Genetics: Early Online, published on October 12, 2017 as 10.1534/genetics.117.300393 1 Dendritic cytoskeletal architecture is modulated by combinatorial transcriptional 2 regulation in Drosophila melanogaster 3 Ravi Das*, Shatabdi Bhattacharjee*, Atit A. Patel*, Jenna M. Harris*, Surajit Bhattacharya*, Jamin 4 M. Letcher*, Sarah G. Clark*, Sumit Nanda‡, Eswar Prasad R. Iyer†, Giorgio A. Ascoli‡, Daniel 5 N. Cox*,1 6 7 Affiliation: 8 * Neuroscience Institute, Georgia State University, Atlanta, GA, 30302 9 † Wyss Institute, Harvard Medical School, Cambridge, MA, 02115 10 ‡ Krasnow Institute for Advanced Study, George Mason University, Fairfax, VA, 22030 11 1 Corresponding author 12 13 Running title: Dendritic cytoskeletal regulation 14 15 Keywords: transcription factors, dendrite, cytoskeleton, Drosophila, neurogenomics 16 17 Corresponding author 18 Daniel N. Cox, Ph.D. 19 Neuroscience Institute 20 Georgia State University 21 P.O. Box 5030 22 Atlanta, GA 30302-5030 23 Office: 404-413-5222 24 Lab: 404-413-5255 25 FAX: 404-413-5446 26 email: [email protected] 27 1 Copyright 2017. 28 Abstract 29 Transcription factors (TFs) have emerged as essential cell autonomous mediators of 30 sub•type specific dendritogenesis, however, the downstream effectors of these TFs remain 31 largely unknown, as are the cellular events that TFs control to direct morphological change. As 32 dendritic morphology is largely dictated by the organization of the actin and microtubule (MT) 33 cytoskeletons, elucidating TF•mediated cytoskeletal regulatory programs is key to understanding 34 molecular control of diverse dendritic morphologies. Previous studies in Drosophila 35 melanogaster have demonstrated that the conserved TFs Cut and Knot exert combinatorial 36 control over aspects of dendritic cytoskeleton development, promoting actin• and MT•based 37 arbor morphology, respectively. To investigate transcriptional targets of Cut and/or Knot 38 regulation, we conducted systematic neurogenomic studies, coupled with in vivo genetic screens 39 utilizing multi-fluor cytoskeletal and membrane marker reporters. These analyses identified a 40 host of putative Cut and/or Knot effector molecules and a subset of these putative TF targets 41 converge on modulating dendritic cytoskeletal architecture and are grouped into three major 42 phenotypic categories, based upon neuromorphometric analyses: complexity enhancer, 43 complexity shifter, and complexity suppressor. Complexity enhancer genes normally function to 44 promote higher order dendritic growth and branching with variable effects on MT stabilization 45 and F-actin organization, whereas complexity shifter and complexity suppressor genes normally 46 function in regulating proximal-distal branching distribution or in restricting higher order 47 branching complexity, respectively, with spatially restricted impacts on the dendritic 48 cytoskeleton. Collectively, we implicate novel genes and cellular programs by which TFs 49 distinctly and combinatorially govern dendritogenesis via cytoskeletal modulation. 2 50 Introduction 51 Neurons are highly polarized cells comprised of two structurally and functionally distinct 52 processes, the axon, which relays signals to other neurons, and the dendrites, which receive 53 signals from other neurons. Since dendrites are the primary site of synaptic input and signal 54 integration, with dendritic size and the range of arborization patterns impacting connectivity, the 55 regulation of dendritic growth and branching is extremely important for the establishment of 56 functional neuronal networks (Lefebvre et al. 2015). 57 Genetic and molecular studies have demonstrated that the acquisition of class-specific 58 dendrite morphologies is mediated by complex regulatory programs involving intrinsic factors 59 and extrinsic cues (Jan and Jan, 2010; Puram and Bonni 2013; Tavosanis 2014; Nanda et al. 60 2017). Many of these factors are part of, or activate, signaling pathways that eventually converge 61 on the neuronal actin and MT cytoskeletons. These cytoskeletal elements form the scaffold 62 around which cell shape is built, and the tracks along which intracellular components are 63 transported (Rodriguez et al. 2003). Despite recent progress in dissecting the roles of TF activity 64 in regulating dendritic cytoskeletal architecture (Jinushi-Nakao et al. 2007; Iyer et al. 2012; Ye et 65 al. 2011; Nagel et al. 2012), much remains unknown regarding the molecular mechanisms via 66 which TFs spatio-temporally modulate cytoskeletal dynamics to direct developing and mature 67 arbor morphologies (Santiago and Bashaw, 2014). Understanding how such changes in 68 cytoskeletal control lead to specific changes in emergent neuron shape can be facilitated by 69 computational simulations (Samsonovich and Ascoli 2005), especially if directly and bi- 70 directionally linked with imaging-driven, systems-level molecular investigations (Megason and 71 Fraser 2007). 3 72 Intriguingly, two transcription factors, Cut (Ct) and Knot (Kn), have been shown to 73 synergize in promoting class IV (CIV) da neuron-specific arbor morphology by each exerting 74 distinct regulatory effects on the dendritic cytoskeleton (reviewed in Nanda et al. 2017). Ct, a 75 member of the evolutionarily conserved CUX family of transcription factors, is a homeodomain 76 containing molecule with functional roles in external sensory organ cell fate specification 77 (Blochlinger et al. 1988; Blochlinger et al. 1990; Bodmer et al. 1987), class-specific da neuron 78 dendrite morphogenesis (Grueber et al. 2003a), and dendritic targeting of olfactory projection 79 neurons (Komiyama & Luo 2007). Ct regulates dendritic diversity among da sensory neurons in 80 an expression level dependent manner. Ct protein expression in da neurons is highest in class III 81 (CIII) neurons, followed by medium and low expression levels in CIV and class II (CII) neurons, 82 respectively, and is undetectable in class I (CI) neurons (Grueber et al. 2003a). Genetic 83 disruption of ct leads to severe reductions in dendritic arbor complexity, particularly the 84 formation of actin-rich structures such as short, unbranched dendrites. Conversely, ectopic 85 misexpression of Ct in CI neurons results in supernumerary branching and the de novo formation 86 of F-actin rich dendritic filopodia converting typical CI dendritic morphology toward the 87 characteristic features of CIII neurons (Grueber et al. 2003a). In mammals, Cux1/Cux2, the 88 vertebrate homologs of Ct, also function in regulating dendritic branching, spine morphology and 89 synaptogenesis in the mammalian cortex revealing the Ct/Cux molecules have evolutionarily 90 conserved roles in dendritic development and maturation (Li et al. 2010; Cubelos et al. 2010). 91 Similarly, the Collier/Olf1/EBF (COE) family transcription factor Kn, which is 92 exclusively expressed in CIV neurons, endows these neurons with an expansive and highly 93 branched dendritic arbor by promoting MT-dependent branching and elongation. As with ct 94 defects, loss of kn function in CIV neurons leads to significant reductions in dendritic growth and 4 95 branching resulting in rudimentary arbor complexity, and conversely, ectopic misexpression of 96 Kn in CI da neurons promotes supernumerary higher-order branches coupled with excessive 97 dendrite branch elongation (Jinushi-Nakao et al. 2007; Hattori et al. 2007; Crozatier and Vincent 98 2008). 99 The combinatorial action of Ct and Kn in specifying class-specific arbor shapes is 100 achieved, at least in part, by differential regulatory effects on the F-actin and MT cytoskeletons 101 (Jinushi-Nakao et al. 2007). Furthermore, Kn and Ct exert their effects on the dendritic 102 cytoskeleton through primarily parallel pathways. Ct, acting via Rac1, promotes the formation of 103 actin-rich dendritic filopodia, whereas Kn promotes the expression of the MT severing protein 104 Spastin which is thought to generate new sites for MT polymerization thereby promoting branch 105 initiation, elongation and arbor complexity (Jinushi-Nakao et al. 2007). Interestingly, the 106 Krüppel-like transcription factor Dar1, which is expressed in all da neuron subclasses, is required 107 for Kn-mediated dendritogenesis and appears to restrict Spastin expression to achieve proper 108 levels of this molecule in promoting dendritic growth (Ye et al. 2011). In CIV neurons, Kn 109 suppresses Ct-induced actin-rich dendritic filopodial formation contributing to cell-type specific 110 arborization, whereas in CIII neurons, Ct promotes the formation of these structures (Jinushi- 111 Nakao et al. 2007). Moreover, Kn does not function in regulating Ct protein levels, however Ct 112 controls the amplitude of Kn expression (Jinushi-Nakao et al. 2007). Despite recent advances, 113 much remains unknown regarding the identity and function of putative targets of Ct and/or Kn, 114 and while these molecules exert combinatorial synergistic effects on sculpting the dendritic 115 cytoskeleton and promoting dendritic diversity, there are, as yet, no identified convergent 116 transcriptional targets, nor do we have a complete picture of the potential cellular programs that 117 these transcription factors modulate to direct cell-type specific dendrite development. 5 118 Here, we address these knowledge gaps by specifically focusing on transcriptional 119 programs that are directed by Ct and/or Kn via combined neurogenomic analyses, 120 bioinformatics, genetic screens, and cytoskeletal reporter studies of putative target function in 121