Takefumi Kondo

Small Peptides Switch the Transcriptional Activity of Shavenbaby During Drosophila Embryogenesis

Transcription On and sORF Eukaryotic transcriptomes include numerous RNAs that are presumed noncoding because they include only short open reading frames (sORFs). However, some sORF RNAs actually produce small peptides with unknown activity. Now Kondo et al. (p. 336; see the Perspective by Rosenberg) report the function of peptides containing 11 to 32 amino acids that are encoded by the gene polished-rice (pri). pri triggers N-terminal truncation of the transcription factor Shavenbaby that controls epidermal differentiation in Drosophila. Following pri expression, Shavenbaby is converted from a transcriptional repressor to an activator. Thus, sORF peptides can control transcriptional programs during embryonic development. A conserved locus in insects encodes four small peptides that shift a transcription factor from repressor into activator. A substantial proportion of eukaryotic transcripts are considered to be noncoding RNAs because they contain only short open reading frames (sORFs). Recent findings suggest, however, that some sORFs encode small bioactive peptides. Here, we show that peptides of 11 to 32 amino acids encoded by the polished rice (pri) sORF gene control epidermal differentiation in Drosophila by modifying the transcription factor Shavenbaby (Svb). Pri peptides trigger the amino-terminal truncation of the Svb protein, which converts Svb from a repressor to an activator. Our results demonstrate that during Drosophila embryogenesis, Pri sORF peptides provide a strict temporal control to the transcriptional program of epidermal morphogenesis.

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.

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.

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.

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.

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.

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.

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.

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.

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.