1 Istituto Scientifico H San Raffaele, DIBIT, via Olgettina 58, 20132 Milan, Italy and 2 Department of Medical Biotechnology, University of Padua, viale Colombo 3, 35100 Padua, Italy
The present address for J. M. Soria is Hospital general universitario Avenida Tres Cruces s/n, 46014 Valencia, Spain. Address correspondence to Antonello Mallamaci, Istituto Scientifico H San Raffaele, DIBIT, via Olgettina 58, 20132 Milan, Italy. Email: a.mallamaci{at}hsr.it.
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Abstract |
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Key Words: areal sizing Emx2 Wnt signalling cell cycle genes proneural genes
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Introduction |
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Materials and Methods |
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Mutant embryos were generated starting from Emx2 null (Pellegrini et al., 1996) and Bat-gal (Maretto et al., 2003
) founder mice and genotyped by PCR (see supplementary material available on-line). When appropriate, they were exposed to chronic lithium, by i.P. injection of pregnant dams (see supplementary material available on-line).
Population Kinetics Profiling
Total cell cycle length, TC, and S-phase length, TS, were determined via the cumulative S-phase labeling method (Takahashi et al., 1993). The fractions of neuroblasts exiting the cell cycle and differentiating into neurons, fDN, were determined by anti-bromodeoxyuridine (BrdU)/anti-class III-neurospecific-betatubulin immunofluorescences (see supplementary material available on-line).
Gene Expression Profiling
In situ hybridization, immunofluorescence/immunohistochemistry, Western blotting and quantitative retrotranscription polymerase chain reaction (qRT-PCR) were performed according to standard techniques, as described elsewhere (Briata et al., 1996; Muzio et al., 2002b
; see also supplementary material available on-line).
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Results |
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At first approximation, the tangential expansion rate of the cortical PVE can be considered a function of a three parameters, describing the basic kinetic behaviour of its components: cycling time (TC), neuronal differentiation rate (fND) and apoptosis rate. To assess if and how Emx2 controls tangential expansion of the PVE, these parameters were carefully compared in wild type and Emx2/ mutant embryos, at different tangential locations as well as at different stages of cortical morphogenesis. As assessed by the cumulative S-phase labeling method (Takahashi et al., 1993), TC, normally 8.68.7 h at E10.5, increased to 12.0 and 10.0 h in the caudal and rostral cortex of Emx2/ mutants, respectively, largely due to slowing down of DNA synthesis [TS(wt) = 3.33.4 h; TS(ko/caudal) = 7.3 h; TS(ko/rostral) = 5.8 h]. A similar increase of TC was also detectable at E13.5, even if less dramatic and restricted to caudal medial cortex [TC(ko,caudalmedial) = 14.0; TC(wt,caudalmedial) = 12.0 h] (Fig. 1a). Neuronal differentiation rates, fND, measured by labelling E11 neuroblasts in S-phase with BrdU and calculating, 10 h later, the fraction of them expressing the early post-mitotic neuronal marker ß3-tubulin, were more than doubled in the Emx2 null caudal cortex (0.084 versus 0.040) and increased by about one-third in the rostral cortex (0.060 versus 0.045). Noticeably, the BrdUß3-tubulin double labeling assay gave similar results also at E13; however, in this case, changes of fND were less dramatic (0.087 versus 0.053 and 0.074 versus 0.060 in caudal and rostral cortices, respectively) (Fig. 1c). Finally, apoptosis rates, assayed by anti-activated-caspase3 immunohistochemistry, did not display any statistically relevant change: at E12.5, activated-caspase3+ cells were 54 400 ± 23 200 and 50 100 ± 19 600 per mm3, in mutant and wild type brains, respectively, with n = 6 and P > 0.5. Coherent results were obtained by TUNEL, at E13.5 (not shown). In synthesis, in the absence of Emx2, tangential expansion rates of the cortical primordium were reduced, due to the slowing-down of DNA synthesis and the exaggerated exit of neuroblasts from the cell cycle. Moreover, these phenomena were much more pronounced in the earlier stages, as well as around the caudalmedial pole of the telencephalic vesicle, i.e. just when and where Emx2 is expressed most intensely.
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To clarify molecular mechanisms leading to these abnormalities, we looked at the expression of four gene panels encoding for the molecular machineries involved in the control of population kinetics, i.e. cyclin/cdk/cki-, proneural/antineural-, lateral inhibition- and canonical Wnt signalling-machineries. Among genes regulating early-to-late G1 progression, cyclinD1 and cyclinD2, normally expressed within the cortical ventricular zone along a latero-ventralhigh-to-dorso-mediallow gradient, were up-regulated (Fig. 2ad, Supplementary Fig. 4), Cdk4, Ink4p18 and Ink4p19 were unaffected (Supplementary Fig. 1ae). Kip1p27 and Kip2p57, both inhibiting G1S transition and S-phase progression and normally expressed along a latero-ventralhigh-to-dorso-mediallow gradient, were strongly up-regulated in the dorso-medial cortex (Fig. 2eh, Supplementary Fig. 5), possibly accounting for slowing down of DNA synthesis and exaggerated neuronogenesis occurring in this region. Other genes involved in the control of the same processes were unaffected (Cdk2, cyclinE, Cip1p21 and cyclinA1, Supplementary Fig. 1fl,o,p) or slightly reinforced (cyclinA2, Supplementary Fig. 1m,n). Concerning proneural genes, Ngn1 and Ngn2 were both up-regulated within the medial pallium (Fig. 2il, arrowheads). In particular, on 10-µm-thick sections, within the caudalmedial pallium of E11.5 Emx2/ and wild type embryos, we could count 13.8 ± 2.8 and 8.3±2.9 Ngn1on cells/100µm of ventricular profile, respectively (n = 6 + 6 and P < 0.025). Among antineural genes, Id3, normally confined to this region, collapsed and its paralog Id4, normally displaying a complementary expression pattern, was down-regulated as well, but less dramatically (Fig. 2nq). As a consequence, within the medial pallium, the proneural/antineural products ratio specifically increased, thus possibly over-boosting neuronogenesis in this region. Relevant changes were also found in the lateral inhibition machinery, supposed to be activated by newborn neurons expressing Delta and to inhibit differentiation of adjacent neuroblasts to neurons. Whereas Notch1 was not affected (not shown), the frequency of Delta1 expressing cells specifically dropped within presumptive archicortex (0.195 ± 0.003 versus 0.119 ± 0.004, n = 4 + 4, P < 0.005) (Fig. 2r,s) and the dorsomedial Hes5 expression subdomain disappeared (Fig. 2t,u), possibly contributing to exaggerated neuronogenesis taking place in this region. Finally, dramatic changes occurred to canonical Wnt signalling, also playing a pivotal role in regulating the balance between proliferation and differentiation in the developing cerebral cortex (Chenn and Walsh, 2002). As suggested by the expression pattern of the nuclear beta-catenin-responsive Bat-gal transgene (Maretto et al., 2003
), this pathway, normally active within the pallium along a caudalmedialhigh-to-rostrallaterallow gradient, almost collapsed (Fig. 3a,b). This phenomenon possibly arose from mis-regulation of distinct classes of molecules involved in Wnt signalling, including ligands, membrane receptors, cytoplasmic modulators and nuclear modulators (Fig. 3q). Four ligand genes, Wnt3a, Wnt8b, Wnt5a and Wnt2b, normally expressed in the cortical hem as well as in the pallium, were dramatically down-regulated (Muzio et al., 2002a
; Fig. 3cf). Receptor genes Fzd9 and Fzd10, both normally restricted to the dorso-medial cortex (Kim et al., 2001
), were also strongly down-regulated (Fig. 3gj), ubiquitous Lrp5 and Lrp6 (He et al., 2004
) were not impaired and the laterally confined (Kim et al., 2001
) Fzd8 was only slightly weakened (Supplementary Fig. 2af). Axin2, encoding for a key-component of the cytoplasmic complex promoting beta-catenin degradation (Jho et al., 2002
), was almost completely switched off (not shown). Tsh3, the archicortex-restricted mouse paralog of the armadillo cofactor gene Tsh (Gallet et al., 1998
), was not affected (Supplementary Fig. 2g,h). Of the genes encoding for HMG-proteins bridging beta-catenin to its DNA targets and expressed in complementary cortical domains (Galceran et al., 2000
), Lef1 was dramatically down-regulated while Tcf3 was only barely affected (Fig. 3m,n and Supplementary Fig. 2i,j). Groucho, encoding for the major co-repressor competing with beta-catenin for binding to these HMG-proteins (Cavallo et al., 1998
), was ectopically activated in the archicortical anlage (Fig. 3o,p). Finally, Reptin and Pontin, exerting opposite effects on beta-catenin signalling (Bauer et al., 2000
), were only slightly reinforced (Supplementary Fig. 2kn). To sum up, canonical Wnt signalling collapsed, possibly because less ligand was synthesized, less receptor was available on the surface of cortical neuroblasts and the signal was relayed from their surface to the chromatin less efficiently.
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Discussion |
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Acknowledgments |
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References |
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