A. Gontijo Lab | Insulin-like hormone triggers pupariation development in Drosophila

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[From left to right: Dr. Juliane Menezes, Dr. Fabiana Heredia, Dr. Cesar Mendes, Dr. Magdalena Fernandez-Acosta and Dr. Alisson Gontijo]

Fabiana Herédia, Yanel Volonté and Joana Pereirinha are all first authors in the brand new Nature Communications article from research co-led by Dr. Alisson Gontijo and Dr. Andres Garelli in the lab Integrative Biomedicine in CEDOC. They described the hormone ecdysone triggers the Dilp8-Lgr3 pathway to induce pupariation development. The article is titled “The steroid-hormone ecdysone coordinates parallel pupariation neuromotor and morphogenetic subprograms via epidermis-to-neuron Dilp8-Lgr3 signal induction” and is available here.

What discoveries led you to the research described in your publication?

We and others had previously demonstrated that Dilp8, a relaxin-like peptide found in Drosophila flies, had a key role in promoting developmental stability via its neuronal receptor, Lgr3. Relaxins are a large group of insulin-like peptides, which are structurally similar to insulin, but act via different receptors. Human relaxins had been implicated in reproduction, stress signaling, and cancer metastasis.

While studying flies lacking the Dilp8-Lgr3 pathway, we noticed, quite serendipitously, the aberrant shape of their pupal case, also known as “puparium”. This structure protects the animal from predators and desiccation during fly metamorphosis (when the larva transforms into an adult form). The puparium is formed by the animal’s hardened and remodeled larval cuticle, in a multi-step “innate” behavioral process called pupariation. Innate behaviors do not require previous experience and are typically executed to completion upon triggering, such as the sucking reflex of newborns, the nest-building of birds, and the web-spinning of spiders.

The motor subprograms of innate behaviors are many times coupled to drastic morphogenetic changes, especially when they occur during animal maturation. In the case of puparium formation, the cuticle is swiftly hardened after the animal executes the body-remodeling contractions and associated behaviors of pupariation, such as the expulsion and spreading of “glue”, a proteinaceous mix produced by the salivary gland that firmly attaches the puparium to its substrate. If the cuticle hardens before or while these behaviors are being executed, puparium remodeling and/or glue expulsion fail(s), leading to decreased survival rates. How the coupling between morphogenetic and motor programs is achieved during innate behaviors was unclear.

What were you trying to understand and what is the main discovery of this work?

In a curiosity-driven effort, we set out to understand whether the role of Dilp8 and Lgr3 in pupal case formation was spatially and temporally identical or distinct from their previously-described role in developmental stability promotion. Of note, pupariation had been shown to be triggered by a surge in the steroid-hormone ecdysone, the major insect molting (cuticle shedding) hormone, at the end of the larval growth phase of flies. Each of the multiple steps of pupariation, however, were hypothesized to be carried out by the so-called “pupariation factors”, which are factors of a neuropeptidic nature that had been isolated as biochemical activities several decades ago by the insect physiologist Gottfried Fraenkel, but which remain to be defined genetically. We wondered if Dilp8 would be one of Fraenkel’s pupariation factors. If the pupal case phenotype arose due to a problem occurring earlier in development due to the function of the Dilp8-Lgr3 pathway in developmental stability, that would be unlikely.

We were able to use several developmental genetics tricks to show that this was not the case. A key finding was that the population of Lgr3 neurons mediating the developmental stability promotion and the pupal case morphogenesis had no overlap. There are unique neuronal populations regulating each process separately. The Dilp8 signal was also coming from different cells at a different developmental stages. Namely, during pupariation, Dilp8 is developmentally activated in the cuticle epidermis by ecdysone. Dilp8 then acts at a distance on six previously-undescribed Lgr3-positive neurons in the central nervous system to transiently delay the hardening of the cuticle and to switch the behavioral subprograms of puparium formation. This epidermis-to-neuron interorgan communication event is critical for the animal to execute the innate behaviors associated with pupal case formation, including its proper remodeling and the glue expulsion and spreading behavior. Hence, this new role played by Dilp8 during pupariation was indeed compatible with it being one of Fraenkel’s pupariation factors. We hypothesize that Dilp8 is ARF/PIF, which stands for anterior-retraction factor/puparium immobilization factor, a factor that remodels puparium shape and was described in the early 80s by Jan Zdarek and Gottfried Fraenkel.

Why is this important?

Our work contributes to the understanding of how neuropeptides and their neuromodulatory effects are paramount for the proper execution of complex innate behaviors. There were few examples of molecules that couple the morphogenetic and motor aspects of an innate behavior. Our findings placing Dilp8 and Lgr3 in time and space in this behavior provide a unique opportunity to dissect this process in greater detail at the cellular and molecular level.

A second important aspect of the work is that it strengthens the notion that the relaxin signaling pathway, implicated in reproduction, stress signaling, and cancer invasion in humans, has critical roles in regulating the timing of developmental processes. We suggest that many of these processes can be revisited under this mechanistic paradigm.

A third aspect is the identification and description of the stereotyped ~1-min-long caterpillar-like crawling-behavior we called “Glue (Expulsion and) Spreading Behavior” (GSB). The function of this behavior is to promote attachment of the future pupal case onto a substrate. To our surprise, and to the best of our knowledge, this behavior had not been yet fully characterized. When we started this work, we were neither expecting to find an unreported behavior in this highly-studied model organism, nor to gain insight into the molecular and cellular requirements to “release” the behavior.

Can you use an analogy to help us understand your work?

Maybe we can use sleep as an analogy. Sleep is also a complex, multi-step innate behavior. Dreams most often occur during a deep sleep stage, also known as “REM” (rapid eye movement) sleep. At this stage, body muscle tone and reflexes become absent, which probably evolved to prevent animals from “acting out” their dreams. How sleep stages transition during sleep progression and how REM and muscle tone depression are precisely coupled in time (with other physiological changes too), are analogous types of questions as the ones we addressed in the pupariation behavior context.

As studying the molecular genetics of innate behaviors in vertebrates and humans is complicated and limited due to technical and ethical issues, an attractive and valuable alternative is to study the shorter and predictable behaviors in genetically-tractable organisms. By studying pupariation in Drosophila, we found clear evidence that behavioral subprograms happening in distinct organs, in an analogous way as the dreams and muscle tone depression of REM sleep, are interconnected via relaxin peptide signaling. We hypothesize that similar peptides or neuromodulatory mechanisms must couple subprograms in these and other innate behaviors.

What questions remain to be asked?

It is not clear how the Dilp8 peptide reaches Lgr3 interneurons deep in the ventral nerve cord. How does it transverse the blood (hemolymph) brain barrier? It is also unclear what occurs following Lgr3 activation in the neurons. Namely, what do Lgr3 neurons do molecularly to unlock behavioral progression and remodel the pupal case?

Finally, we think our findings will interest biologists in general, neurophysiologists, ethologists, geneticists, developmental biologists, physiologists, muscle biologists, entomologists, and scientists in other areas. This includes quantitative biologists and computational modelers interested, for instance, in the regulation of the very sharp peak of transcription that we define for dilp8 at pupariation, or in dissecting the behavioral profiles we obtained by monitoring muscle contraction activity directly using novel genetic tools and behavior arenas.

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