Combined in Situ Hybridization Chain Reaction and Immunostaining to Visualize Gene Expression in Whole-Mount Drosophila Central Nervous Systems

Duckhorn, Julia C.; Junker, Ian P.; Ding, Yun; Shirangi, Troy R.

doi:10.1007/978-1-0716-2321-3_1

Cited by 6 publications

(7 citation statements)

References 16 publications

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“…TN1 neurons are one of the anatomically distinct dsx-expressing neuronal clusters 22,26 , allowing the quantification of TN1 cell number by probing dsx mRNA or protein. Both in situ hybridization chain reaction (HCR) 27,28 using dsx mRNA probes and immunostaining (IHC) using Dsx antibodies 29 confirmed the presence of a TN1 cluster in D. yakuba, but with ~10 fewer neurons than in D. melanogaster (Fig. 1c).…”

Section: Yakuba Has Fewer Sine-song-essential Tn1 Neuronsmentioning

confidence: 82%

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Changes in the cellular makeup of motor patterning circuits drive courtship song evolution inDrosophila

Ye,

Walsh,

Junker

et al. 2024

Preprint

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How evolutionary changes in genes and neurons encode species variation in complex motor behaviors are largely unknown. Here, we develop genetic tools that permit a neural circuit comparison between the model speciesDrosophila melanogasterand the closely-related speciesD. yakuba, who has undergone a lineage-specific loss of sine song, one of the two major types of male courtship song inDrosophila. Neuroanatomical comparison of song patterning neurons called TN1 across the phylogeny demonstrates a link between the loss of sine song and a reduction both in the number of TN1 neurons and the neurites serving the sine circuit connectivity. Optogenetic activation confirms that TN1 neurons inD. yakubahave lost the ability to drive sine song, while maintaining the ability to drive the singing wing posture. Single-cell transcriptomic comparison shows thatD. yakubaspecifically lacks a cell type corresponding to TN1A neurons, the TN1 subtype that is essential for sine song. Genetic and developmental manipulation reveals a functional divergence of the sex determination genedoublesexinD. yakubato reduce TN1 number by promoting apoptosis. Our work illustrates the contribution of motor patterning circuits and cell type changes in behavioral evolution, and uncovers the evolutionary lability of sex determination genes to reconfigure the cellular makeup of neural circuits.

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Section: Yakuba Has Fewer Sine-song-essential Tn1 Neuronsmentioning

confidence: 82%

“…Samples were mounted similarly as in IHC (Fisher Scientific; Cat.#: P36931). Joint-IHC-HCR used an adapted version of a previously published protocol 28 . Following standard dissection and fixation, samples were blocked using Antibody Buffer (Molecular Instruments) for 4 hours at 4°C.…”

Section: Ihc Hcr and Joint-ihc-hcrmentioning

confidence: 99%

Changes in the cellular makeup of motor patterning circuits drive courtship song evolution inDrosophila

Ye,

Walsh,

Junker

et al. 2024

Preprint

Self Cite

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“…The fixed CNS underwent gradual dehydration and rehydration, followed by standard hybridization and amplification steps of the HCR protocol (Choi et al 2018). For double labeling with antibody, the post-HCR labeled CNS was briefly fixed for 30 minutes prior to standard antibody labeling protocol to identify specific CNS cell type(s) with highly localized ifc RNAs (Lacin et al, 2019; Duckhorn et al, 2022).…”

Section: Methodsmentioning

confidence: 99%

Loss of dihydroceramide desaturase drives neurodegeneration by disrupting endoplasmic reticulum and lipid droplet homeostasis in glial cells

Zhu,

Cho,

Lacin

et al. 2024

Preprint

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Dihydroceramide desaturases convert dihydroceramides to ceramides in the last step of thede novoceramide pathway. Reduction of DEGS1 dihydroceramide desaturase function in humans leads to the rare neurodegenerative disorder hypomyelinating leukodystrophy-18, but the exact mechanism that underlies the disease remains unclear. Through a forward genetic screen, we discovered thatinfertile crescent (ifc), the soleDrosophiladihydroceramide desaturase, governs central nervous system development and morphology. Expressed and genetically active most prominently in glia rather than neurons,ifcprimarily controls nervous system development through a cell autonomous function in glia. Within the nervous system, loss ofifcresults in ceramide depletion, dihydroceramide accumulation, and increased saturation of major membrane phospholipids. At the cellular level, loss ofifcleads to severe glial defects, particularly in cortex glia, including expansion of the endoplasmic reticulum, cell swelling, failure to enwrap neurons, and lipid droplet depletion. Our research supports a model in which inappropriate retention of dihydroceramide in the endoplasmic reticulum (ER) of glial cells drives ER expansion and cell swelling, disrupting glial function and leading to subsequent nervous system dysfunction. Given the conserved nature of thede novoceramide biosynthesis pathway, our findings in the fly system suggest that dihydroceramide-triggered expansion of the ER in the membrane-rich glial cells may be the proximal cause of hypomyelinating leukodystrophy-18 in humans.

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“…One question remains: Could environmental cues alter synaptic/circuit states independent of the sex‐determination pathway to trigger sex‐specific responses? This question can be answered by using either transcriptomics or in‐situ hybridization approaches, which can provide insight into the sex‐specific modulation of shared transcripts in the nervous system [86].…”

Section: Experience‐dependent Sexual Dimorphic Behaviours In Drosophi...mentioning

confidence: 99%

Environmental experiences shape sexually dimorphic neuronal circuits and behaviour

2023

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Dimorphic traits, shaped by both natural and sexual selection, ensure optimal fitness and survival of the organism. This includes neuronal circuits that are largely affected by different experiences and environmental conditions. Recent evidence suggests that sexual dimorphism of neuronal circuits extends to different levels such as neuronal activity, connectivity and molecular topography that manifest in response to various experiences, including chemical exposures, starvation and stress. In this review, we propose some common principles that govern experience‐dependent sexually dimorphic circuits in both vertebrate and invertebrate organisms. While sexually dimorphic neuronal circuits are predetermined, they have to maintain a certain level of fluidity to be adaptive to different experiences. The first layer of dimorphism is at the level of the neuronal circuit, which appears to be dictated by sex‐biased transcription factors. This could subsequently lead to differences in the second layer of regulation namely connectivity and synaptic properties. The third regulator of experience‐dependent responses is the receptor level, where dimorphic expression patterns determine the primary sensory encoding. We also highlight missing pieces in this field and propose future directions that can shed light onto novel aspects of sexual dimorphism with potential benefits to sex‐specific therapeutic approaches. Thus, sexual identity and experience simultaneously determine behaviours that ultimately result in the maximal survival success.

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Combined in Situ Hybridization Chain Reaction and Immunostaining to Visualize Gene Expression in Whole-Mount Drosophila Central Nervous Systems

Cited by 6 publications

References 16 publications

Changes in the cellular makeup of motor patterning circuits drive courtship song evolution inDrosophila

Changes in the cellular makeup of motor patterning circuits drive courtship song evolution inDrosophila

Loss of dihydroceramide desaturase drives neurodegeneration by disrupting endoplasmic reticulum and lipid droplet homeostasis in glial cells

Environmental experiences shape sexually dimorphic neuronal circuits and behaviour

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