ex-vivo-calcium-imaging-for-visualizing-brain-responses-to-endocrine

<em>Ex Vivo</em> Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in <em>Drosophila</em>

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

Kurume University

Organs develop distinctive morphologies to fulfill their unique functions. We used Drosophila embryonic gonads as a model to study how two different cell lineages, primordial germ cells (PGCs) and somatic gonadal precursors (SGPs), combine to form one organ. We developed a membrane GFP marker to image SGP behaviors live. These studies show that a combination of SGP cell shape changes and inward movement of anterior and posterior SGPs leads to the compaction of the spherical gonad. This process is disrupted in mutants of the actin regulator, enabled (ena). We show that Ena coordinates these cell shape changes and the inward movement of the SGPs, and Ena affects the intracellular localization of DE-cadherin (DE-cad). Mathematical simulation based on these observations suggests that changes in DE-cad localization can generate the forces needed to compact an elongated structure into a sphere. We propose that Ena regulates force balance in the SGPs by sequestering DE-cad, leading to the morphogenetic movement required for gonad compaction.

The Drosophila actin regulator ENABLED regulates cell shape and orientation during gonad morphogenesis.

The formation of the Drosophila embryonic gonad, involving the fusion of clusters of somatic gonadal precursor cells (SGPs) and their ensheathment of germ cells, provides a simple and genetically tractable model for the interplay between cells during organ formation. In a screen for mutants affecting gonad formation we identified a SGP cell autonomous role for Midline (Mid) and Longitudinals lacking (Lola). These transcriptional factors are required for multiple aspects of SGP behaviour including SGP cluster fusion, germ cell ensheathment and gonad compaction. The lola locus encodes more than 25 differentially spliced isoforms and we have identified an isoform specific requirement for lola in the gonad which is distinct from that in nervous system development. Mid and Lola work in parallel in gonad formation and surprisingly Mid overexpression in a lola background leads to additional SGPs at the expense of fat body cells. Our findings support the idea that although the transcription factors required by SGPs can ostensibly be assigned to those being required for either SGP specification or behaviour, they can also interact to impinge on both processes. 

Transcriptional regulation of Drosophila gonad formation.

The coordination of growth with nutritional status is essential for proper development and physiology. Nutritional information is mostly perceived by peripheral organs before being relayed to the brain, which modulates physiological responses. Hormonal signaling ensures this organ-to-organ communication, and the failure of endocrine regulation in humans can cause diseases including obesity and diabetes. In Drosophila melanogaster, the fat body (adipose tissue) has been suggested to play an important role in coupling growth with nutritional status. Here, we show that the peripheral tissue-derived peptide hormone CCHamide-2 (CCHa2) acts as a nutrient-dependent regulator of Drosophila insulin-like peptides (Dilps). A BAC-based transgenic reporter revealed strong expression of CCHa2 receptor (CCHa2-R) in insulin-producing cells (IPCs) in the brain. Calcium imaging of brain explants and IPC-specific CCHa2-R knockdown demonstrated that peripheral-tissue derived CCHa2 directly activates IPCs. Interestingly, genetic disruption of either CCHa2 or CCHa2-R caused almost identical defects in larval growth and developmental timing. Consistent with these phenotypes, the expression of dilp5, and the release of both Dilp2 and Dilp5, were severely reduced. Furthermore, transcription of CCHa2 is altered in response to nutritional levels, particularly of glucose. These findings demonstrate that CCHa2 and CCHa2-R form a direct link between peripheral tissues and the brain, and that this pathway is essential for the coordination of systemic growth with nutritional availability. A mammalian homologue of CCHa2-R, Bombesin receptor subtype-3 (Brs3), is an orphan receptor that is expressed in the islet β-cells; however, the role of Brs3 in insulin regulation remains elusive. Our genetic approach in Drosophila melanogaster provides the first evidence, to our knowledge, that bombesin receptor signaling with its endogenous ligand promotes insulin production. 

The Nutrient-Responsive Hormone CCHamide-2 Controls Growth by Regulating Insulin-like Peptides in the Brain of Drosophila melanogaster.

The coupling of growth to nutritional status is an important adaptive response of living organisms to their environment. For this ability, animals have evolved various strategies, including endocrine systems that respond to changing nutritional conditions. In animals, nutritional information is mostly perceived by peripheral organs, such as the digestive tract and adipose tissues, and is subsequently transmitted to other peripheral organs or the brain, which integrates the incoming signals and orchestrates physiological and behavioral responses. In Drosophila melanogaster, adipose tissue, known as the fat body, functions as an endocrine organ that communicates with the brain. This fat body-brain axis coordinates growth with nutritional status by regulating the secretion of Drosophila insulin-like peptides (Dilps) from the brain. However, the molecular nature of the fat body-brain axis remains to be elucidated. We recently demonstrated that a small peptide, CCHamide-2 (CCHa2), expressed in the fat body and gut, directly stimulates its receptor (CCHa2-R) in the brain, leading to Dilp production. Notably, the expression of CCHa2 is sensitive to the presence of nutrients, particularly sugars. Our results, together with the results of previous studies, show that signaling between peripheral organs and the brain is a conserved strategy that couples nutritional availability to organismal physiology. 

Article	Year
The Drosophila actin regulator ENABLED regulates cell shape and orientation during gonad morphogenesis. PloS one\| PubMed ID: 23300733	2012
Transcriptional regulation of Drosophila gonad formation. Developmental biology\| PubMed ID: 24927896	2014
The Nutrient-Responsive Hormone CCHamide-2 Controls Growth by Regulating Insulin-like Peptides in the Brain of Drosophila melanogaster. PLoS genetics\| PubMed ID: 26020940	2015
Coupling of growth to nutritional status: The role of novel periphery-to-brain signaling by the CCHa2 peptide in Drosophila melanogaster. Fly\| PubMed ID: 26980588	2015

Hiroko Sano

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