A cell-autonomous requirement for the cell cycle regulatory protein, Rb, in neuronal migration

Abstract

Precise cell cycle regulation is critical for nervous system development. To assess the role of the cell cycle regulator, retinoblastoma (Rb) protein, in forebrain development, we studied mice with telencephalon-specific Rb deletions. We examined the role of Rb in neuronal specification and migration of diverse neuronal populations. Although layer specification occurred at the appropriate time in Rb mutants, migration of early-born cortical neurons was perturbed. Consistent with defects in radial migration, neuronal cell death in Rb mutants specifically affected Cajal-Retzius neurons. In the ventral telencephalon, although calbindin- and Lhx6-expressing cortical neurons were generated at embryonic day 12.5, their tangential migration into the neocortex was dramatically and specifically reduced in the mutant marginal zone. Cell transplantation assays revealed that defects in tangential migration arose owing to a cell-autonomous loss of Rb in migrating interneurons and not because of a defective cortical environment. These results revealed a cell-autonomous role for Rb in regulating the tangential migration of cortical interneurons. Taken together, we reveal a novel requirement for the cell cycle protein, Rb, in the regulation of neuronal migration.

Figure 1

Laminar patterning is perturbed in the absence of Rb. In situ hybridization of E15.5 sagittal sections of mutant and control embryos demonstrates enhanced expression of neuronal markers, Tbr1 and SCG10, in the Rb mutants. Tbr1 labeling is slightly elevated within the mutant CP, and strongly upregulated within the IZ (arrows) (C, D), relative to control (A, B). Similarly, SCG10 expression is highly elevated within the mutant IZ (arrows) (G, H), relative to control (E, F). The boundary between the mutant CP and IZ lacks the clear definition observed in the control embryos (n=4 control; 5 cond. Rb−/−); bar=100 μm, ge=ganglionic eminence, MZ=marginal zone, CP=cortical plate, IZ=intermediate zone.

Figure 2

Rb-deficient cortical neurons exhibit delayed radial migration. Pregnant females at E13.5 of gestation were injected with a single dose of 20 μg/g body weight BrdU. Embryos were removed 5 days later at E18.5, fixed, and subjected to immunohistochemistry for BrdU (A, B). BrdU-labeled cells were counted across a 620-μm-wide section of dorsal cortex. In contrast to the control sections with only 33.3±9.2 cells, 158±14.3 labeled cells were counted within the IZ of Rb mutants, representing an almost five-fold increase (arrows) (C; P<0.001). This dramatic increase was compensated for by a corresponding decrease of labeled cells that had reached the CP. The controls had 480.2±57.9 cells within the CP compared to only 336.9±30.5 in the Rb mutants (C; P<0.05). These results indicate that although similar numbers of neurons were generated at E13.5 in control and mutant cortices, Rb-deficient cortical neurons are delayed in reaching their ultimate position within the CP. Error denotes standard error (n=3 control; n=4 cond. Rb−/−). Bar: 50 μm. MZ: marginal zone; CP: cortical plate; IZ: intermediate zone; VZ: ventricular zone.

Figure 3

Rb is required for survival of Cajal–Retzius neurons. Coronal sections of control and Rb mutant embryos at E12.5 (A, D) and E16.5 (B, E) were subjected to immunohistochemistry with a Reelin (G10) antibody. Positive cells in each section were quantified along a 1500 μm (E12.5) or 3000 μm (E16.5) length of the MZ. At E12.5, Reelin expression in the cortical MZ appeared similar between mutant and control embryos (A, D, G) (n=3 control; n=3 cond. Rb−/−). Total MZ cells counted within a 245 μm length of the dorsal cortex of E13.5 embryos confirmed similar cell numbers between mutant and controls at this time (H; n=4 control; n=4 cond. Rb−/−). However, by E16.5, Rb mutants contained approximately 50% fewer Reelin-positive neurons as compared to control embryos (B, E, I, P<0.001) (n=5 controls; n=4 cond. Rb−/−). Total MZ cell number quantified within a 245 μm length of the dorsal cortex of E16.5 embryos resulted in a similar reduction in Rb mutants (J, P<0.05) (n=3 control; n=4 cond. Rb−/−). To detect cell death, E13.5 conditional mutant and control littermates were assayed for TUNEL labeling. On each section, positive cells were quantified within the MZ. Rb mutants exhibited significantly increased TUNEL labeling within the MZ (C, F, K, P<0.05; arrows point to representative cells) (n=4 control; n=5 cond. Rb−/−). Error denotes standard error. Bar: 25 μm. MZ: marginal zone.

Figure 4

Rb deficiency does not impact interneuron specification or generation. To examine whether interneurons are properly generated in the absence of Rb, mutant and control E12.5 coronal sections were immunolabeled with a calbindin (D-28) antibody or subjected to in situ hybridization with an Lhx6 riboprobe. The generation of calbindin- (A, B) and Lhx6-positive (C, D) progenitors appeared similar in the mutant and control embryos (n=3 control; n=3 cond. Rb−/−). Bar: 50 μm.

Figure 5

Cortical interneurons are mislocalized in Rb mutants. Rb mutant and control embryo sections were examined at mid-neurogenesis to determine whether specific interneuron populations may be impacted by Rb deficiency. E16.5 sections (coronal) were immunolabeled with a calbindin (D-28) antibody and E15.5 (sagittal) sections were subjected to in situ hybridization with an Lhx6 riboprobe. Although calbindin expression appeared normal in other telencephalic regions (A, B), these cells were dramatically reduced in the mutant MZ (C, D). Similarly, Lhx6 expression was normal along the SVZ/IZ migratory route, but was substantially reduced in the mutant CP and MZ (G, H; n=4 control, n=5 cond. Rb−/− (E15.5); n=5 control, n=5 cond. Rb−/− (E16.5). Calbindin-positive cells were quantified either within the MZ or deeper corresponding to the IZ (arrows point to MZ route; arrowheads denote IZ population). Although the total number of calbindin-positive cells does not differ, there is an approximately 50% reduction in cell number within the Rb mutant MZ (I, P<0.05). The decreased number of calbindin-positive neurons within the Rb mutant MZ is associated with a corresponding increase in these neurons within the mutant IZ (I, P<0.01). Error denotes standard error. Bar: 100 μm (A, B, E–H), 25 μm (C, D). MZ: marginal zone; IZ: intermediate zone.

Figure 6

Slice co-cultures. To assess the requirement for Rb function in interneuron migration, we performed slice co-culture assays. (A) GFP-negative telencephalon sections were plated onto coated filter-membrane inserts in a six-well dish. MGE were removed from GFP-positive littermates and equal-sized pieces were placed directly on sections corresponding to the MGE. Co-cultures were grown in vitro for 72 h prior to fixation and GFP immunohistochemistry. The migratory routes of the GFP-positive cells, specifically the MZ trajectory, were analyzed and classified as follows: ‘cell integration', in which GFP-positive cells integrated into the section (arrow) but did not follow a distinct migratory route (arrowhead) or reach the MZ (B, E, H). ‘MZ stops early' occurred when GFP-positive cells formed an MZ route (arrow) but did not reach the dorsal cortex (arrowhead) (C, F, I) ‘MZ migration' included sections in which a GFP-positive MZ migratory route reaching the dorsal cortex was observed (arrows) (D, G, J). Bar: 100 μm (D, G), 50 μm (J). MGE: medial ganglionic eminence; MZ: marginal zone.

Figure 7

A cell-autonomous requirement for Rb in tangential migration. (A) The MZ migratory routes resulting from GFP-positive MGE explants were compared according to the previously described classifications (Figure 6). Control explants were able to complete MZ migration in 95.9% (47/49) of sections examined, regardless of whether they were placed on mutant or control slices (B). In contrast, only 19.2% (5/26) of mutant explants placed on either mutant or control slices displayed proper MZ migration (C). MZ: marginal zone.

Figure 8

Proposed model of Rb-mediated regulation of neuronal differentiation and migration. In wild-type cells, Id2 is sequestered by Rb and is unable to inhibit basic HLH-mediated transcription of specific neuronal genes, such as TrkB, that are required for neuronal differentiation and migration. In the absence of Rb, Id2 activity is deregulated, allowing inhibition of TrkB transcription, which, in turn, leads to impaired neuronal migration and differentiation.