Primary cilia regulate hippocampal neurogenesis by mediating sonic hedgehog signaling Joshua J. Breunig*†, Matthew R. Sarkisian*†, Jon I. Arellano*, Yury M. Morozov*, Albert E. Ayoub*, Sonal Sojitra‡, Baolin Wang§¶, Richard A. Flavellʈ**, Pasko Rakic*††, and Terrence Town‡ʈ *Department of Neurobiology and Kavli Institute of Neuroscience and §Department of Genetic Medicine, Weill Medical College of Cornell University, 1300 York Avenue, W404, New York, NY 10021; ¶Department of Cell and Developmental Biology, Weill Medical College of Cornell University, 1300 York Avenue, W404, New York, NY 10021; ʈDepartment of Immunobiology and **Howard Hughes Medical Institute, Yale University School of Medicine, 300 Cedar Street, TAC S-569, New Haven, CT 06519-8011; and ‡Departments of Biomedical Science, Neurosurgery, and Medicine, Maxine Dunitz Neurosurgical Institute, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, CA 90048 Contributed by Pasko Rakic, May 10, 2008 (sent for review March 20, 2008) Primary cilia are present on mammalian neurons and glia, but their tivity, we noted that the typical lamination and morphology of function is largely unknown. We generated conditional homozy- glial cells was severely disrupted (Fig. 1 E–H). Glial cell bodies, gous mutant mice for a gene we termed Stumpy. Mutants lack cilia as shown by combined Sox2 and Gfap immunoreactivity, were and have conspicuous abnormalities in postnatally developing located more randomly in NestinCre; Stumpyfl/fl mice (hereto- brain regions, including a hypoplasic hippocampus characterized fore referred to as ⌬Stumpy mice), and there was a notable lack by a primary deficiency in neural stem cells known as astrocyte-like of DG radial glia (Fig. 1 A, B, and E–H). neural precursors (ALNPs). Previous studies suggested that primary ⌬Stumpy mice exhibit hydrocephalus (14), and we initially cilia mediate sonic hedgehog (Shh) signaling. Here, we find that thought that this could be in part responsible for the abnormal- loss of ALNP cilia leads to abrogated Shh activity, increased cell ities observed. However, we recovered several ‘‘control’’ idio- cycle exit, and morphological abnormalities in ALNPs. Processing of pathic spontaneously hydrocephalic mice (NestinCreϪ/ Gli3, a mediator of Shh signaling, is also altered in the absence of Stumpyϩ/ϩ) with a comparable severity of hydrocephalus, but cilia. Further, key mediators of the Shh pathway localize to ALNP which lacked any significant malformations in the cerebellum or cilia. Thus, selective targeting of Shh machinery to primary cilia hippocampus (Fig. S3). In addition, we encountered several confers to ALNPs the ability to differentially respond to Shh other spontaneously hydrocephalic mice from different strains mitogenic signals compared to neighboring cells. Our data suggest with comparable levels of hydrocephalus (Fig. S3). None of these these organelles are cellular ‘‘antennae’’ critically required to animals exhibited the characteristic anatomical phenotypes ob- modulate ALNP behavior. served in ⌬Stumpy mice. Therefore, loss of Stumpy has specific consequences on the number and morphology of neuronal ͉ ͉ ͉ ͉ adult neurogenesis Gli1 Patched Smoothened stem cell precursors in the hippocampus and cerebellum that was inde- pendent of hydrocephalus. ilia are conserved microtubule-based organelles that grow In situ hybridization (ISH) for Stumpy in the postnatal hip- Cfrom basal bodies (a centrosome-derived structure) and pocampus showed a fairly ubiquitous expression pattern (Fig. S4 protrude from the cell surface (1). A growing number of A and B). Using an antibody against Stumpy (14), we observed biological processes and clinical disorders are attributable to Stumpy colocalization with the basal body (Fig. S4 C and D). properly functioning or dysfunctional cilia, respectively (1–4). Using the marker adenylyl cyclase III (ACIII), which localizes to Neurons and astrocytes have been found to harbor a nonmotile, the primary cilium (5), cilia were observed on many cells in the primary cilium (5, 6). Despite this widespread presence of control DG and were completely absent in ⌬Stumpy mice (Fig. primary cilia in the forebrain, their function remains elusive (6). S4 E and FЈ). Somatostatin Receptor 3, another marker of Recent findings suggest that the Shh signaling pathway in neuronal cilia, yielded similar results (Fig. S5). Also, ultrastruc- vertebrates appears to be mediated through the primary cilium tural examination of radial glia confirmed the lack of ciliary (4, 7, 8). Whether this pathway is active in primary cilia in the axonemes in ⌬Stumpy mice (Fig. S6). Therefore, loss of stumpy postnatal brain is unknown. Shh signaling is a key regulator of leads to an absence of ciliary axonemes in the hippocampus. cell proliferation in the external granule cell layer (EGL) of the Astroglial cells, the major neural precursors in hippocampus, cerebellum, dentate gyrus (DG) of the hippocampus, and sub- were markedly disrupted in ⌬Stumpy (Fig. 1 E–H). To determine ependymal zone (SEZ) of the lateral ventricles (9, 10): three whether cilia were present on these precursors, we used an brain regions that continue to develop postnatally (11). Notably, inducible GFP-labeling method to mosaically label them in a it was demonstrated that Shh-responsive postnatal precursors Golgi-like manner (Fig. 2A). We identified ACIIIϩ cilia on these appear to be a unique population that are derived relatively late neurogenic, astrocyte-like neural precursors (ALNPs) in the in embryogenesis (10, 12, 13). The mechanisms governing this hilus and subgranular zone (SGZ) (Fig. 2 B and BЉ). Interest- postnatal responsiveness to Shh are unclear. Therefore, we ingly, these cilia were morphologically distinct—i.e., significantly hypothesize that this responsiveness may occur through cilia shorter—from neuronal cilia (2.6 Ϯ 0.5 ␮m in glial cells vs. 8.2 Ϯ within germinal regions of the postnatal brain. Results Author contributions: J.J.B., M.R.S., Y.M.M., P.R., and T.T. designed research; J.J.B., M.R.S., NEUROSCIENCE Stumpy Is Required for Development of Postnatal Brain Structures and J.I.A., Y.M.M., A.E.A., S.S., and T.T. performed research; J.J.B., B.W., R.A.F., and T.T. Ciliogenesis. During characterization, a conditional mutant contributed new reagents/analytic tools; J.J.B., M.R.S., Y.M.M., P.R., and T.T. analyzed data; mouse for the Stumpy gene, we observed gross olfactory bulb J.J.B., M.R.S., J.I.A., Y.M.M., P.R., and T.T. wrote the paper. (data not shown), hippocampal, and cerebellar abnormalities in The authors declare no conflict of interest. P13 mice [Fig. 1, supporting information (SI) Fig. S1]. In †J.J.B. and M.R.S. contributed equally to this work. particular, the DG granule cell layer was thinner when compared ††To whom correspondence should be addressed. E-mail: [email protected]. to control mice and showed dispersion of NeuNϩ granule cells This article contains supporting information online at www.pnas.org/cgi/content/full/ (Figs. 1 A–D and Fig. S2). When the underlying glial scaffold was 0804558105/DCSupplemental. observed by glial fibrillary acidic protein (Gfap) immunoreac- © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0804558105 PNAS ͉ September 2, 2008 ͉ vol. 105 ͉ no. 35 ͉ 13127–13132 Downloaded by guest on September 29, 2021 Neuronal cilia normally protruded with no evidence of mem- brane invagination (data not shown). We confirmed the findings of glial cilia in vitro using two different radial glia-like cell lines: A B C6-R cells (derived from rat glioma cells) and bipolar adult neural progenitor cells harvested using published techniques (Fig. S7) (15). Both cell lines use Shh signaling to maintain the stem cell population (16, 17). These results suggest that cilia on ALNPs could play an important role in regulating their subse- quent proliferation and/or differentiation into neurons. C D Stumpy Mutants Have Defects in Neuronal Precursor Proliferation and Neurogenesis Associated with Perturbed Shh Signaling. Analysis of P0 hippocampi indicated that proliferation of DG precursors was already altered in ⌬Stumpy mutants, as the DG appeared smaller and had fewer dividing precursor cells (Fig. S8 A and B). When we examined the proliferation of Sox2ϩ glia later at P13, ⌬Stumpy mice showed fewer Sox2ϩ cells and a more random distribution when compared with controls (Fig. 1 A, B, E, and F). There was less proliferation overall as determined by the cell cycle marker Ki67 expression in ⌬Stumpy mice (Fig. S9 A and B). E F Notably, fewer proliferating Sox2ϩ cells were present, as deter- mined by colocalization with Ki67 (ϳ52 Ϯ 5% of Ki67ϩ cells were Sox2ϩ in controls vs. ϳ70 Ϯ 8% Sox2ϩ/Ki67ϩ cells in ⌬Stumpy mice; P Ͻ 0.05 [Student’s t test]) (Fig. S9 A and B). However, it should be noted that there appeared to be fewer weakly Sox2ϩ cells that colocalized with Ki67 (Fig. S9 A and B)—perhaps indicative of a decrease in proliferating transit amplifying cells (18). Neurogenesis, in the form of transit- amplifying cells and migrating neurons, was similarly altered in Stumpy mutants. In particular, we noted ectopic Ascl1, and Tbr2 GH expression—early markers of neurogenesis (19) (Fig. S9 C and DЉ). Immature neurons, stained with doublecortin (Dcx), also displayed abnormal morphology (Fig. S9 CЈ and DЈ). Taken together, Stumpy mutants exhibit profound alterations in early (P0) and later proliferation (P13), leading to subsequent defi- ciencies in neurogenesis. To assay where the primary defect in neurogenesis lay, we birthdated two separate populations of S-phase cells with the DNA synthesis markers chlorodeoxyuridine (CldU) and iodode- oxyuridine (IdU). The CldU was administered at P8, and IdU was given at P12 and animals were killed at P13 (Fig. 3A). In IJ controls, hippocampal proliferation was largely confined to the SGZ. However, in ⌬Stumpy mutants, proliferation was dimin- ished in the SGZ and ectopic proliferating cells were more abundant (Fig.