Transcription factor Sp4 regulates dendritic patterning during cerebellar maturation Bele´ n Ramos, Brice Gaudillie` re, Azad Bonni, and Grace Gill* Department of Pathology, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115 Edited by Huda Y. Zoghbi, Baylor College of Medicine, Houston, TX, and approved April 25, 2007 (received for review March 2, 2007) Integration of inputs by a neuron depends on dendritic arborization diverse cellular functions (26, 27). In contrast to Sp1 and Sp3, patterns. In mammals, the genetic programs that regulate dynamic which are ubiquitously expressed, Sp4 is enriched in heart and remodeling of dendrites during development and in response to skeletal muscle, with the highest levels in the brain (24, 28). Sp4 activity are incompletely understood. Here we report that knock- expression in both cardiomyogenic and neural crest lineages has down of the transcription factor Sp4 led to an increased number of been found to be important for development of the cardiac highly branched dendrites during maturation of cerebellar granule conduction system; however, the role of Sp4 in nervous system neurons in dissociated cultures and in cerebellar cortex. Time-course development remains to be elucidated (24, 28–30). analysis revealed that depletion of Sp4 led to persistent generation of In the nervous system, Sp4 mRNA is highly expressed in the dendritic branches and a failure in resorption of transient dendrites. developing hippocampus and cerebellum, in particular, in cer- Depolarization induced a reduction in the number of dendrites, and ebellar granule neurons (25). Dendritic development in cere- knockdown of Sp4 blocked depolarization-induced remodeling. Fur- bellar granule neurons occurs postnatally after these cells have thermore, overexpression of Sp4 wild type, but not a mutant lacking migrated to their final positions in the internal granule layer. the DNA-binding domain, was sufficient to promote dendritic prun- Several stages in the development of mature dendritic arbors ing in nondepolarizing conditions. These findings indicate that the have been described in granule neurons. In particular, some transcription factor Sp4 controls dendritic patterning during cerebel- intermediate stages show exuberant dendritic arbors, which are lar development by limiting branch formation and promoting activity- later remodeled to reach their mature pattern (9, 31, 32). Many dependent pruning. of the stages of cerebellar granule neuron development, includ- ing neuronal polarity and dendritic growth, are faithfully reca- branching ͉ dendrite ͉ depolarization ͉ pruning ͉ neuron pitulated in culture, which make them a reliable system to study neuronal morphogenesis (20, 33–35). he nervous system is a complex and well coordinated net- We have used cerebellar granule neurons as our experimental Twork that depends on the formation of proper connections model to investigate the role of Sp4 in postmitotic neurons. We among diverse types of neurons. Dendritic arborization patterns have found that depletion of Sp4 from granule neurons using determine the way a neuron integrates inputs. Defects in den- RNA-mediated interference (RNAi) disrupted dendritric pat- dritic patterning correlate with severe neurodevelopmental dis- terning by promoting persistent branching and impairing den- orders (1–3). Although proper dendritic arborizations are crucial dritic pruning. We report that depolarization induced a signif- for correct function of the nervous system, the genetic programs icant reduction in the number of primary and secondary that govern dendritic morphology in mammals remain poorly dendrites, and knockdown of Sp4 blocked depolarization- described. Dendritic development is a highly dynamic process induced dendritic remodeling. Furthermore, overexpression of that involves many events, including neurite outgrowth, branch- Sp4 wild type, but not a mutant lacking the DNA-binding ing, stabilization, and pruning of dendrites (4–6). There is a domain, was sufficient to restore dendritic pruning in nondepo- delicate balance between addition and elimination of neuronal larizing conditions. Our results indicate that Sp4 limits branching projections (7, 8). During development, transient overproduc- and is required for activity-dependent dendritic pruning during tion of branched dendrites appears in most neurons, after which cerebellar development. some neurites are eliminated, whereas other neurites are stabi- lized to achieve the mature pattern (9–11). Recent studies have Results described intracellular signaling pathways that operate locally to Knockdown of Sp4 Leads to Exuberant Dendritic Arborization in regulate cytoskeletal elements important for branch formation Cerebellar Granule Neurons. To investigate the function of Sp4 or elimination (12–14). in postmitotic neurons, we used RNAi to knock down Sp4 in Extrinsic factors are coordinated with cell-intrinsic, gene cerebellar granule neurons. This approach allows us to examine expression programs to determine dendritic morphology. Ge- the effects of Sp4 loss of function during a late developmental netic studies in Drosophila and Caenorhabditis elegans have window in postmitotic cells and avoids potential complications identified transcription factors that regulate diverse aspects of due to Sp4 functions in progenitor or neighboring cells, as well dendritic morphology (15–18). Transcriptional control of the as compensatory mechanisms that may act during ontogeny. fine balance between formation and elimination of processes is Morphogenesis of cerebellar granule neurons has been faithfully illustrated by the finding that, in Drosophila, the homeodomain recapitulated in vitro under depolarizing culture conditions, protein, Cut, promotes branching, whereas the BTB/POZ Zinc finger transcription, Abrupt, limits branch formation (18, 19). In mammals, the transcriptional regulators CREB, NeuroD, and Author contributions: B.R., A.B., and G.G. designed research; B.R. and B.G. performed CREST have been identified to promote dendritic growth and research; B.R. analyzed data; and B.R. and G.G. wrote the paper. branching (20–23). Transcriptional regulators that balance these The authors declare no conflict of interest. activities to restrict dendritic branching and promote pruning This article is a PNAS Direct Submission. have not been previously described in mammals. Abbreviation: DIV, days in vitro. The Zinc finger transcription factor Sp4 is highly expressed in *To whom correspondence should be addressed. E-mail: [email protected]. the developing mammalian brain (24, 25). The highly related This article contains supporting information online at www.pnas.org/cgi/content/full/ Sp1, Sp3, and Sp4 transcription factors bind to a GC box DNA 0701946104/DC1. element important for the regulation of genes that control © 2007 by The National Academy of Sciences of the USA 9882–9887 ͉ PNAS ͉ June 5, 2007 ͉ vol. 104 ͉ no. 23 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0701946104 Downloaded by guest on September 24, 2021 Fig. 1. Knockdown of Sp4 transcription factor alters dendritic patterning in cerebellar granule neurons. (A) Lysates from Neuro 2A cells cotransfected with the indicated RNAi plasmid or empty vector, together with pRC-Flag-Sp4 or pRC/CMV (Vec) and GFP, were immunoblotted with antibodies against Flag and GFP. (B) Cerebellar granule neurons transfected with Scr control RNAi or the indicated RNAi targeting Sp4 and an expression plasmid encoding GFP at 2 DIV were immunostained with antibodies against GFP and Sp4 5 days posttransfection. Nuclei were stained with Hoechst. Arrowheads indicate the nucleus of a transfected neuron. (C–E) Cerebellar granule neurons were transfected as indicated in B and immunostained for GFP. (C) Representative images of neurons transfected with different RNAi constructs. Arrowhead indicates axon and asterisk cell bodies of transfected neuron, respectively. Total number of primary, secondary, and tertiary NEUROSCIENCE dendrites (D) or branching points in primary dendrites (E) was quantified. Values represent mean Ϯ SEM (ANOVA; ***, P Ͻ 0.001; **, P Ͻ 0.01; n ϭ 18–41 per condition, total n ϭ 115). For each parameter measured, neurons transfected with Scr control RNAi did not show differences compared with U6 vector alone. which support maximal health and survival of rat cerebellar tions in the Sp4 cDNA that was resistant to RNAi-mediated granule neurons; we have used these culture conditions in our knockdown (Fig. 2A). When the the Flag-Sp4 rescue vector was analysis (36–38). We generated plasmids encoding two different cotransfected with the Sp4RNAi into granule neurons, the short hairpin RNAs that specifically reduced expression of numbers of dendrites were completely restored to wild-type cotransfected FlagSp4 in Neuro 2A cells (Fig. 1A) and endog- enous nuclear Sp4 protein in cerebellar granule neurons [Fig. 1B and supporting information (SI) Fig. 7]. There was no increase in apoptosis in granule neurons transfected with either Sp4 RNAi or control as judged by the appearance of condensed chromatin 5 days after transfection (Fig. 1B and data not shown). Strikingly, we found that the dendritic morphology of neurons transfected with either of the two Sp4 RNAis was highly aberrant compared with neurons transfected with a control hairpin or RNAi for the related transcription factor Sp1 (Fig. 1C and SI Fig. 8). Immunostaining of cerebellar granule neurons for the axonal tau-1 or dendritic Map-2 proteins revealed that the major alteration in granule cells depleted