The role of Foxg1 and dorsal midline signaling in the generation of Cajal-Retzius subtypes

Abstract

Cajal-Retzius (CR) cells, the earliest-born neurons in the neocortex, arise from discrete sources within the telencephalon, including the dorsal midline and the pallial-subpallial boundary (PSB). In particular, the cortical hem, a region of high bone morphogenetic proteins (BMPs) and Wnt (wingless-type MMTV integration site family) expression but lacking in Foxg1 (forkhead box G1) is a major source of CR neurons. Whether CR cells from distinct origins arise from disparate developmental processes or share a common mechanism is unclear. To elucidate the molecular basis of CR cell development, we assessed the role of both Foxg1 and dorsal midline signaling in the production of cortical hem- and PSB-derived CR cells. We demonstrate that the loss of Foxg1 results in the overproduction of both of these CR populations. However, removal of Foxg1 at embryonic day 13, although expanding the number of CR cells with a PSB phenotype, does not result in an expansion of BMPs or Wnts in the dorsomedial signaling center. Conversely, loss of the dorsal midline ligands as observed in Gli3 (glioma-associated oncogene homolog 3) mutants results in the loss of the cortical hem-derived CR character but does not affect the specification of PSB-derived CR cells. Hence, our findings demonstrate that, although the specification of cortical hem-derived CR cells is dependent on signaling from the dorsal midline, Foxg1 functions to repress the generation of both cortical hem- and PSB-derived CR cells.

Figure 1.

Expansion of the dorsomedial signaling center in Foxg1^−/− mutants. Top, Complementary expression of Foxg1 and Wnt3a, and BMP4 in E11.5 Foxg1^+/− control embryos. Arrows indicate the boundary between the cortical hem and the isocortex. Scale bar, 200 μm. Bottom, Expression of multiple BMPs (BMP4, BMP6) and Wnts (Wnt3a, Wnt2b, Wnt8b) mRNA in E11.5 whole-mount embryos (dorsal view). A–E shows expression of these genes in the Foxg1^+/− control embryos. F–J shows expression of the same genes in Foxg1^−/− mutants. Figures in the insets show the lateral view of the telencephalon of each corresponding embryos (rostral is to the left). BMPs and Wnts are both expanded laterally and rostrally in the Foxg1^−/− telencephalon. Scale bar, 1 mm.

Figure 2.

A, B, D, E, Both cortical hem- and PSB-derived CR cells are overproduced in the absence of Foxg1. Top, Expression of p73 (red) and Reelin (green) in E11.5 Foxg1^+/− control (A, B) and Foxg1^−/− (D, E) embryos. a′–d‴ show the enlarged view of the boxed region of medial (a′, d′), lateral (a″, d″), and ventral (a‴, d‴) pallium indicated in A and D, respectively. The ventricle is to the bottom, and the pial surface is to the top. The p73-expressing cells are detected in both the ventricular zone and postmitotic cells at E11.5. p73 marks CR cells before their expression of Reelin (a′). Reelin-expressing cells in the ventral pallium do not express p73 (a‴). C, F, Wnt3a mRNA expression in sections adjacent to those indicated in B and E. Arrowheads demarcate the boundary of p73-expressing cells at this stage. Scale bars: A–F, 200 μm; a′–d‴, 100 μm. Bottom, Reelin (green) and p73 (red) expression in E13.5 Foxg1^+/− control (G, Ia–IIc) and Foxg1^−/− (H, IIIa–IVc) embryos. Ia–Ic, IIIa–IIIc show the dorsal pallium, and IIa–IIc, IVa–IVc show the ventral pallium. Arrowheads in Ic indicate double p73/Reelin-positive cells in the marginal zone in Foxg1^+/− controls. In Foxg1^+/− cortex, p73 expressing CR cells are distributed in the lateral cortex (I), and many of them coexpress Reelin (Ib, Ic). In the Foxg1^−/− cortex, many Reelin⁺ cells express p73 in the lateral pallium (IIIa–IIIc), but Reelin⁺ cells are p73 negative in the ventral pallium (IVa–IVc). Scale bar, 100 μm.

Figure 3.

Dorsomedial signaling is not expanded in Foxg1 E13 conditional mutants. A–D, Top, Coronal sections showing Wnt3a and Msx1 expression in E14.5 Foxg1^tTA/+ heterozygote control (A, B) and Foxg1 conditional (removed at E13) mutants (C, D). Arrows indicate that the expression of both genes is restricted to the cortical hem and the choroid plexus. a′–d′ show an enlarged view of the region indicated in A–D. Middle, Wnt3a, Msx1, and reelin expression at E16.5. E–G show Foxg1^tTA/+ control embryos. In H–J, Foxg1 conditional (removed at E13) mutants are shown. Insets in E, F, H, and I show an enlarged view of the region indicated by the arrows. The increased production of CR cells is indicated by the expansion in reelin expression in both the medial and lateral cortex of Foxg1 conditional (removed at E13) mutants (J), whereas expression is confined to the marginal zone in the lateral cortex in Foxg1^tTA/+ control mice (G). g′ and j′ show an enlarged view of the boxed region in G and J, respectively. Bottom, Expression of Ebf2 (K, O), Reelin (L, P), and p73 (M, Q) in Foxg1^tTA/+ (K–N) and Foxg1 E13 conditional mutants (O–R). Pia is to the top. The ectopic and supernumerary CR cells in the cortical plate express reelin but do not coexpress p73 in the conditional mutants (R). Note the background staining in the pia in L–N, which do not represent CR cells. cp, Cortical plate; iz, intermediate zone. Scale bars: A–D, E–J, 0.5 mm; a′–d′, g′, j′, K–R, 100 μm.

Figure 4.

Supernumerary production of Cajal-Retzius cells in the absence of the cortical hem. A–G, Coronal sections showing the expression of Wnt3a, Wnt2b (cortical hem Wnts), and Wnt8b in E13.5 Gli3^+/− control embryos (A–C) and Gli3^−/− mutants (E–G). Expression of Wnt3a and Wnt2b expression is absent in the dorsal midline of Gli3^−/− mutants (E, F), whereas Wnt8b expression is restricted the medial most dorsal telencephalon (G). D and H show reelin expression, indicating the presence of CR cells in both control (D) and Gli3^−/− mutant mice (H). However, reelin-expressing CR cells are clustered rather than being evenly distributed throughout the cortex in Gli3^−/− mutants (shown at arrowheads). I, J, Expression of Reelin and p73 in control and Gli3^−/− cortex. i′–i‴ and j′-j‴ show an enlarged view of the boxed region indicated in I and J (pial surface is to the top). Many Reelin⁺ are detected in the dorsal pallium in Gli3^−/− mutants (J, j′), and most of these do not express p73 (j″, j‴). Scale bars: A–J, 200 μm; i′–j‴, 100 μm. Q–T, PSB is preserved in Gli3^−/− mutants, as indicated by the normal expression of Dbx1 (Q, S). CR cells are present in both wild-type and mutant embryos as indicated by reelin expression (R, T) in Gli3^+/− control (Q, R) and Gli3^−/− (S, T) E10.5 embryos. Dbx1 is expressed in the ventrolateral telencephalon (shown at asterisk), which is not perturbed in the Gli3^−/− mutants (S). The earliest reelin-expressing CR cells were detected in the ventral telencephalon at this stage in both control and Gli3^−/− mice. R and T show sections that are situated anterior to the Dbx1-expressing regions shown in Q and S. Insets show an enlarged view of reelin-expressing cells as indicated with arrowheads. U–Z, Expression of Dbx1, Ebf2, and reelin in E14.5 control (U–W) and Gli3^−/− (X–Z) mice. Arrowheads in U indicate the restricted expression of Dbx1 within the corticostriatal boundary in controls, whereas Dbx1 expression is expanded in Gli3^−/− nulls (X). Asterisk indicates the cluster of supernumerary CR cells in the Gli3^−/− cortices. Scale bars: Q–T, 100 μm; U–Z, 0.5 mm.

Figure 5.

Cajal-Retzius cells are produced in the absence of BMP signaling. A, B, Reelin expression in both control (A) and Foxg1^Cre/+; Bmpr1B^−/−; Bmpr1A^flox/flox (BmprIa/b dKO) mice (B) at E10.5 (coronal view). Embryos carrying the Foxg1^Cre/+;Bmpr1a^flox/+;Bmpr1b^n/+ genotype are referred to as controls. a′ and b′ show the enlarged view of the boxed region indicated in A and B, respectively. vz, Ventricular zone. Scale bars: A, B, 200 μm; a′, b′, 100 μm.

Figure 6.

Model of the genetic interactions mediating CR cell development in the telencephalon. The current study demonstrates that Foxg1 antagonizes both CR cell fate and dorsomedial signaling center (BMPs, Wnts), but these two events can be dissociated. Moreover, the loss of Foxg1 gene function results in overproduction of both cortical hem- and PSB-derived CR cell subtypes, demonstrating that it is universally involved in the production of CR cells, regardless of their site of origin. However, although Foxg1 is essential to repress CR cell production in all contexts, specification of distinct subclasses may depend on local cues such as Wnt proteins (in the Hem) or Dbx1 expression (in the PSB).