Endosome-associated Rab GTPases control distinct aspects of neural circuit assembly

Reviewer #1 (Public review):

Summary:

Dong et al. present an in-depth analysis of mutant phenotypes of the Rab GTPases Rab5, Rab7, and Rab11 in Drosophila second-order olfactory neuron development. These three Rab GTPases are amongst the best-characterized Rab GTPases in eukaryotes and have been associated with major roles in early endosomes, late endosomes, and recycling endosomes, respectively. All three have been investigated in Drosophila neurons before; however, this study provides the most detailed characterization and comparison of mutant phenotypes for axonal and dendritic development of fly projection neurons to date. In addition, the authors provide excellent high-resolution data on the distribution of each of the three Rabs in developmental analyses.

Strengths:

The strength of the work lies in the detailed characterization and comparison of the different Rab mutants on projection neuron development, with clear differences for the three Rabs and by inference for the early, late, and recycling endosomal functions executed by each.

Weaknesses:

Some weakness derives from the fact that Rab5, Rab7, and Rab11 are, as acknowledged by the authors, somewhat pleiotropic, and their actual roles in projection neuron development are not addressed beyond the characterization of (mostly adult) mutant phenotypes and developmental expression.

Reviewer #2 (Public review):

Summary:

This study by Dong et al. characterizes the roles of highly-expressed Rab GTPases Rab5, Rab7, and Rab11 in the development and wiring of olfactory projection neurons in Drosophila. This convincing descriptive study provides complementary approaches to Rab expression and localization profiling, conventional dominant-negative mutants, and clonal loss-of-function mutants to address the roles of different endosomal trafficking pathways across circuit development. They show distinct distributions and phenotypes for different Rabs. Overall, the study sets the stage for future mechanistic studies in this well-defined central neuron.

Strengths:

Beautiful imaging in central neurons demonstrates differential roles of 3 key Rab proteins in neuronal morphogenesis, as well as interesting patterns of subcellular endosome distribution. These descriptions will be critical for future mechanistic studies. The cell biology is well-written and explanatory, very accessible to a wide audience without sacrificing technical accuracy.

Weaknesses:

The Drosophila manipulations require more explanation in the main text to reach a wide audience.

Reviewer #3 (Public review):

Summary:

The authors aimed at a comprehensive phenotypic characterization of the roles of all Rab proteins expressed in PN neurons in the developing Drosophila olfactory system. Important data are shown for a number of these Rabs with small/no phenotypes (in the Supplements) as well as the main endosomal Rabs, Rab5, 7, and 11 in the main figures.

Strengths:

The mosaic analysis is a great strength, allowing visualization of small clones or single neuron morphologies. This also allows some assessment of the cell autonomy of the observed phenotypes. The impact of the work lies in the comprehensiveness of the experiments. The rescue experiments are a strength.

Weaknesses:

The main weakness is that the experiments do not address the mechanisms that are affected by the loss of these Rab proteins, especially in terms of the most significant cargos. The insights thus do not extend far beyond what is already known from other work in many systems.

Author response:

Reviewer #1 (Public review):

Summary:

Dong et al. present an in-depth analysis of mutant phenotypes of the Rab GTPases Rab5, Rab7, and Rab11 in Drosophila second-order olfactory neuron development. These three Rab GTPases are amongst the best-characterized Rab GTPases in eukaryotes and have been associated with major roles in early endosomes, late endosomes, and recycling endosomes, respectively. All three have been investigated in Drosophila neurons before; however, this study provides the most detailed characterization and comparison of mutant phenotypes for axonal and dendritic development of fly projection neurons to date. In addition, the authors provide excellent high-resolution data on the distribution of each of the three Rabs in developmental analyses.

Strengths:

The strength of the work lies in the detailed characterization and comparison of the different Rab mutants on projection neuron development, with clear differences for the three Rabs and by inference for the early, late, and recycling endosomal functions executed by each.

We would like to thank Reviewer #1 for their appreciation of our characterization of distinct Rab mutants.

Weaknesses:

Some weakness derives from the fact that Rab5, Rab7, and Rab11 are, as acknowledged by the authors, somewhat pleiotropic, and their actual roles in projection neuron development are not addressed beyond the characterization of (mostly adult) mutant phenotypes and developmental expression.

Prior to mid-pupal stage (around 48 hours after puparium formation), glomeruli in the antennal lobe have not yet assumed their stereotyped positions, which complicates analyses and interpretation; thus, many of our analyses are conducted at the adult stage. For Rab11 mutants we did perform many developmental analyses to evaluate the origins of the axonal development (Figure 6—figure supplement 1) and dendrite elaboration phenotypes (Figure 5 J–L) we observed at the adult stage. We realize that the development axonal analyses are in supplemental material where they could be missed. Given the reviewer’s comments, we will move these data to the main figures.

Further, we will extend our Rab5 analyses to evaluate the function of this protein during development in experiments we will add to the revised manuscript.

Reviewer #2 (Public review):

Summary:

This study by Dong et al. characterizes the roles of highly-expressed Rab GTPases Rab5, Rab7, and Rab11 in the development and wiring of olfactory projection neurons in Drosophila. This convincing descriptive study provides complementary approaches to Rab expression and localization profiling, conventional dominant-negative mutants, and clonal loss-of-function mutants to address the roles of different endosomal trafficking pathways across circuit development. They show distinct distributions and phenotypes for different Rabs. Overall, the study sets the stage for future mechanistic studies in this well-defined central neuron.

We appreciate Reviewer #2’s analysis of our work and thank them for their suggestions to improve the clarity of our manuscript.

Strengths:

Beautiful imaging in central neurons demonstrates differential roles of 3 key Rab proteins in neuronal morphogenesis, as well as interesting patterns of subcellular endosome distribution. These descriptions will be critical for future mechanistic studies. The cell biology is well-written and explanatory, very accessible to a wide audience without sacrificing technical accuracy.

Weaknesses:

The Drosophila manipulations require more explanation in the main text to reach a wide audience.

In our revised manuscript we will clarify the fly-specific manipulations and terminology to make our work more accessible to a broader audience.

Reviewer #3 (Public review):

Summary:

The authors aimed at a comprehensive phenotypic characterization of the roles of all Rab proteins expressed in PN neurons in the developing Drosophila olfactory system. Important data are shown for a number of these Rabs with small/no phenotypes (in the Supplements) as well as the main endosomal Rabs, Rab5, 7, and 11 in the main figures.

We appreciate Reviewer #3’s assessment and appreciation of our work.

Strengths:

The mosaic analysis is a great strength, allowing visualization of small clones or single neuron morphologies. This also allows some assessment of the cell autonomy of the observed phenotypes. The impact of the work lies in the comprehensiveness of the experiments. The rescue experiments are a strength.

Weaknesses:

The main weakness is that the experiments do not address the mechanisms that are affected by the loss of these Rab proteins, especially in terms of the most significant cargos. The insights thus do not extend far beyond what is already known from other work in many systems.

We understand this critique and are also interested in the specific cargos regulated by each Rab during development. We attempted to use antibodies to evaluate changes in cell-surface protein localization in response to disrupting individual Rabs but were unable to reliably distinguish(?) shifts in association with specific endosomal compartments. Many available antibodies label cell-surface proteins expressed in antennal lobe cells beyond projection neurons (such as olfactory receptor neurons, glia, or local interneurons) which complicates analyses. Further, although we have produced multiple ‘flp-on’ tags for PN cell-surface proteins, they cannot be used with the MARCM system. This prevents us from simultaneously perturbing individual Rabs and tracking corresponding changes in surface-protein localization with single cell resolution. Moreover, for proteins that are not highly endocytosed, it is difficult to separate plasma-membrane from endosomal localization, and we currently do not know which cell-surface proteins are most robustly endocytosed. Thus, while we share the reviewer’s interest in identifying candidate cargos, technological limitations make it difficult to achieve this goal within the scope of the current study.