Department of Biological and Technological Research (DIBIT), Istituto Scientifico H. San Raffaele, via Olgettina 58, 20132 Milano, Italy
Address correspondence to Antonello Mallamaci, Molecular Biology of Cortical Development, DIBITHSR, via Olgettina 58, 20132 Milan, Italy. Email: a.mallamaci{at}hsr.it.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The specification of area identities in the cerebral cortex is a complex process, beginning at mid-gestational ages and completed after birth. Early phases of this process occur before axons coming from the thalamus reach the cortex, on the basis of cortex-autonomous cues; late phases occur after the arrival of the first thalamo-cortical projections and are partly influenced by them (Ragsdale and Grove, 2001; OLeary and Nakagawa, 2002
). The graded and complementary expression of the homeogenes Emx2 and Pax6 in the ventricular zone of the cerebral cortex since early stages of its development (Walther and Gruss, 1991
; Gulisano et al., 1996
; Mallamaci et al., 1998
) (Fig. 1A
) suggested these genes could control early, thalamus-independent phases of cortical arealization, in an antagonistic way. By using a variety of experimental approaches (in situ hybridization-immunohistochemistry analysis of early area-specific markers, histochemistry of the area-specific ß-galactosidase activity encoded by the transgene H2Z1, DiI profiling of thalamo-cortical connections, study of area-specific BrdU uptake patterns), we previously found that, in the absence of Emx2, the normal spectrum of areal identities is still encoded, but a relevant reduction of cortical areas with more caudal medial identities, together with an expansion of anteriorlateral territories takes place (Mallamaci et al., 2000
) (Fig. 1B
). Similar findings were reported by the OLeary group, which also detected a complementary areal phenotype in Pax6Sey/Sey mutants, hereafter reported as Pax6/ mutants (Bishop et al., 2000
, 2002
) (Fig. 1B
). This analysis left three relevant questions open. First, the original characterization of Emx2 and Pax6 null areal phenotypes was performed at late gestational stages, i.e.
5/6 days after areal commitment of cortical neuroblasts (Barbe and Levitt, 1991
; Arimatsu et al., 1992
; Gitton et al., 1999
). Therefore, the question arose as to whether these phenotypes reflect a coherent distorsion of the early regional profile of the cortical primordium, prior to its areal commitment, or are consequences of a distorsion of its tangential growth profile, after this commitment; obviously, these two mechanisms are not mutually exclusive. Secondly, it remained to clarify which functional relationship, if any, does take place between Emx2 and Pax6 in arealization mechanisms. Thirdly, do Emx2 and Pax6 do all the job or does any other functional pathway exist that is relevant for cortical arealization but does not involve Emx2 and Pax6?
|
Concerning the second question, analysis of null mutants showed that Emx2 and Pax6 pathways inhibit each other. Moreover, the EMX2 protein sustains Emx2 expression in the medial cortical field and the PAX6 protein is necessary to achieve the Emx2-dependent repression of Pax6 in the same area (Muzio et al., 2002a) (Fig. 1F
). Molecular details of these interactions are currently under analysis in our laboratory.
Concerning the third question, two other genes, Fgf8 and Coup-tf1, have recently been shown to be involved in cortical arealization. Ectopic expression of Fgf8 in the rostral cortical primordium results in size-reduction of caudal areas and inhibition of Fgf signaling into a complementary phenotype (Fukuchi-Shimogori and Grove, 2001). Absence of Coup-tf1, leads to uniform rostro-caudal expression of regional markers Cad8, ROR-b and Id2, as well as to improper connection of caudal cortex to the somatosensory thalamic ventro-basal complex (Zhou et al., 2001
). However, both Fgf8 and Coup-tf1 are likely to act along the same pathway of Emx2, upstream (Crossley et al., 2001
; Fukuchi-Shimogori and Grove, 2001
) and possibly downstream of it (Zhou et al., 2001
) (Fig. 1D
), respectively. To see if there exist other arealization pathways, not involving either Emx2 or Pax6, we generated mice double-knockout for both two these genes and looked for possible residual signs of regionalization in their cortical primordium.
Emx2, Pax6 and Cortical Specification
Unfortunately, analysis of these double mutants did not allow us to answer the question, because the specification itself of cortical identity was abolished in their brains. However, this study was fruitful because it provided us with intriguing results about molecular mechanisms underlying the early partition of the telencephalic wall into its main subdivisions, namely cortical hem, pallium, medial and lateral ganglionic eminences (Muzio et al., 2002b). Results of this study can be summarized here as follows.
At E14.5, the cortex of Emx2Pax6 double knockouts, size-reduced and not showing any sign of lamination, did not express a set of six transcription factor genes peculiar to the developing pallium (Emx1, Emx2, Pax6, Tbr2, Ngn1, Ngn2; Fig. 2A), with the exception of a few marginally located Emx1+ presumptive neurons. This cortex expressed a set of basal forebrain markers (Vax1, Gsh2, Islet1, Gad65/67, Calbindin, Ebf1 among them; Fig. 2A
) along the normal ventricular-to-marginal progression they exhibit in the lateral ganglionic eminence, suggesting it acquired properties of this structure (Fig. 2B
). Expression profiles of some of the genes demarcating the boundaries among choroid plexus, cortical hem and pallial field properly called (Msx1, Otx2, Bf1 and Id3, but not Lhx2, Ttr and Wnt8b; Fig. 2A
), were also altered in these mutants, suggesting that some spreading of cortical hem identity into their pallial field took place place as well (Fig. 2B
). Moreover, the lateral ganglionic eminence was converted into medial ganglionic eminence (Fig. 2B
), as indicated by the confinement of Ebf1 to dorsal telencephalon and the spreading of Nkx2.1 up to the cortico-striatal notch. Remarkably, only one of either of the Emx2 or Pax6 functional alleles was able to support cortical specification, in sectors of dorsal telencephalon where the spared allele is more intensely expressed. Monitoring molecular features of Emx2/Pax6/ telencephalons at E11.5 revealed changes similar to E14.5. The three pallial markers Emx1, Tbr2 and Pax6 were dramatically, even if not completely, down-regulated and the double mutant cortex expressed the five pan-basal markers Mash1, Dlx1, Dlx2, Islet1 and Gad65/67 (Fig. 2C
), indicating that, at this age, dorsal-to-ventral transformation of this structure was already in progress (Fig. 2D
). Boundaries between ganglionic eminences and between cortical hem and pallial field were, conversely, not misplaced (Fig. 2D
), as suggested by expression patterns of Shh, Nkx2.1, Wnt3a and Wnt8b (Fig. 2C
). Thus, in the absence of both Emx2 and Pax6, between E11.5 and E14.5, boundaries among all the main telencephalic morphogenetic fields are not fixed, like the pallialsubpallial boundary in Gsh2 null mutants at similar gestational ages (Corbin et al., 2000
). This is particularly remarkable, because it could be symptomatic of latent, unexpected plasticity of the system, becoming apparent in the absence of specific molecular constraints.
|
|
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
New Results and Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Thus, Emx2 and Pax6 control two very basic aspects of cerebral cortex development, commitment of dorsal telencephalon neuroblasts to cortical fates (Muzio et al, 2002b) and execution by these neuroblasts of antero-lateral or caudo-medial regional areal morphogenetic programs, cooperating and antagonizing each other, respectively (Bishop et al., 2000
, 2002
; Mallamaci et al., 2000
; Muzio et al., 2002a
,b
). We wondered whether the Emx2 paralog Emx1 would also be involved in these processes, maybe in a way similar to Emx2. In fact, Emx1 products are specifically detectable in presumptive pallium, since ages at which ganglionar versus cortical commitment is going on (Simeone et al., 1992
; Briata et al., 1996
); graded expression of Emx1 in the pallial ventricular zone is highly similar to that of Emx2 (Briata et al., 1996
; Mallamaci et al., 1998
); Emx1 and Emx2 are structurally very similar (Simeone et al., 1992
). Actually, Emx1 has been described as controlling specific cortical morphogenetic processes, such as early neocortical lamination, development of corticofugal, cortico-cortical and thalamo-cortical projections and migration of GABAerging interneurons from basal forebrain (Yoshida et al., 1997
; Shinozaki et al., 2002
). However, no impairment of cortical committment and arealization has been found in mice knockout for this gene (Yoshida et al., 1997
). It is reasonable to hypothesize that, if such phenomena occurred, they could be very subtle. Therefore, we re-addressed the problem on sensitized genetic backgrounds by generating mouse embryos of different ages, mutant for Emx1, Emx2 and Pax6 in different combinations and looking at the expression of selected molecular markers in their telencephalons. Results can be summarized as follows.
To assess a possible role of Emx1 in regionalization of the early cortical primordium, we compared mediallateral extent of Tbr2 and Wnt8b expression domains in telencephalons of wild-type, Emx2/ and Emx1/Emx2/ E11.5 embryos. The Tbr2 ventricular domain, normally corresponding to ventral and lateral pallium and enlarged in the absence of Emx2 (Muzio et al., 2002a), coincided with the entire cortical neuroepithelium in Emx1/Emx2/ mutants (Fig. 4A
C). The Wnt8b domain, normally corresponding to cortical hem and medial pallium and restricted to the cortical hem in the absence of Emx2 (Muzio et al., 2002a
), was further shrunken in Emx1/Emx2/ mutants (Fig. 4D
F). To see if this early enlargement of latero-ventral pallium at expenses of dorso-medial pallium would result in a coherent change of late cortical areal profile, we monitored the cerebral cortex of E18.5 wild-type, Emx2/ and Emx1/Emx2/ brains for the expression of two regionally restricted genes, Cad6 and Lamp. The paleocortical Lamp expression domain, medially shifted in Emx2/ embryos (Mallamaci et al., 2000
), was further shifted in the same direction in Emx1/Emx2/ mutants (Fig. 4
GI). The archicortical Lamp expression domain, size-reduced in Emx2/ embryos (Mallamaci et al., 2000
), was undetectable in Emx1/Emx2 double knockouts (Fig. 4
GI, arrows). The main Cad6 pallial expression domain, normally spanning both sides of the cortico-striatal notch and medially shifted in Emx2/ embryos (Mallamaci et al., 2000
), was further shifted in the same direction in Emx1/Emx2/ mutants (Fig. 4
JL). The subicular Cad6 expression domain, barely detectable in Emx2 knockouts (Mallamaci et al., 2000
), was indistinguishable in Emx1/Emx2 double knockouts (Fig. 4
JL, arrows). Thus, early changes in pallial regionalization were followed, at perinatal stages, by enlargement of paleocortex at the expense of medial neocortex and archicortex. However, archicortical fates were not completely abolished in Emx1/Emx2 knockouts, as suggested by the absence of Cad6 transcripts in the dorsomedial-most double mutant cortex (Fig. 4L
), which, conversely, still expressed the dentate gyrusfimbria marker Id3 (not shown). Put in other words, in the absence of Emx2, Emx1 can functionally replace it to a partial extent as a promoter of archicortical morphogenetic programs. What is not clear is whether this is its normal functional role. The absence of obvious areal abnormalities in Emx1/ mutants would suggest that it is not so. However, subtle changes in areal profiles could also have been taking place in these mutants, falling under the detectability threshold of our analysis. Remarkably, during the preparation of this manuscript, Bishop et al. reported that abnormal rostro-caudal distribution of Cad6, p75, EphrinA5 and EphA7 transcripts occurring in Emx2/ mutants is not worsened in the absence of both Emx1 and Emx2 (Bishop et al., 2002
). Together with our findings, this suggests that, unlike Emx2 and Pax6, area-profiling activity of Emx1 takes place along only one tangential axis the mediallateral one and not along its perpendicular.
|
|
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barbe MF, Levitt P (1991) The early commitment of fetal neurons to the limbic cortex. J Neurosci 11:519533.[Abstract]
Bishop KM, Goudreau G, OLeary DDM (2000) Regulation of area identity in the mammalian neocortex by Emx2 and Pax6. Science 228: 344349.
Bishop KM, Rubenstein JLR, OLeary DDM (2002) Distinct actions of Emx1, Emx2, and Pax6 in regulating the specification of areas in the developing neocortex. J Neurosci 22:76277638.
Briata P, Di Blas E, Gulisano M, Mallamaci A, Iannone R, Boncinelli E, Corte G (1996) EMX1 homeoprotein is expressed in cell nuclei of the developing cerebral cortex and in the axons of the olfactory sensory neurons. Mech Dev 57:169180.[CrossRef][ISI][Medline]
Corbin JG, Gaiano N, Machold RP, Langston A, Fishell G (2000) The Gsh2 homeodomain gene controls multiple aspects of telencephalic development. Development 127:50075020.
Crossley PH, Martinez S, Ohkubo Y, Rubenstein JLR (2001) Coordinate expression of FGF8, Otx2, BMP4, and Shh in the rostral prosencephalon during development of the telencephalic and optic vesicles. Neuroscience 108:183206.[CrossRef][ISI][Medline]
Fode C, Ma Q, Casarosa S, Ang SL, Anderson DJ, Guillemot F (2000) A role for neural determination genes in specifying dorso-ventral identity of telencephalic neurons. Genes Dev 14:6780.
Fukuchi-Shimogori T, Grove EA (2001) Neocortex patterning by the secreted signaling molecule FGF8. Science 294:10711074.
Gitton Y, Cohen-Tannoudji M, Wassef M (1999) Specification of somatosensory area identity in cortical explants. J Neurosci 19: 48894898.
Gulisano M, Broccoli V, Pardini C, Boncinelli E (1996) Emx1 and Emx2 show different patterns of expression during proliferation and differentiation of the developing cerebral cortex in the mouse. Eur J Neurosci 8:10371050.[ISI][Medline]
Lee SMK, Shuba T, Grove E, McMahon A (2000) A local Wnt-3a signal is required for the development of the mammalian hippocampus. Development 127:457467.
McLaughlin DA, Price J, Mason J (2000) Wnt8b regulates cellular proliferation in the adult dentate gyrus. Soc Neurosci Abstr XXX:228.1.
Mallamaci A, Iannone R, Briata P, Pintonello ML, Mercurio S, Boncinelli E, Corte G (1998) EMX2 in the developing mouse brain and in the olfactory area. Mech Dev 77:165172.[CrossRef][ISI][Medline]
Mallamaci A, Muzio L, Chan CH, Parnavelas J, Boncinelli E (2000) Area identity shifts in the early cerebral cortex of Emx2/ mutant mice. Nat Neurosci 3:679686.[CrossRef][ISI][Medline]
Muzio L, DiBenedetto B, Stoykova A, Boncinelli E, Gruss P, Mallamaci A (2002a) Emx2 and Pax6 control regionalisation of the pre-neuronogenic cortical primordium. Cereb Cortex 12:129139.
Muzio L, DiBenedetto B, Stoykova A, Boncinelli E, Gruss P, Mallamaci A (2002b) Conversion of cerebral cortex into basal ganglia in Emx2/Pax6Sey/Sey double mutant mice. Nat Neurosci 5:737745.[ISI][Medline]
OLeary DDM, Yasushi Nakagawa Y (2002) Patterning centers, regulatory genes and extrinsic mechanisms controlling arealization of the neocortex. Curr Opin Neurobiol 12:1425.[CrossRef][ISI][Medline]
Ragsdale CW, Grove EA (2001) Patterning the mammalian cerebral cortex. Curr Opin Neurobiol 11:5058.[CrossRef][ISI][Medline]
Shinozaki K, Miyagi T, Yoshida M, Miyata T, Ogawa M, Aizawa S, Suda Y (2002) Absence of CajalRetzius cells and subplate neurons associated with defects of tangential cell migration from ganglionic eminence in Emx1/2 double mutant cerebral cortex. Development 129: 34793492.[ISI][Medline]
Simeone A, Gulisano M, Acampora D, Stornaiuolo A, Rambaldi M, Boncinelli E (1992) Two vertebrate genes related to Drosophila empty spiracles gene are expressed in embryonic cerebral cortex. EMBO J 11:25412550.[Abstract]
Stoykova A, Fritsch R, Walther C, Gruss P (1996) Forebrain patterning defects in Small eye mutant mice. Development 122:34533465.
Stoykova A, Treichel D, Hallonet M, Gruss P (2000) Pax6 modulates the dorsoventral patterning of the mammalian telencephalon. J Neurosci 20:80428050.
Theil T, Alvarez-Bolado G, Walter A, Rüther U (1999) Gli3 is required for Emx gene expression during dorsal telencephalon development. Development 126:35613571.
Tole S, Ragsdale CW, Grove EA (2000) Dorsoventral patterning of the telencephalon is disrupted in the mouse mutant extra-toesJ. Dev Biol 217:254265.[CrossRef][ISI][Medline]
Toresson H, Potter SS, Campbell K (2000) Genetic control of dorsalventral identity in the telencephalon: opposing roles for Pax6 and Gsh2. Development 127:43614371.
Walther C, Gruss P (1991) Pax-6, a murine paired box gene, is expressed in the developing CNS. Development 113:14351449.[Abstract]
Yoshida M, Suda Y, Matsuo I, Miyamoto N, Takeda N, Kuratani S, Aizawa S (1997) Emx1 and Emx2 functions in development of dorsal telencephalon. Development 124:101111.
Zhou C, Tsai SY, Tsai MJ (2001) COUP-TFI: an intrinsic factor for early regionalization of the neocortex. Genes Dev 15:20542059.