The Journal of Neuroscience, August 1995, 75(a): 5727-5738

Expression of in the Mouse Central Nervous System Suggests a Role in Neuronal Maturation

Gary E. Lyons,’ Bruce K. Micales,’ John Schwarz,* dames F. Martin,2 and Eric N. Olson2 ‘Department of Anatomy, The University of Wisconsin Medical School, Madison, Wisconsin 53706 and 2Department of Biochemistry and Molecular Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

Members of the myocyfe enhancer factor 2 (MEF2) mondsonet al., 1994). One other publishedin situ hybridization family are expressed in a dynamic pattern during cievel- result with a MEF2 probe showsthat MEF2C is detectable in opment of the CNS of pre- and postnatal mice. The four several neuronal layers of the frontal cortex in 1 week and 4 MEF2 genes, Mef2A, -B, 4, -0, transcription factors week postnatal rat brains (Leifer et al., 1993). belonging to the MADS (MCMl-agamous-deficiens-serum The MEF2 were originally identified as a muscle- response factor) superfamily of DNA binding proteins. specificDNA binding activity in nuclear extracts that recognized MEF2 factors have previously been shown to be positive a conserved A/T-rich sequenceassociated with numerousmus- regulators of in terminally differentiated cle-specific genes (Gossett et al., 1989; Cserjesi and Olson, muscle cells. To begin to determine the role of MEF2 fac- 1991). The cloning of MEF2 showed it is encodedby four ver- tors in CNS development, we used in situ hybridization tebrate genes,MejZA, -B, -C, and -D, whose productsbelong to with gene-specific cRNA probes to define the expression the MADS (MCMl, agamous,deficiens, serum responsefactor) patterns of each of the four Mef2 mRNAs in the developing family of transcription factors (Chamberset al., 1992; Pollock and mature mouse CNS. Mef2C mRNA was first detected and Treisman, 1992; Yu et al., 1992; Breitbart et al., 1993; Leifer in a ventral portion of the telencephalon at 11.5 d postcoi- et al., 1993; Martin et al., 1993, 1994; McDermott et al., 1993). turn (p.c.). By 13.5 d p.c., each of the four Mef2 genes were The four MEF2 gene productsare highly homologouswithin the expressed in overlapping yet distinct patterns in regions of MADS domain, which mediatesdimerization and DNA binding the frontal cortex, midbrain, thalamus, hippocampus, and to the consensussequence CTA(AiT),TAG/A, but they are di- hindbrain. Temporal and spatial patterns of embryonic vergent in their carboxyl-termini. Additional complexity of this Mef2 gene expression appeared to follow gradients of neu- family of regulatorsarises from alternative splicing of Mej2 tran- ron maturation and suggested that the onset of Mef2 gene scripts, yielding isoforms with common DNA binding domains, expression coincides with withdrawal from the cell cycle but unique carboxyl-termini (Yu et al., 1992; Martin et al. 1993, and initiation of neuronal differentiation. This correlation is 1994). particularly striking for Purkinje cells in the cerebellum. Recently, several laboratorieshave focusedon the role of the Since the molecular mechanisms that regulate neuron dif- Mefl genesin skeletaland cardiac muscle developmentin vitro ferentiation are unknown, we propose that the MEF2 fac- and in vivo (Cserjesi and Olson, 1991; Chamberset al., 1992; tors are likely to play an important role in this process. Edmondsonet al., 1992; Navankasattusaset al., 1992; Yu et al., [Key words: Mef2 mRNAs, genes, 1992; Breitbart et al., 1993; Cheng et al., 1993; Martin et al., mouse embryo, postnatal brain, in situ hybridization, neu- 1993, 1994; McDermott et al., 1993). In skeletalmuscle, MEF2 ron maturation] proteins appearto act in concert with the MyoD family of basic helix-loop-helix (bHLH) myogenic factors to regulate muscle- In the mouse, the Myocyte Enhancer Factor 2 genes, MejL?A, specific gene transcription (Edmondson et al., 1992; Cheng et -B, and -D are expressedin a wide range of adult tissues,where- al. 1993; Yee and Rigby, 1993; Kaushal et al., 1994; Leibham as MeflC expressionappears to be restricted to brain, skeletal et al., 1994). muscle,and spleen(Martin et al., 1993; McDermott et al., 1993). Like their patterns of expressionin muscle,we found that the Our preliminary in situ hybridization results indicate that each four Mef2 genesare expressedin spatially and temporally spe- of the four membersof the Me$? gene family is expressedin cific patterns during brain development.Our resultssuggest that, the embryonic mouse brain by 13.5 d postcoitum (pc.; Ed- similar to its expressionpattern in striated muscle, Mej2 gene expressionin the CNS occurs in neuronsexiting the cell cycle and entering differentiation. As the CNS develops,we observed Jan. 19, 1995; revised March 30, 1995; accepted April 6, 1995. that patterns of expressionof the four Mefl genesin specific G.E.L. thanks Dr. Steve Kornauth and Dr. Tom Sutula for heloful comments regions of the mousebrain followed gradientsof neuronalmat- on the manuscript. This research was supported by grants from the Muscular Dystrophy Association and the NIH (HD29471) to G.E.L.; E.N.O. was sup- uration. Since these patterns correlate with published data on ported by grants from the NIH, Muscular Dystrophy Association and the Robert brain-specific gene expression,this study may also serve to iden- A. Welch Foundation. tify potential target genesthat the MEF2 proteinsregulate. Since Correspondence should be addressed to Gary E. Lyons, Department of Anat- omy, University of Wisconsin Medical School, 1300 University Avenue, Mad- the molecular mechanismsthat regulate neuron differentiation ison, WI 53706. are unknown, we propose that the MEF2 factors are likely to Copyright 0 1995 Society for Neuroscience 0270.6474/95/155727-12$05.00/O play an important role in this process. 5728 Lyons et al. l Me@ Gene Expression in Developing Mouse Brain

Figure 1. Mej2 gene expression in the mouse brain from 11.5 to 14.5 d pc. The dark-field micrograph in A shows a frontal section through the brain of an 11.5 d embryo passing through two ventricles (v). B, A parasagittal section through the head of a second 11.5 d embryo showing the same MeJzC expression (arrows in A and B) in the telencephalon (t). Grains are also detected over neural crest cells in the maxilla (0) in A and B. The dark-field micrographs in C and D represent two consecutive parasagittal sections of 13.5 d embryos that were hybridized to probes for (C) MeflC and (D) Mej2A. (Arrow in C points to the frontal cortex. Arrow in D points to the developing thalamus.) E and F, Dark-field micrographs of parasagittal sections of 14.5 d p.c. embryos that were hybridized with probes for (E) Mej2C, (F) Mej2D. Arrowheads in E and F point to layers of the cerebellum. a, amygdala; c, cochlea; h, hippocampus; m, midbrain; p. choroid plexus; r, retina. Scale bar: A-B, 400 pm; C-F, 800 pm.

Materials and Methods CO-BRL) was linearized with Not1 and transcribed with T7 polymerase for the antisense cRNA probe. The plasmid was linearized with Sal1 Preparation and prehybridization of tissue sections. The protocol that and transcribed with Sp6 polymerase for the sense control probe. (2) was used to fix and embed BALBlc and C57BL/6XDBA/2 embryos, fetuses, and postnatal brains is described in detail in Lyons et al. (1990). Mef2B: 156 mouse cDNA (Martin and Olson, manuscript in Briefly, embryos were fixed in 4% paraformaldehyde in phosphate-buf- preparation) in pAMP1 (GIBCO-BRL) was linearized with Not1 and fered saline (PBS) overnight, dehydrated, and infiltrated with paraffin. transcribed with T7 polymerase for the antisense probe. The plasmid Serial sections (5-7 pm) were mounted on gelatinized slides. One to was linearized with EcoRI and transcribed with Sp6 for the sense con- three sections were mounted/slide, deparaffinized in xylene, rehydrated, trol probe. (3) MeflC: 374 base pair mouse cDNA (Edmondson et al., and postfixed. The sections were digested with proteinase K, postfixed, 1994) in pBSKI1 (Stratagene) was linearized with Hind111 and tran- treated with triethanolamine/acetic anhydride, washed, and dehydrated. scribed with T3 polymerase for the antisense cRNA probe. The plasmid cRNA probe preparation. In order to distinguish between transcripts was linearized with BglI and transcribed with T7 polymerase for the within the MEF2 multigene family, we used probes derived from the sense control probe. (4) Mej2D: 715 base pair mouse cDNA (Edmond- 3’ coding and noncoding regions of the mRNAs that are not conserved son et al., 1994) in pBSKI1 (Stratagene) was linearized with Not1 and between the different genes. cDNAs were subcloned into the vector, transcribed with T7 polymerase for the antisense cRNA probe. The Bluescript (Stratagene). The following probes were used. (1) Mej2A: plasmid was linearized with EcoRI and transcribed with T3 polymerase 388 base pair mouse cDNA (Edmondson et al., 1994) in pAMP1 (GIB- for the sense control probe. The Journal of Neuroscience, August 1995, 15(a) 5729

Figure 2. Transverse and frontal sections of 15.5-16.5 d p.c. mouse brains show the anteroposterior and lateral extents of Mefl gene expression. Frontal sections close to the midbrain of a 15.5 d mouse brain were hybridized with probes to Mej2A (A), 43 (B), and -C (C). Frontal sections through the frontal cortex (f), olfactory bulbs (o), and nasal sinus (s) area of a 15.5 d mouse embryonic head were hybridized with probes to Mef2D (D), 43 (E), and -C (F). Transverse sections through a 16.5 d mouse brain were hybridized with probes to Mej2A (G) and -C (H). Arrows in A and G point to the thalamus. Arrows in H point to the cerebellum. p, parietal cortex; w, whisker follicle. Scale bars: A-H, 800 km. 5730 Lyons et al. l Mef2 Gene Expression in Developing Mouse Brain

Figure 3. Members of the Mej!2 gene family are expressed in overlapping but distinct patterns in the prenatal mouse brain. Dark-field micrographs show parasagittal sections of 16.5 d p.c. embryos that were hybridized with probes for Mej2A (A), Mej2B (B), Mej2C (C), and MeflD (D) mRNAs. Arrow in A indicates the thalamus; arrowhead points to the pontine nuclei. The pigmented layer of the retina (r) in the eye is refractile under dark- field illumination. c, cerebellum;f, frontal cortex; h, hippocampus; m, midbrain; o, oral cavity. Scale bar: A-D, 800 pm.

The cRNA transcripts were synthesized according to manufacturer’s ganglia, smooth muscle cells, and melanocytes. Mef2C mRNAs conditions (Stratagene) and labeled with 35S-UTP (> 1000 Wmmol; Amersham). cRNA transcripts larger than 100 nucleotides were sub- were first detected in the CNS at day 11.5 p.c. within two clus- jected to alkali hydrolysis to give a mean size of 70 bases for efficient ters of cells near the intermediate zone of the preoptic area (Fig. hybridization. lA,B; Schambra et al., 1992) of the telencephalon. This region Hybridization and washing procedures. Sections were hybridized of the embryonic brain is one of the first in which neurons begin overnight at 52°C in 50% deionized formamide, 0.3 M NaCl, 20 mM Tris-HCl pH 7.4, 5 mu EDTA, 10 mu NaPO,, 10% dextran sulfate, to differentiate (Altman and Bayer, 1995). Serial sections hy- 1 X Denhardt’s, 50 kg/ml total yeast RNA, and 50-75,000 cpm/pl Y- bridized with the other Mefl probes resulted in only background labeled cRNA probe. The tissue was subjected to stringent washing at levels of grains at this stage (data not shown). 65°C in 50% formamide, 2X SSC, 10 mu DTT, and washed in PBS At 13.5 d p.c., A4ej2C gene transcripts were expressed in a before treatment with 20 kg/ml RNase A at 37°C for 30 min. Following washes in 2X SSC and 0.1X SSC for 10 min at 37”C, the slides were layer of cells in the intermediate zone of the frontal cortex (Fig. dehydrated and dipped in Kodak NTB-2 nuclear track emulsion and 1C; Schambra et al., 1992) and in cells of the olfactory bulb exposed for 1 week in light-tight boxes with dessicant at 4°C. Photo- (data not shown). In contrast to this pattern, MejZ’A mRNAs graphic development was carried out in Kodak D-19. Slides were coun- were detected predominantly in the region of the developing terstained lightly with Toluidine blue and analyzed using both light- and dark-field optics of a Zeiss Axiophot microscope. Sense control cRNA thalamus (Fig. lD, arrow). Each of these regions in the devel- probes (identical to the mRNAs) always gave background levels of oping brain is an area that contains differentiating neurons (Alt- hybridization signal. Several examples of the sense control hybridiza- man and Bayer, 1995). Cells in the choroid plexus were also tion results are shown in the figures. Embryonic structures were iden- positive for Mef2C expression (Fig. 1 C), but this may reflect the tified with the help of the following atlases: Rugh (1990), Kaufman (1992), Schambra et al. (1992), and Altman and Bayer (1995). presence of some smooth muscle cells in the blood vessels in this structure. Results At 13.5 d p.c., Mef2 gene transcripts were also localized in Mef2 gene expressionin the embryonic CNS and neural crest different regions of the neural tube (data not shown). MefLA In situ hybridization with cRNA probes specific for each of the mRNAs were distributed in a gradient with highest levels in the four A4efl gene transcripts did not detect any of these mRNAs dorsal portion of the neural tube and lowest levels in the ventral in ectoderm or neural tissues from 7.5-10.5 d postcoitum (p.c.), portion. MejX was expressed in a layer of cells only in the with the exception of neural crest cells (Edmondson et al., 1994). dorsal region. MeflD mRNAs were distributed uniformly Neural crest cells arise from the dorsal portion of the neural tube throughout the neural tube (data not shown). and migrate to a number of locations to form a variety of cell Examination of parasagittal sections of 14.5 d p.c. brains re- types including neurons of dorsal root ganglia and sympathetic vealed that Mej2C and -D mRNAs were detected in the embry- The Journal of Neuroscience, August 1995, 75(8) 5731

Figure 4. Localization of Mej2B and -C gene transcripts in the neonatal mouse brain. A, Dark-field image of a frontal section shows th at Mej2B mRNAs are detected in the layers of the frontal cortex (f) and olfactory bulbs (0). B, Dark-field image; C, Phase-contrast image of thi e cortical region of the area indicated by the box in A from a serial section hybridized with the Mef2C probe. 0, Higher magnification of the region enclosed in the box in A. Comparison of B and D shows that MejX is expressed in more cortical layers than Mej2B. E, Parasagittal section of a neonatal brain hybridized with the Me$B probe shows that these transcripts are detected in a region of the inferior colliculus (i), the cerebellum (c), cells in the pontine nuclei (arrowhead), thalamus (t) and a region of the medulla oblongata (arrow). F, Dark-field micrographs; G, Phase:-contrast micrographs of a serial section to that in E hybridized with the Mef2C probe. H, Higher magnification of the cerebellum shown in E. An rowheads in F and G indicate the external granule cell layer outside of the Purkinje cell layer @), which likely contains a mixture of Purkinje cells and differentiated granule cell neurons. Scale bar: A and E, 800 brn; B-D and F-H, 200 pm. 5732 Lyons et al. * Mef2 Gene Expression in Developing Mouse Brain

14.5 Day Embryo

Figure 5. Schematicrepresentation of Me@ geneexpression in the 14.5d p.c. andneonatal mouse brain. The regions of gene expressionare indicatedas shaded areas. The intensityof hybrid- ization signalis indicatedby the den- sity of the shading. onic frontal cortex, hippocampus, amygdala, midbrain (Fig. 16.5 d p.c., MeflA mRNA levels above backgroundwere found l&F), and olfactory bulb (data not shown). In the cerebellar predominantly in cells of the thalamus (Fig. 2A,G). MejX gene primordium, MeflD gene transcripts were localized in the ex- transcripts were detected at high levels in several cell layers of ternal granule cell layer, a proliferating cell population (seeDis- the olfactory bulbs and parietal cortex and at lower levels in the cussion),whereas MeJ2C mRNAs appearedto be in the layer of developing cerebellum (Fig. 2C,F,H). MeflB gene transcripts Purkinje cells that underwent their final mitosesat 12-13 d p.c. (Fig. 2&E) were also presentin the parietal cortex neurons,but (Miale and Sidman, 1961). Mef2B gene transcripts were abun- were much more restricted in their distribution than Mef2C dant in the frontal cortex layers, were present at lower levels in mRNAs. MeflD gene transcripts overlapped with those of the developinghippocampus, amygdala, and midbrain, but were Me&X, but were present at lower levels (Fig. 20). not detectablein the cerebellum(data not shown).Hybridization At 16.5d, low levels of Mef2A mRNAs overlappedwith high- of serialsections with sensecontrol probesrevealed background er levels of MeflC in the hippocampus(Fig. 3A,C), midbrain, level of signal with the cRNA probes used (data not shown). and frontal cortex. Mej2B genetranscripts were also detectedin Expressionpatterns of the Mef2 genesat 14.5 d p.c. are sum- both the frontal and midbrain cortex layers II-VI with Mef2C marized in Figure 5. mRNAs (Fig. 3B,C) at 16.5 d p.c. Mej2D (Fig. 30) was the Transverse and frontal (coronal) sections of embryos were most widely expressedof the four genesin the brain, which was hybridized with each of the four antisensecRNA probesto gain consistentwith its pattern of expressionin the other embryonic a three-dimensionalpicture of MejY2gene expression.At 15.5- tissues(Edmondson et al., 1994). MejZD transcript levels were The Journal of Neuroscience, August 1995, 75(8) 5733

Figh we 6. Spatial patterns of A4efl gene expression in 2 week postnatal brain. Parasagittal sections were hybridized to probes for Mef2A (A), -B (B), -c (0 , and -D (D). Arrowhead points to the lateral aeniculate nucleus in the thalamus (t). c, cerebral cortex; a, horn of Ammon; g, dentate gyms. Scale bar, 800 km.

generally lower than those of the other Mefl genes.MeflA gene were more abundant.Mej2A mRNA levels were slightly above transcript levels were highest in the thalamus but were also background in the cerebral cortex (Fig. 6A), while Mej2B gene found in the region of the pontine nuclei and the frontal cortex transcripts were present at higher levels in the outer half of the (Fig. 3A). cortical neuron layers (Fig. 6B). Mef2A mRNAs appearedto be slightly more abundant in the lateral geniculate nucleus than Postnatal “‘-2 gene expression in the brain those of Mej2C and -D, while MeflB mRNAs were not detect- At birth, the expressionpatterns of Mef2B and Mej2C mRNAs able in this structure. The MeflC gene transcriptswere at slight- overlapped in frontal cortex layers II to VI and in the olfactory ly higher levels in the thalamus than those of MeflA and -D, bulbs, but the hybridization signal with the MeflC probe was while Mej2B gene transcripts were also undetectablein these higher in the more internal layers, V and VI (Fig. 4A-D). Both neurons. of thesegenes were expressedin the pontine nuclei and medulla Between 2 and 6 weeks after birth, changesoccurred in the oblongata(Fig. 4.3. The level of expressionvaried for thesetwo expressionpatterns of Me$? genesin the brain. The moststriking geneswithin regions of the thalamus(data not shown). At this pattern of Mefl gene expressionwas in the mature cerebellum. stage, signal in the inferior colliculus was clearly defined (Fig. Mef2A gene transcriptswere detected at low levels in all cere- 4E). This was the first time that MeflB gene transcripts were bellar cells, but slightly higher levels occurred in the granule detectablein the cerebellum. Both Mej2B and Mef2C mRNAs cell layer (Fig. 7G,L). MeflB mRNAs were undetectablein the were localized in a layer deepto the external granule layer (Fig. cerebellum (Fig. 7H,M, folds in the white matter at the center 4F-H; Altman and Bayer, 1993, which likely consistsof a mix- of H and M appear refractile in dark-field illumination and do ture of Purkinje cells and differentiated granule cell neurons. not representpositive signal). Mej2C gene transcripts were de- Expressionpatterns of the Mej2 genesin the neonatalbrain are summarizedin Figure 5. tected at high levels in the cell bodiesbut were below detectable In the 2 week postnatal brain, each of the four Me&? genes levels in the dendrites of Purkinje cells (Fig. 71,N). Me$?C were expressedin the dentategyrus (Fig. 6). Mef2C and MeflD mRNAs were detected as a series of parasagittally oriented mRNAs were presentat higher levels than were those of Me$?A stripes (data not shown), as indicated by the comparisonof a and Mej2B. MejZC genetranscripts were detected in a subsetof series of parasagittalsections hybridized to this probe. Me$2D pyramidal cells in the horn of Ammon of the hippocampus(Fig. mRNAs were found predominantly in the granule cell layer of 6C), but Mej2B mRNAs were not present in any cells of this the cerebellum (Fig. 7J,O). Since the autoradiographic grains structure (Fig. 6B). Mej2A (Fig. 6A) and MeflD (Fig. 60) were were detectedin the emulsionoverlying the embryo or postnatal expressedat similar levels in neurons of the horn of Ammon, brain sections, the in situ data presented here do not permit but Mej2D levels were higher than thoseof Mej2A in the dentate assignmentof Mej2 gene expression to individual neurons or gyms cells. MejX and -D genes were expressed equally supporting glia. However, the striking localization of Mej2C hy- throughout the layers of the frontal cortex, but Mef2C mRNAs bridization signal over Purkinje cells and large neuronsin the 5734 Lyons et al. l Mef2 Gene Expression in Developing Mouse Brain

Figure 7. Mefz gene transcripts are localized in overlapping but distinct regions in the adult (6 week postnatal) mouse brain. A-E, Parasagittal sections through the olfactory bulb and frontal cortex were hybridized to probes for Mej2A sensecontrol (A), Mej2A (B), 43 (C), -C (D), and -D (E). F-J, Parasagittalsections through the cerebellumwere hybridized with probesfor bright-field(F), Mef2A (G), -B (H), -C(l), and -D (J). The sectionsin F-J areenlarged in micrographsK-O to illustratethe layer-specificexpression of Mef2 genesin the cerebellum.Sections were hybridized with probesto bright-field(K), MeflA (L), -B (M), -C (IV), and -D (0). A rrowheads in N point to Purkinjecells. g, innergranule cell layer; m, molecularlayer; w, white matter.Scale bars: A-E, 600 km; F-J, 800 pm; K-O, 200 km. hippocampusstrongly suggestthat A4ef2 genesare only being 2 weeks. Throughout the adult brain Mej2D mRNAs were most expressedin neuronsin the CNS. widely expressed,and Mef2B gene transcripts were most re- The patternsof overlapping expressionin the dentate gyrus, stricted (data not shown). Expressionpatterns of the Meg genes hippocampus(data not shown), olfactory bulbs, and frontal cor- in the embryonic and postnatalmouse brain are summarizedin tex (Figs. 6, 7) remainedsimilar if not identical to that seenat Figures 5 and 8 and in Table 1. The Journal of Neuroscience, August 1995, 15(8) 5735

Figure 8. Schematic representationof Mef2 gene expressionin the adult mousebrain. The regionsof geneex- pressionare indicated as shaded areas. The intensityof hybridizationsignal is indicatedby the densityof the shading.

Discussion differentiation may also involve interactionsbetween neurogenic Our resultsdemonstrate that membersof the Mej2 gene family bHLH proteins and MEF2 factors. are expressedin a dynamic pattern during pre- and postnatal development of the mouseCNS. Other multigene families of Mef2 expression in the cerebellum transcription factors that have been describedin the developing The correlation of expressionpatterns of the Mefl geneswith CNS such as the (Stoykova and Gruss, 1994) and the morphogeneticevents and neuron differentiation in the cerebel- Otx genes(Frantz et al., 1994) appear to be involved in brain lum was particularly striking. The cerebellumappears at 10 to segmentationand regionalization. The Mej.2 genesappear to be 12 d p.c. as an evaginated thickening in the anterior part of the associatedwith withdrawal of neuronsfrom the cell cycle and fourth ventricle. After extensive cell division and migration, a expressionof a differentiated phenotype. Each of the Mej2 genes substantialcerebellar plate has formed by 14 d p.c. The plate showeddistinct, but often overlapping patternsof expressionin contains a primitive ependymal zone with a granular layer in its different regionsof the brain, which suggeststhat they may per- outer region, a thin molecular layer, and an external granule cell form unique roles at different stagesof neuronalmaturation. The layer (Miale and Sidman, 1961). overlapping expressionpatterns of the MefL genesin the devel- Precursorsof Purkinje cells undergo their final mitotic divi- oping brain are reminiscentof the expressionof thesegenes in sionsbetween 11 and 13 d p.c., which is the first stageat which developing myogenic cell lineages, in which they have been we detect Mej2C mRNAs in the cerebellum.These neurons mi- implicated in the control of muscle-specificgene expression (Ed- grate distally, and by 17 d p.c. are located in the primordium of mondsonet al., 1994). the granule layer of the cerebellarcortex deep to the molecular There are several interesting parallelsbetween differentiation layer (Miale and Sidman, 1961). Our results with MejX gene of musclecells and neurons.Both cell types are electrically ex- expressionin the 16 d p.c. and neonatal cerebellum correlate citable and become postmitotic prior to differentiation, which well with this pattern. Although Purkinje cells form early, they suggeststhat some genesin thesetwo cell types may be regu- do not becomedistinctly larger than neighboringcells until after lated by common sets of transcription factors. The recent find- birth, and are not arrangedin a characteristic single layer until ings of expressionof the myogenic regulatory gene, myf-5, in approximately 10 d after birth (Miale and Sidman, 1961). As certain regions of the embryonic brain and in myogenic cells they do so, it is clear that only Mef2C mRNAs are detectedin derived from the mouse neural tube supports this hypothesis these large cerebellarneurons. (Buckingham and Tajbakhsh, 1993; Tajbakhshet al., 1994). The Mej2D gene expression is first detected in the most distal differentiation of several neuronal cell types in Drosophila and portion of the developing cerebellumat 14.5 d p.c. These cells mammalshas also been shown to be controlled by networks of may representthe external granule cell layer (EGL), which orig- bHLH factors similar to thosein the skeletalmuscle lineage (Jan inates within the rhombic lip of the cerebellumand which gives and Jan, 1993). In light of the fact that MEF2 factors and my- rise to the granule cell neurons(reviewed in Hatten, 1993).Since ogenic bHLH proteinsregulate one another’sexpression in skel- the EGL is a zone of cell proliferation, our data suggeststhat etal muscle cells and cooperateto induce skeletal muscle gene Mej2D gene transcripts can be expressedin cells that are still expression (Cserjesi and Olson, 1991; Lassar et al., 1991; dividing. The granule cell precursorsin the EGL later become Kaushal et al., 1994), it is tempting to speculatethat neuronal postmitotic and begin to differentiate postnatally within the sub- 5736 Lyons et al. * Mef2 Gene Expression in Developing Mouse Brain

Table 1. Relative levels of Mefl gene expression in developing brain

Days postcoitum 12.5 14.5 16.5 Nn 2 week Adult Olfactory bulb 2A - + + + + + 2B - + + ++ ++ +++ 2c - ++ ++ +++ +++ +++ 2D + ++ ++ ++ ++ ++ Amygdala 2A - - - + + 2B - - i-l- - - - 2c + +++ +++ + - ++ 2D +/- ++ ++ + + ++ Cerebral cortex 2A - - + + + + 2B + ++ ++ +++ ++ +++ 2c ++ +++ +++ +++ +++ +++ 2D + + ++ ++ ++ ++ Hippocampus 2A nd - - + +++ +++ 2B nd + + + + +/- 2c nd +++ +++ ++ ++ ++ 2D nd + + + +++ +++ Thalamus 2A ++ ++ +++ +++ +++ +++ 2B - + + + ++ - 2c + + + + + ++ 2D + + + ++ + ++ Midbrain/colliculus 2A - + + + + - 2B - - - + ++ ++ 2c - ++ ++ ++ ++ +++ 2D - ++ ++ ++ ++ ++ Cerebellum 2A nd - - + + + 2B nd - - ++ + - 2c nd + ++ ++ ++ + 2D nd + + + ++ ++ Pontine nuclei 2A nd nd ++ ++ - + 2B nd nd +/- + ++ ++ 2c nd nd ++ ++ ++ +++ 2D nd nd + + + ++ Medulla 2A -+I- + + + nd nd 2B - - + + nd nd 2c - + + + nd nd 2D + + + + nd nd

nd, not determined; Nn, neonatal

EGL. These neurons migrate inward to form the inner granule 11 d p.c. The rate of hippocampalneuron production increases cell layer of the mature cerebellum (reviewed in Hatten, 1993). in the next 24 hr to reach a plateau that continues from 12 to That Mej2D gene transcripts continue to be expressed in the 16 d p-c. The pace then decreases,and only an occasionalcell inner granule cell layer suggestsan important role for this gene is generatedas late as 18 d p.c. Neurons continue to form in the in both dividing and postmitotic neuron development. dentate gyms until the third postnatal week (Caviness,1974). In All four Me@ genesare detected in a cell layer that lies deep rats, most of the granule cells of the dentate gyms form post- to the EGL at birth. These neuronsare likely to be a mixture of natally, and hippocampalneurogenesis continues at a low rate Purkinje cells and differentiated granule cells. MegA, like until at least 8 months of age (Altman and Das, 1965). MefLD, continues to be expressedin the granule cells in the Although we did not detect Mef2 mRNAs in hippocampal adult, but Mef2B gene transcripts decreaseto below the level of cells as early as 12.5 d p.c., the pattern of Mej2 gene expression detection of the in situ hybridization technique. MeJ2C is ex- still has a good correlation with the main times of neuron dif- pressedexclusively in Purkinje cells in the adult. MeflA and -B- ferentiation in this region of the brain as describedabove. In- expressingcells in the early postnatal cerebellum may represent terestingly, the Mef2 gene transcripts showed overlapping ex- granule cell neurons that undergo their final mitosesfrom the pression at prenatal stagesbut postnatally, the patterns of the late embryo to postnatal day 15 (Miale and Sidman, 1961). four genes,particularly in the Horn of Ammon cells, were dis- Mef2 gene expression in the hippocampus tinct. The lack of expressionof MeflB mRNAs and the punctate In the mouseembryo, the earliest neuronsto differentiate in the expressionpattern of Mef2C gene transcripts suggeststhat these hippocampusand dentate gyms undergo their final divisions by genesare either downregulated in certain groups of neuronsor The Journal of Neuroscience, August 1995, 15(8) 5737 that some neurons in the hippocampus undergo programmed cell potential target genes are activated. That MEF2 proteins collab- death. orate with myogenic bHLH proteins to regulate skeletal muscle gene expression raises the possibility that neural bHLH proteins, Mef2 gene expression in the cerebral cortex such as the MASH gene products (Lo et al., 1991) or the ME1 Mej2C is the first gene transcript we were able to detect in the gene product (Neuman et al., 1993), may act in conjunction with developing cerebrum, and its expression level remained high MEF2 proteins to regulate neural genes. until 6 weeks after birth. MejX expression was first detected in The expression of Mej2 genes in the myogenic and neuro- the intermediate zone of the frontal cortex at day 12.5 p.c. Neu- genie lineages raises interesting questions about the cis- and rons in this zone underwent mitosis in the subependymal layer truns-regulatory systems that activate these genes in different of the cortex and migrated into the mantle layer of the cerebral cell types. Analysis of the control regions of the Mef2 genes will cortex as differentiated cells (Smart, 1961). As development pro- undoubtedly yield insight into the mechanisms that specify the ceeds, neurons continue to migrate into the cortex to form six myogenic and neurogenic programs for cell-specific gene ex- distinct layers. This is reflected in the gradual increase in thick- pression during development. ness of the cortical layers positive for Mej2C gene expression. MeflB mRNAs were detected predominantly in the six cortical References layers of neurons, whereas MeflC was expressed in both the Altman J, Bayer SA (1995) Atlas of prenatal rat brain development. cortical layers and deep gray matter (Smart, 1961). Ann Arbor, MI: CRC. Expression of Mej2C in the cortex was also observed in a Altman J, Das GD (1965) Autoradiographic and histological evidence preliminary study in which a human MejX cDNA was used to of postnatal hippocampal neurogenesis in rats. J Comp Neurol 124: 3 19-336. analyze expression in tissue sections of rat brain (Leifer et al., Angevine JB (1970) Time of neuron origin in the diencephalon of the 1993). However, in contrast to our results, they reported that mouse. An autoradiographic study. J Comp Neurol 139: 129-188. Mej2C mRNA levels decreased between 1 and 4 weeks after Breitbart R, Liang C, Smoot L, Laheru D, Mahdavi V, Nadal-Ginard B birth in the rat cerebral cortex. The contrasting results for Mef2C (1993) A fourth human MEF-2 transcription factor, hMEF2D, is an early marker of the myogenic lineage. Development 118: 1095-l 106. gene expression patterns between the two studies may be due to Buckingham ME, Tajbakhsh S (I 993) Expression of the myogenic fac- species differences in the Mef2C gene between rat and mouse tors in the mouse: myf-5, the first member of the MyoDgene family or rat and human, or to differences in the hybridization protocols to be transcribed during skeletal mvogenesis._ - C R Acad Sci III 316: used. 1040-1046. Caviness VS (1974) Time of neuron origin in the hippocampus and Mef;! gene expression in the thalamus dentate gyms of normal and reeler mutant mice: an autoradiographic study. J Comp Neural 15 1: 113-120. In the embryo, the expression of MeflA in the thalamus was Chambers A, Kotecha S, Towers N, Mohun T (1992) Muscle-specific striking. Neurons of the ventral thalamus begin to differentiate expression of SRF-related genes in the early embryo of Xenopus as early as 10 d p.c., but cells in different nuclei within this area laevis. EMBO J 11: 14991. continue to arise through 16 d pc. A lateromedial or “outside- Cheng TC, Wallace M, Merlie J, Olson E (1993) Separable regulatory elements govern transcription in embryonic somites and in” gradient in the time of neuron origin is seen in the dorsal limbbuds. Science 261:215-218. thalamus and epithalamus (Angevine, 1970). Examination of co- Cserjesi P Olson E (1991) Myogenin induces muscle-specific enhancer ronal and transverse sections at 15-16 d p.c. hybridized with the binding factor MEF-2 independently of other muscle-specific gene MepA probe reveal a greater number of grains over the lateral products. Mel Cell Biol 11:4854-4862. Edmondson D, Cheng T Cserjesi P Chakraborty T Olson E (1992) neurons of the thalamus compared to the medial cells. A second Analysis of the myogenin promoter reveals an indirect pathway for caudorostral gradient of neuron origin occurs in the dorsal thal- positive autoregulation mediated by MEF2. Mol Cell Biol 12:3665- amus (Angevine, 1970). A parasagittal section of a 16 d p.c. 3677. mouse brain hybridized with the MefLA probe shows a higher Edmondson DG, Lyons GE, Martin J, Olson E (1994) Mefl gene ex- pression marks the cardiac and skeletal muscle lineages during mouse density of silver grains over the caudal portion of the thalamus embryogenesis. Development 120: 125 1-l 263. compared to the adjacent rostra1 region. Overall, these data sug- Frantz GD, Weimann JM, Levin ME, McConnell SK (1994) Otxl and gest that Mef2A may play an important role in the prenatal dif- Otx2 define layers and regions in the developing cerebral cortex and ferentiation of thalamic neurons. cerebellum. J Neurosci 14:5725-5740. Gossett L, Kelvin D, Sternberg E, Olson E (1989) A new myocyte- Mef2 factors may collaborate with bHLH proteins to induce specific enhancer binding factor that recognizes a conserved element cell-specific transcription associated with multiple muscle specific genes. Mel Cell Biol 9:5022-5033. In light of the restricted expression of Mefl genes to early my- Hatten ME (1993) The role of migration in central nervous system ogenic and neurogenic lineages, it is tempting to speculate that neuronal development. Cm-r Opin Neurobiol 3:3844. Jan YN, Jan LY (1993) HLH proteins, fly neurogenesis, and vertebrate members of the MEF2 family are involved in activating the spe- myogenesis. Cell 75:827-830. cific programs of gene expression associated with these lineages. Kaufman MH (1992) The atlas of mouse development. New York: How might these factors participate in these different develop- Academic. mental programs? We favor the hypothesis that the actions of Kaushal S, Schneider JW, Nadal-Ginard B, Mahdavi V (1994) Acti- MEF2 proteins are influenced by other factors unique to differ- vation of the myogenic lineage by MEF2A, a factor that induces and cooperates with MyoD. Science 266:1236-1240. ent cell lineages. Accordingly, within mesodermal cell types, Lassar AB, Davis RL, Wright WE, Kadesch T, Murre C, Voronova A, MEF2 proteins activate muscle gene expression, whereas in ec- Baltimore D, Weintraub H (1991) Functional activity of myogenic todermal derivatives they activate neural gene expression. In this HLH proteins requires hetero-oligomerization with E 12/E47-like pro- regard, it is well established that the actions of MADS proteins teins in vivo. Cell 66:305-315. Leibham D, Wong M, Cheng T Schroeder S, Weil P, Olson E, Perry M are influenced by other regulatory factors with which they in- (1994) Binding of TFIID and MEF2 to the TATA element activate teract. Thus, muscle and neuronal precursors may express dif- transcription of the Xenopus MyoDn promoter. Mol Cell Biol 14:686- ferent cofactors for MEF2 activity that help determine which 699. 5738 Lyons et al. - MefZ Gene Expression in Developing Mouse Brain

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