Deborah J. Andrew

Morphogenesis, the sculpting of tissues into functional architectures, requires precise orchestration of cell shape changes and rearrangements. In the Drosophila salivary gland (SG), recent studies reveal that Crumbs (Crb) and non‐muscle Myosin II (MyoII) act as collaborative antagonists to drive SG internalization. Crb, a conserved type I transmembrane protein best known as a master regulator of apical‐basal polarity, stabilizes attachments by forming homophilic extracellular bridges between neighboring cells and restricting the distribution of junctional MyoII. Conversely, MyoII is a highly conserved motor protein that drives apical constriction and junctional shrinkage by generating contractile forces on apicomedial and cortical actin and promoting apical membrane removal. During SG invagination, these opposing activities are exquisitely tuned, with Crb‐mediated stabilization balancing MyoII‐mediated destabilization to facilitate apical constriction for internalization and neighbor exchange for tube elongation. The conserved interplay between Crb and MyoII goes beyond the shaping of organs, serving as a critical mechanism for cell extrusion and EMT‐like processes in animal development.

Dissecting gene regulation today relies on many genomic assays – including transcriptional output from RNA-seq, chromatin accessibility from ATAC-seq, and transcription factor (TF) binding from ChIP-seq. Whereas numerous tools exist for each modality and some integrate data across modalities, few allow researchers to interactively explore and visualize how TF binding motifs intersect with transcriptional activity and chromatin accessibility in a tissue-specific context. Here we introduce VIDEO (Visual Integration of Drosophila Enhancer Organization), a web-based analysis tool that enables visualization of conserved TF binding motifs within proximal enhancers of genes differentially expressed in specific tissues. Starting with gene lists derived from in situ hybridization, microarray, and/or scRNA-seq studies of WT or mutant samples, one can identify the TFs expressed in each tissue and learn if and where the consensus binding motifs for those TFs are found within the proximal enhancers of a custom gene set. This pipeline also allows for coincident visualization of active chromatin, as determined from ATAC-seq data, and for the visualization of DNA binding data from ChIP-seq datasets for specific TFs. To demonstrate its utility, we apply VIDEO to the well-characterized regulatory system of CrebA and the secretory pathway in the Drosophila melanogaster salivary gland. We also explore a lesser-known system in the embryonic hindgut to show how utilization of this tool can serve to generate hypotheses regarding regulatory interactions.

The functional gonads, essential for the continuity of a species, have both somatic and germline components. Newly formed germ cells are quiescent and are often physically isolated from the rest of the soma, protecting them from the signals that control somatic specification and differentiation. Nonetheless, the sequestered germ cells must ultimately navigate through the embryo to meet up with the somatic gonadal components. Forward genetic screens conducted in Drosophila have uncovered several crucial factors that generate both attractive and repulsive signals controlling germ cell movement. Efforts to reveal how the range of molecular players coordinate their activities to ensure that navigation is a robust and reproducible process have led to exciting, albeit sometimes contentious, discoveries. Herein, we summarize evidence for Hedgehog functioning in a single pathway from the signal source to signal reception to the downstream cytoskeletal events controlling the directed movement of germ cells to the site of gonad formation.

A bstract: Dissecting gene regulation today relies on many genomic assays – including transcriptional output from RNA-seq, chromatin accessibility from ATAC-seq, and transcription factor (TF) binding from ChIP-seq. Whereas numerous tools exist for each modality and some integrate data across modalities, few allow researchers to interactively explore and visualize how TF binding motifs intersect with transcriptional activity and chromatin accessibility in a tissue-specific context. Here we introduce VIDEO ( V isual I ntegration of D rosophila E nhancer O rganization), a web-based analysis tool that enables visualization of conserved TF binding motifs within proximal promoters of genes differentially expressed in specific tissues. Starting with gene lists derived from in situ hybridization, microarray, and/or scRNA-seq studies of WT or mutant samples, one can identify the TFs expressed in each tissue and learn if and where the consensus binding motifs for those TFs are found within the proximal enhancers of a custom gene set. This pipeline also allows for coincident visualization of active chromatin, as determined from ATAC-seq data, and for the visualization of DNA binding data from ChIP-seq datasets for specific TFs. To demonstrate its utility, we apply VIDEO to the well-characterized regulatory system of CrebA and the secretory pathway in the Drosophila salivary gland. We also explore a lesser-known system in the embryonic hindgut to show how utilization of this tool can serve to generate hypotheses regarding regulatory interactions.

DNA binding assays, expression analyses, and binding site mutagenesis revealed that the Drosophila CrebA transcription factor (TF) boosts secretory capacity in the embryonic salivary gland through direct regulation of secretory pathway component genes (SPCGs). The mammalian orthologues of CrebA, the Creb3L-family of leucine zipper TFs, not only activate SPCG expression in a variety of mammalian tissues but can also activate SPCG expression in Drosophila embryos, suggesting a highly conserved role for this family of proteins in boosting secretory capacity. However, in vivo assays reveal that CrebA binds far more genes than it regulates, and it remains unclear what distinguishes functional binding. It is also unclear if CrebA is the major factor driving SPCG gene expression in all Drosophila embryonic tissues and/or if CrebA also regulates other tissue-specific functions. Thus, we performed single-cell RNA sequencing (scRNA-seq) of CrebA null embryos and compared with scRNA-seq data from existing wild-type samples to explore the relationship between CrebA binding and gene regulation. We find that CrebA binds the proximal promoters of its targets, that SPCGs are the major class of genes regulated by CrebA across tissues, and that CrebA is sufficient to activate SPCG expression even in cells that do not normally express the protein. A comparison of scRNA-seq to other methods for capturing regulated transcripts reveals that the different methodologies identify overlapping but distinct sets of CrebA targets.

DNA binding assays, expression analyses, and binding site mutagenesis revealed that the Drosophila CrebA transcription factor (TF) boosts secretory capacity in the embryonic salivary gland (SG) through direct regulation of secretory pathway component genes (SPCGs). The mammalian orthologues of CrebA, the Creb3L-family of leucine zipper TFs, not only activate SPCG expression in a variety of mammalian tissues but can also activate SPCG expression in Drosophila embryos, suggesting a highly conserved role for this family of proteins in boosting secretory capacity. However, in vivo assays reveal that CrebA binds far more genes than it regulates, and it remains unclear what distinguishes functional binding. It is also unclear if CrebA is the major factor driving SPCG gene expression in all Drosophila embryonic tissues and/or if CrebA also regulates other tissue-specific functions. Thus, we did single cell RNA sequencing (scRNA-seq) of wild-type (WT) and CrebA null embryos to explore the relationship between CrebA binding and gene regulation. We find that CrebA binds the proximal promoters of its targets, that SPCGs are the major class of genes regulated by CrebA across tissues, and that CrebA is sufficient to activate SPCG expression even in cells that do not normally express the protein. A comparison of scRNA-Seq to other methods for capturing regulated transcripts reveals that the different methodologies identify overlapping but distinct sets of CrebA targets.

Precise orchestration of morphogenetic processes generates organs that are optimally positioned and the right size and shape to fit and maximize functionality. Here, we show that Arc, a large apical membrane–associated PDZ domain–containing protein, works through the apical determinant Crumbs to limit non-muscle myosin II (MyoII) activity during tissue invagination in the Drosophila salivary gland (SG). We show that loss of Arc, attenuation of Crumbs, and increased activation of MyoII leads to the simultaneous internalization of more precursor cells than normal. Consequently, mature SGs are shorter with more cells surrounding the lumen all along the tube. Correspondingly, overexpression of Arc or SG-specific knockdown of MyoII leads to longer SGs with fewer cells surrounding the lumen. Our findings support a model wherein plasma membrane (PM)-associated Crumbs stabilizes cellular junctions by limiting apical pools of activated MyoII and countering the destabilizing effects of MyoII at the PM, limiting how many cells internalize at any given time, shaping final tube geometry.

Precise orchestration of morphogenetic processes is required to generate organs that are optimally situated within the organism, and that are of the right size and shape to fit and maximize functionality. Here, we describe the role of Arc, a large apical membrane-associated PDZ domain-containing protein, that works through the apical determinant Crumb (Crb) to limit MyoII activity during tissue invagination in the forming Drosophila salivary gland (SG). We show that loss of Arc, attenuation of Crb function, as well as increased activation of non-muscle Myosin II (MyoII) leads to the simultaneous internalization of more precursor cells than normal. Consequently, mature SGs are significantly shorter with more cells surrounding the lumen at all positions along the tube. Correspondingly, overexpression of Arc or SG-specific knockdown of MyoII leads to the formation of longer SGs with fewer cells surrounding the lumen. We show that both Arc PDZ domains are required for Arc function and that they have distinct activities. Finally, we show that Arc facilitates Crb plasma membrane (PM) localization and suggests a model wherein PM-associated Crb stabilizes cellular junctions countering the destabilizing effects of apical medial and junctional pools of activated MyoII, thus limiting the number of primordial cells internalizing at any given time.

To gain insight into the transcription programs activated during the formation of Drosophila larval structures, we carried out single cell RNA sequencing during two periods of Drosophila embryogenesis: stages 10-12, when most organs are first specified and initiate morphological and physiological specialization; and stages 13-16, when organs achieve their final mature architectures and begin to function. Our data confirm previous findings with regards to functional specialization of some organs – the salivary gland and trachea – and clarify the embryonic functions of another – the plasmatocytes. We also identify two early developmental trajectories in germ cells and uncover a potential role for proteolysis during germline stem cell specialization. We identify the likely cell type of origin for key components of the Drosophila matrisome and several commonly used Drosophila embryonic cell culture lines. Finally, we compare our findings with other recent related studies and with other modalities for identifying tissue-specific gene expression patterns. These data provide a useful community resource for identifying many new players in tissue-specific morphogenesis and functional specialization of developing organs.