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First published online 24 August 2005
doi: 10.1242/dev.02008

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Notch signaling coordinates the patterning of striatal compartments

¹ Developmental Genetics Program and the Department of Cell Biology, The Skirball Institute of Biomolecular Medicine, New York University Medical Center, 540 First Avenue, New York, NY 10016, USA
² The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, USA

View larger version (92K): [in a new window]   Fig. 1. Generation of a telencephalic-specific deletion of Notch1. (A) Coronal sections through the embryonic telencephalon of Foxg1Cre/+; ROSA26-floxed-stop-lacZ reporter mice stained with X-gal to visualize Cre activity. Cre-mediated recombination is detected throughout the entire telencephalon by E9.5 and E10.5. (B) Notch1 and Hes5 are present in the telencephalic VZ in wild-type (WT) embryos, whereas Notch1 mRNA is not detected and Hes5 is substantially diminished in the telencephalon of Foxg1Cre; N1 cKOs at E10.5. Notably, residual Hes5 expression is detected only in the medial ganglionic eminence (asterisk), and not in the striatal anlage, the lateral ganglionic eminence, which is more laterally located. (C) At E12.5, Notch1 protein is not detected in telencephalic lysates from Foxg1Cre; N1 cKOs (N1 cKO), whereas levels of -tubulin are equivalent between N1 cKO and WT forebrain lysates, as assessed by immunoblotting for the cleaved portion of Notch1 and -tubulin. Western blot (WB): anti-Notch1 (N1) and anti--tubulin (tub). (D) At E10.5, Notch2 is expressed at high levels within the epithelium of the choroid plexus but is not detectable in the VZ, whereas Notch3 is present at low levels throughout the VZ of wild-type embryos. Scale bar: 150 µm.

View larger version (123K): [in a new window]   Fig. 2. Neurogenesis is perturbed in Notch1 conditional mutants at E12.5 when compared with wild-type embryos, but largely recovers by E14.5. (A) The ganglionic eminences, the LGE and MGE, are drastically reduced in size in Foxg1Cre; N1 cKOs (asterisks), as seen here in coronal sections through the forebrain at E12.5. Hes1 and Hes5, genes that are activated by the Notch signaling pathway, are substantially decreased in the eye (insets) and telencephalon of Foxg1Cre; N1 cKOs at E12.5. Mash1, a bHLH gene repressed by Hes activity, is increased in Foxg1Cre; N1 cKOs at E12.5, particularly in the SVZ (arrows). In addition, the bHLH gene Neurod is upregulated in the developing retina (insets). Scale bar: 500 µm. (B) Overall brain morphology and neurogenesis is relatively normal at E14.5 in Foxg1Cre; N1 cKOs. Expression of Hes1 and Hes5 appear equivalent in the VZ of Foxg1Cre; N1 cKOs and wild-type embryos. Unlike at E12.5, Mash1 is not noticeably upregulated in the SVZ of conditional knockouts when compared with wild-type embryos at E14.5. Neurogenesis is further impaired in the retina of Notch1 conditional mutants, as Hes1 and Hes5 are virtually absent and Neurod is considerably elevated.

View larger version (103K): [in a new window]   Fig. 3. The patch compartment, as well as its dopaminergic innervation from the midbrain, is significantly impaired in the absence of Notch1. (A) Patch neurons in the developing striatum begin to aggregate and display cell-specific markers at E18.5. Immunostained adjacent coronal forebrain sections show that glutamate receptor1 (Glur1) and Darpp32 are expressed by newly differentiating subcallosal (SCS) and patch neurons (arrows) in wild-type embryos but are abnormally expressed in Foxg1Cre; N1 cKOs. The SCS is thicker in the Foxg1Cre; N1 cKOs than wild-type embryos relative to the overall size of the striatum. Scale bar: 200 µm. (B) Incoming dopaminergic fibers from the substania nigra (SN) express tyrosine hydroxylase (Th) and become selectively localized to SCS and patch neurons in the wild-type striatum (arrows). Foxg1Cre; N1 cKOs, which display an expanded SCS, show greater Th innervation than do wild-type mice at E18.5 (upper panels). In addition, fibers from the SN ectopically project to the cerebral cortex in the absence of Notch1. Excessive Th-positive fibers are present throughout the prefrontal cortex in Foxg1Cre; N1 cKOs whereas Th-positive fibers are rarely observed in the wild-type cortex at E18.5 (lower panels). Scale bar: 100 µm.

View larger version (112K): [in a new window]   Fig. 4. Late-born matrix neurons develop normally in the absence of Notch1. Ebf1 expression and ephrin A4/Fc binding is enriched in the matrix compartment of the striatum, and excluded from the SCS and patch regions. The patterns of Ebf1 and ephrin A4/Fc staining appear to be equivalent in both wild-type embryos and Foxg1Cre; N1 cKOs, as seen here in coronal sections through the striatum at E18.5. The SCS, which can be visualized by the lack of Ebf1 and ephrin A4 staining (shown in green), is expanded in the absence of Notch1 (dashed lines). The patch compartments, which also exclude Ebf1 and ephrin A4, express Darpp32 (shown in red) and are reduced at E18.5 in the mutant (arrows). Scale bar: 200 µm.

View larger version (100K): [in a new window]   Fig. 5. Birthdating analysis of neurons in the striatum. (A) BrdU was administered at different embryonic time-points (E10.5-E15.5), and the striatum was subsequently analyzed at E18.5. BrdU-positive neurons were counted with respect to their localization within the striatal compartments (SCS, patch or matrix). The SCS neurons born at E12.5 are significantly increased in Foxg1Cre; N1 cKOs, whereas neurons born at E12.5 in the patch compartment are significantly decreased in mutants compared when with wild-type littermates. Equivalent numbers of matrix neurons are born in the mutants and in wild-type littermates, except at E11.5, at which time there is a small but statistically significant decrease in the birth of matrix neurons in Foxg1Cre; N1 cKOs. A single asterisk (*) denotes a P-value of <0.05, whereas two asterisks (**) signify a P-value of <0.005. Three Foxg1Cre; N1 cKOs and three wild-type embryos were analyzed for each time-point. Error bars represent s.e.m. (B) Coronal sections of the striatum at E18.5 with three representative ages of BrdU administration in Foxg1Cre; N1 cKOs and wild-type littermates. Tissue was immunostained with antibodies to Darpp32 (red) to visualize the SCS and patch compartments, and with antibodies to BrdU (green) to detect cells that become postmitotic shortly after the pulse of BrdU. Scale bar: 250 µm.

View larger version (87K): [in a new window]   Fig. 6. The entire striatum is severely compromised in Foxg1Cre; Notch1 conditional; Notch3 null double mutants (Foxg1Cre; N1; N3 DKOs). As visualized using in situ hybridization for Ebf1 in E17.5 coronal sections of the striatum, only a small region of the matrix compartment develops in the absence of both Notch1 and Notch3. Scale bar: 500 µm (upper right panel). Similarly, both the SCS and patch regions, immunostained here with antibodies to Darpp32, are substantially reduced in size and are severely disorganized in the double knockouts at E17.5. Scale bar: 500 µm. As seen in the lower panels, which show a higher magnification view than the middle panels, the SCS is disrupted and no characteristic clusters of Darpp32-positive cells are observed in Foxg1Cre; N1; N3 DKOs. Scale bar: 250 µm.

View larger version (78K): [in a new window]   Fig. 7. Notch1 and Notch3 are not required for normal striatal development after cells exit the VZ. (A) A coronal section of an E12.5 Dlx5/6Cre-IRES-EGFP forebrain indicates that the Dlx5/6 enhancer directs the expression of EGFP throughout the ventral SVZ and mantle, but not in the VZ. (B) Dlx5/6Cre induces the recombination of floxed alleles throughout the striatum, including the SCS, patch and matrix compartments. Shown here is in a coromal section of a P1 striatum from a cross between the Dlx5/6Cre-IRES-EGFP transgenic and the Z/EG reporter mouse. Cells that have undergone Cre-mediated recombination permanently express EGFP (shown here in green). EGFP is expressed in all regions of the striatum at P1 in progeny from Dlx5/6Cre and Z/EG reporter mice. Antibodies to Darpp32 mark the SCS and patches (shown here in red). The EGFP-negative regions are fiber tracts passing through the striatum, with the anterior commissure (AC) visible in the lower left corner. (C) The SCS and patch compartments develop normally in Dlx5/6Cre; N1; N3 DKOs, as seen here with antibodies to Darpp32 at P1. (D) The matrix compartment is equivalent in both Dlx5/6Cre; N1; N3 DKOs and wild-type littermates at P1. Scale bar: 250 µm.