Monensin Sensitive 1 Regulates Dendritic Arborization in Drosophila by Modulating Endocytic Flux

Monensin Sensitive 1 (Mon1) is a component of the Mon1:Ccz1 complex that mediates Rab5 to Rab7 conversion in eukaryotic cells by serving as a guanine nucleotide exchange factor for Rab7 during vesicular trafficking. We find that Mon1 activity modulates the complexity of Class IV dendritic arborization (da) neurons during larval development. Loss of Mon1 function leads to an increase in arborization and complexity, while increased expression, leads to reduced arborization. The ability of Mon1 to influence dendritic development is possibly a function of its interactions with Rab family GTPases that are central players in vesicular trafficking. Earlier, these GTPases, specifically Rab1, Rab5, Rab10, and Rab11 have been shown to regulate dendritic arborization. We have conducted genetic epistasis experiments, by modulating the activity of Rab5, Rab7, and Rab11 in da neurons, in Mon1 mutants, and demonstrate that the ability of Mon1 to regulate arborization is possibly due to its effect on the recycling pathway. Dendritic branching is critical for proper connectivity and physiological function of the neuron. An understanding of regulatory elements, such as Mon1, as demonstrated in our study, is essential to understand neuronal function.


INTRODUCTION
Dendritic arbors are complex neuronal structures with distinct morphological features (Cajal, 1999;Garcia-Lopez et al., 2010;Berry and Nedivi, 2017). During neuronal development, morphogenetic processes that are not yet completely understood, lead to formation of arbors with defined size, geometry, innervation, and tiling patterns. The dendritic tree structure is unique to a given neuronal cell type and plays a fundamental role in establishing specific neuronal connectivity. An intrinsic genetic program patterns the arbors using molecular processes that are distinct from those that make axons. These are found to be dependent on both, internal as well as external cues (Parrish et al., 2007;Jan and Jan, 2010). The growth and development of dendritic arbors are also concurrent in time and space with synapse formation with proteins of the post-synaptic density playing an integral role in morphogenesis (Cantallops et al., 2000;Cline, 2001;Peng et al., 2009).
The embryonic and larval peripheral nervous system (PNS) in Drosophila melanogaster has served as an excellent model system for studying mechanisms that govern dendritic arbor complexity and tiling. The PNS consists of 45 sensory neurons per hemisegment which are classified into type I and type II neurons (Grueber et al., 2003;Orgogozo and Grueber, 2005). The type II neurons are multidendritic whose dendrites innervate the epidermis. Dendritic arborization (da) neurons are a type of multidendritic neurons which are further classified into class I to IV on the basis of their dendrite field complexity with class IV da neurons having the most complexity in terms of the number of dendrites and their branching (Grueber et al., 2002). The arbor complexity in da neurons is determined through a combinatorial expression of transcription factors indicating the process is hard-wired and intrinsic to the neuronal class (Jinushi-Nakao et al., 2007).
As in other organisms (Dong et al., 2015;Prigge and Kay, 2018) the process of morphogenesis is regulated by signaling mediated by external cues such as Slit and Semaphorins (Jan and Jan, 2010;Meltzer et al., 2016), kinases such as Tricornered (Emoto et al., 2004) and a range of cellular processes that include intracellular trafficking, translational control and cytoskeletal dynamics Satoh et al., 2008;Delandre et al., 2016).
In this study we demonstrate that Drosophila Monensin Sensitivity 1 (DMon1; Yousefian et al., 2013;Deivasigamani et al., 2015;Dhiman et al., 2019), a core component of the Mon1:CCZ1 complex (Wang et al., 2002;Nordmann et al., 2010;Poteryaev et al., 2010), and central to conversion of early endosomes to late endosomes, regulates morphogenesis of Class IV da (CIVda) neurons. We uncover a role for Mon1 by demonstrating that CIVda patterning can be regulated by increasing or decreasing Mon1 function during embryonic/larval development: loss of Mon1 leads to increased branching while overexpression suppresses it. Consistent with its position in the endocytic pathway, we find that Mon1 functions genetically downstream of Rab5. Surprisingly however, the regulation by Mon1 does not seem to be dependent on the late endosomallysosomal pathway. Rather, the modulation appears to be via the Rab11 mediated recycling pathway. We propose that in the context of the da neurons, Mon1 serves to balance the endocytic flux flowing through the endo-lysosmal and recycling pathways to regulate dendrite morphogenesis.

Mon 1 Modulates Dendritic Branching in Class IV da Neurons
CIVda neurons express pickpocket (ppk), a gene involved in nociception in Drosophila (Adams et al., 1998;Crozatier and Vincent, 2008). We recombined ppk-Gal4 (BL32079; Grueber et al., 2007;Kanamori et al., 2013) with a membrane localized GFP expressed under a ppk regulatory element (ppk-GFP (BL35843) (Kanamori et al., 2013), and generated a reporter line ('R, ' see section "Materials and Methods") that allows visualization of CIVda neuron morphology in response to genetic manipulation either through gene knock-down and overexpression in the third instar larva of Drosophila ( Figure 1A). This reporter ('R') line was used to observe the arborization of CIVda in the Dmon1 181 ( 181) line, a loss of function allele of Mon1 (Deivasigamani et al., 2015). When compared to a wild-type control, CIVda in Dmon1 mutant showed enhanced dendritic branching with a ramification index (R.I), of approximately 80 as compared to 60 in wild-type larvae. On normalization, with the reporter line set to 100, Dmon1 mutant shows 45% increase in R.I (Figures 1B,G). This increase in R.I was however not observed in a heterozygous condition (Figures 1C,G), suggesting that a single copy of Dmon1 is sufficient to regulate dendritic arborization in the CIVda neurons. In order to confirm the result, we quantified R.I in Dmon1 181 /Df(9062), an allelic combination, where Df(9062) is a deficiency that uncovers the Mon1 locus (Deivasigamani et al., 2015). An increase in RI by 47% ( Figure 1G) similar to that in Dmon1 181 homozygote confirmed that it is the loss of Dmon1 that leads increased R.I of CIVda neurons. Other parameters (see section "Materials and Methods"), such as dendritic area (D.A; µM 2 ), dendritic length (D.L; µM) and number of dendritic branch points (D.BP) were also measured for the same set of images. Loss of Dmon1 also led to an increase in average values, as compared to controls for D.A (51843 vs. 65787 µM 2 ), D.L (14549 vs. 17811 µM) and D.BP (492 vs. 829). Normalized values, with the control R/+ set to 100 are displayed in the figures (Figures 1H-J).
Further confirmation for the role for Mon1 in regulating CIVda branching was demonstrated by rescue of the dendritic phenotypes in homozygous Dmon1 181 ( Figure 1E) and Dmon1 181 / Df(9062) animals through expression of DMon1 in the ppk domain ( Figure 1G). In both examples, the R.I, D.A, D.L, and D.BP were restored to wild-type or near wild-type levels (Figure 1).
In addition, overexpression of Mon1 in wild-type animals using ppk-GAL4 led to reduction of all four parameters measured (Figure 1). R.I, D.L, and D.BP were reduced significantly, while the reduction of D.A had lower statistical significance (' * '; Figure 1H). Together, these results demonstrate that arborization of the CIVda neurons during development is sensitive to the dose of Mon1 with decrease in Mon1 function leading to increased branching, dendritic length and area while enhancement of Mon1 function leads to a decrease in the measured parameters.

Rabs Modulate Dendritic Arborization
Mon1/SAND1 regulates Rab conversion in yeast, C. elegans and mammalian cells (Nordmann et al., 2010;Poteryaev et al., 2010;Yousefian et al., 2013). Mon1 in complex with Ccz1 functions as a guanine nucleotide exchange factor for Ypt7, the yeast ortholog of Rab7 (Nordmann et al., 2010). As in other model systems, in Drosophila, the recruitment of Rab7 on late endosomes is mediated by the Mon1-Ccz1 complex (Yousefian et al., 2013). In the study by Yousefian et al. (2013), Mon1 loss of function leads to enlargement/enrichment of Rab5 positive early endosomes and concomitant loss of association of mature endosomes with Rab7, a feature that is replicated in CIVda neurons (Supplementary Figure S1). Rab4 and Rab5 co-localized on early endosomes while Rab11 distributions between mutant and wild-type cells were  Supplementary Table S1. ns, not significant. * p < 0.05 and * * * p < 0.001. Error bars represent standard error.
Given the role of Mon1 in endocytic trafficking, we sought to explore the role of Rab proteins in Mon1 mediated CIVda morphogenesis (Figure 2 and Supplementary Figures S2A,B). An earlier study in Drosophila has implicated Rab5 and the distribution of Rab5 endosomes in the patterning of da neurons (Satoh et al., 2008), while Rab11 mediated recycling has been shown to be important in dendritic branching in rat hippocampal neurons (Satoh et al., 2008;Lazo et al., 2013). In Drosophila, the roles of Rab11 mediated recycling pathway or the Rab7 mediated degradative pathway in dendrite morphology has not been tested. We therefore sought to test this in the context of Mon1 mutants through genetic epistasis, by using loss-of-function and gain-of-function transgenic lines against Drosophila Rab5, Rab7, and Rab11 genes. In agreement with earlier studies (Satoh et al., 2008), expression of Rab5 dominant negative (Rab5DN; Rab5 in a GDP-bound form) or knockdown using RNA interference, using ppk-Gal4, show a drastic reduction in the extent of arborization and branching (Figures 2B,D Figures S2A,B).
Cargo present in Rab5 positive early endosome cycle can be channeled down the degradation pathway involving Rab7 or the recycling pathway, marked by Rab11. We tested the involvement of these pathways by modulating the activity of Rab7 and Rab11. We found that increasing Rab7 activity through expression of a CA form, or decreasing Rab7 function by using a dominant negative (DN) form of Rab7 or through expression of Rab7 RNAi the ppk domain (Figures 2E,F,G,J,K and Supplementary  Figures S2A,B) does not affect the arborization, branching, length or area of CIVda neurons. In contrast, expression of both, Rab11CA and Rab11DN altered arborization patterns in an opposing manner: increase in Rab11 activity increased R.I, D.A, D.L and D.BP, while a decrease in Rab11 activity reduced these parameters ( Figures 2H-K and Supplementary Figures S2A,B). Interestingly, the increase in parameters (15-40%) seen upon expression of Rab11CA (Figures 2H-K) were correlated to and comparable with increase seen in Mon1 mutants, with the exception of D.BP, where Rab11CA has a weaker effect, suggesting that the Rab11 mediated recycling pathway plays a central role in CIVda patterning.

Mon1 Interacts With Rabs to Modulate da
Since the activity of Rab5 and Rab11 strongly modulates arborization of CIVda neurons, we explored the nature of the interaction between these Rabs 5, 7, and 11 and Mon1 to uncover features of vesicular recycling that are important for CIVda morphogenesis (Figure 3 and Supplementary Figures S2C-H). We tested this by modulating activity of Rab5, Rab7, and Rab11 in the Although Rab7 on its own does not seem to participate in the regulation of da (Figures 2F,G,J,K), we found that expression of Rab7CA leads to a significant reduction of R.I, D.A., D.L and D.BP, as compared to control Dmon1 181 larvae ( Figures 3H-K and Supplementary Figures S2D,G; Images not displayed) suggesting that activation of the downstream endo-lysosomal pathway can suppress excess arborization in the mutant. This may be an outcome of a flux change due to modulation of the lysosomal branch. Interestingly, Rab7 RNAi also suppresses Mon1 loss of function phenotype (Figures 3H,K  and Supplementary Figures S2D,G Figure S2. ns, not significant. * p < 0.05 and * * * p < 0.001. Error bars represent standard error. Statistical analysis using Dunnet's multiple comparison test using GraphPad Prism 7 with exact p-values listed in Supplementary Table S1.

DISCUSSION
Mon1 is a conserved eukaryotic protein with a 'longin' domain. The domain has an alpha-beta-alpha sandwich architecture and is a feature of endocytic trafficking proteins (De Franceschi et al., 2014). Protein containing 'longin' domains include SANDs, SNAREs, targetins, adaptins, and sedlins (De Franceschi et al., 2014). The dimeric Mon1:CCZ complex is involved in Rab conversion and is a GEF for Rab7 ( Figure 4A). Additionally, there is evidence that Mon1 can be secreted by neurons, either in membrane bound or unbound form (Deivasigamani et al., 2015). Mon1 may thus regulate anterograde signaling in synapses, both neuron:neuron or neuron:muscle. Recent research from our laboratory (Dhiman et al., 2019) also suggests that Mon1 in Octopaminergic neurons regulates systemic insulin signaling by regulating insulin producing cells. CIVda neurons are sensitive to the dose of DMon1 in the cell (Figure 4B). Decrease in Dmon1 leads to increased complexity of arborization, which includes increase in branching, length and area covered by the axons. Increase in Dmon1 decreases complexity of arborization and reduces the values of the parameters measured. Since Mon1 is primarily known for its role as a Rab converter in eukaryotic cells, we explored functions of Rab proteins Rab5, Rab7, and Rab11 in regulating da by themselves and also in the context of Dmon1 loss of function. Amongst the Rabs tested, Rab7 activity did not appear to affect dendritic morphology, suggesting that perturbation of late endosomal trafficking does not affect morphogenesis of CIVda neurons in larvae. This premise however can be strengthened by using Mosaic analysis with a repressible cell marker, studies. In contrast, Rab11 activity altered arbor complexity with decrease in Rab11 activity leading to a dramatic decrease in arborization and expression of Rab11CA having an opposite effect (Figure 3). This suggests that recycling endosomes play critical roles in determining arbor complexity which is in agreement with the role of Rab11 in dendrite morphogenesis in vertebrate systems (Villarroel-Campos et al., 2014;Peng et al., 2015;Gu et al., 2016). Since reduction of Rab5 activity also decreases arbor complexity, and Rab5 'sorting' endosomes are upstream (Figure 4A) of both recycling endosomes (RE) and late endosomes (LE), we propose that vesicular flux through the RE but not the LE is a central determinant of CIVda morphogenesis (Figure 4C). Loss of Rab5 leads to a decrease in the rate of RE formation, while loss of DMon1 changes the endocytic flux, shunting excess early endosomes (EE's) toward the RE pathway. In our model (Figure 4C), increase in activity of Mon1 enhances Rab5 to Rab7 conversion, reducing RE traffic leading to decreased arborization.
How does increase in RE flux lead to increase in dendritic complexity? Since endosomes marked with Rab11 can be exocytosed, we suggest that enhanced vesicular flux in the RE pathway leads to enhanced exocytosis, which in turn is correlated to increase dendritic arborization. This is in agreement with earlier studies where trafficking of cargo in Rab11 vesicles regulated dendritic complexity. For example, Lazo et al. (2013) demonstrate that Rab11 regulates trafficking of brain derived neurotrophic factor along with its receptor TrkB while Peng et al. (2015) have shown that the Rop-exocyst complex is important for dendritic branching in CIVda neurons. In contrast, data from the Klein lab (Yousefian et al., 2013), with experiments performed in the wing imaginal disk, suggest that Rab11 is not affected in the Dmon1 mut4 lines, and instead find changes in the Rab4 associated fast recycling pathway. In addition to protein and RNA based cargo, exocytosis also provides neuronal membrane that is critical for growth of the arbor, underscoring the importance of RE flux and exocytosis.
The balance of endocytosis and exocytosis is crucial for the growth and maintenance of CIVda arbors. Mon1 activity in the early 'sorting' endosomes may be important for modulating the flux through either the LE or RE pathways which in turn could lead to modulation of neuronal architecture. Our results thus underscore the role of endocytic flux in dendrite morphogenesis. The genetic interactions described here suggest a cell-autonomous role for Mon1. Given the ability of Mon1 to be secreted, it would be interesting, in future studies, to test for possible non-autonomous roles in dendrite development.

Fly Husbandry
Fly lines were maintained at 25 • C on standard cornmeal agar medium. UAS-GAL4 system (Brand and Perrimon, 1993) was used for over-expression of transgenes.

Sholl Analysis
Sholl analysis, to measure the ramification index (R.I), was performed using the NIH ImageJ Sholl Analysis Plugin (v1.0) 1 , as distributed by Fiji. Briefly, the maximum intensity projection for z stacks of each neuron was converted to a segmented grayscale image using ImageJ. Background dendrites extending into the image view from neighboring neurons were manually deleted. Sholl analysis was performed by drawing a straight line from the cell body to the distal tip of the neuron. The area for the analysis was hence defined by this straight line which is considered as the radii for each image. The origin of the concentric radii was set at the midpoint of the longest axis of the soma. Analysis was performed in automated way using the following parameters: starting radius, 1 µm; radius step size, 2 µm; span, 1 µm; span type, median. The number of dendrite intersections for each circle is measured and the highest value is divided by the number of primary dendrites (intersections at the starting radius) to obtain the Schoen ramification index (R.I). This parameter is dependent on maximum number of intersections and the number of primary dendrites. Statistics were performed using the Prism statistical package (GraphPad, San Diego, CA, United States).

Neuromorphometric Analysis of da Neurons
The Filament dendrite tracer plug-in of the IMARIS 7 software was used to trace the dendrites in 3D and generate quantitative data for each genotype. The analysis was performed on dendritic arbor's arising from a single neuron and the branches from neighboring neurons were deleted manually. The cell body was