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Modulation of the notch signaling by Mash1 and Dlx1/2 regulates sequential specification and differentiation of progenitor cell types in the subcortical telencephalon

¹ Nina Ireland Laboratory of Developmental Neurobiology, Department of Psychiatry, LPPI, University of California, San Francisco, 401 Parnassus, Box 0984, San Francisco, CA 94143-0984, USA
² Center for Basic Neuroscience, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA
³ GSF National Research Center for Environment and Health, Institute of Experimental Genetics, Ingolstaedter Landstr. 1, D-85764 Neuherberg, Germany
⁴ Department of Biological Chemistry, UCLA School of Medicine, 33-257 CHS, Box 951737, Los Angeles, CA 90095-1737, USA

View larger version (84K): [in a new window]   Fig. 1. Combinatorial expression of MASH1, DLX2 and markers of proliferation and differentiation provide evidence for distinct stages of cellular maturation in the lateral ganglionic eminence (LGE). Fluorescence microscopic images of wild-type subcortical cells stained with a pair of antibodies (differentially labeled with green or red fluorophors) that bind to the proteins indicated above each panel; yellow cells are double labeled. Coronal sections (10 µm) from E10.5 embryos (A-D) and E11.5 embryos (F-J) show the primordium of the LGE. (E) Dissociated cells prepared from E10.5 subcortical telencephalon (arrows indicate double-positive cells). The boxed areas in Fig. 2 show the approximate regions of the LGE shown in Fig. 1. PCNA labels proliferating cells, and thereby defines the location of the progenitor zones, whereas MAP2 and GABA label postmitotic neurons (arrow in C,D), and thereby define the position of the mantle zones. PH3 labels M-phase cells in the VZ and the SVZ (arrow in H). P1 progenitors (MASH1-/DLX2-) and P2 progenitors (P2, MASH1+/DLX2-) are only in the VZ. P3 progenitors (MASH1+/DLX2+) are in the VZ and SVZ. In A, P1 progenitors are unlabeled, P2 progenitors are green and P3 progenitors are yellow. N1 cells are the earliest born neurons; we propose that they are generated from P2 progenitors (see Fig. 7). N1 neurons are MAP2+ and continue to express DLX2 (see inset in D). The LGE progenitor zone can be divided into at least two parts along the dorsoventral axis: the dorsal LGE (dLGE) and the ventral LGE (vLGE) (Yun et al., 2001). dLGE neurogenesis precedes that of vLGE, and therefore may contribute to a major portion of the N1 population. Some MASH-/DLX2+ cells are observed in the VZ and SVZ (particularly in the dLGE); these are the red cells in the VZ in A. These cells might correspond to another type of progenitor. LV, lateral ventricle; ME, mesenchyme; MZ, mantle zone; VZ, ventricular zone; SVZ, subventricular zone. Scale bars: 100 µm. Arrows in J indicate examples of PCNA/Mash1 double-positive cells.

View larger version (20K): [in a new window]   Fig. 7. Models showing progenitor cell (P) and neuronal (N) maturation in the LGE of wild-type, Mash1-/-, Dll1-/- and Dlx1/2-/- embryos. MASH1-/DLX2- neuroepithelial progenitors (P1) give rise to MASH1+/DLX2- (P2) and MASH1+/DLX2+ (P3) progenitors around E10. An alternate model would have P1 cells separately generate P2 and P3 states. Lineage analysis will be needed to determine the relationships between the postulated progenitor cells and their derivatives. We propose that P2 cells mature into early neurons (N1), while P3 cells proliferate to form the SVZ progenitor zone and produce later-born neurons (N2). Mash1 is required cell-autonomously to specify P2 progenitors and hence N1 neurons. Mash1 is also required non cell-autonomously: increasing Dll1 expression to prevent neighboring cells from acquiring the P3 fate prematurely via Notch-dependent lateral inhibition. In Dll1 mutants, P1, P2 and P3 progenitors are present, but their maturation is accelerated due to decreased Notch signaling. By contrast, early neurogenesis is normal in the Dlx1/2 mutants but P3 progenitors are blocked in their ability to differentiate. This phenotype is attributed to the failure to downregulate Notch signaling during specification and/or differentiation steps of P3 maturation. The arrows between cell states do not necessarily imply that mitosis is required. The enlarged arrows represent hypothesized increases in the rate of the indicated process.

View larger version (123K): [in a new window]   Fig. 2. Mash1 mutants have a defect in the production of early-born (N1) neurons and their VZ cells precociously acquire SVZ-like properties. Fluorescence and darkfield microscopic images of coronal hemi-sections of control (left side) and Mash1 mutant (right side) telencephalons at E10.5 (A-C'), E11.5 (D-I', E15.5 (J-K') and E18.5 (L,L'). The sections were stained with antibodies directed to the antigens, or 35S-labeled probe, listed above each panel. Yellow cells are double-labeled. While Mash1 mutants produce few MAP2+ cells at E10.5 (N1 cells; A-B'), their progenitor zone appears to mature precociously based on the increased number of cells that express DLX2 (C,C',F,F'). These DLX2 cells are mitotically active precursors, based on co-expression with PCNA (arrow in F'). At E11.5, the mutant LGE produces many GABA+ cells (arrow E''), but most of these do not express MAP2. We suggest that a substantial number of the MAP2-expressing cells adjacent to the LGE in D' (arrowhead) are not produced by the LGE, but rather correspond to ventral pallial cells (see arrowhead in G' for pattern of Lhx2 expression, which marks the mantle of the ventral pallium, VP). Note the paucity of MAP2 expression in the MGE mantle (D') while GABA expression on the same section is abundant (E'). Mash1 also has later functions in subcortical development. At E15.5 and E18.5 there is ectopic expression of GABA in the progenitor zone of the LGE. Arrowhead in K' indicates ectopic GABA expression; arrows in L,L' indicate LGE proliferative zones. ctx, cortex; LV, lateral ventricle; MGE, medial ganglionic eminence; st, striatum. Scale bars: 100 µm.

View larger version (102K): [in a new window]   Fig. 3. Notch signaling is compromised in the absence of Mash1 function as early as E10.5. Dark-field photomicroscopic images of coronal hemi-sections of control (left side) and Mash1 mutant (right side) telencephalons at E10.5 (A-F') and E11.5 (G-I') studied using 35S-labeled radioactive in situ hybridization. The cDNAs encoding the riboprobes used in the analysis are listed above each normal/mutant pair of images. The VZ in the Mash1 mutant has molecular characteristics of the SVZ, based on the increased expression of Dlx5, Gad67 and the reduced expression of Hes5 (arrowheads in G-I'). ctx, cortex; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; VZ, ventricular zone. Scale bar: 100 µm.

View larger version (104K): [in a new window]   Fig. 4. Dll1 mutants show increased early neurogenesis and reduced Notch signaling. Dark-field and fluorescence photomicroscopic images of coronal hemi-sections of control (left side) and Dll1 mutant (right side) telencephalons at E10.5 (A-E') and E11.5 (F,F') studied using 35S-labeled radioactive in situ hybridization (A,A') and immunofluorescence (B-F'). Dll1 mutants show reduced Hes5 expression (A,A'), which is indicative of decreased Notch signaling, thinning of the VZ [with reduced RC2 expression (F,F')], and an expansion of the mantle zone that expresses DLX2 (B,B'), ß-III-tubulin (C,C') and GABA (D,D'). These neurons may not have differentiated completely based on the low level of MAP2 expression particularly in the presumptive MGE (arrowhead in E,E'). Similar to the Mash1 mutants, GABA expression appears in maturing neurons before MAP2 expression (compare D,D',E,E'). ctx, cortex; LGE, lateral ganglionic eminence; LV, lateral ventricle; MGE, medial ganglionic eminence; MZ, mantle zone; OE, olfactory epithelium; VZ, ventricular zone. Scale bars: 100 µm.

View larger version (57K): [in a new window]   Fig. 5. Dlx1/2 mutants show normal neurogenesis at E10.5, and begin to show elevations in Mash1, Hes5 and Dll1 expression by E11.5 in the SVZ. Coronal hemi-sections of control (left side) and Dlx1/2 mutant (right side) telencephalons at E10.5 (A-C') studied with immunofluorescence, and at E11.5 (D-F'), studied with radioactive in situ RNA hybridization. At E10.5 early neurons, assessed by expression of GABA and MAP2 (A,A',B,B') and radial glia, assessed by expression of RC2 (C,C'), appear normal in the Dlx1/2 mutants. At E11.5 expression of Mash1, Dll1 and Hes5 (D-F') increase in the SVZ (arrowheads). Scale bars: 100 µm.

View larger version (131K): [in a new window]   Fig. 6. Dlx1/2 mutants show expanded Notch signaling domains and a block in differentiation of SVZ cells. Dark-field and fluorescence photomicroscopic images of coronal hemi-sections of control (left side) and Dlx1/2 mutant (right side) telencephalons at E15.5 (A-I') and E18.5 (J-L') studied using radioactive in situ hybridization (B-E',G-H',J,J') and immunofluorescence (A,A',I,I',K,K',L,L'). (G-H') The Dlx1/2 mutant SVZ shows elevated expression of VZ markers (COUP-TF1; Gsh2). These defects are correlated with increases in MASH1, Dll1 and Hes5 expression (A,A',D-E'), particularly in the SVZ. Note that while the Hes5 and Notch1 (E-F') expression domain expands, Hes1 and Notch3 (B-C') expression remains normal in the Dlx1/2 mutants. The persistence of progenitor molecular properties as late as E18.5 (not shown) is linked with persistent expression of a radial glial marker (RC2; L,L'). The defects in SVZ differentiation are correlated with reduced expression of striatal markers, such as dopamine receptor 2 (Drd2) (J,J'), while a general marker of neurogenesis (MAP2) appears normal in the mantle zone (I-K'). Note, however, that MAP2 expression extends much closer to the ventricle in the mutant at E18.5 (boxed areas in K,K'). ctx, cortex; ic, internal capsule; LV, lateral ventricle; spt, septum; st, striatum. Scale bars: 100 µm.