The people behind the papers – Donald Ready and Henry Chang

Don Ready (L) and Henry Chang (R)

Can you give us your scientific biographies and the questions your labs are trying to answer?

DR: I had the great good fortune to join Seymour Benzer's lab as a graduate student in 1971. It was an amazing collection of friends and characters led by Seymour's virtually clairvoyant insight into what would become important in Drosophila neurogenetics. I was captivated by the beauty of the fly eye and, apart from a postdoctoral fling with the leech nervous system thanks to the generosity and guidance of John Nicholls, I haven't taken my eyes off fly eyes for the past half century. The foundation of my own lab's contribution is work with Andrew Tomlinson, in which we determined the assembly sequence of the ommatidium and demonstrated that sevenless mutants lack the R7 photoreceptor because the cell that should be recruited as R7 instead becomes a lens-secreting cone cell. Soon after sevenless, superb graduate students Ross Cagan and Tanya Wolff led the lab into pupal eye development with analyses of pattern formation, including its resolution via cell death. I'm indebted to the numerous students and postdocs who've carried the lab since, always delivering new insights into Drosophila eye development. I remain fascinated by how the incredible Drosophila compound eye takes form.

HC: My scientific training also crossed path with sevenless in R7 development. I was a graduate student with Gerry Rubin at UC Berkeley from 1992 to 1997, collaborating with a team on a large-scale modifier screen for genes acting downstream of Ras1 in the sevenless cascade. For my postdoc, I wanted to learn cell biology and joined Ira Mellman's lab at Yale University School of Medicine, applying fly genetics to identify components of clathrin-mediated endocytosis. Since coming to Purdue in 2004, I have been interested in how vesicular trafficking influences developmental signalling and cell morphogenesis. We were particularly interested in how ligand endocytosis activates the Notch pathway and have implicated auxilin, a factor well-known for its role in the disassembly of clathrin-coated vesicles, in this process. Using auxilin, we also showed that clathrin-dependent transport is needed for male germ cell differentiation when plasma membrane synthesis is in high demand for spermatid elongation.

Henry, how has your research been affected by the COVID-19 pandemic?

HC: We had to adjust our work routines when the pandemic shut down in-person teaching and closed the labs. Purdue was quick to establish strict protocols allowing essential personnel to work on campus and we were able to complete the experiments. As the school reopened, although most in-person interactions were still scaled back, Don and I, with mask wearing and social distancing, could talk frequently because Don's lab is right down the hall.

Don, before your work, what was known about the upstream regulation of actomyosin contractility across tissues?

DR: The effect of calcium on regulation of actomyosin contraction, especially in muscle cells, has been extensively studied. At the cellular level, calcium oscillations are known to induce micro-contractions of actin fibres in cultured myofibroblasts (Castella et al., 2010). In comparison, the physiological significance of calcium waves is less clear. In mammalian visual systems, calcium waves participate in the establishment of neuronal connectivity. In flies, calcium waves have been implicated in the response to mechanical stress in wing discs and in oocyte activation. Our work suggests another interesting role for calcium waves in coordinating contractile forces in shaping tissues during development. We're not aware of an exactly equivalent developmental role for calcium waves in shaping tissue. An intriguing parallel for calcium waves coordinating tissue-wide contractility is the contraction of the basal end-feet of mammary gland myoepithelial cells; coordinated calcium dependent contractions physically deform alveoli to pump milk into the duct network (Stevenson et al., 2020). Of course, we did not set out to study actomyosin contraction, the phenotype led us there; this is one of the rewards of working with Drosophila.

Henry, can you give us the key results of the paper in a paragraph?

HC: The curvature of fly's retina, composed of 800 repeated units (ommatidia), is important for its panoramic vision. The formation of retinal curvature depends on a four-fold reduction of the basal surface, which is tiled by the endfeet of interommatidial cells (IOCs). During development, this retinal basement area reduction is powered by contractile forces from the actomyosin network in the IOC endfeet, although how these forces are coordinated across the eye field is not known. Using live imaging with a calcium sensor, we demonstrated the presence of IOC waves, characterised by spontaneous and regular bouts of calcium increases propagating across the lattices of IOCs. These IOC waves are independent of phototransduction, but require IP3R, suggesting that calcium increases in IOC waves are mediated by calcium released from the endoplasmic reticulum. Removal of IP3R function disrupts the morphology of actin stress fibres (SFs) in IOC endfeet and increases the basal area, linking the IOC waves to retinal floor contraction. We propose that the regular occurrence of calcium increases in IOC waves coordinate SF contraction to shape the fly retina.

During retinal floor contraction, extracellular matrix (ECM) reorganisation also occurs. Do you think this is solely due to the forces applied via the actomyosin or could calcium have a more direct role on the ECM?

HC: In addition to the IP3R mutation affecting both the SF morphology and Vkg::GFP (Collagen IV) organisation, we observed a strong correlation between Vkg::GFP intensity at the ridges and SF morphology; Vkg::GFP ridges were invariably brighter when SF from adjacent clusters engaged and contracted. Thus, the simplest explanation is that a disruption of actomyosin contractile forces causes the ECM reorganisation defect seen in IP3R mutant patches. We are certainly open to and fascinated by the idea of calcium having direct roles on ECM reorganisation, but do not have data to uncouple ECM reorganisation from actomyosin forces in this context.

DR: Betting against calcium doing things you don't know is a fool's bet, but the ridge network has a decidedly ‘mechanical’ feel. It could be informative to model in-plane compression of a Collagen IV network by a hexagonal array of grommet-like foci. Might this raise a ridge network?

When doing the research, did you have any particular result or eureka moment that has stuck with you?

DR: For me, it would be December 2019. Henry had made a new set of IP3R mosaic eyes with markers that could be more easily visualised at the retinal floor and my imaging technique had improved, as we looked at everything we could think of. Over the Christmas holidays, the SF phenotype presented itself; differences between wild-type and mutant tissue side by side in the highly regular grid of IOC is unmistakable – once you see them. Previously, I'd been in an ‘all-or-nothing’ search mode for SF phenotypes and the new mosaics made it plain that, although present, the SF had not contracted normally.

HC: I agree that December 2019 was a big breakthrough moment for this project. We had known about the occurence of IOC waves, its independence of phototransduction, and its requirement of IP3R for some time, but we weren't clear about the physiological relevance of this phenomenon. As IP3R mutations have been shown to cause photoreceptor degeneration, we first hypothesised that IOC waves might coordinate the retinal accessory cells to provide nutrients for neuronal survival. However, this link was tenuous because although the IOC waves start at the pupal stage, the neurodegeneration phenotype does not occur until weeks after eclosion. When we made fly strains for generating mosaic IP3R mutant clones in pigment-less retinas, the SF defects were right there!

And what about the flipside: any moments of frustration or despair?

DR: Frustration? Before a phenotype, constantly. Despair, no. Flies almost always come through in the end.

HC: I agree. I don't think we were ever in despair. The IOC waves were always fun to look at, and this has been a rewarding and satisfying collaboration. Although it took some time to discover the SF defects, along the way we were confident that we were on to something interesting.

Where will this story take both labs next?

DR: There's much to be done sorting out from where Collagen IV comes from and how it is arranged to form grommets and the ridge network. I look forward to being useful at the microscope and having a great deal of fun working with Henry as my lab ramps down in the next few years.

HC: I would like to address follow-up questions on the IOC waves. For example, what generates the second messengers for the wave initiation and propagation? Is a specific phospholipase C involved? What is the mechanism that insulates IOC waves to specific cell types? Answering these questions will improve our understanding of IOC waves and may generate reagents to investigate other aspects of IOC waves. In addition, we are very excited about the ECM direction. The basement membrane in Drosophila retina is formed during a specific pupal stage and contains distinct morphological features, such as grommets and ridges. The eye will be an excellent in vivo model in which to dissect how these features are formed.

Finally, let's move outside the lab – what do you like to do in your spare time?

DR: I'm a backyard astronomer and after a good day on the microscope I can look forward to a good evening at the telescope. Dark skies are a compensation for being surrounded by corn and soy fields.

HC: I play tennis to exercise and to relieve stress.