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Atypical cadherin, Fat2, regulates axon terminal organization in the developing Drosophila olfactory receptor neurons

June 2024
iScience 27(7):110340

DOI:10.1016/j.isci.2024.110340

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Authors:

Chun Yeung

New York Institute of Technology

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The process of how neuronal identity confers circuit organization is intricately related to the mechanisms underlying neurodegeneration and neuropathologies. Modeling this process, the olfactory circuit builds a functionally organized topographic map, which requires widely dispersed neurons with the same identity to converge their axons into one a class-specific neuropil, a glomerulus. In this article, we identified Fat2 (also known as Kugelei) as a regulator of class-specific axon organization. In fat2 mutants, axons belonging to the highest fat2-expressing classes present with a more severe phenotype compared to axons belonging to low fat2-expressing classes. In extreme cases, mutations lead to neural degeneration. Lastly, we found that Fat2 intracellular domain interactors, APC1/2 (Adenomatous polyposis coli) and dop (Drop out), likely orchestrate the cytoskeletal remodeling required for axon condensation. Altogether, we provide a potential mechanism for how cell surface proteins’ regulation of cytoskeletal remodeling necessitates identity specific circuit organization.

Class-specific fat2 expression levels predict severity of fat2 null mutant phenotypes

…

Fat2 intracellular domain mutant, with intact extracellular domain, phenocopies fat2 null mutant glomerular phenotype in a class-specific manner (A-D) Representative z stack composite images of single antenna lobes. Only panel (A) is stained with anti-GFP and anti-N-Cadherin. All other images were unstained. In(B-D) each ORN class is labeled using the same OrX-GAL4 as Figure 3 but instead drives the expression of 10xUAS-RFP. fat2 DICD brains were from flies homozygous for the fat2 DICD allele. (A 0 -D 0 ) Bar graph quantification of brains with glomerular disruptions (red bar). Fisher exact test for (A 0 ) and (D 0 ), separately, resulted in ****p <0 .0001 and ***p = 0.0009. Scale bar shows 26 mm. See also Figures S2, S7, S8, S11, and S13.

…

Local interneuron expression of fat2 does not contribute to ORN glomerular organization (A) Representative unstained z stack image of both antennal lobes where GH146-GAL4-labeled PNs in magenta and Fat2-3xGFP fusion protein in green. White arrows represent Fat2+ cell bodies that do not colocalize with PN cell bodies. (B) Similar to panel A, Fat2-3xGFP fusion protein is colorized green and 499-GAL4 positive LNs are colorized magenta. White arrows represent Fat2+ cell bodies that colocalize with LN cell bodies. (C) Representative z stack composite images of control antennal lobes and LN-specific knockdown of fat2 (499-GAL4, UAS-fat2 RNAi) stained with anti-NCadherin (magenta) and Or47b axons projecting into corresponding glomeruli (Green) stained with anti-GFP (Or47b-GAL4, UAS-mCD8GFP). Control brains presented with 1/18 brain with disruption (6% phenotypic penetrance), 499-GAL4 UAS-fat2 RNAi presented with 1/16 brain with disruptions (5.75% phenotypic penetrance). Scale bar shows 26 mm. See also Figures S9 and S10.

…

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iScience

Article

Atypical cadherin, Fat2, regulates axon terminal

organization in the developing Drosophila

olfactory receptor neurons

Khanh M. Vien,

Qichen Duan,

Chun Yeung, Scott

Barish, Pelin

Cayirlioglu Volkan

pelin.volkan@duke.edu

Highlights

ORN expression of Fat2-

ICD is necessary for class-

speciﬁc glomerular

organization

Fat2-ICD genetically

interacts with APC for

appropriate glomerular

morphology

Fat2 shows differential

expression and functional

requirement across ORN

classes

Vien et al., iScience 27, 110340

July 19, 2024 ª2024 The

Author(s). Published by Elsevier

Inc.

https://doi.org/10.1016/

j.isci.2024.110340

OPEN ACCESS

iScience

Article

Atypical cadherin, Fat2, regulates axon

terminal organization in the developing

Drosophila olfactory receptor neurons

Khanh M. Vien,

Qichen Duan,

Chun Yeung,

Scott Barish,

1,3

and Pelin Cayirlioglu Volkan

1,2,4,

SUMMARY

The process of how neuronal identity confers circuit organization is intricately related to the mechanisms

underlying neurodegeneration and neuropathologies. Modeling this process, the olfactory circuit builds a

functionally organized topographic map, which requires widely dispersed neurons with the same identity

to converge their axons into one a class-speciﬁc neuropil, a glomerulus. In this article, we identiﬁed Fat2

(also known as Kugelei) as a regulator of class-speciﬁc axon organization. In fat2 mutants, axons belonging

to the highest fat2-expressing classes present with a more severe phenotype compared to axons

belonging to low fat2-expressing classes. In extreme cases, mutations lead to neural degeneration. Lastly,

we found that Fat2 intracellular domain interactors, APC1/2 (Adenomatous polyposis coli) and dop (Drop

out), likely orchestrate the cytoskeletal remodeling required for axon condensation. Altogether, we pro-

vide a potential mechanism for how cell surface proteins’ regulation of cytoskeletal remodeling necessi-

tates identity speciﬁc circuit organization.

INTRODUCTION

In both insects and mammals, odor detection relies upon the topographic map created by diverse classes of olfactory neurons that organize

their axons to converge in a class-speciﬁc manner within the antennal lobe in ﬂies, or the mammalian central brain’s olfactory bulb.

1,2

Given its

intricate association with neurodegeneration and neuronal dysfunction, decoding the mechanisms underlying how diverse classes of neurons

organize their axons represents a signiﬁcant pursuit in the ﬁeld of neurobiology. In ﬂies, olfactory receptor neuron (ORN) classes are deﬁned

by the selective co-expression of generally one to three odorant receptor (and/or ionotropic receptor) combinations.

ORNs belonging to the

same class (one of the around ﬁfty possible classes) converge their axons to a single neuropil, called a glomerulus, and synapse with second-

order projection neurons (PNs). This organizational logic is known colloquially as the ‘‘one olfactory receptor, one glomerulus’’ rule. Consid-

ering the stereotyped shapes, boundaries, and positionings of the glomerular map, each ORN class likely has a unique set of instructions that

is shared across neurons belonging to that class. Previously, we and others have shown that the disruption of several cell surface molecules,

such as Cadherin family proteins, can disrupt class-speciﬁc glomerular organization.

4,5

Leveraging the ﬁeld’s detailed understanding of the

Drosophila olfactory circuit architecture and development, we can reveal these cell surface molecules’ mechanisms of action responsible for

the regulation of large-scale tissue organization.

The adult Drosophila olfactory system is an excellent model for studying neurodevelopment because it utilizes biological processes docu-

mented across most developing multi-cellular neural networks, such as direct the behaviors of neuronal processes via the temporal and

spatial regulation of various cell surface molecules. During metamorphosis, adult ORNs are born within the eye-antennal imaginal disc

and all 1200 ORNs per antenna must extend their axons great distances into the central brain.

Around 16–18 hAPF (hours after pupal for-

mation) ORN axons reach the antennal lobe

and begin to snake along the antennal lobe surface with transient exploratory branches

surveying the environment for their target glomerulus.

Between 20 hAPF and 40 hAPF, the exploratory axons target a glomerulus,

9,10

interact

with PNs and local interneurons (LNs),

11,12

and then begin to simultaneously repel from adjacent glomeruli while converging with class-spe-

ciﬁc axons to further accentuate proto-glomerular boundaries.

4,13

The mechanisms critical to the stabilization of these transient axonal

branches are unknown, and likely involve proteins that link chemoafﬁnity receptor activity to cytoskeletal regulators. The litany of cell surface

proteins implicated in olfactory system development, such as Semaphorins, DSCAM, Tenascins/Teneurins and Toll receptors, suggests that

ORN class-speciﬁc cell surface codes are the main drivers of this topographic circuit assembly.

9,11,13–18

Though there are several examples of

cell surface molecules regulating ORN class-speciﬁc axon organization, it is still unclear when and how ORN class-speciﬁc axon convergence

occurs during olfactory circuit development.

4,15,16,19

Department of Biology, Duke University, Durham, NC 27708, USA

Department of Neurobiology, Duke University Medical Center, Durham, NC 27710, USA

Present address: Colossal Biosciences, Dallas, TX 75247, USA

Lead contact

*Correspondence: pelin.volkan@duke.edu

https://doi.org/10.1016/j.isci.2024.110340

iScience 27, 110340, July 19, 2024 ª2024 The Author(s). Published by Elsevier Inc.

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

OPEN ACCESS

Given the importance of cell surface combinatorial codes, identifying novel cell surface proteins functionally required for olfactory circuit

assembly is critical to decrypting the ‘‘rosetta stone’’ needed to translate cell surface signatures into predictable discrete cellular processes.

Given the close association between the evolution of Cadherin family proteins and the emergence of the synapse,

it is unsurprising that

members of the Cadherin family (N-cadherin and protocadherins) have been shown to regulate aspects of neuronal organization,

and spe-

ciﬁcally insect and mammalian olfactory circuit development.

4,22

While highly expressed in the developing olfactory circuit and considered

most closely related to the common ancestor of modern cadherins,

Fat cadherins and their role in neuronal organization have not been

investigated in the developing olfactory system. Fat cadherins, the largest members in the Cadherin family, have 34–36 cadherin repeats

in their extracellular domain,

4,24

which is more than twice the length of Drosophila N-Cadherin, a well-known organizer of protoglomerular

organization, which has 17 cadherin repeats. Due to the size of Fat2, a major obstacle to studying the in vivo function of this protein is the

difﬁcult and cumbersome molecular biology required to generate functional genetic tools. Recent generation of whole animal null mutants,

domain-speciﬁc mutants, and GFP-tagged Fat2 direct fusion protein, allows us to functionally characterize this highly conserved protein in a

neuronal context.

25–27

Fat cadherins are an appealing target due to their remarkable sequence conservation and their highly speciﬁc expression in the devel-

oping olfactory centers across the animal kingdom.

28,29

Notably, it has been proposed that the contemporary protocadherin

ectodomains may have arisen from the gradual reduction of N-terminal cadherin domains in the ancestral Fat protein, further

underscoring the signiﬁcance of studying these proteins.

Compared to the four Fat cadherins in mammals, Drosophila has two Fat cad-

herins, Fat and Fat2 (also known as Kugelei, or Fat-like cadherin), which share striking homology to Fat4 and Fat1/3 in humans, respec-

tively.

It is important to note that the intracellular domains of Drosophila Fat and Fat2 exhibit substantial divergence, characterized

by distinct sets of interacting partners that do not overlap. These ﬁndings strongly indicate that these two proteins serve distinct

functional roles in biological processes. Fat2, and its vertebrate orthologues Fat1 and Fat3, has been shown, using RNA-seq, qRT-PCR,

and in situ hybridization, to be most highly expressed within the developing olfactory bulb and has been associated with several

human neuropathologies.

30–32

The role of Fat2 has been revealed in several mammalian sensory circuits (such as retinal development

and cochlear morphogenesis) and Drosophila tissue morphogenesis, yet the function of Fat2 in olfactory circuit development is un-

known.

26,27,33,34

Here we demonstrate that the Drosophila Fat2 protein organizes glomerular architecture by promoting class-speciﬁc

ORN axon bundle convergence. In fat2 mutants, we observed that many ORN classes displayed fragmented glomeruli with

varying severity. By utilizing intersectional genetic approaches, we have identiﬁed that Fat2 expression varies across ORN classes during

mid-pupal development and is expressed by a unique subpopulation of antennal lobe local interneurons. We have further characterized

Fat2 protein localization to axon terminals and provided evidence that the intracellular signaling domain is necessary for its function in

glomerular organization. Finally, we present evidence suggesting that Fat2 regulates axon behavior by interacting with cytoskeletal remod-

eling proteins.

RESULTS

Genetic disruptions of Fat2 result in abnormal glomerular organization

Fat3 and Fat1, mammalian orthologues of Drosophila Fat2, are documented to be speciﬁcally and highly expressed in the

olfactory bulbs of developing rats and mice.

31,35,36

Consistent with the expression data, several studies in mice show disruptions in

Fat3/Fat1 result in a faulty neural organization, in both auditory and visual sensory systems.

37,38

In order to derive the direct impact of

Fat2 disruptions on ﬂy olfactory circuit organization, we restrictively knocked down fat2 expression using the ORN-speciﬁc driver

pebbled-GAL4 (peb-GAL4) to drive fat2 RNAi. To assess the effect of fat2 knockdown on class-speciﬁc glomerular architecture, we visu-

alized one of the largest and most easily identiﬁable glomeruli innervated by Or47b ORNs using Or47b-CD2. We found that compared to

peb-GAL4 only controls (13% aberrant glomerular morphology, n= 52) or UAS-fat2 RNAi only controls (10%, n= 54), experimental brains

(37%, n=60) resulted in Or47b ORNs-innervated VA1v glomerulus becoming morphologically deformed, including glomerular rotation,

shape change, and/or fragmentation (Figure 1B; Figure S1). While the parallels between Fat2 and its mammalian counterparts, Fat3

and Fat1, have been implicated in neural circuit development, our study offers direct insights into the consequences of Fat2 disruption

in the olfactory system.

We set out to investigate whether the role Fat2 plays on ORN neuropil organization can also be applied to other glomeruli. To test this, and

corroborate our RNAi knockdown results, we visualized four glomeruli (Or47a, Or23a, Or47b, and Gr21a) in whole animal mutants with various

fat2 null alleles. With this paradigm, we were able to compare the morphology of each glomerulus in control brains (11%, n= 17), homozygous

Fat2 null mutant (fat2

N103-2

) (77%, n= 26), and trans-heterozygous mutant for two separately generated fat2 null alleles (fat2

N103-2

/fat2

58D

)

(37%, n= 32) (Figures 1D–1G; Figure S1). fat2

N103-2

is a null allele containing a premature stop codon at position 3718,

and fat2

58D

is a

null allele that contains a deletion for the amino acids 1–687 including the start codon.

In these whole animal mutants, we observed

three glomeruli targeted by Or47a, Or23a, and Or47b ORNs exhibit organizational disruptions. Supporting our previous data, we found

that whole animal mutants phenocopy ORN-speciﬁc fat2 RNAi knockdown animals in VA1v glomerulus (Figures 1B’, 1D00 , and 1F0). We

found a variety of morphological disruptions to each of the three glomeruli (Figure S2). For clarity, we have designated disruptions to any

glomeruli as a ‘‘disrupted’’ brain and included the more detailed quantiﬁcation in the supplementals (Figure S1). We interpreted the

presence of ectopic glomeruli and ectopic axon projection as possibly a result of a loss of class-speciﬁc axon aggregation. Heterozygous

Fat2 null mutants did not exhibit any glomerular disruptions (Figure 1D’).

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2iScience 27, 110340, July 19, 2024

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To summarize, both ORN-speciﬁc RNAi knockdown of fat2, as well as whole animal mutants for homozygous and transallelic fat2 null

alleles result in disruptions to the organization of multiple glomeruli across the antenna lobe. Consistent with our ﬁnding, a previous

study also reported that pan-neuronal knockdown of fat2 resulted in a disrupted Drosophila olfactory circuit architecture.

The observed

deformations in the olfactory circuitry emphasize the signiﬁcance of Fat2 in maintaining the integrity of class-speciﬁc glomerular

architecture.

Figure 1. Genetic disruptions in fat2 breaks stereotype olfactory glomeruli morphology

(A) Schematic to illustrate the neural architecture of the drosophila olfactory system.

(B-B’’) Representative z stack images of the glomeruli (VA1v) innervated by Or47b axon terminals (green) co-stained with antibody against N-Cadherin (magenta)

to visualize antenna lobe structure. Pan-ORN (peb-GAL4) RNAi knockdown of fat2 results in a split VA1v glomerulus.

(C) Quantiﬁcation of brains with disrupted glomerular morphology for experiment in (B). (No GAL4) Control (n= 52) and (No UAS) Control (n= 54) are not

signiﬁcantly different. Both controls compared to peb>fat2 RNAi (n= 60) using Fisher’s exact test (**p= 0.0027, ***p= 0.0004).

(D-D’’) Representative z stack images of glomeruli innervated by Or47b, Or47a, Or23a, and Gr21a axon terminals (green) co-stained with anti-N-Cadherin

(magenta) in Control (D), heterozygotes for fat2 null (fat2

N103-2

)(D

0), and homozygous fat2 null whole animal mutants (D00 ). Or47b-, Or47a-, Or23a-, and

Gr21a-GAL4, UAS-sytGFP transgenic ﬂies were used. Homozygous fat2 null whole animal mutants break apart the continuous neuropil for Or47b, Or47a, and

Or23a ORNs.

(E) Quantiﬁcation of brains with disrupted glomerular morphology for Control (n= 34), heterozygous for fat2 null (fat2

N103-2

)(n= 41), and homozygous for fat2 null

(n= 26) using Fisher’s exact results in ****p<0.0001.

(F) Trans-heterozygote of two different fat2 null alleles from separate labs phenocopy homozygous fat2

N103-2

mutant phenotype. Or47b, Or47a, Gr21a visualized

via direct fusion to sytGFP and then stained with the same antibodies as (B).

(G) Using Fisher’s exact test to compare Control (n= 23), and fat2

N103-2

/fat2

58D

(n= 32) results in signiﬁcance of **p= 0.0011. Scale bar shows 26 mm.

See also Figures S12 and S13.

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12 iScience 27, 110340, July 19, 2024

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aberrations in glomerular organization. Though not statistically signiﬁcant by deﬁnition, with a p-value of 0.0557 calculated by Fisher’s exact

test, Apc

Apc2

N175K

and fat2

DICD

transheterozygotes (19%, 9/49 brains disrupted) biologically signiﬁcantly rescues the disruptions found in

fat2

DICD

heterozygotes (36%, 16/29 brains disrupted) (Figures 7D and 7F). For Fat2 and Apc/Apc2 to physically interact they would have to be

expressed within the same cells. To investigate whether fat2 and Apc/Apc2 were expressed by the same cells in the olfactory system, we

analyzed available single-cell RNA-seq data to assess the class-speciﬁc expression levels for fat2,Apc, and Apc2 during developmental stages

(Figure S12). The RNA proﬁles suggest that fat2 and apc are broadly expressed across Or-classes during pupal development. Notably, Apc is

expressed at much lower levels compared to fat2 expression levels. Given that Apc and Apc2 encode proteins that play roles in multiple cell

biological processes, it is unclear if Fat2 regulates Apc/Apc2’s role in the regulation of b-catenin/Wingless signaling or the regulation of

microtubule organization. The consistent genetic interaction between Fat2 and Apc/Apc2, strongly suggests that the mechanism of action

for Fat2 relies on Fat2 intracellular domain interactions with intracellular regulators to achieve the desired ORN class-speciﬁc axon behavior.

Additional research is necessary to precisely elucidate the underlying mechanisms, including whether Fat2 modulates Apc/Apc2 activity,

localization, or both. Nonetheless, our study lays a solid foundation for understanding the nuanced role of Fat2 in orchestrating the class-spe-

ciﬁc axon behaviors in olfactory circuitry.

DISCUSSION

The olfactory sensory circuit possesses a distinctive feature present in nearly all neural circuits across the animal kingdom, namely its function-

ally organized topographic map. This map relies on widely dispersed neurons of the same type, which converge their axons into a neuropil

called a glomerulus. The intricate process of how neuronal identity inﬂuences circuit organization is a signiﬁcant area of research in neurobi-

ology due to its close connection to neurodegeneration and neuronal dysfunction. Within the olfactory system, various cell surface proteins,

such as Robo/Slit and Toll receptors, regulate many aspects of circuit organization, including axon guidance and synaptic matching. In this

study, we have discovered an unconventional Cadherin protein called Fat2, which acts as a regulator of axon organization speciﬁc to certain

classes. Fat2 exhibits the highest expression levels during metamorphosis, when the larval olfactory system disintegrates before re-construct-

ing the mature olfactory circuit, with varying levels of Fat2 expression speciﬁc to each class. By studying fat2 null mutants, we observed distinct

effects on each class’s glomerulus, with the most severely affected being those with the highest expression of fat2. Furthermore, we provide

evidence suggesting that the intracellular domain of Fat2 is necessary for its function in organizing the axons of olfactory receptor neurons

(ORNs). Examination of confocal images from early pupal development indicates that Fat2 is crucial for appropriate axon bundle behavior,

which in turn allows for the correct spatial positioning and condensation of class-speciﬁc neuropils. Our research also indicates that the

expression of Fat2 in projection neurons (PNs) and local interneurons (LNs) does not signiﬁcantly contribute to the organization of ORNs.

Finally, we have identiﬁed potential interactors of Fat2’s intracellular domain that regulate ORN axon behavior, likely via coordinating the

necessary cytoskeletal changes, during the early stages of glomerular development. In summary, our ﬁndings lay the groundwork for under-

standing the role of Fat2 in the organization of the olfactory circuit and highlight the critical importance of axon bundle behavior during the

maturation of protoglomeruli.

Glomerular morphology relies on Fat2-dependent ORN axon behaviors

ORN axons arrive at the antennal lobes early in pupal development and undergo a sequence of processes: defasciculating and dispersal

across AL, class-speciﬁc axon convergence to form protoglomerular bundles, followed by an intra-glomerular axon expansion, and ﬁnally sec-

ondary compaction of the axon terminals as glomeruli form boundaries and mature (Figure 5). These axonal behaviors are accompanied by

cell surface protein mediated recognition of ORN-PN-LN terminals and dendrites to allow for proper synapseformation across these varying

components of the antennal lobe glomeruli.

The developmental analysis of fat2 mutants revealed that the major process that seems to be

defective is the ﬁnal stage of glomerular formation, wherein axon terminals collectively migrate as a class-speciﬁc bundle before spreading

throughout the prospective glomerular ﬁeld to establish a cohesive protoglomerulus. Speciﬁcally, this perturbation occurs when the bundle

prematurely halts or when segments of the bundle disengage from the main bundle but nonetheless proceed to execute the remainingaxon

behaviors necessary for glomerular formation. As a consequence, these aberrant bundle behaviors lead to the emergence of gaps between

glomerular ﬁelds that should ideally be contiguous, or a displacement from the stereotyped positioning/orientation. Coincidentally, another

publication pinpointed this time frame (after the axon has innervated its target glomerulus) as a critical transition checkpoint for axon cyto-

skeletal organization. Speciﬁcally, the article found that prior to the innervation of the target glomerulus, axon branches contain predomi-

nantly microtubule networks with occasional actin ﬁlaments found in a minority of branches. However, after the axon innervates the target

glomerulus, the axon will produce interstitial branches to innervate and spread through the glomerulus interior and these interstitial axon

branches all contain both high actin network and microtubule network.

It is conceivable that Fat2 may be playing a role in this cytoskeletal

reorganization post-innervation, possibly by localization APC (which is known to stabilize actin and microtubule networks) to axon terminals.

One might hypothesize that the presence of two cytoskeletal networks leads to a stabilization of the axon branches that when disrupted can

lead to axon instability and aberrant axon behaviors. However, further research is necessary to conﬁrm whether cytoskeletal networks are

differentially organized between control and Fat2 mutants. This phenotype differs from the function of N-Cadherin,

which mostly impacts

the initial elaboration of axon terminals within glomeruli, suggesting the sequential utilization of different Cadherinsin the stepwise progres-

sion of glomerular organization in the antennal lobes.

The pattern of Fat2 expression in the developing Drosophila brain shows very speciﬁc localization largely to ORNs and a subset of LNs, but

not PNs. However, cell type-speciﬁc RNAi knockdowns of fat2 are associated with glomerular defects only when Fat2 function is disrupted in

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iScience 27, 110340, July 19, 2024 13

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ORNs but not in LNs. In whole animal fat2 mutants that exhibit glomerular defects, ORNs still retain their normal PN synaptic targets. These

results suggest that Fat2 mainly functions to mediate ORN axon behaviors rather than establishing connection speciﬁcity with other neuronal

components of olfactory circuit organization within antennal lobe glomeruli. Considering that Fat2’s inﬂuence on ORN axon behaviors is suf-

ﬁcient to perturb glomerular morphology, the subsequent crucial step would involve determining whether Fat2 facilitates ORN-ORN inter-

actions or if it primarily governs the cell-autonomous regulation of ORN axon terminals.

Circuit organization through Fat2 intracellular signaling function is conserved between ﬂies and mammals

Across most neural circuits in any animal, axonal growth cones in developing neurons are highly dynamic and require tight regulation of

the cytoskeleton to drive axon guidance, axon fasciculation, and synapse formation. Utilizing mass spectrometry, several publications have

reported the identiﬁcation of cytoskeletal proteins that interact with the intracellular domain of mammalian orthologue of the Drosophila

Fat2 and Fat3 to regulate neuronal projection behaviors.

38,50

Utilizing RNAi in the background of fat2 null heterozygotes, we found that

axonal phenotypes found in dop, which regulates cytoskeletal organization and membrane growth, RNAi knockdown were enhanced by

reducing the dose of fat2. In contrast, whole animal mutant analysis of Apc/Apc2, which encodes a b-catenin regulators/cytoskeletal an-

chors, resulted in a reduction of glomerular disruptions when fat2 is disrupted in conjunction with Apc/Apc2 disruption. These ﬁndings

indicate that Fat3-ICD interactions in the developing mouse brain are conserved and mirrored with Fat2 in the developing insect brain.

Utilizing Psi-BLAST, we performed an alignment between mouse Fat3 and drosophila Fat2 resulting in 95.2% coverage. We further anno-

tated the mammalian Fat3 sequence for published binding motifs for Kif5, WIRS, and Evh1 (solid red rectangle). The Evh1 binding sites

seem to not be conserved in drosophila Fat2 based on the lack of proline-rich sequence homology,

however several other regions of the

ICD exhibit high sequence similarity (such as the putative PTB-like sequence YHWDxSDW, critical for Fat1 localization

), making them

particularly promising candidates for future domain mutation investigations (Figure S13). Despite the lack of sequence alignment for

WIRS binding sites, both mammalian Fat3

and drosophila Fat2 contain non-conserved WIRS binding sites

which demonstrates that

functionality can be conserved without sequence homology. Even though no reports of the role of Fat3 function in mammalian olfactory

circuit development, multiple sources conﬁrm the high expression of Fat3 in the developing mouse olfactory bulb.

30,31,35,36

Given the intra-

cellular interaction conservation and the high Fat3 expression in the developing olfactory bulb, Fat3-ICD is likely functionally relevant in the

mammalian olfactory circuit development.

In contrast to the effects seen in the Fat3 intracellular domain mutants in mammals, which lead to the formation of an ectopic plexiform

layer in the retina due to individual neurite retraction defects, the ORN class-speciﬁc glomerular splits, though sharing the characteristic of an

ectopic neuropil, are attributed to abnormal axon bundle behavior — behavior not associated with the formation of retinal plexiform

layers.

38,72

This difference in axonal phenotype, suggests Fat2 mechanism of action may not be conserved across sensory systems. It is still

to be determined whether Fat2 mechanism of action is conserved in the same sensory system between mammals and insects. Nonetheless, as

a protein that plays a regulatory role in multiple sensory systems with conserved proteininteractions across species, Fat2, and it’s mammalian

orthologue, is likely instrumental to the organization of human neural circuits.

Aside from circuit organization, Fat2 might play a role in other neuronal processes as well. In mammals, the aberrant neurites failed to form

mature synapses, suggesting defects in axon behaviors are accompanied by defects in synapse formation.

This ﬁnding provides a possible

explanation for the ORN neurodegeneration seen in our fat2 mutants since it is well accepted that disruptions to synaptogenesis/synaptic

function can lead to regressive processes such as neurodegeneration.

Further studies are required to conﬁrm whether ORN synaptic num-

ber is affected in fat2 null animals.

Fat2 function in axon organization versus planar polarity

Previously, Fat2 function in Drosophila was predominantly proposed to be restricted to the regulation of planar cell polarity and collective cell

migration.

25,27,74,75

In this article, we show that Fat2 function in neuronal development and ORN axon terminal organization depends on the

intracellular domain, as fat2 mutants with only the extracellular domain phenocopy null mutants. This contrasts with the planar cell polarity

phenotypes in the egg chambers where the extracellular domain of Fat2 alone can partially rescue the mutant phenotypes observed in

fat2 null mutants. As an atypical Cadherin, Fat2 can mediate cell-cell adhesion, yet membrane proteins that act as ligands or receptors for

Fat2 have not been identiﬁed. Recent studies have implicated putative roles for Sema5c or Lar as potential trans interactors of Fat2 in medi-

ating planar polarity and collective cell migration in the egg chambers

; however, we were unable to detect genetic interactions in glomer-

ular organization using mutations in either gene (data not shown). In addition, mutations in Lar exhibited phenotypes that were distinct from

fat2 mutants (data not shown). Therefore, these results suggest potential differences in mechanisms of Fat2 function in establishing planar cell

polarity versus glomerular organization.

In addition to Fat2, another member of the family, Drosophila Fat cadherin, is a well-established regulator of growth and patterning via the

Hippo pathway.

34,76

Despite their functional similarity, the intracellular domain of Fat and Fat2 are highly divergent and are much more similar

to their mammalian orthologues, Fat4 and Fat3 respectively.

35,77

Furthermore, mammalian Fat1/Fat4 interaction can regulate aspects of neu-

rodevelopment in mice.

Thus, the function of Fat protein family members in supporting neuronal survival might be conserved across protein

family members and species, as fat mutants are associated with ommatidial degeneration in the Drosophila visual system.

It is unclear if Fat

protein function in the nervous system extends outside of the visual system or interacts with Fat2 in axon terminal organization. Another future

avenue to investigate is whether interactions between Fat2 and Fat, or other transmembrane receptors, drive olfactory circuit organization

and neuronal survival.

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14 iScience 27, 110340, July 19, 2024

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Limitations of the study

Our analysis of fat2 expression proﬁle suggests high expression in the local interneurons

during olfactory circuit development. Yet, LN-spe-

ciﬁc knockdown of fat2 did not impact ORN axon organization. Given that the scope of our research focuses on ORN class-speciﬁc regulation

of ORN circuit topography, the role of Fat2 in LN development remains to be investigated. LNs play an integral role in modulating synaptic

sensitivity to appropriately relay inter-glomerular and intra-glomerular signals.

Fat2 regulation of glomeruli morphology and expression in

LNs, a key regulator of olfactory signal processing, raises the fundamental question as to whether fat2 null ﬂies exhibit behavioral defects.

Behavioral analysis and electrophysiological evaluations of fat2 null/ICD mutants or cell type-speciﬁc knockdown of fat2 could elucidate

possible neuro-pathologies related to fat2 dysfunction. This could provide possible mechanistic insights into mutations in mammalian fat3

associated with schizophrenia and bipolar disorder.

While we have shown that the extracellular domain of Fat2 is not sufﬁcient to rescue the fractured class-speciﬁc glomeruli, the extracellular

domain of many cadherin proteins remains integral to their function, and Fat2 is likely no exception.

To directly investigate the role of Fat2

extracellular domain (ECD), we would need to generate a fat2

DECD

mutant with a functional intracellular domain and disrupted extracellular

domain. However, due to the size of the extracellular domain, molecular cloning techniques to precisely remove the ECD have not been suc-

cessful. Similarly, the generation of an insertion mutant, UAS-fat2, is difﬁcult due to the sheer size of the fat2 gene. A GAL4 inducible UAS-fat2

would be a powerful reagent because it would allow us to perform rescue experiments as well as over-expression analysis. Recent advances in

gRNA-based overexpression using modiﬁed transcriptional activator CRISPR proteins can also be useful approaches for rescuing mutant phe-

notypes and overexpression experiments further probing Fat2 function.

Given that Fat2 likely functions during dynamic axon behaviors, available tools for inducible genetic disruptions, including conditional ge-

netic drivers, may not be precise enough to assess Fat2 function. Furthermore, developmental trajectories of ORN classes are highly asyn-

chronous and dependent on the birthdate of a given ORN class,

which means adjacent glomeruli can be at very different developmental

stages at a given time point. Therefore, knocking down fat2 at a speciﬁc time point might affect axon behaviors depending on the develop-

mental phase of each ORN class.

A major limitation of our study is the absence of live single ORN axon imaging of wild-type and fat2 mutants during olfactory system devel-

opment. Our developmental analysis provides snapshots of axonal bundles to investigate general disruptions in axon behavior in fat2 null

mutants (Figure 5), in addition to the spatiotemporal expression proﬁle for Fat2. This coarse perspective may obscure potential roles that

Fat2 plays in more transient axon behaviors, as well as Fat2-dependent variations across single axons belonging to the same ORN class.

To summarize, one general organizational logic found in neural systems across various species is the arrangement of neurons performing

similar functions to project to similar regions of the brain. Our results provide evidence for a conserved Fat2 function in organizing both the

mammalian and insect circuit architecture, thus emphasizing the translational implications

of further research into Fat2 and the Fat cadherin

family. Future live imaging approaches to ORN development will reveal a more dynamic view of the role of Fat2 in ORN axon behaviors. The

results from these studies suggest that in addition to a combinatorial and differentially expressed cell surface receptor code, there likely is a

combinatorial code of intracellular cytoskeletal effectors. Further investigation will be necessary to identify the mechanism behind Fat2-Apc/

Apc2 interactions as well as validate Fat2-Dop interactions in driving ORN axon behaviors during circuit assembly. Once again, live imaging of

Fat2 protein dynamics in conjunction with cytoskeletal markers or intracellular interactors during ORN development, in parallel with biochem-

ical and targeted genetic approaches will establish mechanisms by which Fat2 governs ORN axon behaviors.

STAR+METHODS

Detailed methods are provided in the online version of this paper and include the following:

dKEY RESOURCES TABLE

dRESOURCE AVAILABILITY

BLead contact

BMaterials availability

BData and code availability

dEXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

BGenetics, ﬂy husbandry, and RNAi knockdown

dMETHOD DETAILS

BImmunohistochemistry and image acquisition

B72OK-GAL4 developmental time point analysis

dQUANTIFICATION AND STATISTICAL ANALYSIS

BPhenotypic scoring

BFat2 protein expression analysis and intersectional labeling

BStatistics

BAnalyzing the fat2 expression in the single-cell RNA-seq datasets

SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2024.110340.

OPEN ACCESS

iScience 27, 110340, July 19, 2024 15

iScienc

Article

ACKNOWLEDGMENTS

We are grateful to Sally Horne-Badovinac for sharing fat2 genetic reagents (as well as sema5c and lar mutants), and Christian Dahmann for

sharing fat2

58D

and other fat2-related ﬂy lines. We want to thank Rachel Estrella, Douglas Blackiston, Julie Reynolds, Kavya Raghunathan, La-

tanya Coke, and Volkan lab members for their input on the article. We thank the Bloomington Stock Center for its services.

This study was supported by National Science Foundation award 2006471 to PCV and the National Science Foundation Graduate Research

Fellowship under Grant No. DGE 2139754 to KMV.

AUTHOR CONTRIBUTIONS

Conceptualization: KMV and PCV. Investigation: KMV and CY with QD generating Figure 2A; Figures S11 and S12. SB contributed to the initial

RNAi screen for cell surface receptors that revealed fat2 phenotype in ORNs. Analysis/interpretation of data: KMV, QD, and PCV. Article-orig-

inal draft: KMV, QD, and PCV. Article-revision: KMV, QD, and PCV..

DECLARATION OF INTERESTS

The authors declare that they have no competing interests.

DECLARATION OF AI AND AI-ASSISTED TECHNOLOGIES IN WRITING PROCESS

During the writing of this article the author(s) used ChatGPT3 in order to increase readability and brevity, as well as correct grammatical errors.

After using the tool/service the author(s) reviewed the edited the content as needed and take full responsibility for the contents of the

publication.

Received: December 5, 2023

Revised: April 8, 2024

Accepted: June 19, 2024

Published: June 21, 2024

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18 iScience 27, 110340, July 19, 2024

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STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies

Goat anti-Rabbit IgG (H+L) Highly absorbed

secondary antibody, Alexa Fluor Plus 488

(1:1000)

Invitrogen Ref:A32731 Lot:XH353659; RRID: AB_2633280

Alexa Fluor 546 goat anti-mouse IgG (H+L)

(1:300)

Invitrogen Ref:A11030 Lot:2273717; RRID: AB_2737024

Anti-green ﬂuorescent protein, rabbit IgG

fraction (1:1000)

Invitrogen Ref:A11122 Lot:2477546; RRID: AB_221569

Rat anti-NCAD (1:20) DSHB Cat #: DN-Ex #8; RRID: AB_528121

Mouse anti-rat CD2 (1:500) BioRAD MCA154R Batch No:1611; RRID: AB_321239

Alexa Fluor 647 Goat anti-rat IgG (H+L)

(1:200)

Invitrogen Ref:A21247 Lot:2311802; RRID: AB_141778

Experimental models: Organisms/strains

Drosophila: fat2DICD: fat2DICD-3xGFP

FRT80B

Laboratory of S. Horne-Badovinac

N/A

Drosophila: fat2N103-2: fat2N103-2

FRT80B

Laboratory of S. Horne-Badovinac

N/A

Drosophila: Or47b-GAL4 Or23a-GAL4

Or47a-Gal4 Gr21a-GAL4 UAS-SytGFP

Laboratory of L. Vosshall

N/A

Drosophila: UAS-fat2 RNAi (kug) Bloomington Drosophila Stock Center BDSC 40888

Drosophila: fat2-58D Laboratory of C. Dahmann

N/A

Drosophila: Fat2-3xGFP FRT80B Laboratory of S. Horne- Badovinac

N/A

Drosophila: UAS-mCD8GFP Laboratory of P. Volkan N/A

Drosophila: eyFLP Bloomington Drosophila

Stock Center

BDSC 5580

Drosophila: UAS>STOP>mCD8GFP Bloomington Drosophila

Stock Center

BDSC 30032

Drosophila: Fat2-GAL4 (kug) Bloomington Drosophila

Stock Center

BDSC 77610

Drosophila: Or19a-GAL4 Bloomington Drosophila

Stock Center

BDSC 24617

Drosophila: Or69a-GAL4 Silbering et al.

BDSC 9999

Drosophila: Ir40a-GAL4 Silbering et al.

BDSC 41727

Drosophila: Ir64a-GAL4 Silbering et al.

BDSC 41732

Drosophila: Ir84a-GAL4 Silbering et al.

BDSC 41734

Drosophila: Or22-GAL4 Silbering et al.

N/A

Drosophila: Or88a-mCD8GFP Fishilevich and Vosshall

N/A

Drosophila: 72OK-GAL4 UAS-sytGFP Laboratory of P. Volkan N/A

Drosophila: UAS-sytGFP Laboratory of P. Volkan N/A

Drosophila:10xUAS-mCD8RFP Bloomington Drosophila

Stock Center

BDSC 32219

Drosophila: 499-GAL4 Liou et al.

BDSC 63325

Drosophila: peb-GAL4; Or47b/Or23a/

Gr21a- SytGFP/CYO;TM2/TM6B

Laboratory of P. Volkan N/A

(Continued on next page)

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RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulﬁlled by the lead contact, Pelin Volkan (pelin.

volkan@duke.edu).

Materials availability

This study did not generate new unique reagents.

Data and code availability

Data: Single-cell RNA-seq data was previously published

and the accession number is listed in the key resources table. All data reported in

this paper will be shared by the lead contact upon request.

Code: All original code used to analyze and visualize the single-cell RNA-seq data can be found in https://github.com/volkanlab/Vien_

fat2_iScience2024.

Additional information: Any additional information required to reanalyze the data reported in this paper is available from the lead contact

upon request.

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Genetics, ﬂy husbandry, and RNAi knockdown

Flies were kept at room temperature or 25 degrees. For RNAi experiments, RNAi crosses were reared at 28C and only moved to RT (room

temperature) on the day of dissection. Flies were dissected 3-7 days after eclosion. To restrict background variation, we collected RNAi knock

down only, No-GAL4 control, and genetic interaction ﬂies from the same cross. Males and females are equally included in the sample size

where genetically applicable. All experiments conform to the relevant regulatory standards.

ORN speciﬁc knockdown of fat2 RNAi utilized peb-GAL4; UAS-fat2 RNAi (BDSC:40888); Or47b-mCD8GFP. LN speciﬁc knockdown of fat2

RNAi utilized UAS-kug RNAi (BDSC:40888); Or47b-mCD8GFP/ 449-GAL4 (LN-subtype speciﬁc marker, BDSC:63325).

Continued

REAGENT or RESOURCE SOURCE IDENTIFIER

Drosophila: UAS-Apc RNAi Bloomington Drosophila

Stock Center

BDSC 34869

Drosophila: UAS-CG17528 (zyg8) RNA Bloomington Drosophila

Stock Center

BDSC 42575

Drosophila: UAS-Chb RNAi Bloomington Drosophila

Stock Center

BDSC 35442

Drosophila: UAS-dop RNA Bloomington Drosophila

Stock Center

BDSC 60138

Drosophila: dop1 red1 e1/TM6B, Tb1

(dop1)

Bloomington Drosophila

Stock Center

BDSC 64424

Drosophila: Apc2N175K ApcQ8/TM3 Bloomington Drosophila

Stock Center

BDSC 7211

Deposited data

Single-cell RNA-seq data for developing

ORNs

McLaughlin et al.

GEO: GSE162121

Customized code for single-cell RNA-seq

data analysis and visualization

This paper https://github.com/volkanlab/Vien_fat2_iScience2024

Software and algorithms

GraphPad Prism 9 GraphPad N/A

R 4.3.1 R Core Team https://www.R-project.org/

FV10-ASW Viewer software 04.02 Olympus confocal system software N/A

ChatGPT3 OpenAI https://chatgpt.com/?oai-dm=1

FIJI https://imagej.net/software/ﬁji/ N/A

Adobe Illustrator Adobe https://www.adobe.com/products/illustrator.html

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20 iScience 27, 110340, July 19, 2024

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METHOD DETAILS

Immunohistochemistry and image acquisition

Flies were immobilized by and washed in 70% ethanol for 30 seconds and then moved to .1% PBT (Phosphate Buffer Solution with .1% Triton

X-100). Brains were removed via micro dissection and immediately placed in .4% PFA (paraformaldehyde) onice. Then the brains were ﬁxed in

4% PFA for 15 to 30 minutes at room temperature. Then washed in PBT 3x for 20 minutes each wash.

For immunostaining primary antibodies were used at the following concentrations: rabbit a-GFP 1:1000 (Invitrogen), rat a-Ncad 1:20

(Developmental Studies Hybridoma Bank), mouse a-rat CD2 1:200 (Serotec), Secondary antibodies were used at the following concentrations:

AlexaFluor488 goat a-rabbit 1:1000, AlexaFluor568 goat a-mouse IgG highly cross-adsorbed 1:300, AlexaFluor647 goat a-rat 1:200. Confocal

images taken using an Olympus Fluoview FV1000 at 40x.

Stacks of optical sections 2 mm thick were collected on Olympus Fluoview FV1000 of the entire antennal lobe. Z-stack projections created

on either Fluoview or FIJI and then imported into Adobe Illustrator to generate ﬁgures.

72OK-GAL4 developmental time point analysis

One batch of brains (about 15 brains per batch) between 18 hAPF and 30 hAPF were dissected, imaged, and then visually assessed for

age.

3,4,41

Another batch of brains 24 hAPF to 50 hAPF were dissected, imaged, and then visually assessed for age. Developmental time point

was determined using brain morphology found in both groups indicate brains aged between 24 hAPF to 30 hAPF. Fat2 null mutant brains

presented with the same proportion of phenotypic developing brains as compared to adult mutant brains (about 35% phenotypic penetrance

in developing brains).

QUANTIFICATION AND STATISTICAL ANALYSIS

Phenotypic scoring

For our scoring criteria of brains labeled with a single glomerulus: both antennal lobes are assessed for glomerular disruptions. Each brain is

given a score of 0 (wild type) to 4 (severe disruption). Please see Figure S9 for further clariﬁcation of phenotypes. Severity is determined by

whether the morphology is present in control brains, and if present thenat what percentage. Only brains with a score 3 and/or 4 is considered

disrupted. 0 score is for brains that nearly identically mirrors the most common glomerular morphology present in our control group. 1 or 2

scores are given to glomerular morphologies that are seen in a small minority (less than 15% of control group brains may have this

morphology) of control group brains. The numbers 1 and/or 2 does not indicate an increase in the severity of the phenotype, instead the

two scores are available only when multiple categories of minority morphologies are seen in the control group (such as mild positional rear-

rangement of the glomeruli or mild shape change such as thinning of the glomerulus). Brains marked 1 or 2 are ultimately considered wild-

type/control when we calculate phenotypic penetrance. The scores 3 and 4 are given to glomerular morphologies that are rarely (less than 3%

of control brains have the phenotype), if ever, seen in the control group. Again, two numbers are provided not to indicate differing severity but

to distinguish between categories of morphology such as a drastic positional shift vs. a drastic fragmentation of the glomeruli. Brains with

multiple glomeruli labeled are considered disrupted if at least one glomerulus presents with a score 3 or 4 morphology.

Fat2 protein expression analysis and intersectional labeling

Developmental analysis of Fat2 protein expression was obtained using CRISPR generated Fat2-3xGFP ﬂies graciously gifted to us by the

Horne-Badovinac Lab.

White pupa (staged as 0 hAPF) were collected on a microscope slide, then placed in a closed petri dish with a

damp kim wipe for 24hrs, 48hrs, and 72hrs (may vary G3 hours). Both brains and antenna were dissected, immunostained, and imaged.

To intersectionally label fat2 expressing cells we used cell type speciﬁc FLP/FRT (ey-FLP for ORN speciﬁc labeling, and GH146-FLP for PN

speciﬁc labeling) in conjunction with fat2-GAL4 and UAS>stop>GFP system to intersectional label cell type speciﬁc expression patterns for

fat2. Time point collection, dissection, and imaging performed according to immunohistochemistry methods section.

To categorize Or classes based on ﬂuorescence intensity (Figures 2G and 2H), the brains were imaged at three different laser levels (1%,

3%, and 5%) to visualize dim signals (which might oversaturate bright glomeruli) and bright signals (which could miss dimly ﬂuorescent

glomeruli). After acquiring images and selecting the most representative laser level/brain, we pseudo-colored the Fat2-3xGFP ﬂuorescence

intensity using the ‘‘Thermal’’ LUT in the Olympus Fluoview software. The ‘‘Thermal’’ LUT, also known as ‘‘Spec3’’, assigns the pixel with the

highest intensity throughout the z-projection as 4095 and sets the lowest intensity pixel as 0. It then bins pixel intensity values into three groups

and assigns an intensity value to a corresponding color, with red representing the highest intensity value and black/dark blue representing the

lowest intensity value (background outside the brain). High-expressing glomeruli are categorized as those containing pixels with intensity

levels within the highest one third of the entire intensity dynamic range, visually represented as yellow to red coloration. We observed

that some glomeruli do not maintain the same intensity level throughout, so we decided to categorize based on the highest pixel intensity

present anywhere within each glomerulus. Note that white coloration indicates pixels that have reached the maximum intensity and are likely

oversaturated. Low-expressing glomeruli are categorized as those containing pixels with intensity levels corresponding to the lowest third of

the full intensity range, which corresponds to black to light blue coloration.

To quantify ﬂuorescence intensity, we loaded the entire confocal ﬁles capturing each brain into FIJI (Fiji is just imagej). Then using the Ncad

channel to locate the speciﬁed glomerulus. Or-class selection was based on Or classes with the most stereotyped glomeruli morphology that

was also relatively stable and developed by 50 hAPF. We made a concerted effort to include Or classes in high, medium, and low Fat2

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expressing categories. Once located, we use the free draw tool to outline the glomerulus. Then we identiﬁed how many z-slices the glomer-

ulus spanned and switch to the GFP channel to measure the average ﬂuorescence intensity (within the ROI we’ve drawn) across the appro-

priate z-slices. This process is then repeated for the other antennal lobe. Background ﬂuorescence was established using a square ROI directly

dorsal of the antennal lobe, but outside of the antennal lobe, approximately equal to one-ﬁfth of the area of the entire antennal lobe. Back-

ground was also collected across z slices and from both hemispheres of the brain.

We used PRISM9 to create the aligned scatter plot in Figure S2. Each data point is one measurement, each Or class contains ﬂuorescence

measurements across z-slices as well as from both antennal lobes.

Statistics

As for statistics, P-value was calculated by two-tailed Fisher’s exact test through the built-in functions of GraphPad Prism 9 software. All n

numbers represent biological replicates, each n is an individual ﬂy.

Analyzing the fat2 expression in the single-cell RNA-seq datasets

The annotated single-cell RNA-seq datasets were from McLaughlin et al.

(GSE162121). We extracted the expression values (log

(CPM +1))

of fat2 (kug) across all the cells assigned to ORN classes. We calculated the fraction of fat2-positive cells (deﬁned by log

(CPM +1) R1) and the

mean expression of fat2 of each ORN class at each developmental stage.

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ResearchGate has not been able to resolve any citations for this publication.

The effect of Drosophila attP40 background on the glomerular organization of Or47b olfactory receptor neurons

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Bacteriophage integrase-directed insertion of transgenic constructs into specific genomic loci has been widely used by Drosophila community. The attP40 landing site located on the second chromosome gained popularity because of its high inducible transgene expression levels. Here, unexpectedly, we found that homozygous attP40 chromosome disrupts normal glomerular organization of Or47b olfactory receptor neuron (ORN) class in Drosophila. This effect is not likely to be caused by the loss of function of Msp300, where the attP40 docking site is inserted. Moreover, the attP40 background seems to genetically interact with the second chromosome Or47b-GAL4 driver, which results in a similar glomerular defect. Whether the ORN phenotype is caused by the neighboring genes around Msp300 locus in the presence of attP40-based insertions or a second unknown mutation in the attP40 background remains elusive. Our findings tell a cautionary tale about using this popular transgenic landing site, highlighting the importance of rigorous controls to rule out the attP40 landing site-associated background effects.

Fat2 polarizes the WAVE complex in trans to align cell protrusions for collective migration

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eLife

For a group of cells to migrate together, each cell must couple the polarity of its migratory machinery with that of the other cells in the cohort. Although collective cell migrations are common in animal development, little is known about how protrusions are coherently polarized among groups of migrating epithelial cells. We address this problem in the collective migration of the follicular epithelial cells in Drosophila melanogaster. In this epithelium, the cadherin Fat2 localizes to the trailing edge of each cell and promotes the formation of F-actin-rich protrusions at the leading edge of the cell behind. We show that Fat2 performs this function by acting in trans to concentrate the activity of the WASP family verprolin homolog regulatory complex (WAVE complex) at one long-lived region along each cell's leading edge. Without Fat2, the WAVE complex distribution expands around the cell perimeter and fluctuates over time, and protrusive activity is reduced and unpolarized. We further show that Fat2's influence is very local, with sub-micron-scale puncta of Fat2 enriching the WAVE complex in corresponding puncta just across the leading-trailing cell-cell interface. These findings demonstrate that a trans interaction between Fat2 and the WAVE complex creates stable regions of protrusive activity in each cell and aligns the cells' protrusions across the epithelium for directionally persistent collective migration.

Chemoreceptor co-expression in Drosophila melanogaster olfactory neurons

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Drosophila melanogaster olfactory neurons have long been thought to express only one chemosensory receptor gene family. There are two main olfactory receptor gene families in Drosophila, the odorant receptors (ORs) and the ionotropic receptors (IRs). The dozens of odorant-binding receptors in each family require at least one co-receptor gene in order to function: Orco for ORs, and Ir25a, Ir8a, and Ir76b for IRs. Using a new genetic knock-in strategy, we targeted the four co-receptors representing the main chemosensory families in D. melanogaster (Orco, Ir8a, Ir76b, Ir25a). Co-receptor knock-in expression patterns were verified as accurate representations of endogenous expression. We find extensive overlap in expression among the different co-receptors. As defined by innervation into antennal lobe glomeruli, Ir25a is broadly expressed in 88% of all olfactory sensory neuron classes and is co-expressed in 82% of Orco+ neuron classes, including all neuron classes in the maxillary palp. Orco, Ir8a, and Ir76b expression patterns are also more expansive than previously assumed. Single sensillum recordings from Orco-expressing Ir25a mutant antennal and palpal neurons identify changes in olfactory responses. We also find co-expression of Orco and Ir25a in Drosophila sechellia and Anopheles coluzzii olfactory neurons. These results suggest that co-expression of chemosensory receptors is common in insect olfactory neurons. Together, our data present the first comprehensive map of chemosensory co-receptor expression and reveal their unexpected widespread co-expression in the fly olfactory system.

Fat3 acts through independent cytoskeletal effectors to coordinate asymmetric cell behaviors during polarized circuit assembly

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The polarized flow of information through neural circuits depends on the orderly arrangement of neurons, their processes, and their synapses. This polarity emerges sequentially in development, starting with the directed migration of neuronal precursors, which subsequently elaborate neurites that form synapses in specific locations. In other organs, Fat cadherins sense the position and then polarize individual cells by inducing localized changes in the cytoskeleton that are coordinated across the tissue. Here, we show that the Fat-related protein Fat3 plays an analogous role during the assembly of polarized circuits in the murine retina. We find that the Fat3 intracellular domain (ICD) binds to cytoskeletal regulators and synaptic proteins, with discrete motifs required for amacrine cell migration and neurite retraction. Moreover, upon ICD deletion, extra neurites form but do not make ectopic synapses, suggesting that Fat3 independently regulates synapse localization. Thus, Fat3 serves as a molecular node to coordinate asymmetric cell behaviors across development.

Task-specific roles of local interneurons for inter- and intraglomerular signaling in the insect antennal lobe

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Local interneurons (LNs) mediate complex interactions within the antennal lobe, the primary olfactory system of insects, and the functional analog of the vertebrate olfactory bulb. In the cockroach Periplaneta americana , as in other insects, several types of LNs with distinctive physiological and morphological properties can be defined. Here, we combined whole-cell patch-clamp recordings and Ca ²⁺ imaging of individual LNs to analyze the role of spiking and nonspiking LNs in inter- and intraglomerular signaling during olfactory information processing. Spiking GABAergic LNs reacted to odorant stimulation with a uniform rise in [Ca ²⁺ ] i in the ramifications of all innervated glomeruli. In contrast, in nonspiking LNs, glomerular Ca ²⁺ signals were odorant specific and varied between glomeruli, resulting in distinct, glomerulus-specific tuning curves. The cell type-specific differences in Ca ²⁺ dynamics support the idea that spiking LNs play a primary role in interglomerular signaling, while they assign nonspiking LNs an essential role in intraglomerular signaling.

Single-cell transcriptomes of developing and adult olfactory receptor neurons in Drosophila

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eLife

Recognition of environmental cues is essential for the survival of all organisms. Transcriptional changes occur to enable the generation and function of the neural circuits underlying sensory perception. To gain insight into these changes, we generated single-cell transcriptomes of Drosophila olfactory- (ORNs), thermo-, and hygro-sensory neurons at an early developmental and adult stage using single-cell and single-nucleus RNA sequencing. We discovered that ORNs maintain expression of the same olfactory receptors across development. Using receptor expression and computational approaches, we matched transcriptomic clusters corresponding to anatomically and physiologically defined neuron types across multiple developmental stages. We found that cell-type-specific transcriptomes partly reflected axon trajectory choices in development and sensory modality in adults. We uncovered stage-specific genes that could regulate the wiring and sensory responses of distinct ORN types. Collectively, our data reveal transcriptomic features of sensory neuron biology and provide a resource for future studies of their development and physiology.

Cellular bases of olfactory circuit assembly revealed by systematic time-lapse imaging

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Neural circuit assembly features simultaneous targeting of numerous neuronal processes from constituent neuron types, yet the dynamics is poorly understood. Here, we use the Drosophila olfactory circuit to investigate dynamic cellular processes by which olfactory receptor neurons (ORNs) target axons precisely to specific glomeruli in the ipsi- and contralateral antennal lobes. Time-lapse imaging of individual axons from 30 ORN types revealed a rich diversity in extension speed, innervation timing, and ipsilateral branch locations and identified that ipsilateral targeting occurs via stabilization of transient interstitial branches. Fast imaging using adaptive optics-corrected lattice light-sheet microscopy showed that upon approaching target, many ORN types exhibiting “exploring branches” consisted of parallel microtubule-based terminal branches emanating from an F-actin-rich hub. Antennal nerve ablations uncovered essential roles for bilateral axons in contralateral target selection and for ORN axons to facilitate dendritic refinement of postsynaptic partner neurons. Altogether, these observations provide cellular bases for wiring specificity establishment.

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Jan 2020
CELL

Molecular interactions at the cellular interface mediate organized assembly of single cells into tissues and, thus, govern the development and physiology of multicellular organisms. Here, we developed a cell-type-specific, spatiotemporally resolved approach to profile cell-surface proteomes in intact tissues. Quantitative profiling of cell-surface proteomes of Drosophila olfactory projection neurons (PNs) in pupae and adults revealed global downregulation of wiring molecules and upregulation of synaptic molecules in the transition from developing to mature PNs. A proteome-instructed in vivo screen identified 20 cell-surface molecules regulating neural circuit assembly, many of which belong to evolutionarily conserved protein families not previously linked to neural development. Genetic analysis further revealed that the lipoprotein receptor LRP1 cell-autonomously controls PN dendrite targeting, contributing to the formation of a precise olfactory map. These findings highlight the power of temporally resolved in situ cell-surface proteomic profiling in discovering regulators of brain wiring.

Fat/Dachsous family cadherins in cell and tissue organisation

Article

Nov 2019

Precisely controlled organisation at the cellular and tissue level is crucial to establish and maintain complex organisms. The atypical cadherins Fat (Ft), Fat2 and Dachsous (Ds) contribute to this organisation by regulating growth and planar cell polarity. Here we describe the recent advances in understanding how these large cadherins coordinate these processes, and discuss additional progress extending their function in regulation of microtubules, migration and disease.

Atypical cadherin, Fat2, regulates axon terminal organization in the developing Drosophila olfactory receptor neurons

Abstract and Figures

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