Genetic Evidence That Celsr3 and Celsr2, Together with Fzd3, Regulate Forebrain Wiring in a Vangl-Independent Manner

Genetic evidence that Celsr3 and Celsr2, together with Fzd3, regulate forebrain wiring in a Vangl-independent manner

Yibo Qua, Yuhua Huanga, Jia Fenga, Gonzalo Alvarez-Boladob, Elizabeth A. Grovec, Yingzi Yangd, Fadel Tissire, Libing Zhoua,f,1,2, and Andre M. Goffinete,1,2

aGuangdong–Hong Kong–Macau Institute of CNS Regeneration, Jinan University, Guangzhou 510632, China; bDepartment of Neuroanatomy, Heidelberg University, D-69120 Heidelberg, Germany; cNeuroscience, The University of Chicago, Chicago, IL 60637; dNational Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892; eWELBIO - Walloon Excellence in Life Sciences and Biotechnology and Institute of Neuroscience, University of Louvain, B1200 Brussels, Belgium; and fState Key Laboratory of Brain and Cognitive Sciences, The University of Hong Kong, Hong Kong

Edited* by Jeremy Nathans, The Johns Hopkins University, Baltimore, MD, and approved June 18, 2014 (received for review February 3, 2014) Celsr3 and Fzd3, members of “core planar cell polarity” (PCP) AC contains commissural axons from the anterior olfactory nu- genes, were shown previously to control forebrain axon guidance clei and from the temporal cortex, which cross the midline at and wiring by acting in axons and/or guidepost cells. Here, we embryonic day 13.5 (E13.5) to E14.5 (11–14). The IC contains show that Celsr2 acts redundantly with Celsr3, and that their com- three main axonal components. Thalamocortical axons (TCA) bined mutation mimics that of Fzd3. The phenotypes generated emerge from the thalamus—formerly called “dorsal” thalamus upon inactivation of Fzd3 in different forebrain compartments are (15)—at E12.5. They run through the prethalamus (former similar to those in conditional Celsr2-3 mutants, indicating that “ventral” thalamus), turn and cross the diencephalon–telen- Fzd3 and Celsr2-3 act in the same population of cells. Inactivation cephalon junction, progress through a corridor in the ventral of Celsr2-3 or Fzd3 in thalamus does not affect forebrain wiring, telencephalon, and cross the pallial–subpallial boundary to reach and joint inactivation in cortex and thalamus adds little to cortical the cortical anlage from E14.5 (16–19). Corticothalamic axons inactivation alone in terms of thalamocortical projections. On the (CTA) emerge initially from neurons in the subplate and future other hand, joint inactivation perturbs strongly the formation of cortical layer 6, around E13.5. They cross the pallial–subpallial the barrel field, which is unaffected upon single cortical or tha- boundary and progress in the ventral telencephalon, in opposite lamic inactivation, indicating a role for interactions between tha- – lamic axons and cortical neurons in cortical arealization. Unexpectedly, direction to TCA. They cross the diencephalon telencephalon forebrain wiring is normal in mice defective in Vangl1 and Vangl2, junction and begin to enter the thalamus at E14.5 (17, 19, 20). showing that, contrary to epithelial PCP, axon guidance can be Subcerebral projections such as the CST begin to leave the Vangl independent in some contexts. Our results suggest that cortical plate at E14.5–E15.5 to enter the IC, and diverge from Celsr2-3 and Fzd3 regulate axonal navigation in the forebrain CTA to form the cerebral peduncle, en route to their subcortical by using mechanisms different from classical epithelial PCP, and targets such as the spinal cord (21). require interacting partners other than Vangl1-2 that remain to Previous work showed that Celsr3 is required for the de- be identified. velopment of the AC, IC, and CST (2), and Cre-mediated regional

Cre | anterior commissure | internal capsule | cortical barrels Significance

rom a functional perspective, the appropriate wiring of con- Connections are crucial to brain function and a variety of Fnections is arguably the most important aspect of brain de- molecular systems direct axonal growth during development velopment. The initial step in wiring is the directed extension of and regeneration. An important system involves Celsr2, Celsr3, axons through a complex environment. Axonal growth cones are and Fzd3, membrane proteins that also regulate epithelial guided by a wide variety of attractive and repulsive cues. Some planar cell polarity (PCP). Here, we show genetically that Celsr2 are secreted, diffusible, and act at a distance, whereas others are and Celsr3 guide axons redundantly, in collaboration with Fzd3 anchored locally to the extracellular matrix or to the surface of in the same cell populations. However, unlike in epithelial PCP, so-called guidepost cells or intermediate targets. Regulators of their action is Vangl1 and Vangl2 independent. Furthermore, axon guidance include ligand/receptor partners such as Eph/ expression of Celsr2-3 and Fzd3 in thalamocortical axons and ephrins, Slit/Robo, Semaphorins/Plexins-Neuropilins, Netrins/ cortical cells is required for the fine mapping of cortical areas. Unc5-Dcc (1), and the membrane proteins Celsr3 and Frizzled3, Our findings that Celsr2, Celsr3, and Fzd3 regulate axonal whose role was identified more recently (2–4). The seven-pass guidance using mechanisms different than epithelial PCP transmembrane proteins Celsr1-3 (Cadherin EGF LAG seven- have implications for brain wiring during normal development pass G-type receptors 1–3) and Frizzled (Fzd) 3 and 6 belong to and regeneration. a set of proteins that regulate planar cell polarity (PCP) in epithelial sheets, where they work with Van Gogh-like (Vangl) 1 Author contributions: Y.Q., F.T., L.Z., and A.M.G. designed research; Y.Q., Y.H., and J.F. – performed research; G.A.-B., E.A.G., Y.Y., and F.T. contributed new reagents/analytic and 2, the cytoplasmic adaptors Dishevelled (Dvl) 1 3, Prickle tools; Y.Q., Y.H., J.F., F.T., L.Z., and A.M.G. analyzed data; and Y.Q., L.Z., and A.M.G. wrote (Pk) 1–4, and others whose role is less clearly defined (5–10). In the paper. epithelial PCP, expression of Frizzed-Celsr on one side of cells, The authors declare no conflict of interest. and Vangl-Celsr on the other side, imparts a vectorial organi- *This Direct Submission article had a prearranged editor. zation to the epithelium responsible, among other, for the co- Freely available online through the PNAS open access option. ordinated growth of body hairs and cilia. 1L.Z., and A.M.G. contributed equally to this work. In this study, we analyzed genetically the contribution of 2To whom correspondence may be addressed. Email: [email protected] or Celsr2, Celsr3, Fzd3, and Vangl1, Vangl2 to axonal development [email protected]. in the forebrain, by focusing on the anterior commissure (AC), This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the corticospinal tract (CST), and the internal capsule (IC). The 1073/pnas.1402105111/-/DCSupplemental.

E2996–E3004 | PNAS | Published online July 7, 2014 www.pnas.org/cgi/doi/10.1073/pnas.1402105111 Downloaded by guest on September 30, 2021 inactivation indicated that axon navigation requires Celsr3 ex- subcerebral targets, yet can reach the thalamus. We examined PNAS PLUS − pression in neurons of origin and/or in guidepost cells along the Celsr2gt/gt;Emx1-Cre;Celsr3f/ mice, in which Celsr2 is mutated in pathway (22). The Celsr3 mutant axonal phenotype is quite the whole embryo and Celsr3 in cortical progenitors and pro- − − similar to that in Fzd3 mutant mice (3, 23), hinting that a PCP- jection neurons, as well as Emx1-Cre;Celsr2f/ ;Celsr3f/ mice, in like mechanism may regulate axon progression via interactions which both genes are inactivated by Emx1-Cre. Brains were between growth cones and guidepost cells. To understand fur- studied at P0, a stage when hydrocephalus—which develops ther the role of membrane-associated core PCP proteins in axon postnatally in those mutants (24)—does not perturb analysis. All guidance, we used a panel of mutant mice and show genetically components of the IC were well defined and of normal size in − − that Celsr2 and 3 regulate the formation of forebrain axon Celsr2gt/gt and Celsr2 / animals. In contrast, in both Celsr2gt/gt; − − − bundles in a redundant manner, and are required in the same Emx1-Cre;Celsr3f/ and Emx1-Cre;Celsr2f/ ;Celsr3f/ mutants, the cell populations as Fzd3. Unlike epithelial PCP, however, the IC was diminutive and abnormal looping fibers were seen in action of Celsr2-3 and Fzd3 on forebrain axonal fascicles is the external tier of the basal telencephalon, in the vicinity of the Vangl1,2 independent. Inactivation of Celsr2-3 or Fzd3 in thal- external capsule (Fig. 2 A–C). These looping axons were con- amus generates no evident phenotype, showing that the derailed sistently labeled upon 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindo- TCA phenotype in constitutive mutants is non-cell autonomous. carbocyanine perchlorate (DiI) placement in the diencephalon Furthermore, joint inactivation of Celsr2-3 or Fzd3 in thalamus (Fig. 2 G–I), but not upon dye injection in cortex (Fig. 2 D–F), and cortex perturbs the development of cortical maps. indicating that they contained mostly derailed TCA, which failed to progress to cortex, but few CTA fibers. Thus, Celsr3 and Results Celsr2 act redundantly in cortical neurons during development Celsr2 and Celsr3 Function Redundantly in Axon Guidance. Inasmuch of the IC. Compared with the full defect in constitutive mutants, as Celsr2 and Celsr3 have redundant activity during ependymal the partial IC phenotype observed upon cortical inactivation ciliogenesis (24) and facial branchiomotor neuron migration shows that the action of Celsr2 and 3 in cortical neurons, like (25), and both genes are coexpressed in postmitotic neurons, we that of Fzd3 described below (see Fig. 4), are partly non-cell wondered whether they also work together in axon guidance. autonomous. Axonal bundles in Celsr2gt/gt mutant mice were similar to controls (Fig. 1 A and E). In contrast, examination of brains at postnatal Celsr2 and Celsr3 Act Independently of Vangl1 and Vangl2 in day 0 (P0) showed that, in addition to the anomalies described Forebrain Wiring. Van Gogh is a key partner of Frizzled and − − previously in Celsr3 / mutants (Fig. 1 B and F) (2), additional Flamingo in Drosophila epithelial PCP (7). Similarly, in verte- − − defects were prominent in double-mutant Celsr2gt/gt;Celsr3 / brate epithelia such as the neuroepithelium, inner ear, epidermis, − − − − or Celsr2 / ;Celsr3 / samples, particularly the absence of stria or ependyma, Vangl2 collaborates with Celsr1 or 2, and Fzd3 or 6 medullaris and mammillothalamic tract, and a very diminutive (24, 30–32). Furthermore, guidance defects of mesencephalic fasciculus retroflexus (Fig. 1 C and G). Those features were also monoaminergic and spinal commissural fibers are present in − − seen in Fzd3 / mutants (Fig. 1 D and H), confirming a study Looptail Vangl2 mutant mice (33, 34).To assess whether Van with diffusion tensor imaging (26), and indicating that the wiring Gogh-like proteins are implicated in forebrain axonal guidance, anomalies in Fzd3 mutants are very similar to those in Celsr2+3 we inactivated both Vangl1 and Vangl2, using a constitutive double-mutant mice. This observation suggests strongly that Vangl1 genetrap allele known to be a null (35), and a conditional Celsr3 and Celsr2, on one hand, and Fzd3, on the other hand, act “floxed” Vangl2 allele (36). That Cre inactivation of the floxed similarly during axonal development, as they do during the mi- Vangl2 allele proceeds as predicted was demonstrated by im- gration of facial branchiomotor neurons (25) and in the steering munofluorescence (Fig. S1 A and B) and Western blot analysis at of peripheral motor axons (27). E13.5 (Fig. S1C), showing that no Vangl2 protein is detected in − We next investigated the redundancy between Celsr2 and the forebrain of Foxg1-Cre;Vangl2f/ mice, and by analysis of − − Celsr3 using Emx1-Cre mice, which express Cre in cortical pro- Vangl1 / ;Emx1-Cre;Vangl2f/f mice in which hair whorls—a typi- jection neurons and their progenitors (28) as well as in limb cal PCP phenotype (37)—are present in the hindfeet (Fig. S1E). − − ectoderm (22) (Fig. S1D), and Nex-Cre mice in which Cre is We examined brains of Vangl1 / ;Foxg1-Cre;Vangl2f/f mice, in expressed in cortical projection neurons but not progenitors (29). which Vangl1 is constitutively mutated and Vangl2 is inactivated We showed previously that TCA and CTA axonal components of in the whole forebrain except dorsal thalamus (38), at P0, a stage the IC are preserved, and that subcerebral projections such as when all major axonal bundles are easily visualized. In those − the CST are absent in Emx1-Cre;Celsr3f/ mice (22), indicating mutant animals, the AC was similar to that in controls (Fig. 3A), that Celsr3-deficient corticofugal axons are unable to project to the IC, with CTA and TCA bundles, had a normal size and shape

Fig. 1. Redundancy between Celsr2 and Celsr3 in constitutive mutants at P0. Whereas no anomaly of axonal bundles is seen in Celsr2gt/gt brains (A and E)(n = − − 4), profuse defects are present in Celsr3 / (B and F) as described (2). Celsr2 and 3 double mutants (C and G)(n = 6) display additional defects that mimic those − − in Fzd3 / mice (D and H)(n = 3). (A–D) H&E staining; (E–H) neurofilament immunohistochemistry. fr, fasciculus retroflexus; ml, medial lemniscus; mt, NEUROSCIENCE mammillothalamic tract; sm, stria medullaris. (Scale bar: 500 μm.)

Qu et al. PNAS | Published online July 7, 2014 | E2997 Downloaded by guest on September 30, 2021 This phenotype is similar to that described above in Emx1-Cre; − − − − Celsr2f/ ;Celsr3f/ and Nex-Cre;Celsr2f/ ;Celsr3f/ mutant mice (Fig. 2). When Fzd3 was inactivated in the basal forebrain using Dlx5/6-Cre, the AC developed normally (Fig. 4C) but all components of the IC were absent. Prominent neurofilament-rich CTA bundles were derailed in the basal telencephalon. TCA failed to turn at the diencephalon–telencephalon junction and were also misrouted to the ventral aspect of the basal telencephalon; some crossed to the other side in the preoptic and hypothalamic region (Fig. 4F), and no CST was present in the − spinal cord (Fig. 4I). The Dlx5/6-Cre;Fzd3f/ axonal phenotype appears somewhat more marked than that described previously − in Dlx5/6-Cre;Celsr3f/ samples. It also differs in some aspects such as a less prominent whorl of neurofilament-rich axons in the external tier of the basal forebrain. Despite those differences, the overall similarity of axonal bundle malformations in Dlx5/6-Cre; − − Fzd3f/ and Dlx5/6-Cre;Celsr3f/ mutant mice is remarkable. To- gether with an independent study using conditional Fzd3 inactivation (23), our observations that the regional inactivation of Celsr2+3,orFzd3, using different Cre strains, generates similar phenotypes suggests strongly that Celsr2, 3, and Fzd3 proteins are both required in the same cells.

Inactivation of Celsr2 and 3,orFzd3 in Thalamus Does Not Affect Forebrain Wiring. To further evaluate the role of Celsr2, Celsr3, and Fzd3 in thalamocortical wiring, we searched for mouse strains that express Cre in thalamus, and ideally not in prethalamus and more rostral areas. We first used Wnt3a-Cre and Nkx6.2-Cre, which are expressed early in thalamus in a complementary manner (41, 42). However, Nkx6.2 is also expressed focally in the ganglionic Fig. 2. Redundancy between Celsr2 and Celsr3 in cortical neurons. (A–C) Neurofilament immunohistochemistry (NF) at P0. Compared with control (A), mutant mice with regional inactivation of Celsr3 on Celsr2 mutant background (B)(n = 3) and with regional inactivation of Celsr2 and Celsr3 under Emx1-Cre (C)(n = 6) have significant atrophy of the IC (arrows), and looping fibers around the external capsule (arrowheads). (D–I) DiI injection at P0. Tracer administration in cortex (Ctx) (D–F) labels thalamic (Th) neurons retrogradely in control (n = 3) and mutants (n = 4 and 5). DiI in diencephalon (Dien) (G–I) labels TCA and cortical neurons in control brains (G)(n = 3); in mutants (H and I)(n = 4 and 5), it reveals abnormal looping axons (arrowheads). (Scale bars: 500 μm.)

(Fig. 3B), and the CST, visualized in the hindbrain (Fig. 3C) and spinal cord, was indistinguishable from that in control mice. The observation that Vangl1 and Vangl2 are not required for the formation of forebrain axonal bundles was quite unexpected and stands in sharp contrast to the profuse anomalies in mice with forebrain-specific inactivation of Celsr3 (22) and Fzd3 mice described below (Fig. 4 and Fig. S2).

Regional Inactivation of Fzd3 and Celsr3 Generates Similar Axonal Phenotypes. In Drosophila, Frizzled and Flamingo (Celsr) are thought to work in cis and can interact physically upon trans- fection in S2 cells (39). To test genetically whether an analogous action may account for the role of Fzd3 and Celsr2-3 in axon guidance, we examined the AC and IC in mice with regional inactivation of Celsr2+3 and Fzd3 in the whole forebrain using Foxg1-Cre, in the cortex using Emx1-Cre or Nex-Cre, and in the ventral forebrain using Dlx5/6-Cre (40). In comparison with control mice (Fig. 4 A, D, and G), inactivation of Fzd3 upon Foxg1-Cre expression resulted in complete absence of AC and IC (Fig. S2 A and B), and inactivation upon Emx1-Cre or Nex- Cre expression resulted in the following: (i) absence of the AC Fig. 3. Forebrain axon bundles develop independently of Vangl1 and Vangl2. Comparison between wild type (WT) (Left) and Vangl−/−;Foxg1-Cre; (Fig. 4B); (ii) strongly reduced size but normal trajectory of CTA Vangl2f/f mutant brains at P0 show normal development of the anterior and TCA fiber bundles in the IC, except for the presence of commissure (AC in A; H&E staining), normal IC (arrowheads in B; neurofila- aberrant looping fibers in basal forebrain (Fig. 4E and Fig. S2D); ment immunohistochemistry), and normal CST (arrows in C; neurofilament and (iii) complete absence of CST in the spinal cord (Fig. 4H). immunohistochemistry). n = 3. [Scale bars: 500 μm(A and B); 200 μm(C).]

E2998 | www.pnas.org/cgi/doi/10.1073/pnas.1402105111 Qu et al. Downloaded by guest on September 30, 2021 TCA, mediated by other molecules, palliate unilateral Celsr2-3 PNAS PLUS or Fzd3 deficiency. The third model, reminiscent of the “hand- shake” or “rendezvous” (44, 45), predicts that joint inactivation in neurons of origins of CTA and TCA should yield a more severe phenotype than the addition of phenotypes observed upon single inactivation in cortex or thalamus. We tested this latter hypothesis by inactivating Celsr3 upon Emx1, Wnt3a, and Nkx6.2-Cre expression, on a Celsr2 mutant − background (Celsr2gt/gt;Triple Cre;Celsr3f/ ) (Fig. 6). The axonal phenotype in those mice was nearly similar to that observed in mice with Emx1-Cre–induced inactivation alone (Fig. 2), except that derailed looping fibers in the laterobasal telencephalon could be labeled not only by DiI administration in diencephalon, but also in cortex (Fig. 6 D and F). We then used Foxb1-Cre in conjunction with Nex-Cre to inactivate Celsr2+3 or Fzd3 in both − cortex and thalamus. Brains of Foxb1-Cre;Nex-Cre;Celsr2f/ ; − − Celsr3f/ and Foxb1-Cre;Nex-Cre;Fzd3f/ mice were characterized by anomalies related to cortical-specific inactivation, particularly absence of AC and CST and atrophy of IC (Fig. 5 B, F, and J, and D, H, and L, and Table 1). To study this further, we examined E14.5 embryos using DiI injection in cortex or di- Fig. 4. Regional inactivation of Fzd3 phenocopies that of Celsr2+3. Com- encephalon. In control embryos, both TCA and CTA reached − pared with control brains (A, D, and G), inactivation of Fzd3 in cortex (B, E, their target area as expected (Fig. 7 A and D). In Nex-Cre;Celsr2f/ ; and H) results in absence of anterior commissure (AC), diminutive IC (arrow f/− in E) with external looping axons (arrowhead in E), and in absence of CST in Celsr3 embryos, DiI in cortex labeled cortical efferent fibers the cervical spinal cord (white arrow in G, absent in H). Inactivation in basal that did not pass the level of the lateral ganglionic eminence, and forebrain does not affect AC formation but impairs development of all TCA labeled upon diencephalic injection did not extend beyond components of the IC with derailed thalamocortical axons (arrow in F), the pallial–subpallial boundary (Fig. 7 B and E). Inactivation in − balling of cortical efferent fibers (white asterisk in F, black asterisk in C) and thalamus in addition to cortex (Foxb1-Cre;Nex-Cre;Celsr2f/ ; − absence of CST (I). (A–C) H&E staining; (D–F) neurofilament immunohisto- Celsr3f/ ) resulted in a slightly more pronounced IC phenotype, chemistry; (G–I) Prkcg (protein kinase Cγ) immunohistochemistry. n = 3for – μ – μ – with no CTA passing the pallial subpallial boundary and no each mutant genotype. [Scale bars: 500 m(A F); 200 m(G I).] TCA leaving the basal telencephalon (Fig. 7 C and F). This

eminences, around the sulcus that separates the lateral and medial ganglionic eminences, and Wnt3a in the hippocampal hem. − Brains from Celsr2gt/gt;Wnt3a-Cre;Nkx6-Cre;Celsr3f/ mice, with constitutive inactivation of Celsr2 and inactivation of Celsr3 in thalamus upon Wnt3a and Nkx6.2-Cre, were similar to control. To palliate difficulty associated with Wnt3a and Nkx6.2 expression outside thalamus, we obtained more specific Cre expression using Foxb1-Cre, which is active in the dorsal thalamus from an early developmental stage (E9.5–E10.5), but not expressed in prethalamus or telencephalon (43) (Fig. S3). Again, all forebrain axonal bundles and the CST developed normally in Foxb1-Cre; − − Celsr2f/ ;Celsr3f/ (Fig. 5 A, E,andI, and Table 1) and Foxb1-Cre; − Fzd3f/ mutants (Fig. 5 C, G,andK, and Table 1). Together with previous observations using Rora-Cre mice (22), and given the restrictions related to the timing and efficiency of Cre-induced recombination, the present data provide strong evidence that, although TCA are prominently derailed in constitutive Celsr3 and Fzd3 mutants, and upon conditional inactivation in ventral telencephalon (Dlx5/6-Cre), deletion of Celsr2+3 or Fzd3 in their thalamic neurons of origin has no impact on their trajectory.

Joint Inactivation in Cortex and Thalamus Enhance Wiring Phenotypes Marginally. As described above, the action of Celsr2-3 and Fzd3 is nonautonomous in thalamic neurons and TCA, and partially nonautonomous in cortical neurons of origin of CTA. How can Fig. 5. Inactivation in thalamus does not affect wiring, and joint in- we explain a fully cell-autonomous action in neurons of origin of activation in cortex and thalamus affects it mildly. At P0, hematoxylin–eosin the AC and CST, and non–cell-autonomous effect on CTA and (H&E) (A–D), neurofilament immunostaining (NF) (E–H), and protein kinase TCA? First, a trivial explanation that the Cre drivers used are Cγ immunofluorescence (Prkcg) (I–L) show that, upon inactivation in thala- not fully active in the context of early cortical and thalamic mus using Foxb1-Cre (A, C, E, G, I, and K)(n = 5 and 3, respectively), the anterior commissure (AC), the IC (arrows in E and G), and the CST (arrows in neurons cannot be formally excluded but appears rather unlikely, = as discussed later. Second, unlike AC and CST neurons, ex- I and K) are present. Joint inactivation in thalamus and cortex (n 7 and 3, respectively) results in absence of AC crossing (B and D), atrophy of the IC pression of Celsr2-3 and Fzd3 may not be required in cortical (arrows in F and H) with some looping of axons close to the external capsule and thalamic neurons, and CTA and TCA projections could rely (arrowhead in F), and absence of CST in the spinal cord (J and L). These

more than others on expression in guidepost cells. A third pos- anomalies are similar to those obtained upon cortex-specific inactivation. NEUROSCIENCE sibility could be that a reciprocal interaction between CTA and [Scale bars: 500 μm(A–H); 200 μm(I–L).]

Qu et al. PNAS | Published online July 7, 2014 | E2999 Downloaded by guest on September 30, 2021 Table 1. Summary of cortical and thalamic projection phenotypes Genotype Wiring phenotype

Celsr2gt/gt No phenotype (Fig. 1 A and E) − − Celsr2gt/gt;Celsr3 / Full defect of IC (Fig. 1 C, D, G,andH) − − − − Celsr2 / ;Celsr3 / Fzd3−/− Celsr2gt/gt;Emx1-Cre;Celsr3f/− Decreased IC with partially blocked/delayed TCA (Figs. 2 and 4 B and E) − − Emx1-Cre;Celsr2f/ ;Celsr3f/ Looping of TCA in external basal forebrain (Fig. 2 and Fig. S2D) − − Nex-Cre;Celsr2f/ ;Celsr3f/ Absence of CST (Fig. 4H) Emx1-Cre;Fzd3f/− Layer 5 axons routed to thalamus (Fig. 7H) − Nex-Cre;Fzd3f/ Normal barrel field (Fig. S5 A, B, E, and F) − − Foxb1-Cre;Celsr2f/ ;Celsr3f/ No phenotype (Fig. 5 A, C, E, G, I, and K and Fig. S5 C, D, G,andH) Foxb1-Cre;Fzd3f/− Nex-Cre;Foxb1-Cre;Celsr2f/−;Celsr3f/− Decreased IC with partially blocked/delayed TCA (Fig. 5 B, D, F,andH) − Nex-Cre;Foxb1-Cre;Fzd3f/ Looping of TCA and CTA in external basal forebrain (Figs. 5 and 6) Absence of CST (Fig. 5 J and L) Layer 5 axons routed to thalamus (Fig. 7I) Abnormal barrel field (Fig. 8)

modest synergistic effect of inactivation upon both Nex-Cre but never went to thalamus, and a small number of labeled fibers and Foxb1-Cre was confirmed in P30 animals labeled with the from layer 6 ran to the thalamus (Fig. 7G). In Thy1;Nex-Cre; − − Thy1-YFP transgene (46). In control brains, most layer 5 and some Celsr2f/ ;Celsr3f/ mice, layer 5 was diminutive, presumably due layer 6 neurons were labeled, cortical efferent fibers from layer 5 to defective subcerebral projections and resulting neuronal descended in the cerebral peduncle as subcerebral projections death, yet a significant number of labeled axons from layer 5 ran abnormally to the thalamus (Fig. 7H). The phenotype was some- − − what accentuated in Thy1;Nex-Cre;Foxb1-Cre;Celsr2f/ ;Celsr3f/ mice, in which the atrophy of layer 5 was more pronounced, and derailed fibers to thalamus less abundant that in Thy1;Nex-Cre; − − Celsr2f/ ;Celsr3f/ animals (Fig. 7I). Altogether, these observations show that the wiring defects generated upon cortical inactivation of Celsr2+3 and Fzd3 are only marginally increased when target genes are also inactivated in thalamus, arguing against a rescue via CTA–TCA interactions through Celsr2-3/Fzd3 and for the importance of expression in guidepost cells.

Celsr2-3 and Fzd3 Are Required in TCA and Cortical Neurons for Fine Cortical Arealization. We then considered whether the expression of Celsr2-3 and Fzd3 in thalamus might influence the formation of cortical areas, which are known to depend on thalamocortical input (47). To assess this, we studied the barrel field, an area in primary somatosensory cortex corresponding to whisker-related input, where thalamic afferents project to barrel centers and synapse with dendrites of excitatory spiny stellate and pyramidal neurons in cortical layer 4 (48). We compared barrel fields in P8 − − mice of the following genotypes: control, Nex-Cre;Celsr2f/ ;Celsr3f/ , − − − − Foxb1-Cre;Celsr2f/ ;Celsr3f/ , Nex-Cre;Foxb1-Cre;Celsr2f/ ;Celsr3f/ , − − − Nex-Cre;Fzd3f/ , Foxb1-Cre;Fzd3f/ ,andNex-Cre;Foxb1-Cre;Fzd3f/ . All mice had a similar organization of whiskers on the snout (Fig. S4). We found that barrel fields were similar to control in all brains with inactivation of Celsr2-3 or Fzd3 upon single Nex or Foxb1-Cre expression (Fig. 8 A and B,andFig. S5). In contrast, although the barrel field was present in its expected position, it was diminutive − − in Nex-Cre;Foxb1-Cre;Celsr2f/ ;Celsr3f/ and Nex-Cre;Foxb1-Cre; − Fzd3f/ mice (Fig. 8 C–F). In tangential and coronal sections, a pattern of rows and columns was present in the primary sensory field, but the fine organization was diffuse, fuzzy, and less sharply Fig. 6. Inactivation in cortex and thalamus using triple Cre confirm addition defined than in control and single Cre brains. Intriguingly, this of phenotypes. (A and B) At P0, neurofilament immunohistochemistry (NF) architectonic disorganization looked more pronounced in the gt/gt shows that, compared with Celsr2 brains (n = 4)—which are normal— anterolateral than the posteromedial barrel subfields (Fig. 8 C and there is clear atrophy of the IC with looping fibers in triple Cre mutants E). These data indicate that Celsr2-3 and Fzd3 mediate inter- (arrowhead in B)(n = 4). (C–F) DiI injections at P0 show that looping axons are labeled in triple Cre mutants following tracer injection both in cortex actions between thalamocortical afferents and their cortical tar- (Ctx) (arrow in D)(n = 4) and in diencephalon (Dien) (arrowhead in F)(n = 4); gets, which are required for the fine mapping of TCA after they compare with Fig. 2 D–I. (Scale bars: 500 μm.) reach appropriate cortical areas.

E3000 | www.pnas.org/cgi/doi/10.1073/pnas.1402105111 Qu et al. Downloaded by guest on September 30, 2021 affect dendrite development in opposite manner, while being PNAS PLUS redundant in the control of axon guidance? Presumably, downstream intracellular pathways are context and/or time dependent, and may differ in dendritic and axonal compartments of a same neuron. Also, signals that promote and inhibit neurite growth in vitro may end up having similar global effects on axon guidance in vivo, where growing axons encounter a more complex environment. Studies of the downstream signaling events controlled by Celsr/Fzd complexes are needed to understand that issue further.

Forebrain Axon Guidance Is Independent of Vangl1 and Vangl2. Our results show that forebrain axonal bundles require Celsr2, Celsr3, and Fzd3, but not Vangl1 and Vangl2. During epidermal and eye development in flies, Fmi, Fz, Dsh, Van Gogh, and Pk interact closely (51–53). Mutations in Van Gogh and Fz also affect branching of axons of mushroom body neurons, albeit with independent effects (54). Similarly, there is ample evidence that Celsr1, Fzd6, and Vangl1,2 act together to regulate skin development (30, 32, 37) and that Celsr1, Fzd6, Dvl, and Vangl2 collaborate in the regulation of neural tube closure (31, 55, 56), and in the planar organization of cilia in the mouse ependyma (24, 57), inner ear (31, 58), and Xenopus skin (59). Furthermore, Looptail (Lp) mice, which have a G1391A missense mutation in Vangl2, display abnormal growth of mesencephalic monoaminergic axons in homozygous mutant fetuses (33), and of spinal commissural fibers in homozygous and to a lesser extent in heterozygous embryos (34). Our finding that axon guidance in the anterior commissure, thalamocortical, corticothalamic, and corticospinal tracts proceeds normally in the absence of both Vangl1 and Vangl2 was thus unexpected. A first note of caution is that, although the validation data (Fig. S1) make it rather unlikely, the Fig. 7. Connection patterns upon inactivation of Celsr2+3 in both cortex Emx1-Cre and Foxg1-Cre drivers may not be fully active in the and thalamus. In control E14.5 embryos (A and D), DiI in cortex labels CTA Vangl2 floxed genomic context. Second, given its complex regu- and retrogradely fills neurons in thalamus (Th), whereas DiI in diencephalon lation, axon guidance may require Vangl proteins in some (Dien) prominently labels TCA. Upon cortical inactivation (B and E), DiI in – anatomic systems but not, or less, in others. However, we want to cortex labels CTA that cross the pallial subpial boundary (PSPB) (dotted line) emphasize that the Lp allele is known to have strong dominant- but do not extend much beyond it, whereas DiI in Dien labels TCA in basal forebrain that do not reach cortex. The blunted growth of both CTA and negative activity (36, 60). An abnormally folded Vangl2 protein TCA is more severe upon joint inactivation in cortex and thalamus (C and F). may have not only dominant-negative but also some gain- n = 3 for each DiI experiment. Using the Thy1-YFP transgene in P30 animals, of-function activity, for example by perturbing folding and/or control brains (G)(n = 7) show prominent labeling of cortical layer 5 (Ctx) hampering membrane targeting of Celsr and/or Fzd proteins. and subcerebral projections (arrows), none of which reaches the thalamus Furthermore, homozygous Lp embryos have a fully open neural (Th). In cortical mutants (H)(n = 10), layer 5 is atrophic and some of its axons are derailed to the thalamus (arrowheads). The phenotype is more severe upon joint inactivation in cortex and thalamus (I)(n = 7). [Scale bars: 200 μm (A–F); 500 μm(G–I).]

Discussion Celsr2 and Celsr3 Act in Redundant Manner in Axon Guidance. The phenotype in double Celsr2 and Celsr3 mutants is more pronounced than the single-mutant phenotypes, indicating that both genes act in a partly redundant manner in the PCP-related pathway that regulates axon guidance. Further arguments are provided by observations that Celsr2+3 and Fzd3 inactivation also generates similar anomalies during migration of facial branchiomotor neurons (25) and in the patterning of peripheral motor nerves (27). At first sight, this is in contradiction with previously published data attributing opposite roles to Celsr2 Fig. 8. Abnormal organization of the barrel field upon combined inactivation in cortex and thalamus. (A and B) In control mice, the somatosen- and Celsr3 in the control of neurite growth (49, 50). Using RNA sory map, studied using vGlut2 immunostaining (“presynaptic” component, interference in brain slices, Celsr2 down-regulation in neurons green) and DAPI (“postsynaptic” component, blue) at P8, appears normal, resulted in shorter and less profuse basal dendrites, whereas with five rows of barrels (A–E) in the posteromedial subfield corresponding to down-regulating Celsr3 had opposite effects. Reciprocal results whiskers (A, tangential section) and vGlut2-positive TCA terminal clusters in were observed when normal neurons were cultured in contact layer IV (B, coronal section). In contrast, the barrel fields are diminutive and + = with Celsr2- or Celsr3-expressing cells. Experiments carried out architectonically disorganized when Celsr2 3 (C and D)(n 11) or Fzd3 (E and F)(n = 3) are jointly inactivated in both cortex (Nex-Cre) and thalamus by swapping of Celsr2 and 3 ectodomains showed that the (Foxb1-Cre). The vGlut2-positive TCA terminals do not form clear clusters in

transmembrane domains and C terminus determine the dendrite layer IV in both mutants (D and F). Asterisks: anterolateral fields; II/III, IV, V: NEUROSCIENCE enhancing or suppressing action (49). How can Celsr2 and Celsr3 cortical layers. [Scale bars: 500 μm(A, C, and E); 200 μm(B, D, and F).]

Qu et al. PNAS | Published online July 7, 2014 | E3001 Downloaded by guest on September 30, 2021 tube, and some axonal abnormalities in the spinal cord and rescue each other in case of unilateral Celsr2, 3, and Fzd3 in- midbrain could be secondary to the dysraphic malformation activation. Upon inactivation in thalamus, nothing should prevent itself, rather than reflect the direct action of Vangl2 in axon CTA from initiating their growth out of the cortex, in the ventral guidance. In Drosophila, Van Gogh cooperates closely with the corridor (18) toward the thalamus. Reciprocally, cortical in- Prickle adaptor (61). The observation that Fzd3 and Celsr2, 3 activation should not perturb the initial growth and turning of guide forebrain axons in a Vangl1,2-independent manner leads TCA. Thus, in “single-side” mutants, CTA and TCA could meet at to the testable prediction that this role is also independent of some point along their trajectory and assist progression of the Prickle-like proteins. Van Gogh and Fz affect neurite growth mutant partner (44, 45). The observation that the joint in- independently in Drosophila (54), and Van Gogh, Prickle, and activation of Celsr2, 3,orFzd3 in both cortex and thalamus adds Dishevelled mutant neurons in Caenorhabditis elegans display little to single inactivation and fails even remotely to recapitulate supernumerary neurites (62). An additional role of Vangl2 in the constitutive or conditional Dlx5/6-Cre phenotypes, argues synapse formation was recently demonstrated (63, 64). These against a crucial role of TCA–CTA fiber interactions. We there- observations suggest that Vangl1,2 are likely to affect brain fore believe that interactions of axons with intermediate guidepost development by acting in different pathways, in addition to cells are particularly important and need to be investigated fur- epithelial PCP. ther, using more specific Cre driver strains and in vitro culture systems (17, 44, 67). Celsr2, 3, and Fzd3 Act in the Same Cell Populations. The fact that Whereas it does not impact much on axonal wiring, inac- Celsr and Fzd regulate axon guidance independently of Vangl tivation of Celsr2, 3, or Fzd3 in thalamus in addition to cortex suggests that mechanisms are different from those that regulate results in a disorganization of the barrel field. This is not ob- PCP in epithelial sheets. Phenotypes generated upon conditional served upon single inactivation in cortex or thalamus: expression inactivation of Celsr2 and 3 show that axonal growth cones need of Celsr2, 3, or Fzd3 in one partner, either TCA or cortical to interact with guidepost cells and that this interaction requires neurons, is sufficient for fine cortical arealization. The pheno- expression of Celsr2 and/or Celsr3 in guidepost cells or in growth type observed is reminiscent of that generated by constitutive cones, depending on the pathway (22). Together with another inactivation of Bhlhb5 (68), by inactivation of Ebf1 in basal study (23), our present work confirms the essential role of Fzd3 forebrain (69), or by cortex-specific inactivation of Lmo4 (70). in axons, guidepost cells, or both (3). The fact that inactivation of As far as we know, however, areal anomalies generated specifi- Fzd3 with different Cre-expressing strains consistently mimics cally upon joint gene inactivation in thalamus, the presynaptic + that of Celsr2 3 suggests that both proteins act in the same cell component, and cortical neurons, the postsynaptic partner, have populations, indicating that Fzd3 may participate in a complex in not been reported. Whether the tangential organization of other the membrane, together with Celsr2/3. This would be in line with cortical areas is also affected was not tested but would appear genetic and biochemical data in Drosophila, particularly the ob- likely. That a barrel field does form at the right location in servation that Fz and Fmi coimmunoprecipitate when expressed double conditional mutants shows that TCA are still able to in S2 cells (39). Like in Drosophila, the formation of a complex ascend to the cortex and find their way through deep cortical containing Celsr2, 3, and Fzd3 does not preclude expression layers. The simplest explanation is therefore that a reciprocal of Celsr2, 3 in other cells and/or other sectors of the plasma interaction between thalamic afferents and target neurons in membrane. However, the fact that Celsr and Fzd act probably layer 4 is involved. The absence of phenotype upon unilateral in cis and independently of Vangl, raises the crucial question of inactivation suggests that, besides Celsr2, 3, and Fzd3, other un- interacting partners in trans. identified molecules participate in that interaction. Interestingly, Another question concerns the signal generated at the growth a top-down interaction between sensory cortical neurons and cone when it encounters guidepost cells, and how this signal thalamus was recently demonstrated (71). steers the growth cone in the right direction. Both Celsr2 and Celsr3 are able to trigger calcium signals in transfected cells, with Materials and Methods different kinetics leading to activation of different target genes Animals. Experiments were carried out according to guidelines from the Animal (49). It would be interesting to assess whether analogous calcium Ethics Committees of the University of Louvain and Jinan University. In addition signals are induced by Fzd3, and to test whether calcium waves to the Celsr2 gene trap (Celsr2gt) allele used previously (24), we generated triggered by Celsr2, 3–Fzd3 complexes can differ in dendritic and constitutive and conditional Celsr2 mutant mice. A Neo cassette flanked by axonal compartments. LoxP and Frt sites was inserted into intron 15, and a LoxP site into intron 28 to generate allele Celsr2fn. This allele proved to be a null, and the Neo cassette What Mechanisms Could Account for Non–Cell-Autonomous Action in was removed by crosses with ROSA-Flp mice (72), yielding a conditional floxed f − Thalamus and Cortex? Our results demonstrate that inactivation of Celsr2 allele. The null allele Celsr2 was obtained by crosses with PGK-Cre Celsr2+3 or Fzd3 in dorsal thalamus has no effect on reciprocal germline deleter mice (73) (Fig. S6A). Due to the deletion and resulting thalamocortical projections, and that inactivation in cortex pre- frameshift, the predicted mutated protein should be truncated before the GPS domain and the seven-transmembrane segments. No Celsr2 protein was vents the formation of subcerebral projections and leads to at- detected using a C-terminal antibody (Fig. S6B), and the migration of facial rophy of the IC, yet does not generate the full defect that occurs branchiomotor neurons was similarly affected in Celsr2−/− and in Celsr2gt/gt in mice with constitutive mutations or conditional inactivation in embryos (Fig. S6 D and G), showing that this allele is a null, although the basal forebrain (22). A rather similar situation was described expression of an N-terminal partial protein sequence cannot be excluded. That in ubiquitin ligase Phr1 mutant mice, in which constitutive in- the Celsr2f allele behaves as predicted upon Cre-mediated recombination is − activation leads to complete absence of IC, whereas cortical also shown by the abnormal migration of FBM neurons in Isl1-Cre;Celsr2f/ inactivation with Emx1-Cre yields a partial thalamocortical mutant embryos (Fig. S6H). We also generated a “knockout first” Fzd3 allele phenotype (65, 66). As in all studies with the Cre-loxP system, an using a targeting vector from the Eucomm Consortium (Project 78503), which intrinsic limitation and concern is that Cre expression may not proved a hypomorph. A conditional allele was derived by crosses with ROSA- occur early enough with sufficient activity to generate a full phe- Flp females, and a null allele was then obtained by crosses with PGK-Cre females. In the null allele, exon 3 (197 nt) is deleted (Fig. S2E), generating notype. As the activity of the Cre drivers used was validated in a frameshift and a reading frame that encodes 67 N-terminal amino acids of several studies in addition to our data, this is rather unlikely. Non- the Fzd3 protein sequence, or 43 residues after removal of the signal peptide. cell autonomy upon inactivation in cortex or thalamus could be Other mouse strains were described and validated in the following pub- explained by postulating that Celsr2, 3, and Fzd3 have an crucial lications: Celsr3 (22), Dlx5/6-Cre (40), Emx1-Cre (28), Foxb1-Cre (43), Foxg1-Cre role in cortical and thalamic neurons, like in neurons of origin of (38), Nex-Cre (29), Nkx6.2-Cre (74), Wnt3a-Cre (75), Vangl1 and Vangl2 (35, 36), the AC and CST, but that CTA and TCA interact reciprocally and and Thy1-YFP transgenics (46). Primers for genotyping are listed in Table S1.

E3002 | www.pnas.org/cgi/doi/10.1073/pnas.1402105111 Qu et al. Downloaded by guest on September 30, 2021 To palliate issues related to potential perturbations due to Cre expression, Sections were mounted in medium with DAPI and examined with PNAS PLUS Cre drivers were always heterozygotes and control mice with Cre expression fluorescence microscopy. were consistently used. For example, Emx1-Cre;Celsr3f/− mice were compared f/+ + + − − with controls that included Emx1-Cre;Celsr3 mice. We also verified that Western Blot. Whole brains of Celsr2 / , Celsr2 / , Celsr2gt/gt,orforebrain Cre expression does not generate apoptosis, by immunostaining for acti- and hindbrain regions of Foxg1-Cre;Vangl2f/− embryos aged E13.5 were vated Caspase3 (Fig. S7). For comparison of genotypes and DiI experiments, homogenized in NuPAGE LDS sample buffer (Thermo Fisher) and cleared at least three animals were sectioned serially and examined, and phenotypes by centrifugation at 16,000 × g for 15 min at 4 °C. Samples containing were fully penetrant. 30 μg of total protein were analyzed on 4–15% Mini-PROTEAN TGX Gel (Bio-Rad) and transferred to nitrocellulose membrane (BioScience) by Histological Procedures. Brains were fixed by intracardial perfusion with 4% electroblotting (Invitrogen). Membranes were blocked with 5% (wt/vol) (vol/vol) paraformaldehyde (PFA) in PBS, followed by fixation in the same skimmed milk and 0.1% Tween 20, in PBS, for 30 min, and incubated with mixture, overnight for adult and P0 brains, and for a few hours for E14.5 a mouse antibody to Celsr2 (76) or goat polyclonal antibody N-13 to Vangl2 embryos. Brains were embedded in paraffin and sectioned serially at 8 μm. (1:500; Santa-Cruz; sc-46561) at 4 °C overnight. Signal was detected using Paraffin sections were stained with hematoxylin–eosin (H&E) or by immu- an HRP-conjugated goat anti-mouse Ig (DAKO) followed by chem- nohistochemistry, with detection using an ABC kit (DAKO). For cryostat sections, dissected brains were cryoprotected in 30% (wt/vol) sucrose and iluminescence using a SuperSignal West Pico kit (Pierce) and Hyperfilm ECL embedded in OCT. For study of barrels, just after PFA perfusion, hemispheres (Amersham Biosciences). were dissected, flattened between two slides, and further fixed overnight at 4 °C. Seventy-micrometer-thick sections were prepared using a vibratome, ACKNOWLEDGMENTS. WethankJeanHébert,KevinJones,NicolettaKessaris, stained by immunohistochemistry with antibody to Vglut2, and counter- and Klaus Amin Nave for Cre mice; Nicolas Parmentier for help with Western blots; and Esther Paitre, Isabelle Lambermont, Valérie Bonte, and Rachid El stained with DAPI, before photography under a fluorescence or a confocal Kaddouri for technical assistance. This work was supported by grants from microscope. For immunohistochemistry, we used the following primary the National Natural Science Foundation of China [31200826 (to Y.Q.) and antibodies: guinea pig polyclonal antibody to Vglut2 [1:1,000 (vol/vol); Mil- 31070955 (to L.Z.)], National Basic Research Program of China [973 Program, lipore], mouse monoclonal antibody to neurofilament 2H3 (1:500; De- 2014CB542205 (to Y.Q.) and 2011CB504402 (to L.Z.)], Guangdong Natural velopmental Studies Hybridoma Bank), rabbit monoclonal antibody to PKCγ/ Science Foundation [S2012040006744 (to Y.Q.)], Guangzhou Science and Prkcg (1:400; Abcam), goat polyclonal antibody N-13 to Vangl2 (1:50; Santa- Technology Project [13200068 (to Y.Q.)], and Jinan University Research and Cruz; sc-46561), rabbit polyclonal antibody to cleaved (activated) caspase3 Innovation Foundation [21612345 (to Y.Q.)], and by Belgian grants Actions (1:200; Cell Signaling). Signal detection was carried out using goat anti- de Recherches Concertées (ARC-10/15-026), Fonds de la Recherche Scientifique Medicale 3.4550.11, Fonds National de La Recherche Scientifique (FNRS) guinea pig-Fluorescein (1:500; Vector) or donkey anti-rabbit-Alexa Fluor 488 T0002.13, Interuniversity Poles of Attraction (Services Fédéraux des Affaires (1:1,000; Molecular Probes). DiI crystals (D282; Molecular Probes) were Scientifiques, Techniques, et Culturelles, PAI p6/20 and PAI7/20), Fondation implanted in target areas of PFA-fixed P0 or E14.5 brains using tungsten Médicale Reine Elisabeth, Fondation JED-Belgique, and WELBIO-CR-2012A-07 needles. Brains were subsequently incubated at 37 °C for 4 wk for P0 brains from the Région Wallonne (to F.T. and A.M.G.). F.T. is a senior research asso- or 3 wk for E14.5 brains, and sectioned in the coronal plane using a vibratome. ciate of the Belgian FNRS.

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