The Journal of Neuroscience, July 8, 2020 • 40(28):5413–5430 • 5413 Development/Plasticity/Repair Modular and Distinct Plexin-A4/FARP2/Rac1 Signaling Controls Dendrite Morphogenesis Victor Danelon,1* Ron Goldner,2* Edward Martinez,1 Irena Gokhman,2 Kimberly Wang,1 Avraham Yaron,2 and Tracy S. Tran1 1Department of Biological Sciences, Rutgers University, Newark, New Jersey 07102, and 2Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, 76100, Israel Diverse neuronal populations with distinct cellular morphologies coordinate the complex function of the nervous system. Establishment of distinct neuronal morphologies critically depends on signaling pathways that control axonal and dendritic devel- opment. The Sema3A-Nrp1/PlxnA4 signaling pathway promotes cortical neuron basal dendrite arborization but also repels axons. However, the downstream signaling components underlying these disparate functions of Sema3A signaling are unclear. Using the KRK-AAA novel PlxnA4 knock-in male and female mice, generated by CRISPR/cas9, we show here that the KRK motif in the PlxnA4 cytoplasmic domain is required for Sema3A-mediated cortical neuron dendritic elaboration but is dispensable for inhibitory axon guidance. The RhoGEF FARP2, which binds to the KRK motif, shows identical functional specificity as the KRK motif in the PlxnA4 receptor. We find that Sema3A activates the small GTPase Rac1, and that Rac1 activity is required for dendrite elaboration but not axon growth cone collapse. This work identifies a novel Sema3A-Nrp1/PlxnA4/FARP2/Rac1 signaling pathway that specifi- cally controls dendritic morphogenesis but is dispensable for repulsive guidance events. Overall, our results demonstrate that the divergent signaling output from multifunctional receptor complexes critically depends on distinct signaling motifs, highlighting the modular nature of guidance cue receptors and its potential to regulate diverse cellular responses. Key words: axonal repulsion; dendritic branching; guidance receptors; neural development; Semaphorin signaling Significance Statement The proper formation of axonal and dendritic morphologies is crucial for the precise wiring of the nervous system that ultimately leads to the generation of complex functions in an organism. The Semaphorin3A-Neuropilin1/Plexin-A4 signaling pathway has been shown to have multiple key roles in neurodevelopment, from axon repulsion to dendrite elaboration. This study demonstrates that three specific amino acids, the KRK motif within the Plexin-A4 receptor cytoplasmic domain, are required to coordinate the downstream signaling molecules to promote Sema3A-mediated cortical neuron dendritic elaboration, but not inhibitory axon guid- ance. Our results unravel a novel Semaphorin3A-Plexin-A4 downstream signaling pathway and shed light on how the disparate functions of axon guidance and dendritic morphogenesis are accomplished by the same extracellular ligand in vivo. Received Nov. 17, 2019; revised Apr. 29, 2020; accepted May 26, 2020. Introduction Author contributions: V.D., R.G., E.M., I.G., and K.W. performed research; V.D., R.G., A.Y., and T.S.T. The development of precise neuronal connections critically analyzed data; V.D. and R.G. wrote the first draft of the paper; V.D., R.G., A.Y., and T.S.T. edited the paper; depends on proper extension of axons and elaboration of den- A.Y. and T.S.T. designed research; A.Y. and T.S.T. wrote the paper. The authors declare no competing financial interests. drites. Studies of neurite outgrowth and axon guidance are well *V.D. and R.G. contributed equally to this work. established (Barnes and Polleux, 2009; Kolodkin and Tessier- This work was support by National Science Foundation Integrative Organismal Systems BOI Grant 1556968 Lavigne, 2011; Stoeckli, 2018); and while numerous studies were and BSF Grant 2013052 to T.S.T. and A.Y. Research in the laboratory of T.S.T. was also supported by the New conducted in invertebrates (Jan and Jan, 2010), much less is Jersey Governor’s Council for Medical Research and Treatment of Autism Grant CAUT17BSP022, the New Jersey known about the signaling mechanisms controlling dendritic ’ Commission on Spinal Cord Research Grant CSCR16IRG013, and Rutgers University Newark Chancellor s morphogenesis in the mammalian nervous system. Interestingly, Initiative for Multidisciplinary Research Teams Award. Research in the laboratory of A.Y. was supported by the Jeanne and Joseph Nissim Center for Life Sciences Research at the Weizmann Institute of Science and the molecular cues initially described as axonal guidance cues also Nella and Leon Benoziyo Center for Neurologic Diseases. A.Y. is an incumbent of the Jack and Simon Djanogly control later events in dendrite development, suggesting that Professorial Chair in Biochemistry. We thank Alex Kolodkin and Oren Schuldiner for providing critical feedback these cues are multifunctional (Whitford et al., 2002; Yaron et to this work; and Adefemi Baderinwa and Michelle Davis for excellent technical support with mouse al., 2005; Tran et al., 2009; Smith et al., 2012; Mlechkovich et al., genotyping. 2014; Nagel et al., 2015; Anzo et al., 2017). Correspondence should be addressed to Tracy S. Tran at [email protected] or Avraham Yaron at [email protected]. The Semaphorin family of multifunctional cues has been https://doi.org/10.1523/JNEUROSCI.2730-19.2020 mainly studied in the context of axon guidance (Jin and Copyright © 2020 the authors Strittmatter, 1997; Zanata et al., 2002; Huber et al., 2003; 5414 • J. Neurosci., July 8, 2020 • 40(28):5413–5430 Danelon, Goldner et al. · Modular and Distinct Plexin-A4/FARP2/Rac1 Signaling Toyofuku et al., 2005; Tong et al., 2007; Tran et al., 2007). guide RNA (59-CGCCATTGTCAGCATCGCGG-39) was designed to Semaphorins can repel or attract axons by engaging different target exon 20 of PlxnA4 with minimal off-target effects. The ssDNA oli- coreceptors (Castellani et al., 2000; Chauvet et al., 2007) or inter- gonucleotide had 76-79 bp homology arms around the KRK.AAA point mutation, with disruption of the TGG PAM sequence to TAG. In acting with molecules in the local environment (Kantor et al., m m 2004). Interestingly, the Class 3 secreted Semaphorin-3A vitro transcribed Cas9 RNA (100 ng/ l), the sgRNA (50 ng/ l), together with the ssDNA (200 ng/ml), were injected into one cell-fertilized (Sema3A) can repel axons and promote dendrite branching in embryos isolated from superovulated CB6F1 hybrid mice (F1 of BALB/ different neuronal populations both in vitro and in vivo (Gu et cAnNHsd inbred female  C57BL/6NHsd inbred male) mated with al., 2003; Fenstermaker et al., 2004; Yaron et al., 2005; Tran et al., CB6F1 males from Harlan Biotech Israel. Injected embryos were trans- 2009; Mlechkovich et al., 2014). However, the intracellular events ferred into the oviducts of pseudopregnant ICR females. Screening of orchestrating these divergent outputs, elicited by the same tail tip DNAs was conducted by PCR genotyping and confirmed by Sema3A cue, are largely elusive. sequencing. Heterozygous males were backcrossed with females of CD1 Sema3A signaling requires the binding to its obligate partner (ICR) genetic background. Neuropilin-1 (Nrp1) and formation of a complex with one of the For generation of the Farp2 KO mouse line, two CRISPR sgRNAs Type-A Plexins (Plxns) (Tran et al., 2007). However, little was were designed flanking a 18,167 bp sequence in the Farp2 locus on Chr1 known about the specific Plxn signaling receptor involved in (GRCm38-mm10), including the promoter, the first utr exon and the second exon (with ATG). One sgRNA (59-TCTTAAGGACTTATTG dendrite development until we showed the reduced dendritic CCAA-39, chromosome 1: 93511211-93511211) was targeted to a region complexity of PlxnA4 KO deep cortical neurons in vivo (Tran et 9 sema- upstream of the Farp2 gene promoter, and a second sgRNA (5 - al., 2009) that phenocopied those observed in the Nrp1 GTGAGGGCCTCATTCCGAAA-39, chromosome 1: 93529378-93529397) mutants (Gu et al., 2003). We demonstrated in vitro that the abil- to intron 2. The sgRNAs were designed using several CRISPR designing ity of Sema3A to mediate growth cone collapse in DRG neurons tools, and optimized for the best guides, including the following: the MIT versus dendritic arborization of cortical neurons is embedded in CRISPR design tool (Hsu et al., 2013) and sgRNA Designer, Rule Sets 1 and a modular nature of the PlxnA4 receptor, within its distinct cyto- 2(Doench et al., 2014, 2016), in both the original sites and later in the plasmic domains (Mlechkovich et al., 2014). We showed that the Benchling implementations (www.benchling.com), SSC (Xu et al., 2015), KRK motif in PlxnA4 can associate with the RhoGEF FARP2 and sgRNA scorer (Chari et al., 2015), in their websites. In vitro transcribed m m and is required for dendrite elaboration in vitro. However, the Cas9 RNA (100 ng/ l) and the sgRNAs (50 ng/ l) were injected into one cell-fertilized embryos isolated from superovulated CB6F1 hybrid mice (F1 downstream signaling elements controlling dendrite elaboration of BALB/cAnNHsd inbred female  C57BL/6NHsdinbredmale)mated versus axon guidance remained elusive. with CB6F1 males from Harlan Biotech Israel. Injected embryos were trans- Regulators of small GTPases (GEFs and GAPs) may represent ferred into the oviducts of pseudopregnant ICR females. Screening of tail signaling-specificity elements for divergent Semaphorin func- tip DNAs was conducted by PCR genotyping and RT-PCR (see Fig. 1). tions. The