Information provided by the skeletal muscle and associated neurons is necessary for proper brain development

Int. J. Devl Neuroscience 20 (2002) 573–584

Information provided by the skeletal muscle and associated neurons is necessary for proper brain development Boris Kablar a,∗ , Michael A. Rudnicki b a b

Department of Anatomy and Neurobiology, Dalhousie University, 5859 University Avenue, Halifax, NS, Canada B3H 4H7 Department of Molecular Medicine, Ottawa Hospital Research Institute, 501 Smyth Road, Ottawa, ON, Canada K1H 8L6 Received 8 April 2002; received in revised form 20 June 2002; accepted 23 July 2002

Abstract Previously, motor cortex of term Myf5−/− :MyoD−/− fetuses (e.g. have ablated skeletal myogenesis and consequent early loss of lower motor and proprioceptive neurons) was found to lack giant pyramidal cells. We further investigated how the absence of the extrinsic stimuli from the lacking structures influences brain development. Apparently normal motor cortex of mutant fetuses was found to have dramatically reduced presence of nestin-expressing processes of neural precursors, calretinin-expressing pyramidal neurons and calbindin-expressing neurons. Consistently, some areas of the extrapyramidal tract had significantly decreased number of differentiated neurons in mutant brains. Surprisingly, we were unable to detect any change in proliferation or cell death in the mutant neuroepithelium. Together, it appears that the information provided by the lacking structures influences the ratios of the differentiated neuronal types and their progenitor cells. © 2002 ISDN. Published by Elsevier Science Ltd. All rights reserved. Keywords: Myogenesis; Neuroepithelium; Immunohistochemistry

1. Introduction In mammals, myogenesis occurs as a two phase event (Harris et al., 1989). The first phase in mice begins prior to embryonic day (E) 12.5, resulting in formation of primary muscle fibers. The second phase begins during E14.5 and gives rise to approximately 80% of neonatal muscle (e.g. secondary muscle fibers). This phase coincides with the peak of naturally occurring motor neuron cell death in the spinal cord. Abbreviations: Fr, motor (or frontal) neocortex; Cc, cingulate cortex; Par, somatosensory (or parietal) neocortex; In, insular cortex; Il, infralimbic cortex; Pir, piriform cortex; Rs, rhinal sulcus; Lot, lateral olfactory tract; OT, olfactory tubercule; OV, olfactory ventricule; Tca, thalamocortical axons; Svz, subventricular zone or striatum; CP, caudoputamen; Ic, internal capsule; Cc, corpus callosum; TT, tenia teca; CxP, cortical plate; Cl-End, claustrum or endopiriform nucleus; LV, lateral ventricle; SP, subplate; Spt, septum; Sptn, septal neuroepithelium; Ec, external capsule; IG, induseum griseum; Iz, intermediate zone; Acb, nucleus accumbens; Chp, choroids plexus primordium; F, fornix; LVsf, lateral ventricle-septal fork; Ncn, neocortical neuroepithelium; St, stria terminalis; Svzc, subventricular zone-neocortex; Tca, thalamocortical axons; GP, globus pallidus; Nsp, nigrostriatal pathway; ATN, anterior thalamic nuclear complex; TH, tyrosine hydroxylase; CR, calretinin; NF-160, neurofilament-160; PCNA, proliferating cell nuclear antigen; Ache, acetylcholinesterase ∗ Corresponding author. Tel.: +1-902-494-3169; fax: +1-902-494-1212. E-mail addresses: [email protected] (B. Kablar), [email protected] (M.A. Rudnicki).

The myogenic regulatory factors are transcription factors consisting of MyoD, myogenin, Myf5 and MRF4. Mice with null mutation in myogenin fail to form secondary muscle fibers (Hasty et al., 1993). Mutation in MyoD, Myf5 or MRF4 gene, results in essentially normal patterning and skeletal muscle volume (reviewed in Kablar and Rudnicki, 2000). Strikingly, in compound-mutant animals lacking both Myf5 and MyoD the entire embryonic lineage that gives rise to skeletal muscle never forms (Rudnicki et al., 1993). Embryos lacking both Myf5 and MyoD were used to examine neurogenesis of the mammalian motor system in the complete absence of skeletal myogenesis (Kablar and Rudnicki, 1999). Spinal and brain stem motor neurons and proprioceptive sensory neurons in the dorsal root ganglia are normally induced, followed by their progressive and total loss by apoptosis. Strikingly, pyramidal neurons in the E17.5 motor cortex appear completely ablated without any corresponding evidence of apoptosis, suggesting that their precursors fail to undergo normal developmental program and that compartmentalization in the motor cortex is therefore altered. The vertebrate cerebral cortex (pallium) is organized into several functional subdivisions: the medial, dorsal, lateral and ventral pallium. In mammals, the medial pallium (archiocortex) includes the hippocampal and subicular

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regions; the dorsal pallium corresponds to the neocortex; the lateral pallium (paleocortex) includes the primary olfactory cortex; and the ventral pallium includes the claustrum. Each of these regions has further subdivisions. For instance, within the neocortex, there are the prefrontal, motor and sensory regions, each with further subdivisions (reviewed in Rubenstein et al., 1999). There are two schools of thought about genetic and developmental processes that produce diverse regions in the cortex. The Protomap model (Rakic, 1988) postulates that neocortical regionalization is primarily controlled by molecular determinants that are intrinsic to the proliferative zone of the neocortex. In contrast, the Protocortex model (O’Leary, 1989) suggests that neocortical regionalization is primarily controlled by extrinsic influences, such as thalamocortical axons (e.g. receive information from proprioceptive sensory neurons via cerebellum) or by the targets of cortical axons (e.g. spinal and brain stem motor neurons, directly or via interneurons). It appears that cortical differentiation is a progressive sequence of events that starts within forebrain, followed by the telencephalon, the dorsal pallium and lastly the neocortex (reviewed by Pallas, 2001). In this report, by employing Myf5−/− :MyoD−/− embryos that completely lack skeletal myogenesis and consequently all spinal or brain stem somatic motor neurons and proprioceptive sensory neurons (e.g. a massive and abrupt process of motor neuron cell death in the mutant spinal cord starts as early as E13.5, Kablar and Rudnicki, 1999), we further investigated how the absence of extrinsic stimuli (e.g. indirect afferent inputs from proprioceptive neurons and direct or indirect trophic support from skeletal muscle and motor neurons) affected neuronal differentiation in various structures and compartments of the brain. Our data suggest that neuronal precursor cells do not produce sufficient number of differentiated descendent neuronal types and subtypes in the absence of external stimuli. 2. Experimental procedures 2.1. Interbreeding and collection of embryos Embryos lacking both Myf5 and MyoD (designated as Myf5−/− :MyoD−/− ) were derived by a two generation breeding scheme. First, MyoD−/− mice (Rudnicki et al., 1993) were bred with Myf5+/− mice to generate Myf5+/− :MyoD+/− mice. Second, Myf5+/− :MyoD+/− mice were interbred to obtain embryos of nine different genotypes. Embryos and the fetal portion of the placenta were collected by cesarean section on the required embryonic day (E) and embryos prepared for Nissle cresyl violet, Golgi silver impregnation, immunohistochemistry staining and whole-mount TUNEL analysis, as described below. Three embryos of each genotype were examined on E15.5, E17.5 and E18.5. Genomic DNA was isolated from the fetal portion of the placenta using the procedure of

Laird et al. (1991). Embryos were genotyped by Southern analysis (Sambrook et al., 1989) of placental DNA using Myf5 and MyoD specific probes as described previously (Rudnicki et al., 1993). Care of animals was in accordance with institutional guidelines. 2.2. Immunohistochemistry Immunohistochemistry was performed as previously described (Kablar et al., 1997) on paraffin-embedded or frozen 4 ␮m sections, with mouse monoclonal anti-nestin antibody Rat-401 (1:4, Developmental Studies Hybridoma Bank), rabbit polyclonal anti-calretinin antibody (1:500, Chemicon), mouse monoclonal anti-calbindin D antibody (1:200, Sigma), mouse monoclonal anti-tyrosine hydroxylase antibody NCL-TH (1:20, Novocastra), mouse monoclonal anti-neurofilament antibody NF-160 (1:20, Sigma), mouse monoclonal anti-Islet1/2 antibody 39.4D5 (1:4, Developmental Studies Hybridoma Bank) and mouse monoclonal anti-proliferating cell nuclear antigen (PCNA) antibody (1:400, DAKO). 2.3. Acetylcholinesterase (Ache) histochemistry Ache activity was analyzed using a histochemical protocol described elsewhere (Jung et al., 2000). In brief, sections were incubated in 30 ␮M iso-N,N -bis (1-methylethyl) pyrophosphorodiamidic anhydride/0.2 M Tris–maleate (pH 5.7) for 30 min, followed by a 30 min dark incubation in an Ache solution containing 130 mM Tris–maleate (pH 5.7), 5 mM sodium citrate, 3 mM cupric sulphate, 0.5 mM potassium ferricyanide and 25 mg/50 ml of acetylthiocholine iodide. Sections were washed with PBS, gel-mounted and examined with bright-field microscopy. 2.4. In situ TUNEL analysis To detect apoptotic nuclei in situ by TUNEL (Gavrieli et al., 1992), we employed the ApopTag detection system (Genzyme). Transverse sections of embryos were incubated with terminal deoxynucleotydil transferase (TdT enzyme) diluted in the reaction buffer. The enzyme catalyzes a template independent addition of deoxyribonucleotide triphosphate to the 3 -OH ends of double- or single-stranded DNA. The incorporated nucleotides form a random heteropolymer of digoxygenin-11-dUTP and dATP that has a reaction site for anti-digoxigenin antibody carrying a conjugated peroxidase enzyme. The localized peroxidase enzyme was visualized by a chromogenic reaction with AEC (3-amino-9-ethylcarbazole, Sigma) and sections were counter-stained with hematoxylin. 2.5. Morphometric analysis Morphometric analysis was performed as previously described (Kablar and Rudnicki, 1999). In brief, this analysis

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is done on frontal serial sections through the level of the caudoputamen, on calbindin- and Islet1/2-immunostained sections, at the level of globus pallidus, on TH-immunostained sections and on PCNA/TUNEL stained sections. Counts were made under high magnification (400×) in every 10th section and thus the data presented are the actual counts multiplied by 10 and then divided by the total number of sections per region to yield cells per section. The PCNA proliferative index was determined as percentage of positive nuclei (i.e. number of red immunostained nuclei versus hematoxylin-stained blue nuclei; 1000 cells per section were counted).

3. Results 3.1. Development of brain anatomical milestones is unaffected in mutant mice To further investigate how the absence of extrinsic information from the periphery affects neocortical differentiation, regionalization and lamination we compared wild-type and Myf5−/− :MyoD−/− brains of term embryos (e.g. Myf5 and MyoD proteins could not be detected in the brain of wild-type embryos; Daubas et al., 2000; Kablar, 2002; data not shown). Previously, by performing hematoxylin and eosin staining, we have described the absence of morphologically recognizable giant pyramidal cells in E17.5 mutant motor cortex (Kablar and Rudnicki, 1999). In this report, to more precisely characterize different brain regions, we performed Nissle staining (e.g. a special staining procedure to demonstrate rough endoplasmatic reticulum) and immunohistochemistry against NF-160 antibody. We analyzed structural appearance of developing cerebral cortex and other brain regions, according to Jacobowitz and Abbott (1997). In both, wild-type and compound-mutant brains, in frontal serial sections of the region that contained motor (or frontal) neocortex (Fr), we were also able to detect cingulate cortex (Cc), somatosensory (or parietal) neocortex (Par), insular cortex (In), infralimbic cortex (Il), piriform cortex (Pir), rhinal sulcus (rs), lateral olfactory tract (lot), olfactory tubercule (OT), olfactory ventricule (OV), thalamocortical axons (tca), subventricular zone or striatum (svz), caudoputamen (CP), internal capsule (ic), corpus callosum (cc) and tenia teca (TT) (Fig. 1A and B). In the more posterior frontal sections of wild-type and compound-mutant brains containing the primary motor cortex, we were able to detect, in addition to what described in the previous figure, the following structures: cortical plate (CxP), claustrum or endopiriform nucleus (Cl-End), lateral ventricle (LV), subplate (SP), septum (Spt), septal neuroepithelium (sptn), external capsule (ec), induseum griseum (IG) and intermediate zone (iz) (Fig. 1C and D). In the most posterior frontal sections of wild-type and compound-mutant primary motor cortex, we were able to additionally identify: nucleus accumbens (Acb), choroids

Fig. 1. Neocortical regionalization and lamination appears to be retained in the absence of peripheral external stimuli. In E18.5 wild-type (A) and compound-mutant (B) brains, in frontal serial sections of the region of motor (or frontal) neocortex (Fr), we can also detect: cingulate cortex (Cc), somatosensory (or parietal) neocortex (Par), insular cortex (In), infralimbic cortex (Il), piriform cortex (Pir), rhinal sulcus (rs), lateral olfactory tract (lot), olfactory tubercule (OT), olfactory ventricule (OV), thalamocortical axons (tca), subventricular zone or striatum (svz), caudoputamen (CP), internal capsule (ic), corpus callosum (cc) and tenia teca (TT). In the more posterior frontal sections of wild-type (C) and compound-mutant (D) brains additional structures are identifiable: cortical plate (CxP), claustrum or endopiriform nucleus (Cl-End), lateral ventricle (LV), subplate (SP), septum (Spt), septal neuroepithelium (sptn), external capsule (ec), induseum griseum (IG) and intermediate zone (iz). In the most posterior frontal sections of wild-type (E) and compound-mutant (F) primary motor cortex, we can additionally identify: nucleus accumbens (Acb), choroids plexus primordium (Chp), fornix (f), lateral ventricle-septal fork (LVsf), neocortical neuroepithelium (ncn), stria terminalis (st), subventricular zone-neocortex (svzc) and anterior commissue (ac). Magnification 25×.

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plexus primordium (Chp), fornix (f), lateral ventricle-septal fork (LVsf), neocortical neuroepithelium (ncn), stria terminalis (st), and subventricular zone-neocortex (svzc) (Fig. 1E and F). It is recently demonstrated that elimination of thalamic input to the neocortex in the Gbx−2 mouse does not alter neocortical regionalization and lamination (Miyashita-Lin et al., 1999). To determine whether thalamocortical axons were intact in our compound-mutant embryos, we performed immunohistochemistry against NF-160 protein on serial frontal sections of brains (Fig. 2). In both, rostral (Fig. 2A and B) and posterior (Fig. 2C and D) sections of the frontal (motor) neocortex, it was clearly demonstrated that thalamocortical axons developed and maintained equally well in both wild-type and compound-mutant brains. Taken together, these data suggest that anatomical milestones of developing brain frontal neocortex are not altered by the absence of stimuli from skeletal muscle and associated neurons (e.g. spinal or brain stem motor neurons and proprioceptive sensory neurons). This provides evidence that patterning mechanisms intrinsic to the frontal neocortex specify the basic organization of its functional subdivisions. 3.2. Immunohistochemical analysis of primary motor cortex reveals impairment of neural progenitor cells and differentiated cell types To investigate the ratios of differentiated cells and their precursors in the frontal neocortex, we examined the expression pattern of nestin, calretinin and calbindin proteins (e.g. the absence of specific molecular markers for pyramidal neurons and their progenitors hinders the investigation of this issue), followed by Golgi silver impregnation on frontal serial sections through the brain of E15.5-E18.5 wild-type and Myf5−/− :MyoD−/− mutant embryos (Fig. 3 and not shown). Surprisingly, the expression pattern of all three proteins appeared to be altered in compound-mutant motor cortex, although we were unable to observe a significant difference in the pattern of silver impregnation staining between wild-type and mutant brains (Fig. 3A and B). During neurogenesis, proliferating cells of the developing mouse neocortex couple together into clusters (Bittman

Fig. 2. Thalamocortical axons are not affected in compound-mutant brains. In rostral (A, B) and posterior (C, D) sections of the frontal (motor) neocortex, it is clearly demonstrated by immunohistochemistry against NF160, that thalamocortical axons (tca) develop and maintain equally well in both wild-type (A, C) and compound-mutant brains (B, D) of term embryos. Magnification 25×.

and Lo Turco, 1999). These clusters contain both radial glial cells and neural precursors. As neural precursors differentiate, they extend nestin-expressing processes from the cluster to the pial surface. Through differential regulation of connexins, neocortical precursors compartmentalize as they progress through the cell cycle (i.e. differentiate). In frontal serial sections through the brain of wild-type and Myf5−/− :MyoD−/− mutant embryos, the nestin-expressing (Fig. 3C and D) processes of neural precursors were found to be well defined in wild-type (Fig. 3C, arrowhead) and

䉴 Fig. 3. Nestin-, calretinin- and calbindin-expressing clusters of neuronal precursors and differentiated neurons are not well defined in compound-mutant brains. In frontal serial sections through the brain of E18.5 wild-type (A, C, E, G, I) and My5−/− :MyoD−/− mutant (B, D, F, H, J) embryos, it is not possible to observe a significant difference in the pattern of Golgi silver impregnation staining (A, B) between wild-type (A) and mutant (B) brains. However, density of differentiated cells in the cortical plate (CxP) appears greater in wild-type than mutant motor cortex. The nestin-expressing processes of neural precursors (e.g. in the subventricular zone, arrows in C and D) appear well defined in wild-type (arrowhead in C) and inexistent in mutant motor cortex (arrowhead in D; nestin-expressing cells in the mutant motor cortex are rounded in shape, have weak expression of nestin in their cytoplasm and show no nestin-positive processes). The calretinin-expressing (red immunohistochemical signal in E and F) inhibitory neurons, interneurons and pyramidal neurons are dramatically less apparent in mutant (arrowhead in F) than in wild-type motor cortex (arrowhead in E). The expression of calbindin (red immunohistochemical signal in G and H) is well defined in wild-type (arrowhead in G) and almost abolished in mutant (arrowhead in H) motor cortex. It is not possible to detect any significant difference in the amount of PCNA-expressing nuclei (red nuclear staining in I for wild-type and in J for mutant motor cortex) or TUNEL-labeled nuclei (arrowhead in K for wild-type and in L for mutant motor cortex). Arrows in C and D show the subventricular zone and in E-H show cortical layers II and III. Note that in A-J all figures are positioned to show CxP on the right, whereas in K and L CxP is at the top of the figure. Magnification, 200× (A–J), 400× (K, L), 1000× (insets in C–H).

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essentially inexistent in mutant motor cortex (Fig. 3D, arrowhead). This feature of neural precursors is best visible in the subventricular zone (Fig. 3C and D, arrows). Therefore, the process of neural precursor cell differentiation may be affected in compound mutant frontal neocortex. This, in turn, may affect neocortical compartmentalization, since neural precursors compartmentalize as they differentiate (Bittman and Lo Turco, 1999). The two calcium-binding proteins, calretinin (Fig. 3E and F) and calbindin (Fig. 3G and H), are found in morphologically distinct classes of inhibitory interneurons and pyramidal neurons in the mammalian neocortex (Hof et al., 1999). Moreover, depletion of neocortical monoamine afferents (e.g. serotonin, catecholamine) results in a permanent alteration of the dendritic arborization of calretinin-containing interneurons and transient delay of calbindin expression in a number of cortical neurons (Durig and Hornung, 2000). In the frontal serial sections through the brain of wild-type embryos, calbindin- and calretinin-immunoreactive neurons were found to be predominantly situated in layers II and III (Fig. 3E–H, arrows), but were present across all cortical layers (Fig. 3E–H). In the frontal serial sections through the brain of Myf5−/− :MyoD−/− mutant embryos (Fig. 3F), the calretinin-expressing inhibitory neurons, interneurons and pyramidal neurons were dramatically less apparent (Fig. 3F, arrowhead; the morphometric analysis was not performed because of the lack of a clear anatomical border of the primary motor cortex and the pattern of calretinin immunostaining) in comparison to the wild-type motor cortex (Fig. 3E, arrowhead). In addition, the expression of calbindin was well defined in wild-type (Fig. 3G, arrowhead) and essentially inexistent in mutant motor cortex (Fig. 3H, arrowhead). While the expression of calbindin in the wild-type cortex was diffusely and uniformly apparent across all cortical layers, in the mutant cortex calbindin was virtually absent (or delayed; postnatal analysis of the calbindin expression is not possible because Myf5−/− :MyoD−/− fetuses die upon delivery). This suggests that changes in the double mutant cortex resemble conditions of neocortical monoamine deprivation. To assess proliferating status and rate of cell death of the neuroepithelium, immunohistochemistry using proliferating cell nuclear antigen (PCNA) and in situ programmed cell death detection (TUNEL) was performed, respectively. In frontal serial sections through the brain of E15.5 to E18.5 wild-type and Myf5−/− :MyoD−/− mutant embryos, it was not possible to detect any significant difference in the amount of PCNA-expressing (Fig. 3I and J and not shown) or TUNEL-labeled nuclei (Fig. 3K and L and not shown). For instance, the PCNA proliferative index (the counts were performed in the region of the motor cortex containing numerous red stained nuclei, i.e. in the subventricular zone, the intermediate zone and the subplate; Fig. 3I and J) was 37% in E18.5 wild-type motor cortex and 40% in E18.5 mutant motor cortex, but the difference was not statistically significant (P > 0.05, t-test). More superficial layers of the

motor cortex contained virtually no PCNA-positive nuclei in both wild-type and mutant embryos. Similarly insignificant was the difference in the number of apoptotic nuclei, where the number of TUNEL-labeled nuclei per section was 3 ± 2 in wild-type E18.5 motor cortex and 3 ± 1 in mutant E18.5 motor cortex. Virtually no difference in number of PCNA/TUNEL-positive nuclei was observed between E18.5, E17.5 and E15.5 embryos in the motor cortex. The number and distribution of neurons appeared normal in the region of the olfactory cortex, that was used as a control region as it should not be influenced by the loss of skeletal muscle (Kablar and Rudnicki, 1999 and data not shown). Together, these findings suggest that precursor cell cycle kinetics and survival is not altered in compound-mutant motor cortex. 3.3. Distribution of calbindin-expressing neurons in caudoputamen is dramatically affected in mutant brains In addition to the precentral region and the pyramidal tract, many other regions and tracts influence motor activity. They are known collectively as the extrapyramidal motor system (e.g. caudoputamen, globus pallidus, etc). To further investigate how the absence of extrinsic cues from the periphery affects differentiation of neurons in the extrapyramidal motor system, we compared wild-type and Myf5−/− :MyoD−/− brains. We examined the expression pattern of acetylcholinesterase (Ache), nestin, calretinin, calbindin, tyrosine hydroxylase (TH) and Islet1/2 proteins on frontal serial sections through the caudoputamen (or striatum) of E15.5-E18.5 wild-type and compound-mutant embryos (Fig. 4 and not shown). A distinct expression pattern of the calcium-binding protein calbindin characterizes the neurochemical differentiation of the developing and mature striatum (Liu and Graybiel, 1992a,b). The heterogeneously distributed calbindin immunoreactivity is confined to the striatal matrix compartment, where it is concentrated within the medium-sized spiny neurons (the principal projecting neurons of the neostriatum) and their projecting axons terminating in the globus pallidus, entopeduncular nucleus and substantia nigra (DiFiglia et al., 1989; Drago et al., 1994). The major subcellular compartment of calbindin expression is the matrix of the cytoplasm of these cells. In wild-type embryos, a dense calbindin expression was found in the ventromedial region of the caudoputamen and a lesser density of calbindin-immunoreactive neuropil was detected dorsally (Fig. 4A and B). This result is consistent with previous findings of a gradient of calbindin expression in the rat (Liu and Graybiel, 1992b) and mouse (Jung et al., 2000) caudoputamen. However, the number of calbindin-expressing neurons and the cellular distribution of calbindin expression differed substantially between wild-type and compound-mutant embryos. Wild-type caudoputamen contained 112 ± 6 calbindin-expressing neurons per section, whereas mutant caudoputamen contained only

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Fig. 4. Calbindin-expressing spiny neurons of compound-mutant caudoputamen are affected. In frontal serial sections of wild-type (A, C, E, G, I) and compound-mutant (B, D, F, H, J) term embryos (E18.5), a dense calbindin expression is found in the ventromedial region (vm in A) of the caudoputamen and a lesser density of calbindin-immunoreactive neuropil is detected dorsally (d in A). In medium spiny neurons of wild-type embryos, calbindin expression is prominent in both, the nucleus (arrowheads in A) and the cytoplasm. By contrast, spiny neurons of mutant caudoputamen express calbindin mostly in the cytoplasm (arrowheads in B) and the number of calbindin-expressing neurons is dramatically reduced (B). However, it appears that the gradient of calbindin expression is retained even in the mutant caudoputamen, since more calbindin-expressing neurons are found in its ventromedial (vm in B) than in its dorsal region (d in B). The expression pattern of TH (C, D) is found to be indistinguishable between wild-type (C) and compound-mutant (D) caudoputamen (ac, anterior commissure). A histochemical method employed to localize Ache in the caudoputamen of wild-type (E) and compound-mutant (F) embryos reveals a mature-like histochemical staining pattern of Ache in both genotypes. An abundant Ache expression is found in matrix (m in E and F) of caudoputamen and a low Ache expression is found in striosomes (s in E and F) of caudoputamen. The expression patterns of CR (G, H) and Islet1/2 (I, J) are also found unaffected in compound-mutant caudoputamen (H, J, respectively). Magnification, 400×, 1000× (insets in A and B).

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26 ± 2 calbindin-expressing neurons per section (77% less than in the wild-type, P < 0.001, t-test). In medium spiny neurons of wild-type embryos, calbindin expression was prominent in both, the nucleus and the cytoplasm (Fig. 4A; the nuclear expression is indicated by arrowheads). By contrast, spiny neurons of mutant caudoputamen expressed calbindin mostly in the cytoplasm (Fig. 4B, arrowheads). However, it appeared that the gradient of calbindin expression was retained even in the mutant caudoputamen, since more calbindin-expressing neurons were found in its ventromedial than in its dorsal region (Fig. 4B). The expression pattern of TH was found to be indistinguishable between wild-type (Fig. 4C) and compound-mutant caudoputamen (Fig. 4D). The diffuse staining throughout the striatum seen in both grenotypes at E18.5 is similar to what described for more mature striatum in mammals (Graybiel, 1984; Jung et al., 2000). A histochemical method employed to localize Ache in the caudoputamen of wild-type (Fig. 4E) and compound-mutant (Fig. 4F) embryos revealed a mature-like histochemical staining pattern (Graybiel et al., 1981; Graybiel, 1984; Jung et al., 2000) of Ache in both genotypes. An abundant Ache expression was found in Ache-rich areas (e.g. matrix) of caudoputamen and a low Ache expression was found in Ache-poor areas (e.g. striosomes) of caudoputamen (Fig. 4E and F). The expression patterns of CR (Fig. 4G and H) and Islet1/2 (Fig. 4I and J) were also found unaffected in compound-mutant caudoputamen. The diffuse staining of CR throughout the striatum seen in both grenotypes is similar to what described for TH, whereas nuclear expression of Islet1/2 appeared to be indistinguishable between wild-type and compound-mutant caudoputamen (i.e. wild-type caudoputamen contained 29±7 Islet1/2-expressing neurons per section, whereas mutant caudoputamen contained 26 ± 5 Islet1/2-expressing neurons per section; P > 0.05, t-test). To understand the reasons for decreased calbindinexpressing spiny neuron number in mutant caudoputamen, proliferating status and rate of cell death of the neurons in the caudoputamen was assessed by immunohistochemistry using proliferating cell nuclear antigen (PCNA) and in situ programmed cell death detection (TUNEL), respectively. In frontal serial sections through the brain of E15.5-E18.5 wild-type and Myf5−/− :MyoD−/− mutant embryos, it was not possible to detect any significant difference in the amount of PCNA-expressing or TUNEL-labeled nuclei. The PCNA proliferative index was 5% in E18.5 wild-type motor cortex and 6% in E18.5 mutant motor cortex, but the difference was not statistically significant (P > 0.05, t-test). The number of TUNEL-labeled nuclei per section was 2 ± 2 in wild-type E18.5 motor cortex and 2 ± 1 in mutant E18.5 motor cortex (P > 0.05, t-test). These findings suggest that cell cycle kinetics and survival of spiny neurons is not altered in compound-mutant caudoputamen. Together these results suggest that the observed decrease in number of differentiated neuronal cell types in the mu-

tant caudoputamen is due to a lack of the external stimuli from the periphery and not to a decrease in precursor cell proliferation or increase in cell death. 3.4. Globus pallidus and nigrostriatal pathway are changed in mutant brains To further investigate how the absence of peripheral extrinsic cues affects differentiation of neurons in the extrapyramidal motor system, we compared different extrapyramidal areas in wild-type and Myf5−/− :MyoD−/− E15.5-E18.5 embryos. We examined the expression pattern of Ache, nestin, calretinin, calbindin and tyrosine hydroxylase (TH) on frontal serial sections through: thalamocortical axons (tca), subventricular zone (svz), globus pallidus (GP), nigrostriatal pathway (nsp) and anterior thalamic nuclear complex (ATN) (Fig. 5 and not shown). Whereas the expression of nestin was unaffected in tca (Fig. 5A and B), the svz displayed a dramatic shift in localization of nestin-expressing neuroepithelial cells (Fig. 5C and D). In wild-type embryos, sections throught svz showed the nestin-expressing cells to be mostly localized in deep layers of svz, away from the lumen of the lateral ventricle (Fig. 5C). By contrast, in compound-mutant embryos, nestin-expressing cells were predominantly localized in the layer of svz that corresponds to the wall of the lateral ventricle (Fig. 5D). This difference in nestin expression suggests a shift in localization of neuroepithelial precursor cells in the svz of mutant embryos. In contrast to the well-established dopaminergic innervation of the neostriatum, the existence of dopaminergic innervation of GP is controversial (Hedreen, 1999). Consistently with findings in human GP (Hedreen, 1999), TH-expressing axons and axonal swellings were detected in wild-type (Fig. 5E) and Myf5−/− :MyoD−/− (Fig. 5F) term mouse embryo GP Fig. 5E and F) and nsp Fig. 5G and H). In both genotypes, the TH-positive axons ascended from the substantia nigra and passed above and, to a small extent, through the subthalamic nucleus, internal capsule and GP to reach the neostriatum (e.g. nsp in Fig. 5G and H) (Hanley and Bolam, 1997). Surprisingly, in addition to the axons, some TH-positive neuronal perikarya were also observed to be present in the GP of wild-type (Fig. 5E) and mutant (Fig. 5F) embryos. However, the number of TH-positive axons, axonal swellings and neuronal perikarya was significantly larger in wild-type (Fig. 5E and G) in comparison to the compound-mutant GP and nsp Fig. 5F and H). Wild-type GP contained 113 ± 3 TH-expressing neurons per section, whereas mutant GP contained only 52 ± 3 TH-expressing neurons per section (54% less than in the wild-type, P < 0.001, t-test). The observed deficiency of TH immunoreactivity in compound-mutant GP and nsp was not correlated to any significant difference in the amount of PCNA-expressing or TUNEL-labeled nuclei. The PCNA proliferative index was 2% in E18.5 wild-type motor cortex and 1% in E18.5 mutant motor cortex, but

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Fig. 5. Globus pallidus (GP) and nigrostriatal pathways (nsp) are affected in mutant brains. In frontal serial sections of wild-type (A, C, E, G, I) and compound-mutant (B, D, F, H, J) term embryos, the expression of nestin (A–D) is unaffected in tca (tca in A and B). Nestin expression in svz (svz in C and D) is shifted in localization; in wild-type embryos (C), sections throught svz shows the nestin-expressing neurons to be mostly localized in deep layers of svz (d in C), away from the lumen of the lateral ventricle (LV). By contrast, in compound-mutant (D) embryos, nestin-expressing neurons are predominantly localized in the layer of svz that corresponds to the wall of the lateral ventricle (w in D). TH-expressing (E–H) axons and axonal swellings are detected in wild-type (E, G) and Myf5−/− :MyoD−/− (F, H) term mouse embryos GP (E, F) and nsp (G, H). The number of TH-positive axons, axonal swellings and neuronal perikarya is significantly larger in wild-type in comparison to the compound-mutant GP (E, F, respectively) and nsp (G, H, respectively). Calretinin (I, J) immunoreactivity is found to be similar between wild-type (I) and mutant (J) ATN complex. Magnification, 200× (A, B, E, F, G, H), 400× (C, D), 100× (I, J).

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the difference was not statistically significant (P > 0.05, t-test). The number of TUNEL-labeled nuclei per section was 1 ± 1 in wild-type E18.5 motor cortex and 2 ± 1 in mutant E18.5 motor cortex (P > 0.05, t-test). In addition, the pattern of calretinin immunoreactivity was similar between wild-type (Fig. 5I) and mutant (Fig. 5J) ATN complex.

4. Discussion To further investigate how the lack of the extrinsic cues from skeletal muscle and associated neurons affects neuronal differentiation, we performed various structural and molecular comparisons between wild-type and Myf5−/− :MyoD−/− brains. We found that, in spite of normal appearance of developing brain architecture, many neuronal subtypes did not differentiate in a sufficient number in Myf5−/− :MyoD−/− embryos. For example, the nestin-, calretinin- and calbindin-expressing clusters of neural precursor cells and neurons appeared significantly less defined in mutant motor cortex area. Consistently, some areas of the extrapyramidal tract were also affected in mutant brains (e.g. calbindin-expressing neurons in caudoputamen, nestin- and TH-expressing structures in globus pallidus and nigrostriatal pathway). Surprisingly, we were unable to detect any change in proliferation or cell death in the mutant neuroepithelium. Together, these results suggest that patterning mechanisms intrinsic to different brain regions specify the basic organization of its subdivisions. However, extrinsic cues seem to change intrinsic properties of neural progenitors and influence the ratios of the cell types they produce. Myf5 and MyoD proteins and MyoD transcripts are not detectable in the brain of wild-type mouse embryos at various stages of development (Asakura et al., 1995; Daubas et al., 2000; Kablar, 2002). In the most extensive study on this issue (Daubas et al., 2000), some restricted domains of Myf5 transcription are found in the regions of the embryonic mouse brain that are caudal (and therefore do not correspond) to the regions observed to be affected in Myf5−/− :MyoD−/− mutant. Most importantly, the authors do not observe any neural phenotype in homozygous null mutants for Myf5 and conclude that regulation of gene expression at the level of protein synthesis or stability prevents the functional consequences of Myf5 in neurogenesis. Whereas the idea that genes instruct neural elements to differentiate into particular cell types and to make connections is straightforward, the role extrinsic factors play in development is more complicated (e.g. there can be many kinds of influences). For instance, there is ample evidence that early motor activity in the embryonic and fetal stages has an effect not only on the timing of spinal motor neuron death (reviewed by Oppenheim, 1991; Kablar and Rudnicki, 1999), but perhaps also on central connectivity, such as multi-synaptic innervation of cerebellar Purkinje cells (Mariani, 1983), differentiation of giant pyramidal

cells (Kablar and Rudnicki, 1999) and differentiation of cholinergic amacrine cells (B. Kablar, unpublished data). Moreover, manipulations that alter the anatomy (e.g. ablation of whisker pads) or function of the thalamic inputs to the somatosensory cortex (e.g. mutations or pharmacological disruptions of monoamine oxidase, adenylate cyclase or serotonin levels), disrupt the development of the barrels (Killackey et al., 1995; Cases et al., 1996; Welker et al., 1996; Abdel-Majid et al., 1998), suggesting that anatomical and functional changes in axonal inputs to the neocortex play a role in modifying existing and generating new neocortical subdivisions. By contrast, the consequence of elimination of thalamic inputs to the neocortex (Rubenstein et al., 1999) in the Gbx-2 mouse mutants (Bulfone et al., 1993; Wasserman et al., 1997) clearly demonstrates that neocortical regionalization does not require extrinsic information from the thalamus (Miyashita-Lin et al., 1999). With this in mind, we performed Nissle staining and immunohistochemistry against NF-160 antibody and analyzed structural appearance of developing cerebral cortex and other brain regions of wild-type and Myf5−/− :MyoD−/− embryos. We were unable to detect any major disturbance in the architecture of the compound-mutant brain, suggesting that anatomical milestones of developing brain were not altered by the absence of external stimuli. This provides evidence that patterning mechanisms intrinsic to the examined brain regions specify the basic organization of its functional subdivisions, supporting the findings from Gbx-2 mutants. A fundamental issue in neuronal development is to understand what factors influence the differentiation of a neuron. At one extreme, all differentiated characteristics could be intrinsic properties with which a cell becomes endowed by its lineal antecedents. At the other extreme, each differentiated characteristic could be developed as the response to an extrinsic signal. However, recent findings suggest that neural progenitors go through a series of changes in intrinsic properties that control their competence to make different cell types and that extrinsic cues influence the ratios of the cell types that they produce (Belliveau and Cepko, 1999). To assess differentiation of neuronal precursors and neurons in the motor cortex compartment, we examined the expression pattern of nestin, calretinin and calbindin proteins in wild-type and mutant brains. The areas of nestin-expressing neuroepithelial cells was found to be larger and better defined in wild-type than in mutant motor cortex; the calretinin-expressing inhibitory neurons, interneurons and pyramidal neurons were dramatically less apparent in mutant in comparison to the wild-type motor cortex; the expression of calbindin in neural precursors was also found to be larger and better defined in wild-type motor cortex. However, it was not possible to detect any significant difference between wild-type and mutant structures in the amount of PCNA-expressing or TUNEL-labeled nuclei. Taken together, these results suggest that the extrinsic information from the lacking peripheral structures (e.g. muscle, motor and sensory neurons) have an influence on

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the intrinsic properties (e.g. competence) and amounts of neural progenitors in the motor cortex (e.g. down regulated expression of nestin and calbindin). Moreover, the extrinsic information influences the ratios of the cell types that neuronal precursor cells produce (e.g. a fewer number of calretinin-expressing pyramidal neurons were present in the mutant cortex). Together, it appears that pyramidal cells are indeed born in the mutant motor cortex (i.e. some expression of calretinin is retained), but they fail to fully differentiate (e.g. giant pyramidal cells are not recognizable morphologically at E15.5–E18.5 in mutant brains; Kablar and Rudnicki, 1999 and data not shown) in the absence of their normal targets. It is clear, for example, that Purkinje cells in the cerebellum do not fully differentiate (i.e. grow to their full size and elaborate a mature dendritic tree) unless they both receive normal innervation from granule cells and find their normal output synaptic target (Mariani, 1983). In fact, from the data presented, it appears that pyramidal neurons have even extended their axons, since both the internal capsule and corpus callosum are intact. On the other hand, the external stimuli do not influence basic organization of neocortical functional subdivisions (see Fig. 1) and do not alter cell cycle kinetics of neocortical neuronal precursor cells (e.g. PCNA/TUNEL data do not differ between wild-type and mutant brain). During voluntary movements of one limb, there is simultaneous stimulation of muscle groups in other limbs and trunk, in order to maintain balance and the upright posture, as well as to permit the movements to occur smoothly (Kahle et al., 1993). These simultaneous muscular actions are not under voluntary control. They are controlled by the extrapyramiadal motor system, consisted of caudoputamen (or striatum), globus pallidus, substantia nigra, nigrostriatal pathway, etc. To investigate how the absence of external axonal inputs effects differentiation of neurons in the extrapyramidal motor system, we analyzed the expression pattern of Ache, nestin, calretinin, calbindin, TH and Islet1/2 in different extrapyramidal areas of wild-type and Myf5−/− :MyoD−/− embryos. Consistently with findings in mice with targeted disruption of dopamine D2 and D3 receptor genes (Jung et al., 2000), the expression pattern of calbindin protein was clearly and prominently altered in compound-mutant caudoputamen. The expression pattern of other markers that characterize the neurochemical differentiation of the striatum (e.g. mostly acetylcholinesterase and tyrosine hydroxylase) appeared unaffected in compound-mutant caudoputamen. While the expression pattern of Ache and calbindin were unaffected in tca, svz, GP, nsp and ATN the expression of nestin was not properly localized in mutant brain and the expression of TH was deficient in compound-mutant GP and nsp (without a correlation to any difference in the amount of PCNA-expressing or TUNEL-labeled nuclei). Therefore, similarly to what observed in the motor cortex, it appears that the basic organization of the developing caudoputamen, tca, GP, nsp and ATN complex is not altered by the absence of external stimuli (e.g. caudoputamen

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retained expression of Ache and TH, as well as the gradient of calbindin expression). However, the external stimuli most likely change the intrinsic properties of neural progenitors and influence the ratios of the cell types they produce (e.g. low numbers of spiny neurons and TH-expressing neurons, without associated programmed cell death or decreased cell proliferation). Consequently, it appears that the extrinsic stimuli are not required for differentiation of all the cells in the examined area (e.g. 33% of calbindin-expressing neurons were present in the mutant caudoputamen). In conclusion, it appears that that the competence of each neuronal progenitor cell type is defined by both extrinsic and intrinsic cues, supporting the recent findings in the neural retina (Cepko, 1999) and the generality of strategies used throughout in the CNS.

Acknowledgements This work was supported by a Natural Sciences and Engineering Research Council of Canada (NSERC), The Banting Research Foundation and The Hospital for Sick Children Foundation research grant to B.K. and a Canadian Institutes of Health Research (CIHR) grant to M.A.R. M.A.R. holds the Canada Research Chair in Molecular Genetics.

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