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. 2018 Oct 17;38(42):9105-9121.
doi: 10.1523/JNEUROSCI.0375-18.2018. Epub 2018 Aug 24.

DMRT5, DMRT3, and EMX2 Cooperatively Repress Gsx2 at the Pallium-Subpallium Boundary to Maintain Cortical Identity in Dorsal Telencephalic Progenitors

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Free PMC article

DMRT5, DMRT3, and EMX2 Cooperatively Repress Gsx2 at the Pallium-Subpallium Boundary to Maintain Cortical Identity in Dorsal Telencephalic Progenitors

Elodie Desmaris et al. J Neurosci. .
Free PMC article

Abstract

Specification of dorsoventral regional identity in progenitors of the developing telencephalon is a first pivotal step in the development of the cerebral cortex and basal ganglia. Previously, we demonstrated that the two zinc finger doublesex and mab-3 related (Dmrt) genes, Dmrt5 (Dmrta2) and Dmrt3, which are coexpressed in high caudomedial to low rostrolateral gradients in the cerebral cortical primordium, are separately needed for normal formation of the cortical hem, hippocampus, and caudomedial neocortex. We have now addressed the role of Dmrt3 and Dmrt5 in controlling dorsoventral division of the telencephalon in mice of either sex by comparing the phenotypes of single knock-out (KO) with double KO embryos and by misexpressing Dmrt5 in the ventral telencephalon. We find that DMRT3 and DMRT5 act as critical regulators of progenitor cell dorsoventral identity by repressing ventralizing regulators. Early ventral fate transcriptional regulators expressed in the dorsal lateral ganglionic eminence, such as Gsx2, are upregulated in the dorsal telencephalon of Dmrt3;Dmrt5 double KO embryos and downregulated when ventral telencephalic progenitors express ectopic Dmrt5 Conditional overexpression of Dmrt5 throughout the telencephalon produces gene expression and structural defects that are highly consistent with reduced GSX2 activity. Further, Emx2;Dmrt5 double KO embryos show a phenotype similar to Dmrt3;Dmrt5 double KO embryos, and both DMRT3, DMRT5 and the homeobox transcription factor EMX2 bind to a ventral telencephalon-specific enhancer in the Gsx2 locus. Together, our findings uncover cooperative functions of DMRT3, DMRT5, and EMX2 in dividing dorsal from ventral in the telencephalon.SIGNIFICANCE STATEMENT We identified the DMRT3 and DMRT5 zinc finger transcription factors as novel regulators of dorsoventral patterning in the telencephalon. Our data indicate that they have overlapping functions and compensate for one another. The double, but not the single, knock-out produces a dorsal telencephalon that is ventralized, and olfactory bulb tissue takes over most remaining cortex. Conversely, overexpressing Dmrt5 throughout the telencephalon causes expanded expression of dorsal gene determinants and smaller olfactory bulbs. Furthermore, we show that the homeobox transcription factor EMX2 that is coexpressed with DMRT3 and DMRT5 in cortical progenitors cooperates with them to maintain dorsoventral patterning in the telencephalon. Our study suggests that DMRT3/5 function with EMX2 in positioning the pallial-subpallial boundary by antagonizing the ventral homeobox transcription factor GSX2.

Keywords: Dmrt; Emx2; Gsx2; dorsoventral patterning; pallium–subpallium boundary; telencephalon.

Figures

Figure 1.
Figure 1.
Cortices of Dmrt3;Dmrt5 double KO embryos are more severely reduced in size than either single KO embryos and contain a prominent OBLS. A, Dorsal views of E18.5 brains of the indicated genotypes. B, Quantification of dorsal cortical surface area compared with WT controls (red) or as indicated. **p < 0.01, ***p < 0.001. n ≥ 5. C, Diagram showing the telencephalon of E18.5 WT embryos and the size reduction and absence of the OBs observed in Dmrt3−/−;Dmrt5−/−. D, Sagittal sections through E18.5 brains processed by IF or ISH for Tbr1, Tbr2, and Tbx21 OB mitral projection neuron markers. In the cortex, Tbr2 is also detected in SVZ progenitors and Tbr1 in layer VI neurons. For WT and double KO embryos, high-magnification views of the cortex (a′, d′, m′, q′) and OBs (a′, d′, l′, p′, u′) are shown. For the double KO brains, two sections are shown (i, j, m, n), taken at the levels indicated in the diagram. In the Dmrt3+/−;Dmrt5−/− and double KO embryos, the presence of a prominent OBLS occupying most of the reduced cortex (shown schematically in the diagram compared with controls) in which Tbr1, Tbr2, and Tbx21 are expressed and form a cluster or appear as a disorganized layer (l′, p′, u′). Note also in the cortex the disappearance of cortical Tbr2+ basal progenitors (compare a′ and m′) and more diffuse Tbr1 expression (compare d′ and q′). *Region of strong ectopic expression of Tbr2 outside the cortex in the double KO embryos. AOB, Accessory OB; AON, anterior olfactory nucleus; BP, basal progenitor; Ctx, cortex, OB-L, OB-like structure; Hip, hippocampus; MCL, mitral cell layer; OB, olfactory bulb. Scale bars, 50 μm.
Figure 2.
Figure 2.
Genome-wide transcriptome analysis reveals that DMRT3 and DMRT5 cooperate to regulate cortical gene expression and play a role in early telencephalon DV patterning. A, Dissected dorsal telencephalic tissues analyzed by RNA-seq and Venn diagram showing the overlap of differentially expressed genes (both upregulated and downregulated) identified in Dmrt3 KO, Dmrt5 KO, and double KO E12.5 embryos. B, Heatmap for the 50 most significantly regulated genes (according to p value) in a comparison between WT and double KOs. Yellow represents upregulated genes. Blue represents downregulated genes. C, Examples of identified differentially expressed dorsal and ventral genes with log2-fold changes observed in each genotype.
Figure 3.
Figure 3.
Downregulation of dorsal determinants and expansion of dLGE markers in the lateral telencephalic neuroepithelium of Dmrt3;Dmrt5 double KO embryos. A, B, Coronal brain sections of E12.5 embryos hybridized with the indicated markers. Arrowheads indicate the region of the PSB. Arrows point to the shifted dorsal limit of ventral markers expressed ectopically in the pallium of the double KO embryos. Open arrows indicate the strongly downregulated expression of Emx1, Emx2, and Ngn2 that remains detectable only in the dorsomedial telencephalon and the absence of Dbx1. Inset in the Isl1 panel of the Dmrt double KO represents a high-magnification view of the boxed region where some Isl1 ectopic staining in the pallium is observed. C, Diagram showing the expression domain of the different markers used in the telencephalon of E12.5 WT embryos and the reduction of the ventral pallium and an expansion of the dLGE domain as observed in Dmrt3−/−;Dmrt5−/−. Scale bar, 200 μm.
Figure 4.
Figure 4.
Subpallial gene expression expands in the rostral telencephalon of Dmrt3;Dmrt5 double KO embryos. Sagittal sections of E12.5 brains processed by ISH or IF with the indicated markers, with the rostral part to the right. Arrowheads indicate PSB. Arrows indicate the rostral or caudal boundaries of the gene expression domains. These boundaries are shifted dorsally in the Dmrt3;Dmrt5 double KO embryos. Open arrows indicate stronger Pax6 staining in a stream of postmitotic cells that derive from dLGE progenitors in the mantle zone of the rostral part of cortical neuroepithelium of the single and double KO embryos. Scale bar, 200 μm.
Figure 5.
Figure 5.
Dorsal telencephalon cells express ventral markers in Dmrt3;Dmrt5 double KO embryos. A, Coronal section though E12.5 brains of the indicated genotypes processed by IF for Pax6 and Gsx2. Bottom, high-magnification views of the PSB. B, Histograms showing the number of double-positive cells among Pax6-positive cells in the boxed area. ***p < 0.001.
Figure 6.
Figure 6.
Expression of GABAergic, glutamatergic, and OB interneuron markers in the cortex of E18.5 Dmrt3 and Dmrt5 single KO and in double KO embryos. a, o, Sagittal sections through brains of the indicated genotypes processed by ISH or IF for the indicated markers. p, The line in the schematic of the brain of WT and double KO embryos indicates the positions of the sections. For the double KO brains, two sections are shown for each marker. e′, j′, A high magnification of the Math2 and Gad67 staining in the residual cortex of the double KO. There is slight upregulation of Gad67 in both single and double mutants (*) and a dramatic loss of Math2 in the cortex of the double KO embryos. TH-positive cells that are not correctly laminated and form a large cluster in the OB-L structures of the double KO that extend more caudally in the dorsal telencephalon than in WT embryos (arrowheads). Ctx, Cortex; MFB, medial forebrain bundle; Acbn, accumbens nucleus. Scale bars: 500 μm; h, p, 50 μm. OB, olfactory bulb; OB-L, olfactory bulb like.
Figure 7.
Figure 7.
Repression of ventral and expansion of dorsal telencephalic and ventral pallium markers in the subpallium of Dmrt5Tg/+;Foxg1-IRES-Cre embryos. A, B, Coronal sections through the telencephalon of transgenic and control embryos at E12.5 (A) and E13.5 (B) processed by ISH for the indicated markers. A, Top 8 panels, Arrowheads indicate the boundary of the MGE and LGE, a constant landmark between control and mutant mice, used for quantification of Emx1 expansion. Arrows point to the ventral limit of Emx1 and Ngn2 expression, or the dorsal limit of Gsx2 and Dlx2 expression. Notably, in Foxg1-IRES-Cre embryos, the arrows and arrowheads are closer together than in controls, indicating the dorsalization of the telencephalon when Dmrt5 is overexpressed. A, Bottom 6 panels, arrowheads indicate the PSB, arrows indicate the mis-located expression of sFrp2 and Tgfa when Dmrt5 is overexpressed, and the near loss of Sp8 expression. B, Arrowheads indicate the PSB. Arrows indicate restored expression of sFrp2 and Tgf-α to the PSB, and upregulated expression of Dbx1. Asterisk marks continued absence of Sp8. C, Diagram showing the ventral expansion of DP and VP markers as observed in the telencephalon of Dmrt5Tg/Tg;Foxg1-IRES-Cre embryos. D, Dorsal view of P6 brains and eosin staining of OBs. Arrowheads point to the RMS. Arrow indicates the OV. RMS, Rostral migrating stream; OV, olfactory ventricle; GL, glomerular layer; Igr, internal granule layer; LGE, lateral ganglionic eminence; dLGE, dorsal lateral ganglionic eminence; MGE, medial ganglionic eminence; OB, olfactory bulb; NP, neocortical primordium.
Figure 8.
Figure 8.
Repression of ventral and expansion of dorsal and ventral pallium markers in the subpallium of Dmrt5Tg/Tg;Gsx2-Cre embryos. A, B, Coronal sections through the telencephalon of E12.5 embryos processed by IF (A) or ISH (B) with the indicated markers. A, *The ectopic expression of Dmrt5 and downregulation of Ascl1 and Gsx2 in the ventral telencephalon. B, Arrowheads indicate the region of the PSB. Arrows point to the expansion and shifted ventral limit of Ngn2, Emx1, and Dbx1 and downregulation and shifted dorsal limit of Dlx2 expression in the subpallium of the transgenics. C, Diagram showing the expansion of the ventral pallium as observed in Dmrt5Tg/Tg;Gsx2-CIE embryos. Scale bar, 200 μm.
Figure 9.
Figure 9.
DMRT5 and EMX2 cooperate in telencephalon DV patterning. A, Dorsal views of the head of E12.5 embryos. H&E staining of E12.5 brain coronal sections of the telencephalon, at rostral and caudal levels. Arrows point to the caudomedial cortex of the Dmrt5;Emx2 double mutants, more severely reduced than in single mutants. B, Coronal brain sections of E12.5 embryos processed by ISH with the indicated markers. Arrowheads indicate the region of the PSB. Arrows point to the dorsal limit of Gsx2, Dlx2, and Ascl1 expression, shifted dorsally in the Dmrt5−/−;Emx2−/− double KO embryos. *Dramatic reduction of Ngn2 in the cortex of the double KO embryos. Scale bar, 200 μm.
Figure 10.
Figure 10.
DMRT5 forms a boundary with GSX2 at the PSB, and the overexpression of Gsx2 represses Dmrt5 expression. A, Coronal sections of the head of E14.5 Gsx2GFP/+ knock-in embryos processed by IF with DMRT5 and GFP antibodies showing that, at the PSB, cells expressing DMRT5 do not express GFP, hence, GSX2. Boxed area is shown at a high magnification on the left. Oe, Olfactory epithelium. Scale bar, 200 μm. B–E, Coronal sections through the telencephalon of E12.5 WT or Foxg1tTA/+; tet-O-Gsx2-IRES-EGFP double-transgenic embryos immunostained with the indicated antibodies. In these transgenic embryos, Gsx2 is misexpressed throughout the embryonic telencephalon and Dmrt5 is reduced. B, E, Arrows point to the ventral limit of high Dmrt5 expression.
Figure 11.
Figure 11.
DMRT5 and EMX2 bind a Gsx2 ventral-specific telencephalon enhancer. A, UCSC genome browser view of the Gsx2 locus with the location of the two cloned fragments tested in transgenic embryos. The identified putative DMRT3/5 and EMX2 BSs are shown. B, Left, A lateral view and a coronal section of the head of a E12.5 Gsx2 1.8 kb enhancer-LacZ reporter transgenic embryo (Construct A). Scale bars: lateral view, 500 μm; coronal section, 200 μm. Dashed line indicates the level of the section. Right, A coronal section of the brain of a Gsx2 1.3 kb enhancer-GFP reporter transgenic embryo (Construct B) processed by DAB immunostaining for GFP and a high-magnification view of the LGE region processed by IF for both GSX2 (red) and GFP (green). C, EMSA showing in vitro binding of cellular extracts containing DMRT3, DMRT5, and EMX2 or control extracts to BS3 of the Gsx2 enhancer. DMRT3/5 and EMX2 complex formation is competed by WT enhancer oligonucleotides but not by oligonucleotides containing mutations in the DMRT and EMX2 BSs. Arrowhead indicates a nonspecific band.

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