preparing-developing-peripheral-olfactory-tissue-for-molecular

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in <em>Drosophila</em>

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and ...

Duke University

Detection of a broad range of chemosensory signals is necessary for the survival of multicellular organisms. Chemical signals are the main facilitators of foraging, escape, and social behaviors. To increase detection coverage, animal sensory systems have evolved to create a large number of neurons with highly specific functions. The olfactory system, much like the nervous system as a whole, is astonishingly diverse. The mouse olfactory system has millions of neurons with over a thousand classes, whereas the more compact Drosophila genome has approximately 80 odorant receptor genes that give rise to 50 neuronal classes and 1300 neurons in the adult.(4) Understanding how neuronal diversity is generated remains one of the central questions in developmental neurobiology. Here, we review the current knowledge on the development of the adult Drosophila olfactory system and the progress that has been made toward answering this central question.

Mechanisms of olfactory receptor neuron specification in Drosophila.

The zinc-finger protein Rotund (Rn) plays a critical role in controlling the development of the fly olfactory system. However, little is known about its molecular function in vivo. Here, we added protein tags to the rn locus using CRISPR-Cas9 technology in Drosophila to investigate its subcellular localization and the genes that it regulates . We previously used a reporter construct to show that rn is expressed in a subset of olfactory receptor neuron (ORN) precursors and it is required for the diversification of ORN fates. Here, we show that tagged endogenous Rn protein is functional based on the analysis of ORN phenotypes. Using this method, we also mapped the expression pattern of the endogenous isoform-specific tags in vivo with increased precision. Comparison of the Rn expression pattern from this study with previously published results using GAL4 reporters showed that Rn is mainly present in early steps in antennal disc patterning, but not in pupal stages when ORNs are born. Finally, using chromatin immunoprecipitation, we showed a direct binding of Rotund to a previously identified regulatory element upstream of the bric-a-brac gene locus in the developing antennal disc. 

Examination of Endogenous Rotund Expression and Function in Developing Drosophila Olfactory System Using CRISPR-Cas9-Mediated Protein Tagging.

Sensory neuron diversity is required for organisms to decipher complex environmental cues. In Drosophila, the olfactory environment is detected by 50 different olfactory receptor neuron (ORN) classes that are clustered in combinations within distinct sensilla subtypes. Each sensilla subtype houses stereotypically clustered 1-4 ORN identities that arise through asymmetric divisions from a single multipotent sensory organ precursor (SOP). How each class of SOPs acquires a unique differentiation potential that accounts for ORN diversity is unknown. Previously, we reported a critical component of SOP diversification program, Rotund (Rn), increases ORN diversity by generating novel developmental trajectories from existing precursors within each independent sensilla type lineages. Here, we show that Rn, along with BarH1/H2 (Bar), Bric-à-brac (Bab), Apterous (Ap) and Dachshund (Dac), constitutes a transcription factor (TF) network that patterns the developing olfactory tissue. This network was previously shown to pattern the segmentation of the leg, which suggests that this network is functionally conserved. In antennal imaginal discs, precursors with diverse ORN differentiation potentials are selected from concentric rings defined by unique combinations of these TFs along the proximodistal axis of the developing antennal disc. The combinatorial code that demarcates each precursor field is set up by cross-regulatory interactions among different factors within the network. Modifications of this network lead to predictable changes in the diversity of sensilla subtypes and ORN pools. In light of our data, we propose a molecular map that defines each unique SOP fate. Our results highlight the importance of the early prepatterning gene regulatory network as a modulator of SOP and terminally differentiated ORN diversity. Finally, our model illustrates how conserved developmental strategies are used to generate neuronal diversity. 

A Functionally Conserved Gene Regulatory Network Module Governing Olfactory Neuron Diversity.

Drosophila uses 50 different olfactory receptor neuron (ORN) classes that are clustered within distinct sensilla subtypes to decipher their chemical environment. Each sensilla subtype houses 1-4 ORN identities that arise through asymmetric divisions of a single sensory organ precursor (SOP). Despite a number of mutational studies investigating the regulation of ORN development, a majority of the transcriptional programs that lead to the different ORN classes in the developing olfactory system are unknown. Here we use transcriptional profiling across the time series of antennal development to identify novel transcriptional programs governing the differentiation of ORNs. We surveyed four critical developmental stages of the olfactory system: 3rd instar larval (prepatterning), 8 hours after puparium formation (APF, SOP selection), 40 hrs APF (neurogenesis), and adult antennae. We focused on the expression profiles of olfactory receptor genes and transcription factors-the two main classes of genes that regulate the sensory identity of ORNs. We identify distinct clusters of genes that have overlapping temporal expression profiles suggesting they have a key role during olfactory system development. We show that the expression of the transcription factor distal antenna (dan) is highly similar to other prepatterning factors and is required for the expression of a subset of ORs.

Transcriptional profiling of olfactory system development identifies distal antenna as a regulator of subset of neuronal fates.

Organisms have evolved strikingly parallel phenotypes in response to similar selection pressures suggesting that there may be shared constraints limiting the possible evolutionary trajectories. For example, the behavioral adaptation of specialist Drosophila species to specific host plants can exhibit parallel changes in their adult olfactory neuroanatomy. We investigated the genetic basis of these parallel changes by comparing gene expression during the development of the olfactory system of two specialist Drosophila species to that of four other generalist species. Our results suggest that the parallelism observed in the adult olfactory neuroanatomy of ecological specialists extends more broadly to their developmental antennal expression profiles, and to the transcription factor combinations specifying olfactory receptor neuron (ORN) fates. Additionally, comparing general patterns of variation for the antennal transcriptional profiles in the adult and developing olfactory system of the six species suggest the possibility that specific, non-random components of the developmental programs underlying the Drosophila olfactory system harbor a disproportionate amount of interspecies variation. Further examination of these developmental components may be able to inform a deeper understanding of how traits evolve.

Article	Year
Mechanisms of olfactory receptor neuron specification in Drosophila. Wiley interdisciplinary reviews. Developmental biology\| PubMed ID: 26088441	2015
Examination of Endogenous Rotund Expression and Function in Developing Drosophila Olfactory System Using CRISPR-Cas9-Mediated Protein Tagging. G3 (Bethesda, Md.)\| PubMed ID: 26497147	2015
A Functionally Conserved Gene Regulatory Network Module Governing Olfactory Neuron Diversity. PLoS genetics\| PubMed ID: 26765103	2016
Transcriptional profiling of olfactory system development identifies distal antenna as a regulator of subset of neuronal fates. Scientific reports\| PubMed ID: 28102318	2017
Patterns of transcriptional parallelism and variation in the developing olfactory system of Drosophila species. Scientific reports\| PubMed ID: 28821769	2017

Scott Barish

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