Pluripotency of a founding field: rebranding developmental biology.

The field of developmental biology has declined in prominence in recent decades, with off-shoots from the field becoming more fashionable and highly funded. This has created inequity in discovery and opportunity, partly due to the perception that the field is antiquated or not cutting edge. A 'think tank' of scientists from multiple developmental biology-related disciplines came together to define specific challenges in the field that may have inhibited innovation, and to provide tangible solutions to some of the issues facing developmental biology. The community suggestions include a call to the community to help 'rebrand' the field, alongside proposals for additional funding apparatuses, frameworks for interdisciplinary innovative collaborations, pedagogical access, improved science communication, increased diversity and inclusion, and equity of resources to provide maximal impact to the community.

Membrane potential regulates Hedgehog signalling in the Drosophila wing imaginal disc.

While the membrane potential of cells has been shown to be patterned in some tissues, specific roles for membrane potential in regulating signalling pathways that function during development are still being established. In the Drosophila wing imaginal disc, Hedgehog (Hh) from posterior cells activates a signalling pathway in anterior cells near the boundary which is necessary for boundary maintenance. Here, we show that membrane potential is patterned in the wing disc. Anterior cells near the boundary, where Hh signalling is most active, are more depolarized than posterior cells across the boundary. Elevated expression of the ENaC channel Ripped Pocket (Rpk), observed in these anterior cells, requires Hh. Antagonizing Rpk reduces depolarization and Hh signal transduction. Using genetic and optogenetic manipulations, in both the wing disc and the salivary gland, we show that membrane depolarization promotes membrane localization of Smoothened and augments Hh signalling, independently of Patched. Thus, membrane depolarization and Hh-dependent signalling mutually reinforce each other in cells immediately anterior to the compartment boundary.

Regeneration and transdetermination in Drosophila imaginal discs.

The study of regeneration in Drosophila imaginal discs provides an opportunity to use powerful genetic tools to address fundamental problems pertaining to tissue regeneration and cell plasticity. We present a historical overview of the field and describe how the application of modern methods has made the study of disc regeneration amenable to genetic analysis. Discs respond to tissue damage in several ways: (a) Removal of part of the disc elicits localized cell proliferation and regeneration of the missing tissue. (b) Damage at specific locations in the disc can cause cells to generate disc-inappropriate structures (e.g., wing instead of leg), a phenomenon known as transdetermination. (c) Diffuse damage to imaginal discs, results in compensatory proliferation of surviving cells. Candidate-gene approaches have implicated the JNK, Wingless, and Hippo pathways in regeneration. Recently developed systems will enable extensive genetic screens that could provide new insights into tissue regeneration, transdetermination and compensatory proliferation.

Size regulation blossoms in Kobe.

Coincident with the blossoming of the sakura was the 14th annual CDB Symposium hosted by the RIKEN Center for Developmental Biology in Kobe, Japan. This year's meeting, 'Size in Development: Growth, Shape and Allometry' focused on the molecular and cellular mechanisms underlying differences in size and shape and how they have evolved. On display was the power of using diverse approaches ranging from the study of organoids to whole organisms.

CoinFLP: a system for efficient mosaic screening and for visualizing clonal boundaries in Drosophila.

Screens in mosaic Drosophila tissues that use chemical mutagenesis have identified many regulators of growth and patterning. Many of the mutant phenotypes observed were contingent upon the presence of both wild-type and mutant cells in the same tissue. More recently, large collections of RNAi lines or cDNAs expressed under Gal4/UAS control have been used to alter gene expression uniformly in specific tissues. However, these newer approaches are not easily combined with the efficient generation of genetic mosaics. The CoinFLP system described here enables mosaic screens in the context of gene knockdown or overexpression by automatically generating a reliable ratio of mutant to wild-type tissue in a developmentally controlled manner. CoinFLP-Gal4 generates mosaic tissues composed of clones of which only a subset expresses Gal4. CoinFLP-LexGAD/Gal4 generates tissues composed of clones that express either Gal4 or LexGAD, thus allowing the study of interactions between different types of genetically manipulated cells. By combining CoinFLP-LexGAD/Gal4 with the split-GFP system GRASP, boundaries between genetically distinct cell populations can be visualized at high resolution.

TIE-DYE: a combinatorial marking system to visualize and genetically manipulate clones during development in Drosophila melanogaster.

Two types of information are particularly valuable in understanding the development of a tissue or an organ from a small population of founder cells. First, it is useful to know the composition of the final structure in terms the contribution of individual founder cells. Second, it is important to understand cell-cell interactions. To facilitate the study of both of these aspects of organ development at a tissue-wide level, we have developed a method, TIE-DYE, that allows simultaneous lineage tracing of multiple cell populations as well as the genetic manipulation of a subset of these populations. Seven uniquely marked categories of cells are produced by site-directed recombination of three independent cassettes. We have used the TIE-DYE method to estimate the number of founder cells that give rise to the wing-imaginal disc during normal development and following compensatory growth caused by X-ray irradiation of the founder cells. We also show that four out of the seven types of marked clones can be genetically manipulated by gene overexpression or RNAi knockdown, allowing an assessment of the consequences of these manipulations on the entire wing disc. We demonstrate the utility of this system in studying the consequences of alterations in growth, patterning and cell-cell affinity.

Differences in levels of the transmembrane protein Crumbs can influence cell survival at clonal boundaries.

The survival and growth of individual cells in a tissue can be nonautonomously regulated by the properties of adjacent cells. In mosaic Drosophila imaginal discs, for example, wild-type cells induce the elimination of adjacent slow-growing Minute cells by apoptosis, while, conversely, certain types of faster-growing cells are able to eliminate adjacent wild-type cells. This process, known as cell competition, represents one example of a diverse group of phenomena in which short-range heterotypic interactions result in the selective elimination of one type of cell by another. The mechanisms that designate "winner" and "loser" genotypes in these processes are not known. Here we show that apoptosis is observed preferentially at boundaries that separate populations of cells that express different levels of the transmembrane protein Crumbs (Crb). Cells that express higher levels of Crb tend to be eliminated when they are near cells that express lower levels of Crb. We also observe distortions in the structure of epithelia on either side of boundaries between populations of cells that differ in Crb expression. Thus, while previous studies have focused mostly on the cell autonomous functions of Crb, we show that Crb can regulate cell survival and tissue morphology nonautonomously. Moreover, we find that the extracellular domain (ECD) of Crb, which seems to be dispensable for some of the other characterised functions of Crb, is required to elicit the nonautonomous effects on cell survival. The ECD can also regulate the subcellular localisation of Hippo pathway components, and possibly other proteins, in adjacent cells and may therefore directly mediate these effects. Several genetic lesions alter Crb levels, including loss-of-function mutations in hyperplastic tumour suppressors in the Hippo-Salvador-Warts pathway and in neoplastic tumour suppressor genes, such as scribble. Thus, Crb may be part of a "surveillance mechanism" that is responsible for the cell death that is observed at the boundaries of mutant clones in these cases.

A buoyancy-based screen of Drosophila larvae for fat-storage mutants reveals a role for Sir2 in coupling fat storage to nutrient availability.

Obesity has a strong genetic component, but few of the genes that predispose to obesity are known. Genetic screens in invertebrates have the potential to identify genes and pathways that regulate the levels of stored fat, many of which are likely to be conserved in humans. To facilitate such screens, we have developed a simple buoyancy-based screening method for identifying mutant Drosophila larvae with increased levels of stored fat. Using this approach, we have identified 66 genes that when mutated increase organismal fat levels. Among these was a sirtuin family member, Sir2. Sirtuins regulate the storage and metabolism of carbohydrates and lipids by deacetylating key regulatory proteins. However, since mammalian sirtuins function in many tissues in different ways, it has been difficult to define their role in energy homeostasis accurately under normal feeding conditions. We show that knockdown of Sir2 in the larval fat body results in increased fat levels. Moreover, using genetic mosaics, we demonstrate that Sir2 restricts fat accumulation in individual cells of the fat body in a cell-autonomous manner. Consistent with this function, changes in the expression of metabolic enzymes in Sir2 mutants point to a shift away from catabolism. Surprisingly, although Sir2 is typically upregulated under conditions of starvation, Sir2 mutant larvae survive better than wild type under conditions of amino-acid starvation as long as sugars are provided. Our findings point to a Sir2-mediated pathway that activates a catabolic response to amino-acid starvation irrespective of the sugar content of the diet.

Coordinated growth of linked epithelia is mediated by the Hippo pathway.

An epithelium in a living organism seldom develops in isolation. Rather, most epithelia are tethered to other epithelial or non-epithelial tissues, necessitating growth coordination between layers. We investigated how two tethered epithelial layers of the Drosophila larval wing imaginal disc, the disc proper (DP) and the peripodial epithelium (PE), coordinate their growth. DP growth is driven by the morphogens Hedgehog (Hh) and Dpp, but regulation of PE growth is poorly understood. We find that the PE adapts to changes in growth rates of the DP, but not vice versa, suggesting a "leader and follower" mechanism. Moreover, PE growth can occur by cell shape changes, even when proliferation is inhibited. While Hh and Dpp pattern gene expression in both layers, growth of the DP is exquisitely sensitive to Dpp levels, while growth of the PE is not; the PE can achieve an appropriate size even when Dpp signaling is inhibited. Instead, both the growth of the PE and its accompanying cell shape changes require the activity of two components of the mechanosensitive Hippo pathway, the DNA-binding protein Scalloped (Sd) and its co-activator (Yki), which could allow the PE to sense and respond to forces generated by DP growth. Thus, an increased reliance on mechanically-dependent growth mediated by the Hippo pathway, at the expense of morphogen-dependent growth, enables the PE to evade layer-intrinsic growth control mechanisms and coordinate its growth with the DP. This provides a potential paradigm for growth coordination between different components of a developing organ.

The cell adhesion molecule Echinoid promotes tissue survival and separately restricts tissue overgrowth in Drosophila imaginal discs.

The interactions that cells in Drosophila imaginal discs have with their neighbors are known to regulate their ability to survive. In a screen of genes encoding cell surface proteins for gene knockdowns that affect the size or shape of mutant clones, we found that clones of cells with reduced levels of echinoid (ed) are fewer, smaller, and can be eliminated during development. In contrast, discs composed mostly of ed mutant tissue are overgrown. We find that ed mutant tissue has lower levels of the anti-apoptotic protein Diap1 and has increased levels of apoptosis which is consistent with the observed underrepresentation of ed mutant clones and the slow growth of ed mutant tissue. The eventual overgrowth of ed mutant tissue results not from accelerated growth, but from prolonged growth resulting from a failure to arrest growth at the appropriate final size. Ed has previously been shown to physically interact with multiple Hippo-pathway components and it has been proposed to promote Hippo pathway signaling, to exclude Yorkie (Yki) from the nucleus, and restrain the expression of Yki-target genes. We did not observe changes in Yki localization in ed mutant tissue and found decreased levels of expression of several Yorkie-target genes, findings inconsistent with the proposed effect of Ed on Yki. We did, however, observe increased expression of several Yki-target genes in wild-type cells neighboring ed mutant cells, which may contribute to elimination of ed mutant clones. Thus, ed has two distinct functions: an anti-apoptotic function by maintaining Diap1 levels, and a function to arrest growth at the appropriate final size. Both of these are unlikely to be explained by a simple effect on the Hippo pathway.

Imaginal Disc Regeneration: Something Old, Something New.

Imaginal discs are simple epithelial sacs found in Drosophila larvae, which generate adult structures including wings and legs. The first studies of imaginal disc regeneration involved technically challenging transplantation experiments. Yet despite the difficulty, many aspects of regeneration including wound healing, blastema formation, and the repatterning of regenerated tissue were characterized. An important discovery was the phenomenon of transdetermination, where a small group of cells in regenerating tissue collectively switch fate ("collective cell reprogramming"). The development of genetic tissue-ablation systems over the last 12 years has energized this field, by making experiments less technically challenging, more reproducible, and by incorporating additional genetic analysis. Recent progress includes defining mechanistic links between early responses to wounding and the signaling pathways that drive proliferation, uncovering a role for localized silencing of damage-responsive enhancers to limit regenerative capacity as tissues mature, and identifying genes that maintain cellular plasticity within acceptable limits during regeneration.

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.

The Drosophila melanogaster Apaf-1 homologue ARK is required for most, but not all, programmed cell death.

The Apaf-1 protein is essential for cytochrome c-mediated caspase-9 activation in the intrinsic mammalian pathway of apoptosis. Although Apaf-1 is the only known mammalian homologue of the Caenorhabditis elegans CED-4 protein, the deficiency of apaf-1 in cells or in mice results in a limited cell survival phenotype, suggesting that alternative mechanisms of caspase activation and apoptosis exist in mammals. In Drosophila melanogaster, the only Apaf-1/CED-4 homologue, ARK, is required for the activation of the caspase-9/CED-3-like caspase DRONC. Using specific mutants that are deficient for ark function, we demonstrate that ARK is essential for most programmed cell death (PCD) during D. melanogaster development, as well as for radiation-induced apoptosis. ark mutant embryos have extra cells, and tissues such as brain lobes and wing discs are enlarged. These tissues from ark mutant larvae lack detectable PCD. During metamorphosis, larval salivary gland removal was severely delayed in ark mutants. However, PCD occurred normally in the larval midgut, suggesting that ARK-independent cell death pathways also exist in D. melanogaster.

Single-cell transcriptomics of the Drosophila wing disc reveals instructive epithelium-to-myoblast interactions.

In both vertebrates and invertebrates, generating a functional appendage requires interactions between ectoderm-derived epithelia and mesoderm-derived cells. To investigate such interactions, we used single-cell transcriptomics to generate a temporal cell atlas of the Drosophila wing disc from two developmental time points. Using these data, we visualized gene expression using a multilayered model of the wing disc and cataloged ligand-receptor pairs that could mediate signaling between epithelial cells and adult muscle precursors (AMPs). We found that localized expression of the fibroblast growth factor ligands, Thisbe and Pyramus, in the disc epithelium regulates the number and location of the AMPs. In addition, Hedgehog ligand from the epithelium activates a specific transcriptional program within adjacent AMP cells, defined by AMP-specific targets Neurotactin and midline, that is critical for proper formation of direct flight muscles. More generally, our annotated temporal cell atlas provides an organ-wide view of potential cell-cell interactions between epithelial and myogenic cells.

Melting fat away in flies.

Two important proteins that function in regulating cell growth are the transcriptional regulator FOXO and the protein kinase Tor. A recent publication shows that the Melted protein can modulate both FOXO and Tor activity and can also regulate fat levels in Drosophila.

Capicua regulates cell proliferation downstream of the receptor tyrosine kinase/ras signaling pathway.

Signaling via the receptor tyrosine kinase (RTK)/Ras pathway promotes tissue growth during organismal development and is increased in many cancers [1]. It is still not understood precisely how this pathway promotes cell growth (mass accumulation). In addition, the RTK/Ras pathway also functions in cell survival, cell-fate specification, terminal differentiation, and progression through mitosis [2-7]. An important question is how the same canonical pathway can elicit strikingly different responses in different cell types. Here, we show that the HMG-box protein Capicua (Cic) restricts cell growth in Drosophila imaginal discs, and its levels are, in turn, downregulated by Ras signaling. Moreover, unlike normal cells, the growth of cic mutant cells is undiminished in the complete absence of a Ras signal. In addition to a general role in growth regulation, the importance of cic in regulating cell-fate determination downstream of Ras appears to vary from tissue to tissue. In the developing eye, the analysis of cic mutants shows that the functions of Ras in regulating growth and cell-fate determination are separable. Thus, the DNA-binding protein Cic is a key downstream component in the pathway by which Ras regulates growth in imaginal discs.

Ras and Rap: are former enemies now friends?

The small GTPase Rap1 was originally thought to function as an antagonist of Ras. A recent paper by provides evidence that Ras and Rap1 function in parallel to activate Raf downstream of the Torso receptor tyrosine kinase in Drosophila.

Mutations in erupted, the Drosophila ortholog of mammalian tumor susceptibility gene 101, elicit non-cell-autonomous overgrowth.

The reproducible pattern of organismal growth during metazoan development is the product of genetically controlled signaling pathways. Patterned activation of these pathways shapes developing organs and dictates overall organismal shape and size. Here, we show that patches of tissue that are mutant for the Drosophila Tsg101 ortholog, erupted, cause dramatic overproliferation of adjacent wild-type tissue. Tsg101 proteins function in endosomal sorting and are required to incorporate late endosomes into multivesicular bodies. Drosophila cells with impaired Tsg101 function show accumulation of the Notch receptor in intracellular compartments marked by the endosomal protein Hrs. This causes increased Notch-mediated signaling and ectopic expression of the Notch target gene unpaired (upd), which encodes the secreted ligand of the JAK-STAT pathway. Activation of JAK-STAT signaling in surrounding wild-type cells correlates with their overgrowth. These findings define a pathway by which changes in endocytic trafficking can regulate tissue growth in a non-cell-autonomous manner.

The Hippo pathway coactivator Yorkie can reprogram cell fates and create compartment-boundary-like interactions at clone margins.

During development, tissue-specific patterns of gene expression are established by transcription factors and then stably maintained via epigenetic mechanisms. Cancer cells often express genes that are inappropriate for that tissue or developmental stage. Here, we show that high activity levels of Yki, the Hippo pathway coactivator that causes overgrowth in Drosophila imaginal discs, can also disrupt cell fates by altering expression of selector genes like engrailed (en) and Ultrabithorax (Ubx). Posterior clones expressing activated Yki can down-regulate en and express an anterior selector gene, cubitus interruptus (ci). The microRNA bantam and the chromatin regulator Taranis both function downstream of Yki in promoting ci expression. The boundary between Yki-expressing posterior clones and surrounding wild-type cells acquires properties reminiscent of the anteroposterior compartment boundary; Hedgehog signaling pathway activation results in production of Dpp. Thus, at least in principle, heterotypic interactions between Yki-expressing cells and their neighbors could activate boundary-specific signaling mechanisms.

Damage-responsive, maturity-silenced enhancers regulate multiple genes that direct regeneration in Drosophila.

Like tissues of many organisms, Drosophila imaginal discs lose the ability to regenerate as they mature. This loss of regenerative capacity coincides with reduced damage-responsive expression of multiple genes needed for regeneration. We previously showed that two such genes, wg and Wnt6, are regulated by a single damage-responsive enhancer that becomes progressively inactivated via Polycomb-mediated silencing as discs mature (Harris et al., 2016). Here we explore the generality of this mechanism and identify additional damage-responsive, maturity-silenced (DRMS) enhancers, some near genes known to be required for regeneration such as Mmp1, and others near genes that we now show function in regeneration. Using a novel GAL4-independent ablation system we characterize two DRMS-associated genes, apontic (apt), which curtails regeneration and CG9752/asperous (aspr), which promotes it. This mechanism of suppressing regeneration by silencing damage-responsive enhancers at multiple loci can be partially overcome by reducing activity of the chromatin regulator extra sex combs (esc).