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, 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, , Cut, promotes branching, whereas the BTB/POZ 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 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 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 revealed that the major alteration in granule cells depleted for Sp4 was in the dendrites (SI Fig. 7 B and C). We quantified the changes in dendritic patterning by counting the number of primary, secondary, and tertiary dendrites per neuron. Both Sp4 RNAi constructs increased the number of dendrites at least 2-fold compared to control-transfected neu- rons (Fig. 1D). The frequency of branch points on the shaft of a primary dendrite for neurons depleted of Sp4 was 3- to 4-fold higher than control-transfected neurons, supporting the observation that Sp4 knockdown neurons have more complex dendritic arbors (Fig. 1E). Total dendritic length was not Fig. 2. RNAi-resistant Sp4 rescues the altered dendritic patterning induced significantly affected after knocking down Sp4 (SI Fig. 8B). by Sp4 knockdown. (A) Lysates from Neuro 2A cells cotransfected with U6/Scr Similarly, total axonal growth was not reduced after depletion of or U6/Sp4 #1 RNAi plasmid, together with an expression vector encoding an Sp4 (data not shown). Thus, depletion of Sp4 led to altered RNAi-resistant Sp4 protein (Sp4 Rescue) or pRC/CMV vector (Vec) and an dendritic patterning, but did not compromise the growth of expression vector encoding GFP, were immunoblotted with the indicated neuronal processes. antisera. (B) Cerebellar granule neurons were analyzed after transfection with U6/Scr or U6/Sp4 #1 RNAi plasmid, together with an expression vector encod- To confirm that the observed exuberant dendritic arborization ing GFP and RNAi-resistant Sp4 Rescue or pRC/CMV vector, as described in Fig. was due specifically to the depletion of Sp4 and not because of 1. Values represent mean Ϯ SEM of number of primary, secondary, or tertiary nonspecific off-target effects of the RNAi pathway, we gener- dendrites per neuron (ANOVA; ***, P Ͻ 0.001; **, P Ͻ 0.01; n ϭ 16–21 per ated a Flag-Sp4 ‘‘rescue’’ vector bearing two silent point muta- condition, total n ϭ 79).

Ramos et al. PNAS ͉ June 5, 2007 ͉ vol. 104 ͉ no. 23 ͉ 9883 Downloaded by guest on September 24, 2021 Fig. 3. Sp4 is required for dendritic maturation in intact cerebellar cortex. Organotypic cerebellar slices from P9 rat pups were transfected after 3 days in culture using gene gun-delivery methodology with U6/Scr, U6/Sp4 #1, or U6/Sp1 RNAi plasmids, together with expression vectors encoding Bcl-xL and GFP. After 5 days, slices were fixed and immunostained with a monoclonal antibody against GFP. (A) Image shows layers of cerebellar cortex stained with Hoechst 33258 (Upper) and a representative cerebellar granule neuron (Lower). Arrowhead indicates axon, and asterisk indicates cell bodies of transfected neuron. (Scale bar: 10 ␮m.) (B) Representative images of cerebellar granule neurons transfected in slice with the indicated plasmids. Arrowhead indicates axon, and asterisk indicates cell bodies of transfected neuron. (C and Fig. 4. Sp4 is required for dendritic remodeling and limiting branching D) Quantification of the number of primary, secondary, and tertiary dendrites during morphogenesis. Cerebellar granule neurons were transfected after 2 (C) and branching points in 100 ␮m of a primary dendritic shaft (D). Values DIV with U6/Scr or U6/Sp4 #1 RNAi plasmid and an expression vector encoding GFP, inmunostained and analyzed as in Fig. 1. (A) Representative images of represent mean Ϯ SEM (ANOVA; ***, P Ͻ 0.001; **, P Ͻ 0.01; n ϭ 104–136 neurons per condition, total n ϭ 352). For each parameter measured, neurons transfected neurons at the indicated DIV. Arrowhead indicates axon and, transfected with Scr control RNAi did not show differences compared with U6 asterisk cell body of transfected neuron. (B–D) Quantitation of the number of vector alone. primary dendrites (B), secondary dendrites (C), and total branching points per 100 ␮m of primary dendritic shaft (D) were performed in neurons transfected with U6/Scr or Sp4 RNAi at the indicated times (ANOVA comparing with U6/Scr Ͻ levels (Fig. 2B). Taken together, these data show that the more control or Sp4 RNAi vector-transfected neurons after 3 days; ***, P 0.001; **, P Ͻ 0.01; *, P Ͻ 0.05; n ϭ 33–69 per condition, total n ϭ 344 neurons). Values complex dendritic arbors induced by Sp4 RNAi are the result of represent mean Ϯ SEM. specific depletion of Sp4.

Sp4 Is Required for Dendritic Maturation of Granule Neurons in remodeling and/or restricting branching associated with matu- Cerebellar Cortex. The finding that depletion of Sp4 leads to more ration of cerebellar granule neurons. complex dendritic trees in primary granule neurons led us to investigate Sp4 function in dendritic development in the context Sp4 Knockdown Impairs Dendritic Pruning and Leads to Persistent of the cerebellum cortex. We used sagittal cerebellar slices Branching. Dendritic development involves highly dynamic and maintained in nondepolarizing concentrations of potassium, choreographed events, including the formation and elimination where tissue is preserved and dendrites develop their physio- of primary dendrites and branches (4–6, 39). To determine the logical morphology (20, 35). We introduced the control, Sp4, or steps of dendritic development controlled by Sp4, we performed Sp1 RNAi constructs into cerebellar slices using biolistic method a time-course analysis. We transfected primary cerebellar gran- of gene delivery. We confirmed that tissue structure was intact ule neurons with control or Sp4 RNAi and monitored the by staining with the DNA dye Hoechst (Fig. 3A). Control- number of primary and secondary dendrites over time. Consis- transfected granule neurons displayed mature dendritic arbors tent with the pruning that occurs during development in vivo,as with two or three robust primary dendrites with few branches described by Ramon y Cajal and others (9, 31), in control (Fig. 3A). Neurons depleted of Sp4 exhibited exuberant dendritic neurons, we observed a decrease in the number of dendrites from day 4 to day 6 in vitro. In contrast, pruning of dendrites was trees, with more numerous and highly branched dendrites blocked in neurons depleted for Sp4 (Fig. 4 A–C and SI Fig. 9). around the soma compared with control Scr or Sp1 RNAi- In fact, we observed that the addition of branches was persistent transfected neurons (Fig. 3B). The appearance of Sp4 knock- in Sp4 RNAi-transfected neurons, thereby accumulating more down neurons closely resembled classic descriptions of granule than twice as many branches and resulting in a 4-fold increase of neurons at an intermediate stage of dendritic maturation, ex- branching points compared to control neurons (Fig. 4 C and D). hibiting numerous, highly branched, primary dendrites that are Total dendritic length was not affected on depletion of Sp4 (SI later remodeled (9, 31). Quantitative analysis confirmed that Sp4 Fig. 9B). These studies reveal that Sp4 is required for pruning knockdown led to an increase in the number of dendrites and the and limiting branching during dendritic maturation. frequency of branching compared to control and Sp1 RNAi, consistent with the phenotype observed in dissociated primary Sp4 Is Required for Dendritic Remodeling Regulated by Neuronal neurons (Fig. 3 C and D). Taken together, these results suggest Activity. It has been observed that maturation of cerebellar that Sp4 contributes to dendritic patterning by controlling granule neurons in vivo is concomitant with the arrival of mossy

9884 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0701946104 Ramos et al. Downloaded by guest on September 24, 2021 Fig. 5. Sp4 is essential for dendritic remodeling regulated by depolarization in cerebellar granule neurons. Cerebellar granule neurons were transfected Fig. 6. Overexpression of Sp4 promotes dendritic pruning in nondepolariz- with the indicated RNAi plasmid, together with expression vectors encoding ing conditions. Neuro 2A cells (A) or granule neurons at 2 DIV (B) cells were GFP and Bcl-xL to prevent cell death. At 4 DIV, cerebellar granule neurons transfected with expression vectors encoding full-length FlagSp4 (Sp4), a maintained in conditioned media (COND) with 25 mM KCl were switched to deletion mutant of FlagSp4 lacking the Zinc finger DNA-binding domain fresh culture medium containing 25 mM or 5 mM KCl. At the indicated DIV, NEUROSCIENCE (Sp4⌬ZnF) or empty vector (Vec) together with GFP and Bcl-xL (A). The figure cells were immunostained as described above. (A) Representative images of illustrates domains in full-length Sp4 and the Sp4⌬ZnF deletion mutant. AD, transfected neurons at 4 DIV in conditioned medium with 25 mM KCl (Left), activation domain; S/T, serine/threonine-rich region. Relative luciferase activ- and at 6 DIV in fresh culture medium with 25 mM KCl (Middle)or5mMKCl ity after cotransfection of the indicated expression vectors, together with a (Right). Arrowhead indicates axon, asterisk indicates cell body, and arrows p35-promoter luciferase reporter plasmid and pRL-TK renilla reporter, are indicate primary dendrites of transfected neuron. (B and C) Quantitation of shown. Values represent mean Ϯ SE. Immunoblot analysis with anti-flag the number of primary and secondary dendrites per neuron was performed in antisera confirmed that the inactive Sp4⌬ZnF protein was expressed (data not neurons transfected and maintained as described above at the indicated times shown). (B) At 4 DIV, neurons were switched to fresh medium with 5 mM or 25 (B)orat7DIV(C) [ANOVA analysis comparing to transfected neurons at 4 DIV; mM KCl. Quantitation of primary and secondary dendrites per neuron was Ͻ Ͻ Ͻ ϭ ϭ ***, P 0.001; **, P 0.01; *, P 0.05; n 18–40 per condition, total n 321 performed in neurons at 6 DIV. Values represent mean Ϯ SEM (ANOVA analysis ϭ ϭ Ϯ (B); n 18–20 per condition, total n 79 (C)]. Values represent mean SEM. comparing to control-transfected neurons in depolarizing conditions; a two- Ͼ In C, the indicated P values ( 0.05) as determined by two-tailed t test are not tailed t test was also performed to compare differences between two differ- considered significant. ent conditions; ***, P Ͻ 0.001; **, P Ͻ 0.01; *, P Ͻ 0.05; n ϭ 21–50 per condition, total n ϭ 154). fibers, raising the possibility that dendritic remodeling in these cells is regulated by neuronal activity, as shown in many other depolarizing or nondepolarizing conditions to investigate the types of neurons (9, 40–44). We therefore investigated, first, contribution of Sp4 function to activity-dependent remodeling. whether depolarization regulates remodeling in cerebellar gran- In control RNAi-transfected neurons, the number of dendrites ule neurons, and, second, whether Sp4 contributes to activity- was reduced in neurons maintained in depolarizing compared to dependent remodeling in these cells. Neurons transfected with nondepolarizing conditions (Fig. 5C). In contrast, in neurons GFP were maintained in conditioned medium with 25 mM depleted of Sp4, there was not a significant difference in the potassium up to 4 days in vitro (DIV) to allow dendritic growth number of dendrites on membrane depolarization (Fig. 5C). and branching. Then granule neurons were switched to fresh Thus, Sp4 knockdown blocked depolarization-induced remod- medium containing depolarizing (25 mM) or nondepolarizing (5 eling. These findings support the conclusion that Sp4 is required mM) concentrations of KCl for 3 days. A vector expressing Bclx L for activity-dependent remodeling in cerebellar granule neurons. was included in these studies to prevent apoptosis induced by low potassium (38). At 4 DIV, we observed complex dendritic trees, Overexpression of Sp4 Promotes Dendritic Pruning in Nondepolarizing which were remodeled to simpler arbors when neurons were maintained in high potassium concentrations for the next 3 days Conditions. To investigate the contribution of Sp4 transcriptional (Fig. 5A). When switched to nondepolarizing media, however, activity to activity-dependent dendritic pruning, we generated a dendrites were not pruned, showing more complex trees (Fig. 5A deletion mutant of Sp4 lacking the Zinc finger DNA-binding ⌬ ⌬ and SI Fig. 10). Quantitation revealed a significant reduction of domain (Sp4 ZnF). Wild-type Sp4, but not Sp4 ZnF, activated dendrites under depolarizing concentrations of potassium, expression of a cotransfected Sp4-responsive reporter, p35- whereas the number of dendrites was maintained in nondepo- luciferase (45) (Fig. 6A). Transfection with wild-type Sp4 led to larizing conditions (Fig. 5B). Consistent with these findings, a significant decrease in the number of dendrites in granule pharmacological inhibition of L-type voltage-sensitive calcium neurons maintained in nondepolarizing conditions (Fig. 6B). In channels (L-VSCC) with nimodipine in cerebellar slices from P9 contrast, expression of transcriptionally inactive Sp4⌬ZnF did pups delayed dendritic pruning (SI Fig. 11). Thus, dendritic not lead to a change in dendrite number (Fig. 6B). These results remodeling in cerebellar granule neurons is regulated by depo- demonstrate that high levels of Sp4 are sufficient to promote larization and voltage-sensitive calcium channels. dendritic pruning in these cells when calcium signaling is We next induced knockdown of Sp4 in neurons maintained in blocked, and, furthermore, transcriptional activity of Sp4 is

Ramos et al. PNAS ͉ June 5, 2007 ͉ vol. 104 ͉ no. 23 ͉ 9885 Downloaded by guest on September 24, 2021 required to promote elimination of dendrites during the devel- eration was observed in hippocampus, but not cerebellum of Sp4 opment of cerebellar granule neurons. knockout mice (49). Sp4 continues to be expressed in the adult brain, raising the possibility that Sp4 may regulate dynamic Discussion changes in dendritic patterning in response to synaptic activity in In this study, we identified a function of the transcription factor the mature nervous system. Sp4 in specifying proper dendritic patterning in the developing Our findings indicate that the Zinc finger transcription factor cerebellum. Dendritic development involves the coordination of Sp4 contributes to the simple arborization pattern characteristic several events, including growth, branching, and pruning (re- of mature cerebellar granule neurons. Dendritic arborization viewed in refs. 4–6). We report that knockdown of Sp4 leads to patterns are the result of tightly controlled addition and elimi- highly complex dendritic arborizations of granule neurons in nation of neurites and branches. Despite recent progress, knowl- dissociated culture and in cerebellar cortex (Figs. 1 and 3). edge of the proteins and signaling pathways whose coordinated Increased complexity when Sp4 is depleted is because of both a activity determines final dendritic arborization patterns remains persistent generation of secondary dendrites (Fig. 4C) and a incomplete (7, 14, 44, 50). We propose that the transcription failure of pruning both primary and secondary dendrites during factor Sp4 regulates a program essential for later stages of dendritic development (Fig. 4 B and C). Consis- proper dendritic patterning. Identification of target genes that tent with an essential role of Sp4 in regulating dendritic pruning act downstream of Sp4 to limit dendritic branching and promote during development, we have observed that Sp4 protein levels pruning will provide additional insight into the pathways that peak in the cerebellum 2 weeks after birth, concomitant with the control dendritic morphogenesis. time of dendritic maturation in vivo (data not shown). Together these experiments support the hypothesis that Sp4 promotes the Materials and Methods transition from complex to simple dendritic trees during matu- Plasmids. Flag-Sp4 and Flag-Sp1 were expressed from the CMV ration of cerebellar granule neurons. promoter in pRC-Flag-Sp4 and pRC-Flag-Sp1. Short hairpin We also observed that remodeling of dendrites during devel- RNAs were expressed from the U6 promoter in pBSSK (51). opment of cerebellar granule neurons is regulated by depolar- Sequences targeted by the short hairpin RNAs were: Scrambled ization in dissociated cultures and by L-VSCC in cerebellar Sp3 control (gggaattaatatgcacacaggcc), Sp4 #1 nucleotides 1551– cortex (Fig. 5 and SI Fig. 11). Depolarization-induced dendritic 1571 (gggctccaactttaacacctt), Sp4 #2 nucleotides 1804–1824 (gggt- pruning was blocked when Sp4 was depleted (Fig. 5). Overex- gctgcgggtgttcaagt), and Sp1 nucleotides 881–901 (gggaacatcacctt- pression of wild-type Sp4, but not a mutant lacking the DNA- gctacct) [accession nos. NM 003112 (Sp4) and XM 028606 binding domain, promoted dendritic pruning in nondepolarizing (Sp1)]. Silent mutations resistant to Sp4 RNAi hairpin #1 were conditions, revealing that high levels of Sp4 are sufficient to introduced into Flag-Sp4 by PCR, and nucleotides were mutated to promote remodeling in developing cerebellar granule neurons, gggctccaacACtaacacctt. The Sp4⌬ZnF mutant was generated by an and transcriptional activity of Sp4 is required for this process inframe deletion of Sp4 amino acids 625–775. The reporter plasmid (Fig. 6B). Notably, we did not observe a reduction in dendrite containing p35 promoter sequence Ϫ467 upstream of the luciferase number when Sp4 was overexpressed in depolarizing conditions gene has been described (45). (data not shown), consistent with the idea that other factors act to stabilize some dendrites and limit the extent of pruning. Thus, Cell Culture and Transfection. Cerebellar granule neurons were these studies demonstrate that the transcription factor Sp4 is obtained from postnatal day 6 rat pups as described (52). Briefly, essential for activity-dependent remodeling during cerebellar neurons were maintained in BMEM supplemented with 10% development. Activity-dependent signaling pathways have been FCS, 25 mM KCl, penicillin (50 units per ml), streptomycin (50 shown to regulate several transcription factors (20–22); it re- ␮g/ml), and 2 mM glutamine at 4.2 ϫ 103 cells per mm2 density. mains to be determined how Sp4 activity is coupled to calcium After 18 h, 10 ␮M cytosine arabinofuranoside was added. Cells signaling pathways that regulate dendritic morphology. were transfected by calcium phosphate precipitation at DIV 2 as Recent studies in both vertebrates and invertebrates highlight described (37). In some experiments, cerebellar granule neurons the important role of cell-intrinsic genetic programs in control- were switched to BEM with 5 mM KCl or 25 mM KCl 2 days after ling diverse aspects of dendritic morphology. Previous studies in transfection. Where indicated, a vector expressing the antiapo- the mammalian nervous system have identified transcription ptotic protein Bcl-xL was included. factors that function to induce dendritic growth and branching, Neuro2A cells were plated at 3.6 ϫ 104 cells per cm2 24 h but transcription factors that act to balance these activities by before transfection with Lipofectamine (Invitrogen, Carlsbad, restricting branching and/or promoting pruning have previously CA). The ratio of Flag-Sp4 vector and U6/RNAi hairpin vector been described only in invertebrates (17, 19–21). Our analysis of was 1:5. Relative luciferase assays were performed as indicated Sp4 function in dendritic maturation provides evidence of a by the manufacturer (Promega, Madison, WI). genetic program controlling limiting of branching and activity- dependent pruning in the mammalian nervous system. Under- Cerebellar Slice Cultures and Transfection. Slice cultures and trans- standing how opposing transcriptional programs are coordinated fections were done as described (20). Briefly, cerebella from to balance growth, elimination, and maintenance of neurites postnatal day 9 rats were cut sagitally into 400-␮m sections, during development is an exciting new area of investigation. transferred onto a porous membrane (Millipore Corporation, Aberrant dendritic patterning is associated with neurological Billerica, CA), and maintained in ␣-MEM medium (5 mM KCl) disorders and defects in learning and memory (2, 3, 46). Sp4 supplemented with 12.5 mM Hepes, 30 mM glucose, 0.25% knockout mice exhibit pleiotropic phenotypes, including high HBSS, 25% Horse serum, 2 mM glutamine, penicillin (50 units mortality in the first postnatal month, which has complicated per ml), and streptomycin (50 ␮g/ml). Individual neurons were cellular and behavorial analyses in these animals (24, 28, 47, 48). transfected after 3 days by using biolistics (Helios gene gun; Interestingly, viable mice with significantly reduced expression Bio-Rad, Hercules, CA) with 1 ␮m gold particle coated with the of Sp4 exhibit memory defects characteristic of neuropsychiatric test plasmids together with GFP and Bcl-xL expression plasmids. disorders (48). Sp4 is highly expressed in many regions of the developing brain, and it is likely that Sp4 contributes to diverse Immunofluorescence and Immunoblotting. At the indicated times, aspects of neuronal development dependent on cellular and cells were fixed in 4% paraformaldehyde. Neurons were immu- developmental context, as shown for Sp4 function in the devel- nolabeled by using a rabbit antibody to GFP (Molecular Probes, oping heart (28). Consistent with this idea, reduced cell prolif- Eugene, OR) or Sp4 (Santa Cruz Technology, Santa Cruz, CA)

9886 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0701946104 Ramos et al. Downloaded by guest on September 24, 2021 or monoclonal antibody against MAP-2 (Sigma–Aldrich, St. by counting total secondary dendrites and total length of all Louis, MO), Tau-1 (Chemicon International, Temecula, CA), or primary dendrites in a neuron. The following formula was GFP (Molecular Probes), followed by goat anti-rabbit or anti- applied: mouse conjugated to Cy2 or Cy3 (Amersham Biosciences, Piscataway, NJ) and stained with the DNA dye bisbenzimide Total branching points/100 ␮m primary dendrites (Hoechst 33258). Neuro2A extracts were prepared in 1% Non- ϭ ϫ idet P-40 lysis buffer for 30 min on ice, followed by sonication TS/TPL 100 and centrifugation at 15,000 ϫ g for 15 min. Lysates were where TS is total number of secondary dendrites and TPL is total resolved by SDS/PAGE and immunoblotted with monoclonal primary dendritic length. anti-flag antibody (Sigma–Aldrich) or polyclonal antibody against GFP (BD Biosciences, San Jose, CA). Statistical differences were determined by ANOVA and a post hoc Tukey test or a two-tailed t test using GraphPad Prism Morphometric Analysis of Dendrites. Images of individual trans- Software. For every condition, quantitation was performed in fected neurons with no overlapping processes from other trans- three independent experiments with similar results. fected neurons were captured randomly in a blinded manner at ϫ400 magnification using a Nikon eclipse TE2000 epifluores- We thank G.G. laboratory members Alvaro Valin and Sarah Ross for the cence microscope with a CCD camera (Diagnostic Instruments, gifts of control (scrambled Sp3) and Sp1 RNAis and for flag-Sp4 and flag-Sp1, respectively; Aryaman Shalizi, Angeles Fernandez-Gonzalez, Sterling Heights, MI). Digital zoom magnification and quanti- and members of the Gill laboratory for their comments on the manu- tation of the length of individual primary, secondary, and script; and Bonni laboratory members for their helpful advice. This work tertiary dendrites were performed by using SPOT imaging was supported by a fellowship from the Spanish Ministry of Education/ software. Total dendritic length corresponds to the sum of the Fulbright (Secretaries of State, Education, and Universities, and the length of all individual dendrites per neuron. The number of European Social Fund) (to B.R.) and National Institutes of Health branching points in 100 ␮m primary dendrites was determined Grants HD043364 (to G.G.) and NS051255 and NS041021 (to A.B.).

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