Zic2 Controls the Migration of Specific Neuronal Populations in the Developing Forebrain

Abstract

Human mutations in ZIC2 have been identified in patients with holoprosencephaly and schizophrenia. Similarly, Zic2 mutant mice exhibit holoprosencephaly in homozygosis and behavioral and morphological schizophrenic phenotypes associated with forebrain defects in heterozygosis. Despite the devastating effects of mutations in Zic2, the cellular and molecular mechanisms that provoke Zic2-deficiency phenotypes are yet unclear. Here, we report a novel role for this transcription factor in the migration of three different types of forebrain neurons: the Cajal-Retzius cells that populate the surface of the telencephalic vesicles, an amygdaloid group of cells originated in the caudal pole of the telencephalic pallium, and a cell population that travels from the prethalamic neuroepithelium to the ventral lateral geniculate nucleus. Our results also suggest that the receptor EphB1, previously identified as a Zic2 target, may mediate, at least partially, Zic2-dependent migratory events. According to these results, we propose that deficiencies in cell motility and guidance contribute to most of the forebrain pathologies associated with Zic2 mutations.

Significance statement: Although the phenotype of Zic2 mutant individuals was reported more than 10 years ago, until now, the main function of this transcription factor during early development has not been precisely defined. Here, we reveal a previously unknown role for Zic2 in the migration of forebrain neurons such as Cajal-Retzius cells, interneurons moving to the ventral lateral geniculate nucleus, and neocortical cells going to the amygdala. We believe that the role of this transcription factor in certain populations of migratory cells contributes to defects in cortical layering and hypocellularity in the ventral LGN and amygdala and will contribute to our understanding of the devastating phenotypes associated with Zic2 mutations in both humans and mice.

Keywords: Cajal–Retzius cells; LOT2 amygdaloid nucleus; Zic2; forebrain; neuronal migration; vLGN.

Figure 1.

Zic2 is expressed in CRCs from hem and septum. A, Whole-mount telencephalic hemisphere of a E12.5 Tg(Zic2eGFP) embryo. B, Calretinin and reelin immunostaining in coronal sections through the rostral telencephalon of E12.5 Tg(Zic2^EGFP). Note eGFP expression in the septum region. C, Calretinin immunostaining in coronal sections through the telencephalon of E12.5 Tg(Zic2^EGFP) mouse embryos revealed that, whereas a large number of calretinin cells are found in the dorsolateral superficial cells, eGFP cells only localize in medial areas, close to the hem region. D–D′′, Reelin and COUP-TF2 stainings in coronal sections through the telencephalon of E12.5 Tg(Zic2^EGFP) mouse embryos show that the majority of Zic2/eGFP cells express these CRC markers. E, E′, Tbr1 and reelin staining in coronal sections of E14.5 Tg(Zic2^EGFP) embryos confirm that Zic2/eGFP cells still express this CRCs marker at later stages. Arrowheads point to Tbr1 positive cells that are also positive for reelin and eGFP. F–G′, Zic2 staining in coronal sections through the telencephalon of E12.5 and E15.5 Tg(Zic2^EGFP) mouse embryos demonstrate that eGFP expression reproduces the endogenous expression of Zic2 in the CRCs (white arrowheads). Note that, at E15.5, Zic2 but not eGFP is expressed in some DAPI-stained meningeal cells (empty arrowheads). R, Rostral; C, caudal; M, medial; L, lateral; PSB, palium subpalium boundary. Scale bars: A–C, 200 μm; D–G′, 20 μm.

Figure 2.

Zic2 mutants exhibit defects in the distribution of CRCs. A, Scheme summarizing the experimental procedure. Telencephalic vesicles were isolated, cut, and flattened on a dish. B, C, Images showing flattened telencephalic preparations from [Zic2^kd/kd; Tg(Zic2^eGFP)] and control E12.5 embryos. Note that, whereas in the control preparation, eGFP cells delineate a mediolateral gradient with homogeneously distributed CRCs, in the mutant vesicle, eGFP cells are unevenly distributed. Yellow-dashed areas in C are devoid of eGFP cells. Red-dashed areas delineate places where green cells had migrated into deeper layers. D, E, Images showing high magnification of cells from flattened telencephalic preparations. Zic2 mutant CRCs show longer processes compared with the control cells. Note that in, some areas, mutant CRCs were grouped or attached to neighbor cells at the expense of other areas devoid of cells. F, G, Time-lapse sequence of the surface of whole-mount telencephalic preparations from [Zic2^kd/kd; Tg(Zic2^eGFP)] and control E12.5 embryos. Cell trackings are indicated in different colors. H, Graph representing the distribution of eGFP CRCs measured by FI in flattened telencephalic vesicles of [Zic2^kd/kd; Tg(Zic2^eGFP)] and control E12.5 embryos. Note the higher FI in the regions close to the hem in the Zic2 mutant embryos compared with the controls and a lower FI in the regions distal to the hem. I, Graph representing the percentage of trapped cells that form aggregates and therefore do not move or move for very short distances in the time-lapse videos. R, Rostral; C, caudal; M, medial; L, lateral. Scale bars: B, C, 500 μm; D–G, 50 μm.

Figure 3.

Zic2 mutants show cortical lamination defects. A, Schematic drawings representing the orientation and level of sectioning in the rest of the figure. B, C, Coronal sections of E12.5 Tg(Zic2^eGFP) embryos showing that Zic2 CRCs are located in the superficial layer of the telencephalic vesicle, whereas in the Zic2 mutant embryos, eGFP cells were not restricted to the most superficial cortical layer. White arrowheads point to mislocated eGFP cells. D, E, Coronal sections of E15.5 Tg(Zic2^eGFP) embryos showing that Zic2 CRCs are located in the telencephalic superficial layer. In contrast, in the Zic2 mutant embryos, many eGFP cells expressing reelin reached deep cortical laminae. White arrowheads point to mislocated eGFP/reelin cells. F–J, Inmunohistochemistry for Tbr1, Tbr2, and reelin in coronal sections of embryos at the indicated stages show that cortical layering, particularly the Tbr1 laminae, are affected from early stages of development (arrows) in the Zic2 mutant mice. Scale bars in B–K indicate 100 μm.

Figure 4.

Zic2 is essential to maintaining proper morphology and migration of CRCs. A, B, Hem explants from E12.5 [Zic2^+/+;Tg(Zic2^eGFP)] or [Zic2^kd/kd; Tg(Zic2^eGFP)] embryos cultured on laminin showed a reduced number of cells migrating away from the explant. A′–B′, High magnification of representative hem explants from E12.5 embryos showing a higher number of eGFP CRCs attached to the explant in the [Zic2^kd/kd; Tg(Zic2^eGFP)] explant than in the controls. Yellow dashed lines include cells that have not completely detached from the explant. C, D, Immunostaining against cofilin and Tuj1 in migrating cells from the explants showing cytoskeleton differences between Zic2 mutant and control cells. E–J, Three representative examples of WT and Zic2 mutant CRCs. K, Drawing representing the analyzed areas in hem explant cultures. The graph shows the percentage of cells located within the 150 μm close to the explant (area 1, yellow) and the percentage of cells located in area 2 (green). Note that, in the Zic2 mutant explants, most cells are located in area 1 and there are very few cells located in area 2. L, Graph quantifying eGFP CRCs not completely detached from the explant measured by FI (in a.u.). M, Schematic drawing representing a control (+/+) and a Zic2 mutant (kd/kd) CRC to clarify the analysis approach. Light blue line represents the SN and the dark blue line the LN. Neurites growing directly from the cell body (yellow) were considered 1ONs (gray) and any other neurites were considered 2ONs (pink). Graphs show the quantification of 1ONs and 2ONs and the average length of the LNs and SNs in CRCs from E12.5 Zic2 mutant and control cells from hem explants. ns, Nonsignificant. Scale bars in A–J indicate 50 μm.

Figure 5.

Zic2 is expressed in CAS cells migrating from the caudal telencephalon to the amygdaloid nucleus. A, Schematic drawing representing the area of CAS cells migrating from the lateral ventricle to nLOT2. B, C, Zic2 and Tbr1 staining in sagittal sections of E15.5 [Zic2^+/+;Tg(Zic2^eGFP)] embryos demonstrating that eGFP expression reproduces the endogenous expression of Zic2 in the area where Tbr1 CAS cells are migrating to nLOT2. D, E, Representative sagittal sections of Zic2 E15.5 [Zic2^+/+;Tg(Zic2^eGFP)] and [Zic2^kd/kd; Tg(Zic2^eGFP)] embryonic brains revealing a defect in the trajectories of eGFP positive cells in the Zic2 mutant mice. D′–E′, Tbr1 immunostaining in sagittal sections of Zic2 E15.5 [Zic2^+/+;Tg(Zic2^eGFP)] and [Zic2^kd/kd; Tg(Zic2^eGFP)] embryonic brains. High-magnification images are shown in the squared areas in D and E. D′′–E′′, High magnification of the squared areas in D and E reveal aberrant morphologies of eGFP cells in the Zic2 mutant mice. Scale bars: B, C, E, 100 μm; D–E′′, 50 μm.

Figure 6.

Zic2 is expressed in thalamus–prethalamus boundary INs. A, Scheme representing a coronal section from an E13.5 mouse brain. The squared area delimits the zone depicted in all the images of this figure. B, Arx immunostaining in coronal sections of E13.5 Tg(Zic2^eGFP) embryos confirm the existence of a population of Zic2/eGFP cells in the thalamic-prethalamic boundary. White dashed lines delimit the thalamic prethalamic boundary (TpTB) Zic2-positive area. C, D, Zic2/eGFP cells in the thalamic–prethalamic boundary at E15.5 and E16.5. Note that Zic2 is also expressed in the thalamus and that thalamocortical axons are also positive for eGFP and visible from E14.5 in advance. E, E′, Immunostaining for RC2 in coronal sections of E15.5 Tg(Zic2^eGFP) embryos demonstrate that Zic2/eGFP cells are not glial cells. F–F′′′, Zic2 and Tuj1 immunostaining in coronal sections of E15.5 Tg(Zic2^eGFP) embryos demonstrate that Zic2/eGFP cells in the TpTB are neurons. Note that eGFP cells present Zic2 staining in the nucleus. G–G′′, Otx2 immunostaining in coronal sections of E15.5 Tg(Zic2^eGFP) embryos demonstrate that Zic2/eGFP cells are INs. Th, thalamus; pTh, prethalamus, Cx, cortex; TCA, thalamocortical axons. Scale bars: B–G, 100 μm; E′–G′′, 50 μm.

Figure 7.

Zic2 thalamus–prethalamus INs are born between E11.5 and E13.5. A, Double staining for BrdU and Zic2 in coronal sections from E14.5 embryos with mothers that were injected with BrdU at E11.5 or E12.5. B, Quantification of the number of double-labeled Zic2/BrdU cells in E14.5 embryos from mothers injected with BrdU at E10.5, E11.5, E12.5, or E13.5. The majority of the Zic2 cells in the thalamic prethalamic boundary (TpTB) area were born between E11.5 and E12.5. Quantifications were performed in at least four sections per embryo. Numbers in the columns represent number of embryos analyzed. C, C′, Time-lapse sequence in the MS of E13.5 Tg(Zic2^eGFP) embryos. At the left, color arrowheads point out the starting (top) and the ending (bottom) points of the video. At the right, the three examples of migrating eGFP cells (turquoise, violet, and green arrowheads) over time demonstrate that these cells move from lateral to medial locations. D, Dorsal; V, ventral; M, medial; L, lateral. Scale bars: A, C–C′, 100 μm; small panels, 20 μm.

Figure 8.

Zic2 mutant mice show lower number of eGFP thalamic prethalamic boundary (TpTB) IN in the vLGN. A, B, Coronal sections from E14.5 [Zic2^+/+;Tg(Zic2^eGFP)] and [Zic2^kd/kd;Tg(Zic2^eGFP)] embryos through the medial diencephalon. Dashed lines delineate the MS from the third ventricle to the vLGN. White arrowheads highlight the reduction in the number of cells in the MS and vLGN of Zic2 mutant mice. Red arrowheads point to cells mislocated in the prethalamus of Zic2 mutant embryos. C, D, Close-up of the ventricular region of E14.5 [Zic2^+/+;Tg(Zic2^eGFP)] and [Zic2^kd/kd;Tg(Zic2^eGFP)] embryos. Red arrowheads point to an area containing accumulation of cells in the ventricular wall of a Zic2 mutant embryo. E, F, Coronal sections from E14.5 control and Zic2 mutant embryos from mothers injected with BrdU at E12.5. A reduction in the number of BrdU-positive cells is observed in the vLGN area, but not in the ventricular areas of Zic2 mutant mice (arrowheads). White lines divide medial and lateral thalamic areas. G, H, Caspase3-positive cells (arrows) in coronal sections from E14.5 [Zic2^+/+;Tg(Zic2^eGFP)] and [Zic2^kd/kd;Tg(Zic2^eGFP)] embryos. I, Graphs representing the quantification of FI from BrdU-positive cells in the MS close to the ventricular zone (medial area) or close to the vLGN (lateral area) in Zic2 mutant and control embryos. J, Graph representing the number of Caspase3-positive cells in Zic2 mutant and control embryos at E14.5. Th, Thalamus; pTh, prethalamus; TCA, thalamocortical axons.

Figure 9.

eGFP thalamic prethalamic boundary (TpTB) INs from Zic2 mutant mice show defects in migration. A–B′, Time-lapse sequence of cells in the MS of [Zic2^+/+;Tg(Zic2^eGFP)] and [Zic2^kd/kd;Tg(Zic2^eGFP)] E13.5 embryos. Color arrowheads in A and B mark the starting point of individual eGFP cells and colored lines in A′ and B′ delineate individual cell trajectories. Note that, in the Zic2 mutant mouse, many cells abandon their stereotyped mediolateral path to the vLGN. As an example, the trajectories of an eGFP-migrating cell in the control and the Zic2 mutant are shown and highlighted with a purple arrowhead. C, Tracks of 10 cells taken from three different embryos evidence aberrant trajectories in Zic2 mutant embryos (right) compared with the controls (left). Cell trajectories in the mutants delineate aberrant directions and cover longer distances. D, Quantification of the percentage of cells that migrate aberrantly and the maximum and average speed of [Zic2^kd/kd;Tg(Zic2^eGFP)] and control cells. D, Dorsal; V, ventral; M, medial; L, lateral; Th, thalamus; pTh, prethalamus; vLGN, ventral lateral geniculate nucleus; 3V, third ventricle. Scale bars: A–B′, 100 μm; small panels, 20 μm.

Figure 10.

EphB1 is expressed in thalamic prethalamic boundary (TpTB) cells and its expression is disrupted in Zic2 mutant mice. A–B′′, In situ hybridization in coronal sections through the diencephalon of E13.5 embryos showing that EphB1 mRNA is expressed in the TpTB area coinciding with the location of Zic2 cells. Arrowheads point to EphB1 expression. B–B′′, Higher-magnification of the squared areas in B. C, D, In situ hybridization in Zic2 mutant and control littermates revealing a reduction in the expression of EphB1 in embryos expressing low levels of Zic2. Arrowheads delineate the expression of EphB1 in the control and its absence in the mutant section. E, In situ hybridization in coronal sections of E13.5 embryos showing that ephrinB2 mRNA is expressed in thalamic areas proximal to the ventricular zone. Zic2 staining reveals the Zic2 MS. F, In situ hybridization in coronal sections of E13.5 embryos showing that ephrinB3 mRNA is highly expressed in thalamic and prethalamic areas proximal to the ventricular zone and at basal levels in lateral areas. Note that the stream of Zic2/eGFP-migrating cells is devoid of ephrinB3 expression. L, Lateral; M, medial; 3V, third ventricle; pTH, prethalamus; Th, thalamus. Scale bars: A–F, 100 μm; B′-B′′, 50 μm.