Target gene responses differ when transcription factor levels are acutely decreased by nuclear export versus degradation.

Defining the time of action for morphogens requires tools capable of temporally controlled perturbations. To study how the transcription factor Dorsal affects patterning of the Drosophila embryonic dorsal-ventral axis, we used two light-inducible tags that result in either nuclear export or degradation of Dorsal when exposed to blue light. Nuclear export of Dorsal results in loss of expression for the high threshold, ventrally-expressed target gene snail (sna) but retention of the low threshold, laterally-expressed target gene short-gastrulation (sog). In contrast, degradation of Dorsal results in retention of sna, loss of sog, and lower nuclear levels than when Dorsal is exported from the nucleus. To elucidate how nuclear export results in loss of sna but degradation does not, we investigated Dorsal kinetics using photobleaching and found it reenters the nucleus even under conditions of blue-light when export is favored. The associated kinetics of being imported and exported continuously are likely responsible for loss of sna but, alternatively, can support sog. Collectively, our results show that this dynamic patterning process is influenced by both Dorsal concentration and nuclear retention.

Two sequential gene expression programs bridged by cell division support long-distance collective cell migration.

The precise assembly of tissues and organs relies on spatiotemporal regulation of gene expression to coordinate the collective behavior of cells. In Drosophila embryos, the midgut musculature is formed through collective migration of caudal visceral mesoderm (CVM) cells, but how gene expression changes as cells migrate is not well understood. Here, we have focused on ten genes expressed in the CVM and the cis-regulatory sequences controlling their expression. Although some genes are continuously expressed, others are expressed only early or late during migration. Late expression relates to cell cycle progression, as driving string/Cdc25 causes earlier division of CVM cells and accelerates the transition to late gene expression. In particular, we found that the cell cycle effector transcription factor E2F1 is a required input for the late gene CG5080. Furthermore, whereas late genes are broadly expressed in all CVM cells, early gene transcripts are polarized to the anterior or posterior ends of the migrating collective. We show this polarization requires transcription factors Snail, Zfh1 and Dorsocross. Collectively, these results identify two sequential gene expression programs bridged by cell division that support long-distance directional migration of CVM cells.

Single-cell transcriptomics illuminates regulatory steps driving anterior-posterior patterning of Drosophila embryonic mesoderm.

Single-cell technologies promise to uncover how transcriptional programs orchestrate complex processes during embryogenesis. Here, we apply a combination of single-cell technology and genetic analysis to investigate the dynamic transcriptional changes associated with Drosophila embryo morphogenesis at gastrulation. Our dataset encompassing the blastoderm-to-gastrula transition provides a comprehensive single-cell map of gene expression across cell lineages validated by genetic analysis. Subclustering and trajectory analyses revealed a surprising stepwise progression in patterning to transition zygotic gene expression and specify germ layers as well as uncovered an early role for ecdysone signaling in epithelial-to-mesenchymal transition in the mesoderm. We also show multipotent progenitors arise prior to gastrulation by analyzing the transcription trajectory of caudal mesoderm cells, including a derivative that ultimately incorporates into visceral muscles of the midgut and hindgut. This study provides a rich resource of gastrulation and elucidates spatially regulated temporal transitions of transcription states during the process.

BMP-gated cell-cycle progression drives anoikis during mesenchymal collective migration.

Tissue homeostasis involves the elimination of abnormal cells to avoid compromised patterning and function. Although quality control through cell competition is well studied in epithelial tissues, it is unknown if and how homeostasis is regulated in mesenchymal collectives. Here, we demonstrate that collectively migrating Drosophila muscle precursors utilize both fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) signaling to promote homeostasis via anoikis, a form of cell death in response to substrate de-adhesion. Cell-cycle-regulated expression of the cell death gene head involution defective is responsible for caudal visceral mesoderm (CVM) anoikis. The secreted BMP ligand drives cell-cycle progression via a visceral mesoderm-specific cdc25/string enhancer to synchronize collective proliferation, as well as apoptosis of cells that have lost access to substrate-derived FGF. Perturbation of BMP-dependent cell-cycle progression is sufficient to confer anoikis resistance to mismigrating cells and thus facilitate invasion of other tissues. This BMP-gated cell-cycle checkpoint defines a quality control mechanism during mesenchymal collective migration.

brinker levels regulated by a promoter proximal element support germ cell homeostasis.

A limited BMP signaling range in the stem cell niche of the ovary protects against germ cell tumors and promotes germ cell homeostasis. The canonical repressor of BMP signaling in both the Drosophila embryo and wing disc is the transcription factor Brinker (Brk), yet the expression and potential role of Brk in the germarium has not previously been described. Here, we find that brk expression requires a promoter-proximal element (PPE) to support long-distance enhancer action as well as to drive expression in the germarium. Furthermore, PPE subdomains have different activities; in particular, the proximal portion acts as a damper to regulate brk levels precisely. Using PPE mutants as well as tissue-specific RNA interference and overexpression, we show that altering brk expression within either the soma or the germline affects germ cell homeostasis. Remarkably, we find that Decapentaplegic (Dpp), the main BMP ligand and canonical antagonist of Brk, is upregulated by Brk in the escort cells of the germarium, demonstrating that Brk can positively regulate this pathway.

NaNuTrap: a technique for in vivo cell nucleus labelling using nanobodies.

In vivo cell labelling is challenging in fast developmental processes because many cell types differentiate more quickly than the maturation time of fluorescent proteins, making visualization of these tissues impossible with standard techniques. Here, we present a nanobody-based method, Nanobody Nuclear Trap (NaNuTrap), which works with the existing Gal4/UAS system in Drosophila and allows for early in vivo cell nuclei labelling independently of the maturation time of the fluorescent protein. This restores the utility of fluorescent proteins that have longer maturation times, such as those used in two-photon imaging, for live imaging of fast or very early developmental processes. We also present a more general application of this system, whereby NaNuTrap can convert cytoplasmic GFP expressed in any existing transgenic fly line into a nuclear label. This nuclear re-localization of the fluorescent signal can improve the utility of the GFP label, e.g. in cell counting, as well as resulting in a general increase in intensity of the live fluorescent signal. We demonstrate these capabilities of NaNuTrap by effectively tracking subsets of cells during the fast movements associated with gastrulation.

Light-dependent N-end rule-mediated disruption of protein function in Saccharomyces cerevisiae and Drosophila melanogaster.

Here we describe the development and characterization of the photo-N-degron, a peptide tag that can be used in optogenetic studies of protein function in vivo. The photo-N-degron can be expressed as a genetic fusion to the amino termini of other proteins, where it undergoes a blue light-dependent conformational change that exposes a signal for the class of ubiquitin ligases, the N-recognins, which mediate the N-end rule mechanism of proteasomal degradation. We demonstrate that the photo-N-degron can be used to direct light-mediated degradation of proteins in Saccharomyces cerevisiae and Drosophila melanogaster with fine temporal control. In addition, we compare the effectiveness of the photo-N-degron with that of two other light-dependent degrons that have been developed in their abilities to mediate the loss of function of Cactus, a component of the dorsal-ventral patterning system in the Drosophila embryo. We find that like the photo-N-degron, the blue light-inducible degradation (B-LID) domain, a light-activated degron that must be placed at the carboxy terminus of targeted proteins, is also effective in eliciting light-dependent loss of Cactus function, as determined by embryonic dorsal-ventral patterning phenotypes. In contrast, another previously described photosensitive degron (psd), which also must be located at the carboxy terminus of associated proteins, has little effect on Cactus-dependent phenotypes in response to illumination of developing embryos. These and other observations indicate that care must be taken in the selection and application of light-dependent and other inducible degrons for use in studies of protein function in vivo, but importantly demonstrate that N- and C-terminal fusions to the photo-N-degron and the B-LID domain, respectively, support light-dependent degradation in vivo.

Dynamic patterning by morphogens illuminated by cis-regulatory studies.

Morphogen concentration changes in space as well as over time during development. However, how these dynamics are interpreted by cells to specify fate is not well understood. Here, we focus on two morphogens: the maternal transcription factors Bicoid and Dorsal, which directly regulate target genes to pattern Drosophila embryos. The actions of these factors at enhancers has been thoroughly dissected and provides a rich platform for understanding direct input by morphogens and their changing roles over time. Importantly, Bicoid and Dorsal do not work alone; we also discuss additional inputs that work with morphogens to control spatiotemporal gene expression in embryos.

Odd-paired is a pioneer-like factor that coordinates with Zelda to control gene expression in embryos.

Pioneer factors such as Zelda (Zld) help initiate zygotic transcription in Drosophila early embryos, but whether other factors support this dynamic process is unclear. Odd-paired (Opa), a zinc-finger transcription factor expressed at cellularization, controls the transition of genes from pair-rule to segmental patterns along the anterior-posterior axis. Finding that Opa also regulates expression through enhancer sog_Distal along the dorso-ventral axis, we hypothesized Opa's role is more general. Chromatin-immunoprecipitation (ChIP-seq) confirmed its in vivo binding to sog_Distal but also identified widespread binding throughout the genome, comparable to Zld. Furthermore, chromatin assays (ATAC-seq) demonstrate that Opa, like Zld, influences chromatin accessibility genome-wide at cellularization, suggesting both are pioneer factors with common as well as distinct targets. Lastly, embryos lacking opa exhibit widespread, late patterning defects spanning both axes. Collectively, these data suggest Opa is a general timing factor and likely late-acting pioneer factor that drives a secondary wave of zygotic gene expression.

FGF Pyramus Has a Transmembrane Domain and Cell-Autonomous Function in Polarity.

Most fibroblast growth factors (FGFs) function as receptor ligands through their conserved FGF domain, but sequences outside this domain vary and are not well studied. This core domain of 120 amino acids (aa) is flanked in all FGFs by highly divergent amino-terminal and carboxy-terminal sequences of variable length. Drosophila has fewer FGF genes, with only three identified to date, pyramus (pyr), thisbe (ths), and branchless (bnl), and all three encoding relatively large FGF proteins (∼80 kDa). We hypothesized that the longer FGF proteins present in Drosophila and other organisms may relate to an ancestral form, in which multiple functions or regulatory properties are present within a single polypeptide. Here, we focused analysis on Pyr, finding that it harbors a transmembrane domain (TMD) and extended C-terminal intracellular domain containing a degron. The intracellular portion limits Pyr levels, whereas the TMD promotes spatial precision in the paracrine activation of Heartless FGF receptor. Additionally, degron deletion mutants that upregulate Pyr exhibit cell polarity defects that lead to invagination defects at gastrulation, demonstrating a previously uncharacterized cell-autonomous role. In summary, our data show that Pyr is the first demonstrated transmembrane FGF, that it has both extracellular and intracellular functions, and that spatial distribution and levels of this particular FGF protein are tightly regulated. Our results suggest that other FGFs may be membrane tethered or multifunctional like Pyr.

Collective Migrations of Drosophila Embryonic Trunk and Caudal Mesoderm-Derived Muscle Precursor Cells.

Mesoderm migration in the Drosophila embryo is a highly conserved, complex process that is required for the formation of specialized tissues and organs, including the somatic and visceral musculature. In this FlyBook chapter, we will compare and contrast the specification and migration of cells originating from the trunk and caudal mesoderm. Both cell types engage in collective migrations that enable cells to achieve new positions within developing embryos and form distinct tissues. To start, we will discuss specification and early morphogenetic movements of the presumptive mesoderm, then focus on the coordinate movements of the two subtypes trunk mesoderm and caudal visceral mesoderm, ending with a comparison of these processes including general insights gained through study.

Twist-dependent ratchet functioning downstream from Dorsal revealed using a light-inducible degron.

Graded transcription factors are pivotal regulators of embryonic patterning, but whether their role changes over time is unclear. A light-regulated protein degradation system was used to assay temporal dependence of the transcription factor Dorsal in dorsal-ventral axis patterning of Drosophila embryos. Surprisingly, the high-threshold target gene snail only requires Dorsal input early but not late when Dorsal levels peak. Instead, late snail expression can be supported by action of the Twist transcription factor, specifically, through one enhancer, sna.distal This study demonstrates that continuous input is not required for some Dorsal targets and downstream responses, such as twist, function as molecular ratchets.

Setting up for gastrulation: D. melanogaster.

Drosophila melanogaster embryos develop initially as a syncytium of totipotent nuclei and subsequently, once cellularized, undergo morphogenetic movements associated with gastrulation to generate the three somatic germ layers of the embryo: mesoderm, ectoderm, and endoderm. In this chapter, we focus on the first phase of gastrulation in Drosophila involving patterning of early embryos when cells differentiate their gene expression programs. This patterning process requires coordination of multiple developmental processes including genome reprogramming at the maternal-to-zygotic transition, combinatorial action of transcription factors to support distinct gene expression, and dynamic feedback between this genetic patterning by transcription factors and changes in cell morphology. We discuss the gene regulatory programs acting during patterning to specify the three germ layers, which involve the regulation of spatiotemporal gene expression coupled to physical tissue morphogenesis.

Coacting enhancers can have complementary functions within gene regulatory networks and promote canalization.

Developmental genes are often regulated by multiple enhancers exhibiting similar spatiotemporal outputs, which are generally considered redundantly acting though few have been studied functionally. Using CRISPR-Cas9, we created deletions of two enhancers, brk5' and brk3', that drive similar but not identical expression of the gene brinker (brk) in early Drosophila embryos. Utilizing both in situ hybridization and quantitative mRNA analysis, we investigated the changes in the gene network state caused by the removal of one or both of the early acting enhancers. brk5' deletion generally phenocopied the gene mutant, including expansion of the BMP ligand decapentaplegic (dpp) as well as inducing variability in amnioserosa tissue cell number suggesting a loss of canalization. In contrast, brk3' deletion presented unique phenotypes including dorsal expansion of several ventrally expressed genes and a decrease in amnioserosa cell number. Similarly, deletions were made for two enhancers associated with the gene short-gastrulation (sog), sog.int and sog.dist, demonstrating that they also exhibit distinct patterning phenotypes and affect canalization. In summary, this study shows that similar gene expression driven by coacting enhancers can support distinct, and sometimes complementary, functions within gene regulatory networks and, moreover, that phenotypes associated with individual enhancer deletion mutants can provide insight into new gene functions.

Sticking to a plan: adhesion and signaling control spatial organization of cells within migrating collectives.

Collective cell migration is required in a vast array of biological phenomena, including organogenesis and embryonic development. The mechanisms that underlie collective cell migration not only involve the morphogenetic changes associated with single cell migration, but also require the maintenance of cell-cell junctions during movement. Additionally, cell shape changes and polarity must be coordinated in a multicellular manner in order to preserve directional movement in the migrating cohort, and often relates to multiple functions of common signaling pathways. In this review, we summarize the current understanding of the mechanisms underlying higher order tissue organization during migration, with particular focus on the interplay between cell adhesion and signaling that we propose can be tuned to support different types of collective movements.

Distinct Roles of Broadly Expressed Repressors Support Dynamic Enhancer Action and Change in Time.

How broadly expressed repressors regulate gene expression is incompletely understood. To gain insight, we investigated how Suppressor of Hairless-Su(H)-and Runt regulate expression of bone morphogenetic protein (BMP) antagonist short-gastrulation via the sog_Distal enhancer. A live imaging protocol was optimized to capture this enhancer's spatiotemporal output throughout the early Drosophila embryo, finding in this context that Runt regulates transcription initiation, Su(H) regulates transcription rate, and both factors control spatial expression. Furthermore, whereas Su(H) functions as a dedicated repressor, Runt temporally switches from repressor to activator. Our results demonstrate that broad repressors play temporally distinct roles and contribute to dynamic gene expression. Both Run and Su(H)'s ability to influence the spatiotemporal domains of gene expression may serve to counterbalance activators and function in this manner as important regulators of the maternal-to-zygotic transition in early embryos.

Migrating cells control morphogenesis of substratum serving as track to promote directional movement of the collective.

In Drosophila embryos, caudal visceral mesoderm (CVM) cells undergo bilateral migration along the trunk visceral mesoderm (TVM) in order to form midgut muscles. Mutation of FGF receptor Heartless (Htl) has been shown to cause CVM migration defects, particularly midline crossing of the bilateral groups. Here, we show that htl mutants also exhibit TVM defects including contralateral merging. Both CVM mismigration and TVM contralateral merging are attenuated by restoring FGF signaling specifically in the CVM, suggesting that migrating CVM cells influence TVM morphogenesis; however, the inverse, supplying FGF to the TVM, does not rescue CVM mismigration. In addition, we show that FGF regulates integrin expression in both tissues, but only providing a source of integrin specifically to the TVM attenuates the contralateral merging phenotype. Finally, we demonstrate that the CVM influences cell shape in the TVM, and a loss of CVM results in TVM morphological defects. In summary, this study provides insight into how a migrating collective of cells can influence their tissue substrate and supports the view that morphogenesis of tissues during development is interdependent.

A Developmental Program Truncates Long Transcripts to Temporally Regulate Cell Signaling.

Rapid mitotic divisions and a fixed transcription rate limit the maximal length of transcripts in early Drosophila embryos. Previous studies suggested that transcription of long genes is initiated but aborted, as early nuclear divisions have short interphases. Here, we identify long genes that are expressed during short nuclear cycles as truncated transcripts. The RNA binding protein Sex-lethal physically associates with transcripts for these genes and is required to support early termination to specify shorter transcript isoforms in early embryos of both sexes. In addition, one truncated transcript for the gene short-gastrulation encodes a product in embryos that functionally relates to a previously characterized dominant-negative form, which maintains TGF-ß signaling in the off-state. In summary, our results reveal a developmental program of short transcripts functioning to help temporally regulate Drosophila embryonic development, keeping cell signaling at early stages to a minimum in order to support its proper initiation at cellularization.

FGF controls epithelial-mesenchymal transitions during gastrulation by regulating cell division and apicobasal polarity.

To support tissue and organ development, cells transition between epithelial and mesenchymal states. Here, we have investigated how mesoderm cells change state in Drosophila embryos and whether fibroblast growth factor (FGF) signaling plays a role. During gastrulation, presumptive mesoderm cells invaginate, undergo an epithelial-to-mesenchymal state transition (EMT) and migrate upon the ectoderm. Our data show that EMT is a prolonged process in which adherens junctions progressively decrease in number throughout the migration of mesoderm cells. FGF influences adherens junction number and promotes mesoderm cell division, which we propose decreases cell-cell attachments to support slow EMT while retaining collective cell movement. We also found that, at the completion of migration, cells form a monolayer and undergo a reverse mesenchymal-to-epithelial transition (MET). FGF activity leads to accumulation of ß-integrin Myospheroid basally and cell polarity factor Bazooka apically within mesoderm cells, thereby reestablishing apicobasal cell polarity in an epithelialized state in which cells express both E-Cadherin and N-Cadherin. In summary, FGF plays a dynamic role in supporting mesoderm cell development to ensure collective mesoderm cell movements, as well as proper differentiation of mesoderm cell types.

Broadly expressed repressors integrate patterning across orthogonal axes in embryos.

The role of spatially localized repressors in supporting embryonic patterning is well appreciated, but, alternatively, the role ubiquitously expressed repressors play in this process is not well understood. We investigated the function of two broadly expressed repressors, Runt (Run) and Suppressor of Hairless [Su(H)], in patterning the Drosophila embryo. Previous studies have shown that Run and Su(H) regulate gene expression along anterior-posterior (AP) or dorsal-ventral (DV) axes, respectively, by spatially limiting activator action, but here we characterize a different role. Our data show that broadly expressed repressors silence particular enhancers within cis-regulatory systems, blocking their expression throughout the embryo fully but transiently, and, in this manner, regulate spatiotemporal outputs along both axes. Our results suggest that Run and Su(H) regulate the temporal action of enhancers and are not dedicated regulators of one axis but, instead, act coordinately to pattern both axes, AP and DV.