Reverse engineering morphogenesis through Bayesian optimization of physics-based models.

Morphogenetic programs coordinate cell signaling and mechanical interactions to shape organs. In systems and synthetic biology, a key challenge is determining optimal cellular interactions for predicting organ shape, size, and function. Physics-based models defining the subcellular force distribution facilitate this, but it is challenging to calibrate parameters in these models from data. To solve this inverse problem, we created a Bayesian optimization framework to determine the optimal cellular force distribution such that the predicted organ shapes match the experimentally observed organ shapes. This integrative framework employs Gaussian Process Regression, a non-parametric kernel-based probabilistic machine learning modeling paradigm, to learn the mapping functions relating to the morphogenetic programs that maintain the final organ shape. We calibrated and tested the method on Drosophila wing imaginal discs to study mechanisms that regulate epithelial processes ranging from development to cancer. The parameter estimation framework successfully infers the underlying changes in core parameters needed to match simulation data with imaging data of wing discs perturbed with collagenase. The computational pipeline identifies distinct parameter sets mimicking wild-type shapes. It enables a global sensitivity analysis to support the regulation of actomyosin contractility and basal ECM stiffness to generate and maintain the curved shape of the wing imaginal disc. The optimization framework, combined with experimental imaging, identified that Piezo, a mechanosensitive ion channel, impacts fold formation by regulating the apical-basal balance of actomyosin contractility and elasticity of ECM. This workflow is extensible toward reverse-engineering morphogenesis across organ systems and for real-time control of complex multicellular systems.

Regenerative growth is constrained by brain tumor to ensure proper patterning in Drosophila.

Some animals respond to injury by inducing new growth to regenerate the lost structures. This regenerative growth must be carefully controlled and constrained to prevent aberrant growth and to allow correct organization of the regenerating tissue. However, the factors that restrict regenerative growth have not been identified. Using a genetic ablation system in the Drosophila wing imaginal disc, we have identified one mechanism that constrains regenerative growth, impairment of which also leads to erroneous patterning of the final appendage. Regenerating discs with reduced levels of the RNA-regulator Brain tumor (Brat) exhibit enhanced regeneration, but produce adult wings with disrupted margins that are missing extensive tracts of sensory bristles. In these mutants, aberrantly high expression of the pro-growth factor Myc and its downstream targets likely contributes to this loss of cell-fate specification. Thus, Brat constrains the expression of pro-regeneration genes and ensures that the regenerating tissue forms the proper final structure.

Protocol for assessing translation in living Drosophila imaginal discs by O-propargyl-puromycin incorporation.

Translation is a fundamental process of cellular behavior. Here, we present a protocol for measuring translation in Drosophila epithelial tissues using O-propargyl-puromycin (OPP), a puromycin derivative. We detail steps for larval dissection, OPP incorporation, fixation, OPP labeling, immunostaining, and imaging. We also provide details of quantification analysis. Significantly, OPP addition to methionine-containing media enables polypeptide labeling in living cells. Here, we study wing imaginal discs, an excellent model system for investigating growth, proliferation, pattern formation, differentiation, and cell death. For complete details on the use and execution of this protocol, please refer to Lee et al. (2018), Ji et al. (2019), and Kiparaki et al. (2022).1,2,3.

Conditional knockdown protocol for studying cellular communication using Drosophila melanogaster wing imaginal disc.

Apicobasal polarity determinants are potential tumor suppressors that have been extensively studied. However, the precise mechanisms by which their misregulation disrupts tissue homeostasis are not fully understood. Here, we present a comprehensive protocol for establishing a conditional RNAi knockdown of scribble in Drosophila wing imaginal disc. We describe steps for generating fly lines, conditional knockdown in host stocks, and sample preparation. We then detail procedures for imaging, image analysis, and verification of wing disc phenotypes by various antibodies. For complete details on the use and execution of this protocol, please refer to Huang et al.1.

Oxidative Stress Is Associated with Overgrowth in Drosophila l(3)mbt Mutant Imaginal Discs.

The loss-of-function conditions for an l(3)malignant brain tumour (l(3)mbt) in larvae reared at 29 °C results in malignant brain tumours and hyperplastic imaginal discs. Unlike the former that have been extensively characterised, little is known about the latter. Here we report the results of a study of the hyperplastic l(3)mbt mutant wing imaginal discs. We identify the l(3)mbt wing disc tumour transcriptome and find it to include genes involved in reactive oxygen species (ROS) metabolism. Furthermore, we show the presence of oxidative stress in l(3)mbt hyperplastic discs, even in apoptosis-blocked conditions, but not in l(3)mbt brain tumours. We also find that chemically blocking oxidative stress in l(3)mbt wing discs reduces the incidence of wing disc overgrowths. Our results reveal the involvement of oxidative stress in l(3)mbt wing discs hyperplastic growth.

Ets21C sustains a pro-regenerative transcriptional program in blastema cells of Drosophila imaginal discs.

An important unanswered question in regenerative biology is to what extent regeneration is accomplished by the reactivation of gene regulatory networks used during development versus the activation of regeneration-specific transcriptional programs. Following damage, Drosophila imaginal discs, the larval precursors of adult structures, can regenerate missing portions by localized proliferation of damage-adjacent tissue. Using single-cell transcriptomics in regenerating wing discs, we have obtained a comprehensive view of the transcriptome of regenerating discs and identified two regeneration-specific cell populations within the blastema, Blastema1 and Blastema2. Collectively, these cells upregulate multiple genes encoding secreted proteins that promote regeneration including Pvf1, upd3, asperous, Mmp1, and the maturation delaying factor Ilp8. Expression of the transcription factor Ets21C is restricted to this regenerative secretory zone; it is not expressed in undamaged discs. Ets21C expression is activated by the JNK/AP-1 pathway, and it can function in a type 1 coherent feedforward loop with AP-1 to sustain expression of downstream genes. Without Ets21C function, the blastema cells fail to maintain the expression of a number of genes, which leads to premature differentiation and severely compromised regeneration. As Ets21C is dispensable for normal development, these observations indicate that Ets21C orchestrates a regeneration-specific gene regulatory network. We have also identified cells resembling both Blastema1 and Blastema2 in scribble tumorous discs. They express the Ets21C-dependent gene regulatory network, and eliminating Ets21C function reduces tumorous growth. Thus, mechanisms that function during regeneration can be co-opted by tumors to promote aberrant growth.

Structural resemblance of the DNAJA-family protein, Tid1, to the DNAJB-family Hsp40.

The specific pair of heat shock protein 70 (Hsp70) and Hsp40 constitutes an essential molecular chaperone system involved in numerous cellular processes, including the proper folding/refolding and transport of proteins. Hsp40 family members are characterized by the presence of a conserved J-domain (JD) that functions as a co-chaperone of Hsp70. Tumorous imaginal disc 1 (Tid1) is a tumor suppressor protein belonging to the DNAJA3 subfamily of Hsp40 and functions as a co-chaperone of the mitochondrial Hsp70, mortalin. In this work, we performed nuclear magnetic resonance spectroscopy to determine the solution structure of JD and its interaction with the glycine/phenylalaninerich region (GF-motif) of human Tid1. Notably, Tid1-JD, whose conformation was consistent with that of the DNAJB1 JD, appeared to stably interact with its subsequent GF-motif region. Collectively with our sequence analysis, the present results demonstrate that the functional and regulatory mode of Tid1 resembles that of the DNAJB1 subfamily members rather than DNAJA1 or DNAJA2 subfamily proteins. Therefore, it is suggested that an allosteric interaction between mortalin and Tid1 is involved in the mitochondrial Hsp70/Hsp40 chaperone system. [BMB Reports 2022; 55(10): 488-493].

Fasciclin 2 engages EGFR in an auto-stimulatory loop to promote imaginal disc cell proliferation in Drosophila.

How cell to cell interactions control local tissue growth to attain a species-specific organ size is a central question in developmental biology. The Drosophila Neural Cell Adhesion Molecule, Fasciclin 2, is expressed during the development of neural and epithelial organs. Fasciclin 2 is a homophilic-interaction protein that shows moderate levels of expression in the proliferating epithelia and high levels in the differentiating non-proliferative cells of imaginal discs. Genetic interactions and mosaic analyses reveal a cell autonomous requirement of Fasciclin 2 to promote cell proliferation in imaginal discs. This function is mediated by the EGFR, and indirectly involves the JNK and Hippo signaling pathways. We further show that Fasciclin 2 physically interacts with EGFR and that, in turn, EGFR activity promotes the cell autonomous expression of Fasciclin 2 during imaginal disc growth. We propose that this auto-stimulatory loop between EGFR and Fasciclin 2 is at the core of a cell to cell interaction mechanism that controls the amount of intercalary growth in imaginal discs.

Epithelial monitoring through ligand-receptor segregation ensures malignant cell elimination.

Animals have evolved mechanisms, such as cell competition, to remove dangerous or nonfunctional cells from a tissue. Tumor necrosis factor signaling can eliminate clonal malignancies from Drosophila imaginal epithelia, but why this pathway is activated in tumor cells but not normal tissue is unknown. We show that the ligand that drives elimination is present in basolateral circulation but remains latent because it is spatially segregated from its apically localized receptor. Polarity defects associated with malignant transformation cause receptor mislocalization, allowing ligand binding and subsequent apoptotic signaling. This process occurs irrespective of the neighboring cells' genotype and is thus distinct from cell competition. Related phenomena at epithelial wound sites are required for efficient repair. This mechanism of polarized compartmentalization of ligand and receptor can generally monitor epithelial integrity to promote tissue homeostasis.

Protocol to study cell death using TUNEL assay in Drosophila imaginal discs.

Cell death maintains tissue homeostasis by eliminating dispensable cells. Misregulation of cell death is seen in diseases like cancer, neurodegeneration, etc. Therefore, cell death assays like TUNEL have become reliable tools, where fragmented DNA of dying cells gets fluorescently labeled and can be detected under microscope. We used TUNEL assay in Drosophila melanogaster third-instar larval eye-antennal imaginal discs to label and quantify cell death. This assay is sensitive to detect DNA fragmentation, an important event, during apoptosis in retinal neurons. For complete details on the use and execution of this profile, please refer to Wang et al. (1999), Tare et al. (2011), and Mehta et al. (2021).

The Drosophila functional Smad suppressing element fuss, a homologue of the human Skor genes, retains pro-oncogenic properties of the Ski/Sno family.

Over the years Ski and Sno have been found to be involved in cancer progression e.g. in oesophageal squamous cell carcinoma, melanoma, oestrogen receptor-positive breast carcinoma, colorectal carcinoma, and leukaemia. Often, their prooncogenic features have been linked to their ability of inhibiting the anti-proliferative action of TGF-ß signalling. Recently, not only pro-oncogenic but also anti-oncogenic functions of Ski/Sno proteins have been revealed. Besides Ski and Sno, which are ubiquitously expressed other members of Ski/Sno proteins exist which show highly specific neuronal expression, the SKI Family Transcriptional Corepressors (Skor). Among others Skor1 and Skor2 are involved in the development of Purkinje neurons and a mutation of Skor1 has been found to be associated with restless legs syndrome. But neither Skor1 nor Skor2 have been reported to be involved in cancer progression. Using overexpression studies in the Drosophila eye imaginal disc, we analysed if the Drosophila Skor homologue Fuss has retained the potential to inhibit differentiation and induce increased proliferation. Fuss expressed in cells posterior to the morphogenetic furrow, impairs photoreceptor axon pathfinding and inhibits differentiation of accessory cells. However, if its expression is induced prior to eye differentiation, Fuss might inhibit the differentiating function of Dpp signalling and might maintain proliferative action of Wg signalling, which is reminiscent of the Ski/Sno protein function in cancer.

Ecdysone exerts biphasic control of regenerative signaling, coordinating the completion of regeneration with developmental progression.

In Drosophila melanogaster, loss of regenerative capacity in wing imaginal discs coincides with an increase in systemic levels of the steroid hormone ecdysone, a key coordinator of their developmental progression. Regenerating discs release the relaxin hormone Dilp8 (Drosophila insulin-like peptide 8) to limit ecdysone synthesis and extend the regenerative period. Here, we describe how regenerating tissues produce a biphasic response to ecdysone levels: lower concentrations of ecdysone promote local and systemic regenerative signaling, whereas higher concentrations suppress regeneration through the expression of broad splice isoforms. Ecdysone also promotes the expression of wingless during both regeneration and normal development through a distinct regulatory pathway. This dual role for ecdysone explains how regeneration can still be completed successfully in dilp8- mutant larvae: higher ecdysone levels increase the regenerative activity of tissues, allowing regeneration to reach completion in a shorter time. From these observations, we propose that ecdysone hormone signaling functions to coordinate regeneration with developmental progression.

Microvilli-derived extracellular vesicles carry Hedgehog morphogenic signals for Drosophila wing imaginal disc development.

Morphogens are secreted molecules that regulate and coordinate major developmental processes, such as cell differentiation and tissue morphogenesis. Depending on the mechanisms of secretion and the nature of their carriers, morphogens act at short and long range. We investigated the paradigmatic long-range activity of Hedgehog (Hh), a well-known morphogen, and its contribution to the growth and patterning of the Drosophila wing imaginal disc. Extracellular vesicles (EVs) contribute to Hh long-range activity; however, the nature, the site, and the mechanisms underlying the biogenesis of these vesicular carriers remain unknown. Here, through the analysis of mutants and a series of Drosophila RNAi-depleted wing imaginal discs using fluorescence and live-imaging electron microscopy, including tomography and 3D reconstruction, we demonstrate that microvilli of the wing imaginal disc epithelium are the site of generation of small EVs that transport Hh across the tissue. Further, we show that the Prominin-like (PromL) protein is critical for microvilli integrity. Together with actin cytoskeleton and membrane phospholipids, PromL maintains microvilli architecture that is essential to promote its secretory function. Importantly, the distribution of Hh to microvilli and its release via these EVs contribute to the proper morphogenesis of the wing imaginal disc. Our results demonstrate that microvilli-derived EVs are carriers for Hh long-range signaling in vivo. By establishing that members of the Prominin protein family are key determinants of microvilli formation and integrity, our findings support the view that microvilli-derived EVs conveying Hh may provide a means for exchanging signaling cues of high significance in tissue development and cancer.

Xrp1 and Irbp18 trigger a feed-forward loop of proteotoxic stress to induce the loser status.

Cell competition induces the elimination of less-fit "loser" cells by fitter "winner" cells. In Drosophila, cells heterozygous mutant in ribosome genes, Rp/+, known as Minutes, are outcompeted by wild-type cells. Rp/+ cells display proteotoxic stress and the oxidative stress response, which drive the loser status. Minute cell competition also requires the transcription factors Irbp18 and Xrp1, but how these contribute to the loser status is partially understood. Here we provide evidence that initial proteotoxic stress in RpS3/+ cells is Xrp1-independent. However, Xrp1 is sufficient to induce proteotoxic stress in otherwise wild-type cells and is necessary for the high levels of proteotoxic stress found in RpS3/+ cells. Surprisingly, Xrp1 is also induced downstream of proteotoxic stress, and is required for the competitive elimination of cells suffering from proteotoxic stress or overexpressing Nrf2. Our data suggests that a feed-forward loop between Xrp1, proteotoxic stress, and Nrf2 drives Minute cells to become losers.

Multiview confocal super-resolution microscopy.

Confocal microscopy1 remains a major workhorse in biomedical optical microscopy owing to its reliability and flexibility in imaging various samples, but suffers from substantial point spread function anisotropy, diffraction-limited resolution, depth-dependent degradation in scattering samples and volumetric bleaching2. Here we address these problems, enhancing confocal microscopy performance from the sub-micrometre to millimetre spatial scale and the millisecond to hour temporal scale, improving both lateral and axial resolution more than twofold while simultaneously reducing phototoxicity. We achieve these gains using an integrated, four-pronged approach: (1) developing compact line scanners that enable sensitive, rapid, diffraction-limited imaging over large areas; (2) combining line-scanning with multiview imaging, developing reconstruction algorithms that improve resolution isotropy and recover signal otherwise lost to scattering; (3) adapting techniques from structured illumination microscopy, achieving super-resolution imaging in densely labelled, thick samples; (4) synergizing deep learning with these advances, further improving imaging speed, resolution and duration. We demonstrate these capabilities on more than 20 distinct fixed and live samples, including protein distributions in single cells; nuclei and developing neurons in Caenorhabditis elegans embryos, larvae and adults; myoblasts in imaginal disks of Drosophila wings; and mouse renal, oesophageal, cardiac and brain tissues.

Tumorigenesis and cell competition in Drosophila in the absence of polyhomeotic function.

Cell competition is a homeostatic process that eliminates by apoptosis unfit or undesirable cells from animal tissues, including tumor cells that appear during the life of the organism. In Drosophila there is evidence that many types of oncogenic cells are eliminated by cell competition. One exception is cells mutant for polyhomeotic (ph), a member of the Polycomb family of genes; most of the isolated mutant ph clones survive and develop tumorous overgrowths in imaginal discs. To characterize the tumorigenic effect of the lack of ph, we first studied the growth of different regions of the wing disc deficient in ph activity and found that the effect is restricted to the proximal appendage. Moreover, we found that ph-deficient tissue is partially refractory to apoptosis. Second, we analyzed the behavior of clones lacking ph function and found that many suffer cell competition but are not completely eliminated. Unexpectedly, we found that nonmutant cells also undergo cell competition when surrounded by ph-deficient cells, indicating that within the same tissue cell competition may operate in opposite directions. We suggest two reasons for the incompleteness of cell competition in ph mutant cells: 1) These cells are partially refractory to apoptosis, and 2) the loss of ph function alters the identity of imaginal cells and subsequently their cell affinities. It compromises the winner/loser interaction, a prerequisite for cell competition.

Evidence for a nuclear role for Drosophila Dlg as a regulator of the NURF complex.

Scribble (Scrib), Discs-large (Dlg), and Lethal giant larvae (Lgl) are basolateral regulators of epithelial polarity and tumor suppressors whose molecular mechanisms of action remain unclear. We used proximity biotinylation to identify proteins localized near Dlg in the Drosophila wing imaginal disc epithelium. In addition to expected membrane- and cytoskeleton-associated protein classes, nuclear proteins were prevalent in the resulting mass spectrometry dataset, including all four members of the nucleosome remodeling factor (NURF) chromatin remodeling complex. Subcellular fractionation demonstrated a nuclear pool of Dlg and proximity ligation confirmed its position near the NURF complex. Genetic analysis showed that NURF activity is also required for the overgrowth of dlg tumors, and this growth suppression correlated with a reduction in Hippo pathway gene expression. Together, these data suggest a nuclear role for Dlg in regulating chromatin and transcription through a more direct mechanism than previously thought.

EGFRAP encodes a new negative regulator of the EGFR acting in both normal and oncogenic EGFR/Ras-driven tissue morphogenesis.

Activation of Ras signaling occurs in ~30% of human cancers. However, activated Ras alone is insufficient to produce malignancy. Thus, it is imperative to identify those genes cooperating with activated Ras in driving tumoral growth. In this work, we have identified a novel EGFR inhibitor, which we have named EGFRAP, for EGFR adaptor protein. Elimination of EGFRAP potentiates activated Ras-induced overgrowth in the Drosophila wing imaginal disc. We show that EGFRAP interacts physically with the phosphorylated form of EGFR via its SH2 domain. EGFRAP is expressed at high levels in regions of maximal EGFR/Ras pathway activity, such as at the presumptive wing margin. In addition, EGFRAP expression is up-regulated in conditions of oncogenic EGFR/Ras activation. Normal and oncogenic EGFR/Ras-mediated upregulation of EGRAP levels depend on the Notch pathway. We also find that elimination of EGFRAP does not affect overall organogenesis or viability. However, simultaneous downregulation of EGFRAP and its ortholog PVRAP results in defects associated with increased EGFR function. Based on these results, we propose that EGFRAP is a new negative regulator of the EGFR/Ras pathway, which, while being required redundantly for normal morphogenesis, behaves as an important modulator of EGFR/Ras-driven tissue hyperplasia. We suggest that the ability of EGFRAP to functionally inhibit the EGFR pathway in oncogenic cells results from the activation of a feedback loop leading to increase EGFRAP expression. This could act as a surveillance mechanism to prevent excessive EGFR activity and uncontrolled cell growth.

Regulated delivery controls Drosophila Hedgehog, Wingless, and Decapentaplegic signaling.

Morphogen signaling proteins disperse across tissues to activate signal transduction in target cells. We investigated dispersion of Hedgehog (Hh), Wnt homolog Wingless (Wg), and Bone morphogenic protein homolog Decapentaplegic (Dpp) in the Drosophila wing imaginal disc. We discovered that delivery of Hh, Wg, and Dpp to their respective targets is regulated. We found that <5% of Hh and <25% of Wg are taken up by disc cells and activate signaling. The amount of morphogen that is taken up and initiates signaling did not change when the level of morphogen expression was varied between 50 and 200% (Hh) or 50 and 350% (Wg). Similar properties were observed for Dpp. We analyzed an area of 150 µm×150 µm that includes Hh-responding cells of the disc as well as overlying tracheal cells and myoblasts that are also activated by disc-produced Hh. We found that the extent of signaling in the disc was unaffected by the presence or absence of the tracheal and myoblast cells, suggesting that the mechanism that disperses Hh specifies its destinations to particular cells, and that target cells do not take up Hh from a common pool.

Interaction between Ras and Src clones causes interdependent tumor malignancy via Notch signaling in Drosophila.

Cancer tissue often comprises multiple tumor clones with distinct oncogenic alterations such as Ras or Src activation, yet the mechanism by which tumor heterogeneity drives cancer progression remains elusive. Here, we show in Drosophila imaginal epithelium that clones of Ras- or Src-activated benign tumors interact with each other to mutually promote tumor malignancy. Mechanistically, Ras-activated cells upregulate the cell-surface ligand Delta while Src-activated cells upregulate its receptor Notch, leading to Notch activation in Src cells. Elevated Notch signaling induces the transcriptional repressor Zfh1/ZEB1, which downregulates E-cadherin and cell death gene hid, leading to Src-activated invasive tumors. Simultaneously, Notch activation in Src cells upregulates the cytokine Unpaired/IL-6, which activates JAK-STAT signaling in neighboring Ras cells. Elevated JAK-STAT signaling upregulates the BTB-zinc-finger protein Chinmo, which downregulates E-cadherin and thus generates Ras-activated invasive tumors. Our findings provide a mechanistic explanation for how tumor heterogeneity triggers tumor progression via cell-cell interactions.