PlexinD1 signaling controls domain-specific dendritic development in newborn neurons in the postnatal olfactory bulb

Newborn neurons show immature bipolar morphology and continue to migrate toward their destinations. After the termination of migration, newborn neurons undergo spatially controlled dendrite formation and change into a complex morphology. The mechanisms of dendritic development of newborn neurons have not been fully understood. Here, we show that in the postnatal olfactory bulb (OB), the Sema3E-PlexinD1 signaling, which maintains bipolar morphology of newborn neurons, also regulates their dendritic development after the termination of migration in a dendritic domain-specific manner. Genetic ablation of Sema3E or PlexinD1 enhanced dendritic branching in the proximal domain of the apical dendrites of OB newborn granule cells, whereas PlexinD1 overexpression suppressed it in a Rho binding domain (RBD)-dependent manner. Furthermore, RhoJ, a small GTPase that directly binds to PlexinD1RBD in vascular endothelial cells, is expressed in migrating and differentiating newborn granule cells in the OB and is also involved in the suppression of proximal branching of their apical dendrites. These results suggest that the Sema3E-PlexinD1-RhoJ axis regulates domain-specific dendrite formation of newborn neurons in the postnatal OB.


Introduction
The brain of postnatal mammals contains neural stem cells (NSCs), which have the ability to generate new functional neurons.In the ventricular-subventricular-zone (V-SVZ) lining the lateral walls of the lateral ventricles, the largest neurogenic niche in the postnatal brain, newborn neurons generated from NSCs migrate toward the olfactory bulb (OB), a primary center for odor information processing (Bressan and Saghatelyan, 2020;Nakajima et al., 2021).After reaching the OB, most of the newborn neurons terminate their migration in the granule cell layer (GCL), develop their complex dendrites to precisely connect with preexisting OB circuits, and differentiate fully into granule cells, the major inhibitory interneurons in the OB (Luskin, 1993;Lois and Alvarez-Buylla, 1994).Eliminating newborn neurons or blocking their synaptic transmission causes disruption of normal olfactory behaviors (Breton-Provencher et al., 2009;Sakamoto et al., 2014;Muthusamy et al., 2017).Thus, the integration of newborn neurons into mature OB circuits is critical for olfactory functions, and could be accomplished by appropriate dendritogenesis.However, its mechanism is still largely unknown.
In the OB, granule cells have short basal dendrites extending in the basal domain and a single long apical dendrite consisting of an unbranched proximal domain and a highly ramified distal domain (Price and Powell, 1970;Petreanu and Alvarez-Buylla, 2002;Kelsch et al., 2008).In the apical dendrite of granule cells, while the proximal domain receives centrifugal inputs from olfactory higher centers, the distal domain receives peripheral inputs from mitral/tufted cells, projection neurons in the OB (Shepherd et al., 2004).Therefore, the dendritic domains of granule cells are integrated into functionally distinct OB circuits, and their morphological regulation could be the basis of granule cell functions.Previous studies suggest that olfactory input (Saghatelyan et al., 2005) and 5 T4 (Yoshihara et al., 2012) promote dendritic branching in the distal domain of the apical dendrites.However, the mechanism for suppression of dendritic branching in the proximal domain of apical dendrites remains unknown.
The secreted protein Sema3E and its receptor PlexinD1 are involved in morphogenesis of the nervous and vascular systems during development (Gay et al., 2011;Oh and Gu, 2013).In the central nervous system (CNS), PlexinD1 signaling is involved in the regulation of migration (Bribian et al., 2014;Sawada et al., 2018), survival (Cariboni et al., 2015), axonal elongation (Chauvet et al., 2007;Deck et al., 2013;Burk et al., 2017), and connections (Pecho-Vrieseling et al., 2009;Ding et al., 2011;Fukuhara et al., 2013;Mata et al., 2018) of neural cells.However, the role of PlexinD1 signaling in dendritogenesis in the CNS remains unclear.We have previously shown that PlexinD1 signaling suppresses the formation of filopodium-like lateral protrusions (FLPs), which branch laterally from the proximal domain of the leading process of migrating newborn neurons, thereby maintaining their immature bipolar morphology (Sawada et al., 2018).Therefore, we hypothesized that PlexinD1 signaling also suppresses dendritic branching in the proximal domain of apical dendrites in newborn granule cells after migration termination.
In this study, we show that PlexinD1 signaling is involved in the suppression of proximal branching of apical dendrites in newborn granule cells.We also show that this domain-specific regulation is controlled by the small GTPase RhoJ, a direct binding partner of PlexinD1.

Animals
Wild-type (WT) C57BL/6 J mice were purchased from Japan SLC.PlexinD1-flox mice (Zhang et al., 2009) were described previously.Sema3E-KO mice (Gu et al., 2005) were provided by Dr. Fanny Mann (Institut de Biologie du Developpement de Marseille) and Dr. Christopher E. Henderson (Columbia University).RhoJ-KO mice were described previously (Kim et al., 2014).Both male and female animals were used in this study.All animals were maintained with their mother mouse during weaning and within 7 mice per cage after weaning on a 12 h light/dark cycle with ad libitum access to food and water.All of the animal experiments were performed in accordance with the guidelines and regulations of Nagoya City University .

In vivo lentiviral infection and electroporation
Injection of lentiviral suspension and plasmid solution was described previously (Ota et al., 2014;Sawada et al., 2018).For lentiviral infection, a 2 μL volume of lentiviral suspension was stereotaxically injected into the V-SVZ (1.8 mm anterior, 1.4 mm lateral to lambda and 1.5-2.0mm deep) of male and female pups at postnatal day 1 (P1).For in vivo electroporation of P1 male and female pups, a 2 μL volume of plasmids (3.5 μg/μL) containing 0.05% Fast Green was stereotaxically injected into the lateral ventricle (1.8 mm anterior, 1.2 mm lateral to lambda and 2.0 mm deep), and electroporation was performed using an electroporator (CUY-21SC, 10.3389/fnins.2023.1143130Frontiers in Neuroscience 03 frontiersin.org Nepagene) with a forceps-type electrode (CUY650P7, Nepagene).Lentivirus-injected or electroporated pups were allowed to recover on a heating pad and returned to their home cage.

Confocal image acquisition, quantification, and dendritic tracing of granule cells
Image acquisition and dendritic tracing of granule cells were performed as previously described (Sawada et al., 2018).Images of labeled granule cells of the postnatal OB were acquired by scanning at 2 μm intervals using an LSM 700 confocal laser-scanning microscope (Carl Zeiss) with a 20× objective lens (NA 0.8).For characterization of RhoJ +/GFP -positive cells, three regions-of-interest (ROIs) were randomly selected from three consecutive sections (one ROI per section) from every sixth 60 μm-thick OB section and image acquisition was performed, and all of the positive cells in the ROIs were counted.The obtained proportions of RhoJ +/GFP -positive cells were reported as mean ± SEM (n = 3 mice).Acquired z-stack images of virally labeled neurons were traced, reconstructed, and quantified using Neurolucida and Neurolucida Explorer software (MBF Bioscience) (Sawada et al., 2018).For analysis of apical and basal dendrites, all of the labeled granule cells showing complete neuronal morphologies observed in every sixth section were traced and analyzed.For analysis of dendritic branching in the proximal domain of the apical dendrite, all of the labeled granule cells showing >100 μm-length apical dendrites from soma were analyzed in this study.Dendritic branching in all domains of granule cells was expressed as branching in the dendritic domain per cell.For validation of PlexinD1 expression in neuronal culture, the signal intensity in the cell surface of labeled cells was measured using ZEN software (Carl Zeiss), and the average signal intensity (per μm), and normalized value (average value of control groups is 1.0) were calculated as reported previously (Sawada et al., 2018).

Statistical analysis
All of the data were two-tailed and analyzed using EZR (Kanda, 2013).The data distribution was analyzed by the Kolmogorov-Smirnov test.The equality of variance between groups was analyzed by the F test.Comparisons between two groups were analyzed by unpaired t-test or Mann-Whitney U-test.Comparisons among multiple groups were analyzed by one-way ANOVA test followed by a post-hoc Tukey-Kramer test, or Kruskal-Wallis test followed by a post-hoc Steel-Dwass test.All the numerical data are presented as the mean ± SEM and p-values less than 0.05 were considered statistically significant.

Results
PlexinD1 signaling specifically suppresses dendritic branching in the proximal domain of the apical dendrite of newborn granule cells in the postnatal OB In the GCL of the postnatal OB, while Sema3E is expressed in mature granule cells, PlexinD1 is expressed in the leading process of migrating newborn neurons and is involved in suppressing FLP formation to maintain their immature morphology (Sawada et al., 2018).In this study, we investigated whether Sema3E-PlexinD1 signaling is also involved in the suppression of dendritic branching in the proximal domain of the apical dendrite of newborn granule cells that have terminated their migration.
First, we injected lentivirus encoding Venus into the V-SVZ of Sema3E +/− (control) and Sema3E −/− (Sema3E-KO) mice (Gu et al., 2005) at postnatal day 1 (P1) and analyzed the dendritic morphology of Venus+ granule cells in the GCL at 10 days post infection (dpi) (Figure 1A).The branch number in the proximal domain of the apical dendrite was significantly increased in Sema3E-KO mice compared with control mice (Figures 1B-D), suggesting that Sema3E suppresses dendritic branching in the proximal domain of the apical dendrite of newborn granule cells.
Next, to investigate the expression and role of PlexinD1 in this process, brain sections were stained with a PlexinD1 antibody.The PlexinD1 signal was observed in the proximal domain of the apical dendrite of differentiating granule cells (Supplementary Figure S1A).For functional analyses of PlexinD1, we used lentivirus encoding tdTomato and Cre recombinase.The decrease of PlexinD1 proteins by these vectors was confirmed in primary neuronal cultures (Figures 1E,F).To examine the function of PlexinD1 in vivo, we injected these lentiviral vectors into the V-SVZ of PlexinD1 +/flox (control) and PlexinD1 flox/flox (PlexinD1-cKO) mice (Zhang et al., 2009) at P1 and analyzed the dendritic morphology of tdTomato+ granule cells (Figure 1G).At 10 dpi, similar to the phenotype observed in Sema3E-KO mice, the branch number in the proximal domain of the apical dendrite was significantly increased by PlexinD1 deficiency (Figures 1H-J).Moreover, at 28 dpi, when granule cells morphologically  mature (Petreanu and Alvarez-Buylla, 2002), the whole dendritic morphology of tdTomato+ granule cells was traced (Figure 1K).We found that the dendritic branch number in the proximal but not the distal or basal domain in tdTomato+ granule cells was significantly increased in PlexinD1-cKO mice compared to that in control mice (Figures 1L-P).The dendritic length of the apical and basal dendrites was not significantly different between control and PlexinD1-cKO mice (Supplementary Figures S1B-E).Taken together, these results suggest that Sema3E-PlexinD1 signaling is specifically involved in the suppression of dendritic branching in the proximal domain of the apical dendrite of newborn granule cells in the postnatal OB.
PlexinD1's RBD is involved in the PlexinD1-mediated suppression of dendritic branching at proximal domain of apical dendrites in newborn granule cells The intracellular domain of Plexins has a Rho binding domain (RBD), which regulates cytoskeletal dynamics through binding to various Rho family small GTPases (Gay et al., 2011) (Figure 2A).To study the role of PlexinD1 and its RBD in dendritic development of newborn granule cells, we generated lentiviral vectors encoding PlexinD1 or its deletion mutant of RBD (PlexinD1ΔRBD), and confirmed their overexpression in primary neuronal cultures (Figures 2A-D).To examine the effects of PlexinD1 overexpression on the dendritic branching in vivo, we injected these lentiviruses into the V-SVZ and analyzed the dendritic morphology of infected granule cells in the OB at 10 dpi (Figure 2E).Overexpression of PlexinD1 decreased the number of dendritic branches in the proximal domain of the apical dendrite (Figures 2F,G,I).This effect was partially diminished when lentiviruses encoding PlexinD1ΔRBD were injected (Figures 2H,I).These results suggest that RBD is involved in the suppression of dendritic branching in the proximal domain of the apical dendrites of newborn granule cells by PlexinD1 overexpression.

Rhoj is expressed in migrating and differentiating granule cells, and suppresses dendritic branching of proximal domain of their apical dendrites
RhoJ, a member of Rho family small GTPases, is highly expressed in vascular endothelial cells and involved in Sema3E's repulsive effect by directly binding to the PlexinD1RBD to promote F-actin depolymerization (Fukushima et al., 2011(Fukushima et al., , 2020)).In the developing retina, RhoJ is expressed in not only vascular endothelial cells but also neurons (Fukushima et al., 2020).However, the function of RhoJ in the CNS remains unknown.Since RBD is involved in the PlexinD1-induced suppression of dendritic branching in the proximal domain of apical dendrites (Figure 2), we hypothesized that RhoJ contributes to this process.
First, to identify RhoJ-expressing cells in the V-SVZ-OB pathway, we analyzed GFP-expressing cell types in RhoJ +/GFP mice (Kim et al., 2014), in which EGFP replaces exon 1 of the RhoJ gene and is expressed under the control of the endogenous RhoJ promoter.EGFP was strongly expressed in the CD31+ vascular endothelial cells not only in the retina (Fukushima et al., 2020) but also in the brain (Figure 3A).We found that EGFP was not expressed in Dcx + cells in the V-SVZ, but was expressed in a subset of Dcx + and NeuN+ cells in the core and GCL of the OB (Figures 3B-E).These results suggest that newborn neurons express RhoJ during migration and maturation in the postnatal OB.
Next, to investigate the effect of forced expression of RhoJ on the dendritogenesis of newborn granule cells, plasmids encoding an EGFP-fused RhoJ were introduced into the V-SVZ by in vivo electroporation, and the dendritic morphology of labeled granule cells in the GCL was analyzed at 10 dpi (Figure 3F).The forced expression of RhoJ in granule cells significantly suppressed both their dendritic branching in the distal and proximal domains (Figures 3G-J) and dendritic length in the distal domain (Figure 3G; control, 340.0 ± 28.5 μm; RhoJ, 224.5 ± 29.0 μm; p = 0.0063, unpaired t-test).These results suggest that RhoJ overexpression has an inhibitory effect on dendritic branching and outgrowth in vivo.
Finally, to study the function of RhoJ on dendritogenesis of newborn granule cells, we introduced plasmids encoding DsRed into the V-SVZ of RhoJ +/GFP (control) and RhoJ GFP/GFP (RhoJ-KO) mice by in vivo electroporation and analyzed their dendritic morphology (Figure 3K).The dendritic branch number in the proximal but not distal or basal domain in DsRed+ granule cells was significantly increased in RhoJ-KO mice (Figures 3L-O).The dendritic length of the apical and basal dendrites was not significantly different between control and RhoJ-KO mice (Supplementary Figures S1B,F-H).Furthermore, the increase of dendritic branching in the proximal domain in RhoJ-KO neurons was partially diminished by PlexinD1 overexpression (Figures 3L-O).Together, these results suggest that RhoJ is specifically involved in the suppression of dendritic branching in the proximal domain of the apical dendrite of newborn granule cells in the postnatal OB.

Discussion
In this study, we demonstrated that Sema3E-PlexinD1 signaling is involved in the domain-specific dendritic branching of newborn granule cells in the postnatal OB.Furthermore, we showed that RhoJ, a small GTPase that directly binds to PlexinD1, is expressed in newborn granule cells during migration and maturation in the postnatal OB and involved in the proximal domain-specific inhibition of dendritic branching.These results provide new insights into the dendritogenesis of newborn neurons in the postnatal brain.
In the postnatal OB, granule cells receive centrifugal inputs in the proximal domain of the apical dendrite (Kaneko et al., 2006;Whitman and Greer, 2007;Yokoyama et al., 2011;Komano-Inoue et al., 2014).The distal domain of the apical dendrite undergoes many branchings, whereas the proximal domain maintains an unbranched state  Frontiers in Neuroscience 06 frontiersin.org(Shepherd et al., 2004).Excessive centrifugal input to newborn granule cells may result in strong GABA-mediated feedback to mitral/tufted cells and excessively downregulated neuronal transmission from the OB toward the olfactory higher centers.Therefore, granule cells need a mechanism to inhibit dendritic branching in the proximal domain of the apical dendrite to prevent them from receiving too much centrifugal input.Our results indicate that PlexinD1 signaling not only determines the final positioning of newborn neurons by suppressing FLP formation during migration (Sawada et al., 2018), but also maintains the proximal domain of the apical dendrite unbranched after migration termination, thereby ensuring proper reception of centrifugal inputs.Previous studies suggested that olfactory input (Saghatelyan et al., 2005) and 5 T4 (Yoshihara et al., 2012) promote dendritic branching in the distal domain of granule cells.In contrast, the mechanism inhibiting their dendritic branching in the proximal domain has remained unknown.In this study, PlexinD1-or RhoJ-deficiency specifically enhanced dendritic branching in the proximal domain of the apical dendrite without affecting overall dendritic morphology or length in newborn granule cells.In newborn neurons migrating in the OB, PlexinD1 signaling suppresses FLP formation without affecting leading process formation (Sawada et al., 2018).Moreover, newborn neurons start to express RhoJ during their migration and maturation in the OB.Thus, our results suggest that Sema3E-PlexinD1-RhoJ signaling is involved in the proximal domain-specific suppression of branching both in the leading process during migration (Sawada et al., 2018) and in the apical dendrite after migration termination (this study).
FLPs are the cellular protrusions that link termination of migration and initiation of dendritogenesis in newborn neurons in the postnatal OB, and the timing of their formation is regulated by local PlexinD1 endocytosis during the process of migration termination (Sawada et al., 2018).Rac1 is locally activated in the proximal domain of the leading process to form FLPs (Sawada et al., 2018).On the other hand, this study showed that RhoJ suppresses dendritic branching in the proximal domain of apical dendrites.Since RhoJ, which is activated by  Sema3E-PlexinD1 signaling, is localized in the perinuclear region and involved in F-actin depolymerization in vascular endothelial cells (Fukushima et al., 2020), it is possible that RhoJ is also localized in the proximal leading process and involved in the proximal domain-specific suppression of branching in migrating and maturing newborn neurons.
Considering that PlexinD1 overexpression effects are partially diminished by RBD deletion (Figure 2I) or RhoJ deficiency (Figure 3N), our findings suggest that in newborn neurons, PlexinD1 activates RhoJ and inactivates Rac1 to suppress FLP formation during maintenance of migration, and its downregulation by local endocytosis relieves Rac1 inhibition and decreases RhoJ activation to promote FLP formation during termination of migration (Figure 3P).Thus, we propose that the PlexinD1-mediated regulation of the two opposing Rho GTPases has an advantage in the efficient transition from maintenance to termination of migration by controlling their timing of the branching of newborn neurons.

Conclusion
In this study, we showed a domain-specific mechanism for dendritic development in newborn granule cells in the postnatal OB.These results contribute to a better understanding of the development and function of newborn neurons in the postnatal OB circuits.