Spatiotemporal remodeling of extracellular matrix orients epithelial sheet folding.

Biological systems are inherently noisy; however, they produce highly stereotyped tissue morphology. Drosophila pupal wings show a highly stereotypic folding through uniform expansion and subsequent buckling of wing epithelium within a surrounding cuticle sac. The folding pattern produced by buckling is generally stochastic; it is thus unclear how buckling leads to stereotypic tissue folding of the wings. We found that the extracellular matrix (ECM) protein, Dumpy, guides the position and direction of buckling-induced folds. Dumpy anchors the wing epithelium to the overlying cuticle at specific tissue positions. Tissue-wide alterations of Dumpy deposition and degradation yielded different buckling patterns. In summary, we propose that spatiotemporal ECM remodeling shapes stereotyped tissue folding through dynamic interactions between the epithelium and its external structures.

Single-cell transcriptome atlas of Drosophila gastrula 2.0.

During development, positional information directs cells to specific fates, leading them to differentiate with their own transcriptomes and express specific behaviors and functions. However, the mechanisms underlying these processes in a genome-wide view remain ambiguous, partly because the single-cell transcriptomic data of early developing embryos containing accurate spatial and lineage information are still lacking. Here, we report a single-cell transcriptome atlas of Drosophila gastrulae, divided into 77 transcriptomically distinct clusters. We find that the expression profiles of plasma-membrane-related genes, but not those of transcription-factor genes, represent each germ layer, supporting the nonequivalent contribution of each transcription-factor mRNA level to effector gene expression profiles at the transcriptome level. We also reconstruct the spatial expression patterns of all genes at the single-cell stripe level as the smallest unit. This atlas is an important resource for the genome-wide understanding of the mechanisms by which genes cooperatively orchestrate Drosophila gastrulation.

Image-based parameter inference for epithelial mechanics.

Measuring mechanical parameters in tissues, such as the elastic modulus of cell-cell junctions, is essential to decipher the mechanical control of morphogenesis. However, their in vivo measurement is technically challenging. Here, we formulated an image-based statistical approach to estimate the mechanical parameters of epithelial cells. Candidate mechanical models are constructed based on force-cell shape correlations obtained from image data. Substitution of the model functions into force-balance equations at the cell vertex leads to an equation with respect to the parameters of the model, by which one can estimate the parameter values using a least-squares method. A test using synthetic data confirmed the accuracy of parameter estimation and model selection. By applying this method to Drosophila epithelial tissues, we found that the magnitude and orientation of feedback between the junction tension and shrinkage, which are determined by the spring constant of the junction, were correlated with the elevation of tension and myosin-II on shrinking junctions during cell rearrangement. Further, this method clarified how alterations in tissue polarity and stretching affect the anisotropy in tension parameters. Thus, our method provides a novel approach to uncovering the mechanisms governing epithelial morphogenesis.

JNK and Yorkie drive tumor malignancy by inducing L-amino acid transporter 1 in Drosophila.

Identifying a common oncogenesis pathway among tumors with different oncogenic mutations is critical for developing anti-cancer strategies. Here, we performed transcriptome analyses on two different models of Drosophila malignant tumors caused by Ras activation with cell polarity defects (RasV12/scrib-/-) or by microRNA bantam overexpression with endocytic defects (bantam/rab5-/-), followed by an RNAi screen for genes commonly essential for tumor growth and malignancy. We identified that Juvenile hormone Inducible-21 (JhI-21), a Drosophila homolog of the L-amino acid transporter 1 (LAT1), is upregulated in these malignant tumors with different oncogenic mutations and knocking down of JhI-21 strongly blocked their growth and invasion. JhI-21 expression was induced by simultaneous activation of c-Jun N-terminal kinase (JNK) and Yorkie (Yki) in these tumors and thereby contributed to tumor growth and progression by activating the mTOR-S6 pathway. Pharmacological inhibition of LAT1 activity in Drosophila larvae significantly suppressed growth of RasV12/scrib-/- tumors. Intriguingly, LAT1 inhibitory drugs did not suppress growth of bantam/rab5-/- tumors and overexpression of bantam rendered RasV12/scrib-/- tumors unresponsive to LAT1 inhibitors. Further analyses with RNA sequencing of bantam-expressing clones followed by an RNAi screen suggested that bantam induces drug resistance against LAT1 inhibitors via downregulation of the TMEM135-like gene CG31157. Our observations unveil an evolutionarily conserved role of LAT1 induction in driving Drosophila tumor malignancy and provide a powerful genetic model for studying cancer progression and drug resistance.

Model-based prediction of spatial gene expression via generative linear mapping.

Decoding spatial transcriptomes from single-cell RNA sequencing (scRNA-seq) data has become a fundamental technique for understanding multicellular systems; however, existing computational methods lack both accuracy and biological interpretability due to their model-free frameworks. Here, we introduce Perler, a model-based method to integrate scRNA-seq data with reference in situ hybridization (ISH) data. To calibrate differences between these datasets, we develop a biologically interpretable model that uses generative linear mapping based on a Gaussian mixture model using the Expectation-Maximization algorithm. Perler accurately predicts the spatial gene expression of Drosophila embryos, zebrafish embryos, mammalian liver, and mouse visual cortex from scRNA-seq data. Furthermore, the reconstructed transcriptomes do not over-fit the ISH data and preserved the timing information of the scRNA-seq data. These results demonstrate the generalizability of Perler for dataset integration, thereby providing a biologically interpretable framework for accurate reconstruction of spatial transcriptomes in any multicellular system.

Two-step regulation of trachealess ensures tight coupling of cell fate with morphogenesis in the Drosophila trachea.

During organogenesis, inductive signals cause cell differentiation and morphogenesis. However, how these phenomena are coordinated to form functional organs is poorly understood. Here, we show that cell differentiation of the Drosophila trachea is sequentially determined in two steps and that the second step is synchronous with the invagination of the epithelial sheet. The master gene trachealess is dispensable for the initiation of invagination, while it is essential for maintaining the invaginated structure, suggesting that tracheal morphogenesis and differentiation are separately induced. trachealess expression starts in bipotential tracheal/epidermal placode cells. After invagination, its expression is maintained in the invaginated cells but is extinguished in the remaining sheet cells. A trachealess cis-regulatory module that shows both tracheal enhancer activity and silencer activity in the surface epidermal sheet was identified. We propose that the coupling of trachealess expression with the invaginated structure ensures that only invaginated cells canalize robustly into the tracheal fate.

Development and Function of the Drosophila Tracheal System.

The tracheal system of insects is a network of epithelial tubules that functions as a respiratory organ to supply oxygen to various target organs. Target-derived signaling inputs regulate stereotyped modes of cell specification, branching morphogenesis, and collective cell migration in the embryonic stage. In the postembryonic stages, the same set of signaling pathways controls highly plastic regulation of size increase and pattern elaboration during larval stages, and cell proliferation and reprograming during metamorphosis. Tracheal tube morphogenesis is also regulated by physicochemical interaction of the cell and apical extracellular matrix to regulate optimal geometry suitable for air flow. The trachea system senses both the external oxygen level and the metabolic activity of internal organs, and helps organismal adaptation to changes in environmental oxygen level. Cellular and molecular mechanisms underlying the high plasticity of tracheal development and physiology uncovered through research on Drosophila are discussed.

Elasticity-based boosting of neuroepithelial nucleokinesis via indirect energy transfer from mother to daughter.

Neural progenitor cells (NPCs), which are apicobasally elongated and densely packed in the developing brain, systematically move their nuclei/somata in a cell cycle-dependent manner, called interkinetic nuclear migration (IKNM): apical during G2 and basal during G1. Although intracellular molecular mechanisms of individual IKNM have been explored, how heterogeneous IKNMs are collectively coordinated is unknown. Our quantitative cell-biological and in silico analyses revealed that tissue elasticity mechanically assists an initial step of basalward IKNM. When the soma of an M-phase progenitor cell rounds up using actomyosin within the subapical space, a microzone within 10 µm from the surface, which is compressed and elastic because of the apical surface's contractility, laterally pushes the densely neighboring processes of non-M-phase cells. The pressed processes then recoil centripetally and basally to propel the nuclei/somata of the progenitor's daughter cells. Thus, indirect neighbor-assisted transfer of mechanical energy from mother to daughter helps efficient brain development.

Mechanisms of cell height changes that mediate epithelial invagination.

Epithelial invagination is a morphogenetic process that converts flat cell sheets into tubular structures and contributes to the formation of three-dimensional organs during development. Because the cells in tubular structures have smaller apical than basal surfaces, apical constriction is thought to be critical for the process of epithelial invagination. In addition, the invagination process is also accompanied by cell elongation, followed by cell shortening and basal expansion. While the mechanisms involved in apical constriction have been well-characterized, recent technical advances are just beginning to unravel the mechanisms involved in cell height control, which include cytoskeletal changes, cortical tension generation, cell adhesion, and cytoplasmic flow. Furthermore, cell height changes associated with mitosis and apoptosis have recently been shown to contribute to epithelial invagination. To develop a comprehensive understanding of epithelial invagination, it is important to elucidate the mechanisms that mediate cell shape changes and facilitate their coordination. In this review, we summarize the recent advances in this field, focusing on the mechanisms that control cell height.

Pri peptides are mediators of ecdysone for the temporal control of development.

Animal development fundamentally relies on the precise control, in space and time, of genome expression. Whereas we have a wealth of information about spatial patterning, the mechanisms underlying temporal control remain poorly understood. Here we show that Pri peptides, encoded by small open reading frames, are direct mediators of the steroid hormone ecdysone for the timing of developmental programs in Drosophila. We identify a previously uncharacterized enzyme of ecdysone biosynthesis, GstE14, and find that ecdysone triggers pri expression to define the onset of epidermal trichome development, through post-translational control of the Shavenbaby transcription factor. We show that manipulating pri expression is sufficient to either put on hold or induce premature differentiation of trichomes. Furthermore, we find that ecdysone-dependent regulation of pri is not restricted to epidermis and occurs over various tissues and times. Together, these findings provide a molecular framework to explain how systemic hormonal control coordinates specific programs of differentiation with developmental timing.

TALEN-induced gene knock out in Drosophila.

We report here a case study of TALEN-induced gene knock out of the trachealess gene of Drosophila. Two pairs of TALEN constructs caused targeted mutation in the germ line of 39% and 17% of injected animals, respectively. In the extreme case 100% of the progeny of TALEN-injected fly was mutated, suggesting that highly efficient biallelic germ line mutagenesis was achieved. The mutagenic efficiency of the TALEN pairs paralleled their activity of single strand annealing (SSA) assay in cultured cells. All mutations were deletion of 1 to 20 base pairs. Merit and demerit of TALEN-based gene knockout approach compared to other genome editing technologies is discussed.

TAG-1-assisted progenitor elongation streamlines nuclear migration to optimize subapical crowding.

Neural progenitors exhibit cell cycle-dependent interkinetic nuclear migration (INM) along the apicobasal axis. Despite recent advances in understanding its underlying molecular mechanisms, the processes to which INM contributes mechanically and the regulation of INM by the apicobasally elongated morphology of progenitors remain unclear. We found that knockdown of the cell-surface molecule TAG-1 resulted in retraction of neocortical progenitors' basal processes. Highly shortened stem-like progenitors failed to undergo basalward INM and became overcrowded in the periventricular (subapical) space. Surprisingly, the overcrowded progenitors left the apical surface and migrated into basal neuronal territories. These observations, together with the results of in toto imaging and physical tests, suggest that progenitors may sense and respond to excessive mechanical stress. Although, unexpectedly, the heterotopic progenitors remained stem-like and continued to sequentially produce neurons until the late embryonic period, histogenesis was severely disrupted. Thus, INM is essential for preventing overcrowding of nuclei and their somata, thereby ensuring normal brain histogenesis.

Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging.

A recent key requirement in life sciences is the observation of biological processes in their natural in vivo context. However, imaging techniques that allow fast imaging with higher resolution in 3D thick specimens are still limited. Spinning disk confocal microscopy using a Yokogawa Confocal Scanner Unit, which offers high-speed multipoint confocal live imaging, has been found to have wide utility among cell biologists. A conventional Confocal Scanner Unit configuration, however, is not optimized for thick specimens, for which the background noise attributed to "pinhole cross-talk," which is unintended pinhole transmission of out-of-focus light, limits overall performance in focal discrimination and reduces confocal capability. Here, we improve spinning disk confocal microscopy by eliminating pinhole cross-talk. First, the amount of pinhole cross-talk is reduced by increasing the interpinhole distance. Second, the generation of out-of-focus light is prevented by two-photon excitation that achieves selective-plane illumination. We evaluate the effect of these modifications and test the applicability to the live imaging of green fluorescent protein-expressing model animals. As demonstrated by visualizing the fine details of the 3D cell shape and submicron-size cytoskeletal structures inside animals, these strategies dramatically improve higher-resolution intravital imaging.

Mitotic cell rounding accelerates epithelial invagination.

Mitotic cells assume a spherical shape by increasing their surface tension and osmotic pressure by extensively reorganizing their interphase actin cytoskeleton into a cortical meshwork and their microtubules into the mitotic spindle. Mitotic entry is known to interfere with tissue morphogenetic events that require cell-shape changes controlled by the interphase cytoskeleton, such as apical constriction. However, here we show that mitosis plays an active role in the epithelial invagination of the Drosophila melanogaster tracheal placode. Invagination begins with a slow phase under the control of epidermal growth factor receptor (EGFR) signalling; in this process, the central apically constricted cells, which are surrounded by intercalating cells, form a shallow pit. This slow phase is followed by a fast phase, in which the pit is rapidly depressed, accompanied by mitotic entry, which leads to the internalization of all the cells in the placode. We found that mitotic cell rounding, but not cell division, of the central cells in the placode is required to accelerate invagination, in conjunction with EGFR-induced myosin II contractility in the surrounding cells. We propose that mitotic cell rounding causes the epithelium to buckle under pressure and acts as a switch for morphogenetic transition at the appropriate time.

Coding vs non-coding: Translatability of short ORFs found in putative non-coding transcripts.

Genome analysis has identified a number of putative non-protein-coding transcripts that do not contain ORFs longer than 100 codons. Although evidence strongly suggests that non-coding RNAs are important in a variety of biological phenomena, the discovery of small peptide-coding mRNAs confirms that some transcripts that have been assumed to be non-coding actually have coding potential. Their abundance and importance in biological phenomena makes the sorting of non-coding RNAs from small peptide-coding mRNAs a key issue in functional genomics. However, validating the coding potential of small peptide-coding RNAs is complicated, because their ORF sequences are usually too short for computational analysis. In this review, we discuss computational and experimental methods for validating the translatability of these non-coding RNAs.

Lilliputians get into the limelight: novel class of small peptide genes in morphogenesis.

Generally, bioactive small peptides are derived from precursors with signal sequences at their N-terminal ends, which undergo modification and proteolysis through a secretory pathway. By contrast, small peptides encoded in short open reading frames (sORF) lack signaling sequences and therefore are released into the cytoplasm, which may result in their having functions distinct from those of secreted peptides. Several small peptides encoded by sORF are involved in the morphogenesis of multicellular organisms. POLARIS, ROTUNDIFOLIA4, and Enod40 are plant peptides that are involved, respectively, in root formation, leaf shape control, and cortical cell division during nodule formation. Brick1/HSPC300 is an evolutionarily conserved component of the actin reorganization complex. polished rice/tarsal-less and mille-pattes encode related small peptides that are required for epithelial morphogenesis in Drosophila and segmentation in Tribolium. There are only a few known examples of small peptides encoded by sORF, and their molecular functions are still largely obscure. Nevertheless, an increasing number of sORF genes is being identified, and further research should reveal their roles in novel molecular mechanisms underlying developmental events.

Small peptide regulators of actin-based cell morphogenesis encoded by a polycistronic mRNA.

Transcriptome analyses in eukaryotes, including mice and humans, have identified polyA-containing transcripts that lack long open reading frames (ORFs; >100 amino acids). These transcripts are believed most likely to function as non-coding RNAs, but their translational capacities and biological activities have not been characterized in detail. Here, we report that polished rice (pri), which was previously identified as a gene for a non-coding RNA in Drosophila, is in fact transcribed into a polycistronic mRNA that contains evolutionarily conserved short ORFs that encode 11 or 32 amino acid-long peptides. pri was expressed in all epithelial tissues during embryogenesis. The loss of pri function completely eliminated apical cuticular structures, including the epidermal denticles and tracheal taenidia, and also caused defective tracheal-tube expansion. We found that pri is essential for the formation of specific F-actin bundles that prefigures the formation of the denticles and taenidium. We provide evidences that pri acts non-cell autonomously and that four of the conserved pri ORFs are functionally redundant. These results demonstrate that pri has essential roles in epithelial morphogenesis by regulating F-actin organization.

Rapid construction of Drosophila RNAi transgenes using pRISE, a P-element-mediated transformation vector exploiting an in vitro recombination system.

RNAi is a gene-silencing phenomenon mediated by double-stranded RNA (dsRNA) and has become a powerful tool to elucidate gene function. To accomplish rapid construction of transgenes expressing dsRNA in Drosophila, we developed a novel transformation vector, pRISE, which contains an inverted repeat of the attR1-ccdB-attR2 cassette for in vitro recombination and a pentameric GAL4 binding site for conditional expression. These features enabled us to construct RNAi transgenes without a complicated cloning scheme. In cultured cells and transgenic flies, pRISE constructs carrying dsRNA transgenes induced effective RNAi against an EGFP transgene and the endogenous white gene, respectively. These results indicate that pRISE is a convenient transformation vector for studies of multiple Drosophila genes for which functional information is lacking.

Identification and expression analysis of putative mRNA-like non-coding RNA in Drosophila.

One of the most surprising results to emerge from mammalian cDNA sequencing projects is that thousands of mRNA-like non-coding RNAs (ncRNAs) are expressed and constitute at least 10% of poly(A)(+) RNAs. In most cases, however, the functions of these RNA molecules remain unclear. To clarify the biological significance of mRNA-like ncRNAs, we computationally screened 11,691 Drosophila melanogaster full-length cDNAs. After eliminating presumable protein-coding transcripts, 136 were identified as strong candidates for mRNA-like ncRNAs. Although most of these putative ncRNAs are found throughout the Drosophila genus, predicted amino acid sequences are not conserved even in related species, suggesting that these transcripts are actually non-coding RNAs. In situ hybridization analyses revealed that 35 of the transcripts are expressed during embryogenesis, of which 27 were detected only in specific tissues including the tracheal system, midgut primordial cells, visceral mesoderm, germ cells and the central and peripheral nervous system. These highly regulated expression patterns suggest that many mRNA-like ncRNAs play important roles in multiple steps of organogenesis and cell differentiation in Drosophila. This is the first report that the majority of mRNA-like ncRNAs in a model organism are expressed in specific tissues and cell types.