. N., Bohlen, J. F., Davis, B. A., Ye, Z., Chen, H. Y., Mayfield, B., et al. (2020).A myelin-related transcriptomic profile is shared by Pitt-Hopkins syndromemodels and human autism spectrum disorder. Nat. Neurosci. 23, 375–385.doi: 10.1038/s41593-019-0578-x

Pickard, B. S., Malloy, M. P., Clark, L., Lehellard, S., Ewald, H. L., Mors, O.,et al. (2005). Candidate psychiatric illness genes identified in patients withpericentric inversions of chromosome 18. Psychiatr. Genet. 15, 37–44. doi:10.1097/00041444-200503000-00007

Rakic, P. (1972). Mode of cell migration to the superficial layers of fetal monkeyneocortex. J. Comp. Neurol. 145, 61–83. doi: 10.1002/cne.901450105

Rakic, P., Ayoub, A. E., Breunig, J. J., and Dominguez, M. H. (2009). Decision bydivision: making cortical maps. Trends Neurosci. 32, 291–301. doi: 10.1016/j.tins.2009.01.007

Rannals, M. D., Hamersky, G. R., Page, S. C., Campbell, M. N., Briley, A., Gallo,R. A., et al. (2016). Psychiatric risk gene transcription factor 4 regulates intrinsicexcitability of prefrontal neurons via repression of SCN10a and KCNQ1.Neuron 90, 43–55. doi: 10.1016/j.neuron.2016.02.021

Rapanelli, M., Frick, L. R., and Pittenger, C. (2017). The role of interneurons inautism and Tourette syndrome. Trends Neurosci. 40, 397–407. doi: 10.1016/j.tins.2017.05.004

Rogers, T. D., McKimm, E., Dickson, P. E., Goldowitz, D., Blaha, C. D., andMittleman, G. (2013). Is autism a disease of the cerebellum? An integration ofclinical and pre-clinical research. Front. Syst. Neurosci. 7:15. doi: 10.3389/fnsys.2013.00015

Rubin, R. D., Watson, P. D., Duff, M. C., and Cohen, N. J. (2014). The role of thehippocampus in flexible cognition and social behavior. Front. Hum. Neurosci.8:742. doi: 10.3389/fnhum.2014.00742

Rudy, B., Fishell, G., Lee, S., and Hjerling-Leffler, J. (2011). Three groups ofinterneurons account for nearly 100% of neocortical GABAergic neurons. Dev.Neurobiol. 71, 45–61. doi: 10.1002/dneu.20853

Schmahmann, J. D., and Caplan, D. (2006). Cognition, emotion and thecerebellum. Brain 129(Pt 2), 290–292. doi: 10.1093/brain/awh729

Sepp, M., Kannike, K., Eesmaa, A., Urb, M., and Timmusk, T. (2011). Functionaldiversity of human basic helix-loop-helix transcription factor TCF4 isoformsgenerated by alternative 5′ exon usage and splicing. PLoS One 6:e22138. doi:10.1371/journal.pone.0022138

Sepp, M., Pruunsild, P., and Timmusk, T. (2012). Pitt-Hopkins syndrome-associated mutations in TCF4 lead to variable impairment of the transcriptionfactor function ranging from hypomorphic to dominant-negative effects. Hum.Mol. Genet. 21, 2873–2888. doi: 10.1093/hmg/dds112

Silva-Santos, S., van Woerden, G. M., Bruinsma, C. F., Mientjes, E., Jolfaei, M. A.,Distel, B., et al. (2015). Ube3a reinstatement identifies distinct developmentalwindows in a murine Angelman syndrome model. J. Clin. Invest. 125, 2069–2076. doi: 10.1172/JCI80554

Sinnett, S. E., Hector, R. D., Gadalla, K. K. E., Heindel, C., Chen, D., Zaric, V., et al.(2017). Improved MECP2 gene therapy extends the survival of MeCP2-nullmice without apparent toxicity after intracisternal delivery. Mol. Ther. MethodsClin. Dev. 5, 106–115. doi: 10.1016/j.omtm.2017.04.006

Skene, N. G., Bryois, J., Bakken, T. E., Breen, G., Crowley, J. J., Gaspar, H. A., et al.(2018). Genetic identification of brain cell types underlying schizophrenia. Nat.Genet. 50, 825–833. doi: 10.1038/s41588-018-0129-5

Steinlin, M. (2008). Cerebellar disorders in childhood: cognitive problems.Cerebellum 7, 607–610. doi: 10.1007/s12311-008-0083-3

Stoner, R., Chow, M. L., Boyle, M. P., Sunkin, S. M., Mouton, P. R., Roy, S., et al.(2014). Patches of disorganization in the neocortex of children with autism.N. Engl. J. Med. 370, 1209–1219. doi: 10.1056/NEJMoa1307491

Straub, C., Saulnier, J. L., Begue, A., Feng, D. D., Huang, K. W., and Sabatini, B. L.(2016). Principles of synaptic organization of GABAergic interneurons in thestriatum. Neuron 92, 84–92. doi: 10.1016/j.neuron.2016.09.007

Taniguchi, H., He, M., Wu, P., Kim, S., Paik, R., Sugino, K., et al. (2011). A resourceof Cre driver lines for genetic targeting of GABAergic neurons in cerebralcortex. Neuron 71, 995–1013. doi: 10.1016/j.neuron.2011.07.026

Thaxton, C., Kloth, A. D., Clark, E. P., Moy, S. S., Chitwood, R. A., and Philpot,B. D. (2018). Common pathophysiology in multiple mouse models of pitt-hopkins syndrome. J. Neurosci. 38, 918–936. doi: 10.1523/JNEUROSCI.1305-17.2017

Wang, F., Flanagan, J., Su, N., Wang, L. C., Bui, S., Nielson, A., et al. (2012).RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J. Mol. Diagn. 14, 22–29. doi: 10.1016/j.jmoldx.2011.08.002

Wang, Q13Y., Lu, Z., Zhang, Y., Cai, Y., Yun, D., Tang, T., et al. (2020). TranscriptionFactor 4 safeguards hippocampal dentate gyrus development by regulatingneural progenitor migration. Cereb. Cortex 30, 3102–3115. doi: 10.1093/cercor/bhz297

Whalen, S., Heron, D., Gaillon, T., Moldovan, O., Rossi, M., Devillard, F., et al.(2012). Novel comprehensive diagnostic strategy in Pitt-Hopkins syndrome:clinical score and further delineation of the TCF4 mutational spectrum. Hum.Mutat. 33, 64–72. doi: 10.1002/humu.21639

Xia, H., Jahr, F. M., Kim, N. K., Xie, L., Shabalin, A. A., Bryois, J., et al. (2018).Building a schizophrenia genetic network: transcription factor 4 regulates genesinvolved in neuronal development and schizophrenia risk.Hum.Mol. Genet. 27,3246–3256. doi: 10.1093/hmg/ddy222

Zollino, M., Zweier, C., Van Balkom, I. D., Sweetser, D. A., Alaimo, J., Bijlsma,E. K., et al. (2019). Diagnosis and management in Pitt-Hopkins syndrome: first

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international consensus statement. Clin. Genet. 95, 462–478. doi: 10.1111/cge.13506

Zweier, C., Peippo, M. M., Hoyer, J., Sousa, S., Bottani, A., Clayton-Smith, J., et al.(2007). Haploinsufficiency of TCF4 causes syndromal mental retardation withintermittent hyperventilation (Pitt-Hopkins syndrome). Am. J. Hum. Genet. 80,994–1001. doi: 10.1086/515583

Zweier, C., Sticht, H., Bijlsma, E. K., Clayton-Smith, J., Boonen, S. E., Fryer, A.,et al. (2008). Further delineation of Pitt-Hopkins syndrome: phenotypic andgenotypic description of 16 novel patients. J. Med. Genet. 45, 738–744. doi:10.1136/jmg.2008.060129

Conflict of Interest: The authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interest.

Copyright © 2020 Kim, Berens, Ochandarena and Philpot. This is an open-accessarticle distributed under the terms of the Creative Commons Attribution License(CC BY). The use, distribution or reproduction in other forums is permitted, providedthe original author(s) and the copyright owner(s) are credited and that the originalpublication in this journal is cited, in accordance with accepted academic practice. Nouse, distribution or reproduction is permitted which does not comply with these terms.

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