The E-protein Tcf4 interacts with Math1 to regulatedifferentiation of a specific subset ofneuronal progenitorsAdriano Flora*†‡, Jesus J. Garcia*†, Christina Thaller§, and Huda Y. Zoghbi*†‡¶�**

*Howard Hughes Medical Institute, Departments of †Molecular and Human Genetics, §Biochemistry and Molecular Biology, ¶Neuroscience, and�Pediatrics, and **Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030

Contributed by Huda Y. Zoghbi, August 8, 2007 (sent for review July 16, 2007)

Proneural factors represent <10 transcriptional regulators requiredfor specifying all of the different neurons of the mammalian nervoussystem. The mechanisms by which such a small number of factorscreates this diversity are still unknown. We propose that proteinsinteracting with proneural factors confer such specificity. To test thishypothesis we isolated proteins that interact with Math1, a proneuraltranscription factor essential for the establishment of a neural pro-genitor population (rhombic lip) that gives rise to multiple hindbrainstructures and identified the E-protein Tcf4. Interestingly, haploin-sufficiency of TCF4 causes the Pitt–Hopkins mental retardation syn-drome, underscoring the important role for this protein in neuraldevelopment. To investigate the functional relevance of the Math1/Tcf4 interaction in vivo, we studied Tcf4�/� mice and found that theyhave disrupted pontine nucleus development. Surprisingly, this se-lective deficit occurs without affecting other rhombic lip-derivednuclei, despite expression of Math1 and Tcf4 throughout the rhombiclip. Importantly, deletion of any of the other E-protein-encodinggenes does not have detectable effects on Math1-dependent neu-rons, suggesting a specialized role for Tcf4 in distinct neural progen-itors. Our findings provide the first in vivo evidence for an exclusivefunction of dimers formed between a proneural basic helix–loop–helix factor and a specific E-protein, offering insight about themechanisms underlying transcriptional programs that regulate de-velopment of the mammalian nervous system.

basic helix–loop–helix � mental retardation � neural development �proneural

The establishment of neural progenitor territories in mammalsrelies on the expression of �10 basic helix–loop–helix (bHLH)

transcription factors, collectively called proneural proteins (1–3).The mechanism by which a few transcriptional regulators controlthe differentiation of hundreds of distinct neuronal subtypes re-mains unclear (4). Distinct neuronal populations can originate fromcells that express a single proneural gene, suggesting that progen-itors with similar expression profiles are highly plastic and can adoptdifferent fates (5–7). The heterogeneity of neurons derived fromthe same progenitor population begs the question of how a singleproneural factor activates diverse genetic programs to regulate thedifferentiation of distinct neurons. Transcription factors typicallyinteract with different cofactors to form distinct functional com-plexes (8). However, only a few cofactors interacting with neuralbHLHs have been identified to date (9–11).

Math1 (also called Atoh1) is a bHLH transcription factor that ishighly expressed in proliferating progenitors of the rhombic lip(RL), a specialized neuroepithelium in the developing dorsalhindbrain (12, 13). Math1 is required for the differentiation of mostcerebellar and precerebellar structures, including the pontine,lateral reticular, and external cuneate nuclei (7, 14). Moreover,Math1�/� mice lack most of the cochlear nucleus and all cerebellargranule neurons. Like other proneural proteins, Math1 dimerizeswith the widely expressed bHLH E-proteins to bind DNA andactivate transcription (3). There are four E-proteins in mammals:Tcf4 (also called E2.2 or ITF2), E12, E47, and HEB, encoded by

three genes, E12 and E47 being different splice isoforms of E2a (3,15–17). Because their bHLH domains are highly homologous, it hasbeen proposed that E-proteins have redundant functions, despiteobvious phenotypes in animals lacking any one of them (18, 19).Although all E-proteins are highly expressed in neural progenitors(20, 21), their role in nervous system development has not beeninvestigated in depth (22). Recently, genetic studies demonstratedthat loss of one copy of TCF4 causes Pitt–Hopkins syndrome (PHS)(23–25), a neurodevelopmental disease characterized by mentalretardation, seizures, and hyperventilation (26, 27), suggesting thatTCF4 is critical for human nervous system development.

In this study we report the identification of Tcf4 as an interactorof Math1, and we demonstrate through genetic studies that Math1/Tcf4 heterodimers have an exclusive role in pontine neuron differ-entiation, thus providing the first evidence that a specific proneuralbHLH/E-protein heterodimer plays a unique function in the de-veloping nervous system.

ResultsMath1 and Tcf4 Form Transcriptionally Active Heterodimers. Toidentify Math1 interactors, we designed a yeast two-hybrid strategyusing the bHLH domain because of its fundamental role (28, 29).However, the Math1 bHLH-Gal4 DNA-binding domain fusionprotein proved to be unsuitable as bait because it was a strongtransactivator in yeast (data not shown). To overcome this problem,we used the highly conserved bHLH domain of Drosophila atonal[supporting information (SI) Fig. 7], the orthologue known torescue phenotypes of Math1-null mice (30), to screen a mouseembryonic cDNA library.

After two rounds of selection, we isolated 73 colonies thatrepresented six different proteins. Seven colonies corresponded tothree independent clones of Tcf4; one of these, comprising theentire coding sequence, was used to confirm the interaction (Fig.1A). Reciprocal coimmunoprecipitation of the two proteins tran-siently expressed in NIH 3T3 cells confirmed the interaction inmammalian cells (Fig. 1B).

To determine whether Tcf4 can heterodimerize with Math1 toform a functional DNA-binding complex, we performed EMSAusing an oligonucleotide that contains two variants of the canonicalE-box consensus sequence, previously demonstrated to interactwith Math1 (12). Incubation of nuclear extracts from GFP-transfected NIH 3T3 cells with the radioactive probe revealed a

Author contributions: A.F. and H.Y.Z. designed research; A.F., J.J.G., and C.T. performedresearch; A.F. analyzed data; and A.F. and H.Y.Z. wrote the paper.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.

Abbreviations: RL, rhombic lip; LC, locus ceruleus; AES, anterior extramural migratorystream; bHLH, basic helix–loop–helix; PHS, Pitt–Hopkins syndrome; P0, postnatal day 0.

‡To whom correspondence may be addressed. E-mail: aflora@bcm.edu or hzoghbi@bcm.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0707456104/DC1.

© 2007 by The National Academy of Sciences of the USA

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very faint band, probably due to binding of endogenous bHLHproteins to the probe (Fig. 1C, lanes 2–4, band a). Transfection ofa vector expressing Tcf4 alone did not alter the mobility of theoligonucleotide (Fig. 1C, lanes 6–8). Incubation of the probe withnuclear extracts of Math1-transfected cells (Fig. 1C, lanes 9–11)caused the formation of a new specific DNA–protein complex(band c), confirmed by competition with cold oligonucleotide andantibody supershifting (Fig. 1C, lanes 10 and 11, band d). BecauseMath1 homodimers do not bind to DNA (12), the complex ob-served in lane 9 likely results from the dimerization of Math1 withendogenous E-proteins. When we incubated the probe with nuclearextracts from cells transfected with both Tcf4 and Math1, a strongband appeared that was supershifted by antibodies against eitherMath1- or Tcf4-tagged proteins (Fig. 1C, lanes 12, 14, and 15, bandsc and d). These data show that Math1/Tcf4 heterodimers bind theDNA target sequence. We also observed a faster-migrating com-plex (Fig. 1C, lane 12, band b), probably due to binding of aMath1/Tcf4 dimer on one of the two E-box sequences of the probe.The specificity of these interactions was verified by competitionwith unlabeled oligonucleotide (Fig. 1C, lane 13).

To determine whether the Math1/Tcf4 heterodimers are tran-scriptionally active, we used a reporter plasmid bearing a minimalpromoter flanked by seven E-box sequences cloned in front of theluciferase reporter gene (12). As expected, transfection of Math1 inNIH 3T3 cells induced the luciferase activity (Fig. 1D), probably by

interacting with endogenous E-proteins. Transfection of Tcf4 byitself did not alter the expression of the reporter gene. However,coexpression of Tcf4 and Math1 increased the Math1-dependentactivation of the reporter gene. Together, these data indicate thatMath1 and Tcf4 interact and form transcriptionally competentheterodimers.

Math1 and Tcf4 Are Coexpressed in the RL. To investigate thephysiological relevance of the Math1/Tcf4 interaction, we sought todetermine whether the two genes are coexpressed during mousedevelopment. Because the expression of Math1 in the embryonicbrain is restricted to the RL (a population of neuronal progenitorslocated in the dorsal aspect of the developing hindbrain) we verifiedthat Tcf4 is expressed in the RL by in situ hybridization of sectionsfrom mouse embryos at embryonic day 14.5 (RL in Fig. 2B and SIFig. 8 and data not shown). The expression profile of Tcf4 overlapswith and extends beyond that of Math1 (SI Fig. 8 A, A�, and Inset).Both genes are expressed in the rostral, or upper, RL (uRL in Fig.2) and in the caudal, or lower, RL (lRL in Fig. 2). Sense probes didnot show any signal (data not shown). The temporal and spatialcoexpression of Math1 and Tcf4 suggests that Tcf4 may play a rolein the differentiation of RL neural progenitors.

Tcf4 Is Required for the Differentiation of a Specific Subset ofMath1-Expressing Progenitors. To analyze the hindbrain develop-ment in Tcf4 mutant mice, we injected embryonic stem cells bearing

Fig. 1. Identification of Tcf4 as a functional partner of Math1. (A) A �-galactosidase assay confirms the interaction between atonal and Tcf4 in yeast. Positive (p53�

T antigen) and negative (p53 or the atonal bait, Ato) controls are shown. (B) Coimmunoprecipitation of Math1 and Tcf4 in NIH 3T3 cells. Samples subjected toimmunoprecipitation are shown on top of the gel; antibodies are indicated at the bottom. Anti-Flag and anti-V5 antibody detected tagged Math1 and Tcf4 proteins,respectively (arrows). Molecular markers are shown. (C) Autoradiography of a nondenaturing acrylamide gel used for EMSA; transfected constructs are indicated atthe bottom. The top indicates which samples were incubated with antibodies against tagged Math1 and Tcf4 proteins or with unlabeled oligonucleotide. The arrowsindicate the complexes formed between nuclear protein extracts and labeled oligonucleotide. Lanes are numbered at the bottom. (D) Bar diagram shows luciferasedata. The expression constructs transfected in NIH 3T3 cells are indicated at the bottom. Bars show the fold induction over luciferase activity from cells transfected withempty vector pCDNA3. The standard deviation is shown.

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a null Tcf4 allele (19) into mouse blastocysts. Tcf4 heterozygousanimals are viable and fertile and show no obvious phenotype.Tcf4-null mice die in the first 24 h after birth, as previously reported.However, in contrast to previous observations (19), we did notobserve embryonic lethality of null animals, and we recovered theexpected Mendelian ratio of Tcf4�/� mice at birth. The brains ofTcf4-null animals were grossly normal and did not show any evidentmorphological defect (data not shown). We crossed Tcf4�/� miceto Math1�/lacZ mice to study the differentiation of RL progenitorsin Tcf4 mutant animals. In Math1�/lacZ mice, the coding region ofone allele of Math1 has been replaced with the lacZ gene, leadingto the expression of �-galactosidase in cells that would normallyexpress Math1, allowing the visualization of Math1-expressingprogenitors and structures derived from them (7, 31).

Two main groups of neuronal structures are derived from theRL: dorsal structures, such as the granule cell layer of thecerebellum (Fig. 3A, Cb) and the external cuneate nucleus (Fig.3A, ECN), and ventral structures, such as the pontine nucleusand the lateral reticulate nucleus (Fig. 3C, PN and LRt) (7, 32).Surprisingly, despite the expression of Tcf4 in the entire RL, itsdeletion caused a differentiation defect only in the pontinenucleus, leaving the other Math1-dependent structures intact(Figs. 3 and 4). This defect resulted in a substantial reduction inthe neurons of the pontine nucleus (compare Fig. 3 C and C�,

arrowheads) as well as ectopic accumulation of �-galactosidase-expressing cells in a dorsal–lateral region of the hindbrain(compare Fig. 3 B and B� with C and C�, arrows), likely becauseof aberrant migration of Math1-dependent neuronal progeni-tors. Math1/Tcf4 doubly heterozygous mice showed a persistentanterior extramural migratory stream (AES), suggesting a delayor interruption in migration (compare Fig. 3 B and B� with C andC�, arrows).

To confirm that the differentiation defect observed in Tcf4�/�

mice is limited to pontine neurons, we compared the phenotype ofTcf4�/� animals to that of Math1�/� mice, which lack all of thestructures derived from Math1-expressing progenitors. To evaluatethe differentiation of the RL progenitors, we analyzed the expres-sion pattern of Pax6, a homeodomain transcription factor expressedby all Math1-expressing progenitors in the hindbrain, and whoseexpression is maintained in the migratory streams that originate inthe RL (32). Analysis of Pax6 staining confirmed that the pontinenucleus is the only structure disrupted by deletion of Tcf4, whereasthe other Math1-dependent structures appear to develop normally(Fig. 4).

Math1 and Tcf4 Interact Genetically. To verify that the differentiationdefects in the Tcf4�/� animals are due to the absence of Math1/Tcf4heterodimers rather than the lack of a Math1-independent functionof Tcf4 in the RL, we tested the genetic interaction between Math1and Tcf4. Nissl staining of coronal sections from brains at postnatalday 0 (P0) revealed very few cells in the region immediately lateralto the caudal pontine nucleus in wild-type animals, demonstratingcompletion of migration of pontine nucleus neurons (Fig. 5A). Asexpected, sections from Math1�/lacZ animals did not differ fromthose of wild-type mice. Similarly, Tcf4�/� mice had very few cellsin this region, suggesting that a single copy of Tcf4 in an otherwise

Fig. 2. In situ hybridization of sequential sagittal sections of a mouse embryoat embryonic day 14.5. The antisense probes are shown on the right. The sectionshown in A has been hybridized with the Math1 antisense probe, and the sectionshown in B has been hybridized with the Tcf4 antisense probe. uRL, upper RL; lRL,lower RL; Cb, cerebellum.

Fig. 3. �-Galactosidase staining of structures derived from the RL of Math1�/lacZ

mice. The pictures show dorsal (A–A�), lateral (B–B�), and ventral (C–C�) views ofP0 hindbrains. The arrows point to the AES, and the arrowhead points to thepontine nucleus (PN). ECN, external cuneate nucleus; Cb, cerebellum; LRt, lateralreticulate nucleus. The genotype of the animals is shown at the top.

Fig. 4. Comparison of the phenotypes of Math1�/� and Tcf4�/� mice. Theoutline of the sections is shown on the left of each row of images, and the framedportions represent the regions shown in the pictures. Arrowheads in A and Bindicate the lateral reticulate (LRt) and external cuneate (ECN) nuclei, respec-tively. The arrowheads in C indicate the external granule layer cells (EGL), and thearrow points to the lower RL (lRL). In D, the arrowheads indicate the pontinenucleus (PN), and the arrows show the location of the AES. Pax6 staining isrevealed as a red signal over the blue color of the nuclear stain TOTO3. Thestructures missing in Math1�/� animals are indicated between parentheses. Thegenotypes of the animals are shown at the top.

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wild-type background is sufficient for the normal migration of theAES cells (Fig. 5D). On the other hand, Math1�/lacZ;Tcf4�/�

animals showed clear accumulation of cells in the region lateral tothe pontine nucleus, a phenotype consistent with the �-galactosi-dase staining data (compare Fig. 5 E and B, arrows). Analysis ofTcf4-null animals revealed a massive accumulation of neuronal

precursors that were unable to reach the pontine nucleus region aswell as a drastic reduction in the size of the pontine nucleus itself(compare Fig. 5 F and B, arrows and PN). Immunofluorescenceanalysis of the caudal portion of the pontine nucleus of P0 animalsshowed that the ectopic cluster of cells present in Math1�/lacZ;Tcf4�/� mice are positive for Pax6, indicating that these cells arepart of the AES that normally gives rise to the pontine nucleus(Fig. 5J).

Other E-Proteins Are Expressed in RL and Can Interact with Math1.Tcf4 is a member of the E-protein family, a group of highlyconserved bHLH proteins with similar biochemical and functionalcharacteristics. The presence of a phenotype in Tcf4-null animalsraises the question as to why other members of the family are notable to compensate for the lack of Tcf4 in the progenitors of thepontine neurons. The simplest explanation is that the other E-pro-tein coding genes are not expressed in the lower RL during mousedevelopment. Alternatively, the E-proteins encoded by these genesmight not be able to interact with Math1 to form functionalDNA-binding dimers. To test these hypotheses, we performed insitu hybridization for the E2a and HEB genes on sagittal sections ofembryos at embryonic day 14.5. Both E2a and HEB are expressedin the RL, suggesting that lack of compensation is not caused bydifferent patterns of expression between E-proteins (SI Fig. 9A). Toconfirm that Math1 can form dimers with E-proteins other thanTcf4 (12, 33), we analyzed the ability of E47, a splice isoform of E2a,to interact with Math1. We transfected NIH 3T3 cells with aconstant amount of a Math1-expressing plasmid and increasingconcentrations of plasmids encoding the V5-tagged version of Tcf4or E47, and immunoprecipitated with an antibody recognizingtagged Math1. Comparable amounts of Tcf4 and E47 coimmuno-precipitated (SI Fig. 9B), suggesting that the two E-proteins have asimilar affinity for Math1. To verify that the Math1/E47 het-erodimers bind to DNA, we performed EMSAs using the E-box-containing DNA sequence. The incubation of nuclear extracts fromNIH 3T3 cells cotransfected with Math1 and E47 with the labeledoligonucleotide resulted in the formation of a DNA–protein com-plex (SI Fig. 9C, lane 11, band a), indicating that the Math1/E47heterodimer binds the DNA target sequence. The ability of theMath1/E47 dimers to bind E-boxes was comparable to that of theMath1/Tcf4 dimers (SI Fig. 9C, lanes 5 and 11).

Tcf4/Math1 Dimers Play a Unique Role in Pontine Nucleus Develop-ment. Having established that the lack of compensation by otherE-proteins in Tcf4�/� mice is not due to absence of E2a and HEBin the RL, or to their inability to interact with Math1, we ponderedwhether the pontine nucleus phenotype of Tcf4�/� mice is due toa reduction in the dosage of genes coding for E-proteins. In theimmune system, deletion of either Tcf4 or E2a causes a similardifferentiation defect in B cells, an effect also present inTcf4�/�;E2a�/� doubly heterozygous animals (19). These datasuggested that the E-proteins have identical functions and that areduction in the total E-protein level is the cause of the observeddifferentiation phenotype. To test for the role of dosage, we crossedTcf4�/� mice with E2a�/� and HEB�/� mice and analyzed thedoubly heterozygous animals by Pax6 staining (Fig. 6). Remarkably,Tcf4�/�;E2a�/� and Tcf4�/�;HEB�/� animals displayed normaldevelopment of the pontine nucleus (compare Fig. 6 C and D withA), which is distinctly different from what is seen in Tcf4�/� mice(Fig. 6B). To rule out compensatory mechanisms due to transcrip-tional up-regulation of the remaining allele of E2a or HEB in doublyheterozygous animals, we analyzed coronal sections obtained fromE2a�/� or HEB�/� mice and observed normal development of thepontine nucleus (Fig. 6 E and F). Analysis of the other Math1-dependent structures in E2a- and HEB-null animals did not revealany differentiation defect (data not shown). These findings stronglysuggest that the defects in Tcf4�/� pontine nucleus cannot beattributed to a simple ‘‘gene dosage’’ effect.

Fig. 5. Genetic interactionbetweenMath1andTcf4.A schematicof thecoronalsections is showninA. Theframedportionrepresents theregionanalyzedbyNissland Pax6 staining. (B–F) Nissl staining of coronal sections at the level of the caudalpontine nucleus of hindbrains collected from P0 mice. The brackets show theregion corresponding to the location of the AES. The arrows indicate the cells ofthe AES. Magnifications of the region between brackets are shown in the Insets.PN, pontine nucleus. At least six animals per genotype were analyzed; genotypesare shown in each panel. (G–J) Pax6 staining of the same region analyzed by Nisslstaining. Pax6 expression is revealed by the red color superimposed on the bluenuclear staining. Arrowheads indicate the position of the AES.

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Math1 Heterodimers Have Different Cell-Specific Transcriptional Ac-tivities. To better understand the molecular basis of the differen-tiation phenotype seen in Tcf4-null mice, we compared the tran-scriptional activity of the different Math1 heterodimers in severalcell types using the E-box–luciferase reporter construct. We co-transfected a Math1-expressing vector along with plasmids encod-ing any one of the four E-proteins in NIH 3T3 fibroblasts, neuro-blastoma line Neuro2a cells, the teratoma cell line P19, and primaryneural precursors purified from postnatal day 5 cerebella. In all celltypes, the transcriptional activity of Math1 was highly dependent onthe E-protein partner. The ratio between the activities of thedifferent heterodimers varied dramatically between different celllines (compare NIH 3T3 and P19 profiles in SI Fig. 10), suggestingthat that the activity of each Math1 heterodimer can be differen-tially modulated in a cell context-specific manner.

Deletion of Tcf4 Does Not Affect the Locus Ceruleus (LC). To furtherevaluate the role of Tcf4 in hindbrain development, we investigatedthe effect of its deletion on Mash1-dependent neural progenitors,which give rise to the main noradrenergic structure of the brain-stem, the LC. We focused our attention on LC neurons becauseTcf4 was reported to interact in vitro with Mash1 (34), and becausethe respiratory defects observed in patients with PHS have beenattributed to defective function of the LC (24, 25). Staining ofcoronal sections of Tcf4�/� mice with an antibody raised againsttyrosine hydroxylase, an enzyme expressed in the noradrenergic

cells that form the LC, did not show any obvious morphologicaldefect of the LC, suggesting that the Mash1-expressing progenitorsof the hindbrain are able to differentiate properly in the absence ofTcf4 (SI Fig. 11).

DiscussionIn this study we identified the interaction between the proneuralprotein Math1 and the E-protein Tcf4 and investigated the role oftheir heterodimers in development of the mouse hindbrain. Weshow that lack of Math1/Tcf4 heterodimers results in the loss of arestricted neuronal population in the hindbrain. We also demon-strate that the E-proteins E47, E12, and HEB do not compensatefor the absence of Tcf4, despite their overlapping expression patternand similar biochemical properties. Our data provide the first invivo evidence for a specialized function of dimers formed betweena proneural bHLH factor and a specific E-protein, revealing a newlayer of complexity to the transcriptional events regulating thedevelopment of the mammalian nervous system.

Our data suggest that two different classes of neural progenitorsexist in the RL. The first class of progenitors, to which mostMath1-dependent progenitors belong, can differentiate normallywhen any one of the three E-protein genes is deleted, presumablybecause of functional compensation by the remaining E-proteins.The differentiation behavior of this class of neural progenitors isconsistent with the current model of bHLH heterodimer functionin the development of the nervous system, which suggests thatproneural proteins mediate the transcriptional specificity of theheterodimers, whereas E-proteins are interchangeable (3, 35). Thesecond class of progenitors, to which the pontine nuclear progen-itors belong, requires the presence of Tcf4/Math1 dimers to activatethe normal differentiation program. The reasons for the failure ofother members of the E-protein family to compensate for thedeletion of Tcf4 in this class of progenitors are not entirely clear.Our analysis of E2a and HEB mutant mice indicates that theinability of other E-proteins to compensate for the absence of Tcf4in pontine nucleus progenitors is due to intrinsic molecular differ-ences between these proteins and not to a generic gene dosageeffect caused by the reduction of the number of alleles coding forE-proteins. The observation that the transcriptional activity ofvarious Math1/E-protein heterodimers is differentially modulatedby the cellular context supports the hypothesis that differentMath1/E-protein dimers might interact with distinct cell-specifictranscriptional cofactors to differentially activate target genes. Inthe developing embryo, factors expressed by pontine nuclear neu-ron progenitors might interact selectively with Math1/Tcf4 dimersto activate a specific differentiation program. The ability of a singleproneural bHLH to interact with E-proteins to form heterodimerswith different transcriptional activity would increase the number ofspecific genetic programs regulated by this class of transcriptionfactors and possibly clarify how �10 proneural proteins coordinatethe differentiation of all of the different progenitor populations ofthe mammalian nervous system.

Our data suggest a possible developmental model to explain howmutations in TCF4 cause the complex features of PHS, character-ized by widespread neurological symptoms without obvious defectsin the anatomical structure of the nervous system. If TCF4 playslimited and specialized roles in different neural progenitor do-mains, its deletion would be expected to affect the differentiationof restricted neuronal populations in several portions of the nervoussystem, causing a wide variety of neurological symptoms. Themental retardation and epilepsy, for example, could be due toaberrant migration of specific groups of cortical neurons, and theintestinal phenotype could be caused by a partially defectivedifferentiation of neural crest cells. Some of the symptoms, like thehyperventilation phenotype, could also be caused by subtle deficitsin different systems that are part of the same functional network.The breathing defects of PHS patients have been previously sug-gested to arise from a deficit in the function of the proneural bHLH

Fig. 6. Analysis of pontine nuclear neuron differentiation in mice lackingdifferent E-proteins. The cells of the anterior migratory stream (AES) arevisualized by immunofluorescence staining using the anti-Pax6 antibody. Pax6expression is represented by the red staining in the pictures. Cell nuclei arestained in blue. The arrowheads are pointing to the AES. The genotype of theanimals is shown in the left bottom part of each panel. PN, pontine nucleus.

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Mash1 leading to aberrant development of the noradrenergicneurons of the LC (24, 25). However, our data show that micelacking Tcf4 have a normal LC, suggesting that the function of theproneural gene Mash1 in the progenitors of these neurons ismaintained in the absence of Tcf4. If this holds true in humans, itwould imply that the hyperventilation phenotype of PHS is notcaused by the absence of the LC, but most likely by the concomitantlack of small subsets of Mash1- and Math1-dependent medullaryneurons involved in breathing control.

In sum, the interaction between Math1 and Tcf4 is necessary forthe normal activation of the differentiation program of a subset ofneural progenitors. We propose that a similar requirement exists inspecific progenitor populations in different regions of the devel-oping neural tube. Therefore, detailed analyses of the developmen-tal function of the heterodimers formed between Tcf4 and thevarious proneural bHLH proteins will be important to define therole of this E-protein in development of the mammalian nervoussystem and to understand the basis of PHS.

Materials and MethodsYeast Two-Hybrid Screen. We screened a pretransformed mouseembryonic 12.5 library using the Matchmaker yeast two-hybridsystem following the supplier’s instructions (Clontech, MountainView, CA). The bait consisted of a portion of Drosophila atonalfrom amino acid 248 to amino acid 312. After the first round ofselection, we isolated and sequenced DNA from yeast. Independentclones were retested for growth on selective medium and for�-galactosidase activity.

Coimmunoprecipitation, EMSA, and Luciferase Assays. For coimmu-noprecipitation, cells were harvested 48 h after transfection andresuspended in lysis buffer (10 mM Tris�HCl, pH 8/140 mMNaCl/0.5% Triton X-100/1 mM iodoacetamide/1 mM PMSF/1%hemoglobin/1� protease inhibitors; all from Sigma, St. Louis,MO). After 4 h of incubation with agarose-conjugated mousemonoclonal antibodies (Sigma), proteins associated to the beadswere subjected to Western blot by using rabbit polyclonalanti-Flag and anti-V5 antibodies (Sigma). EMSAs were per-

formed as previously described (36). Detailed information aboutconstructs and oligonucleotides used can be found in SI Text.

Nonradioactive in Situ Hybridization. Tissue preparation, automatedin situ hybridization, and digital imaging were performed as pre-viously described (37). Probe details can be found in SI Text.

Histochemistry. �-Galactosidase staining was performed as pre-viously described (7). For Nissl staining, P0 brain paraffinsections were incubated in histoclear (Baxter, Deerfield, IL) andgradually rehydrated through a series of ethanol–water solutions.The slides were then incubated in cresyl violet solution (0.5%cresyl violet acetate from Sigma in 0.3% glacial acetic acid inwater) and washed in increasing concentrations of ethanol inwater. After complete dehydration in histoclear, slides weremounted with Cytoseal (Richard-Allan Scientific, Kalamazoo,MI) and analyzed with an Axiocam2 (Zeiss, Berlin, Germany).

Immunofluorescence. P0 brains were collected, fixed in 4% para-formaldehyde, and frozen in OCT (Sakura, Torrance, CA). Sec-tions (25 �m thick) were incubated overnight at 4°C with a 1:500dilution of a rabbit polyclonal anti-Pax6 antibody (Covance, Prince-ton, NJ) or a dilution 1:1,000 of a mouse monoclonal anti-tyrosinehydroxylase antibody (Immunostar). Secondary antibodies conju-gated with Alexa Fluor 488 or Cy3 (Molecular Probes, Carlsbad,CA) were then added for 1 h at room temperature. Cell nuclei werevisualized by using TOTO3 (Molecular Probes). Images wereacquired with a confocal Axiocam microscope (Zeiss). All of theexperiments involving animals were performed following the In-stitutional Animal Care and Use Committee guidelines.

We thank Yuan Zhuang at Duke University (Durham, NC) for the generousgift of embryonic stem cells with a targeted Tcf4 allele and for the HEB- andE2a-null mice; Gabriele Schuster, Sukeshi Vaishnav, Barbara Antalffy, andRichard Atkinson for technical support; members of the H.Y.Z. laboratoryfor critical reading of the manuscript and advice; and Mental Retardationand Developmental Disabilities Research Center Gene Expression andNeuropathology Core Grant 5 P30 HD024064. H.Y.Z. is an investigator andA.F. is a postdoctoral research associate with the Howard Hughes MedicalInstitute.

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