The neurogenic fate of the hindbrain boundaries relies on Notch3-dependent asymmetric cell divisions.

Elucidating the cellular and molecular mechanisms that regulate the balance between progenitor cell proliferation and neuronal differentiation in the construction of the embryonic brain demands the combination of cell lineage and functional approaches. Here, we generate the comprehensive lineage of hindbrain boundary cells by using a CRISPR-based knockin zebrafish transgenic line that specifically labels the boundaries. We unveil that boundary cells asynchronously engage in neurogenesis undergoing a functional transition from neuroepithelial progenitors to radial glia cells, coinciding with the onset of Notch3 signaling that triggers their asymmetrical cell division. Upon notch3 loss of function, boundary cells lose radial glia properties and symmetrically divide undergoing neuronal differentiation. Finally, we show that the fate of boundary cells is to become neurons, the subtype of which relies on their axial position, suggesting that boundary cells contribute to refine the number and proportion of the distinct neuronal populations.

Genome-wide phenotypic RNAi screen in the Drosophila wing: phenotypic description of functional classes.

The Drosophila genome contains approximately 14,000 protein-coding genes encoding all the necessary information to sustain cellular physiology, tissue organization, organism development, and behavior. In this manuscript, we describe in some detail the phenotypes in the adult fly wing generated after knockdown of approximately 80% of Drosophila genes. We combined this phenotypic description with a comprehensive molecular classification of the Drosophila proteins into classes that summarize the main expected or known biochemical/functional aspect of each protein. This information, combined with mRNA expression levels and in situ expression patterns, provides a simplified atlas of the Drosophila genome, from housekeeping proteins to the components of the signaling pathways directing wing development, that might help to further understand the contribution of each gene group to wing formation.

Genome-wide phenotypic RNAi screen in the Drosophila wing: global parameters.

We have screened a collection of UAS-RNAi lines targeting 10,920 Drosophila protein-coding genes for phenotypes in the adult wing. We identified 3653 genes (33%) whose knockdown causes either larval/pupal lethality or a mutant phenotype affecting the formation of a normal wing. The most frequent phenotypes consist of changes in wing size, vein differentiation, and patterning, defects in the wing margin and in the apposition of the dorsal and ventral wing surfaces. We also defined 16 functional categories encompassing the most relevant aspect of each protein function and assigned each Drosophila gene to one of these functional groups. This allowed us to identify which mutant phenotypes are enriched within each functional group. Finally, we used previously published gene expression datasets to determine which genes are or are not expressed in the wing disc. Integrating expression, phenotypic and molecular information offers considerable precision to identify the relevant genes affecting wing formation and the biological processes regulated by them.

The mechanosensitive Piezo1 channel controls endosome trafficking for an efficient cytokinetic abscission.

Mechanical forces are exerted throughout cytokinesis, the final step of cell division. Yet, how forces are transduced and affect the signaling dynamics of cytokinetic proteins remains poorly characterized. We now show that the mechanosensitive Piezo1 channel is activated at the intercellular bridge (ICB) connecting daughter cells to regulate abscission. Inhibition of Piezo1 caused multinucleation both in vitro and in vivo. Piezo1 positioning at the ICB during cytokinesis depends on Pacsin3. Pharmacological and genetic inhibition of Piezo1 or Pacsin3 resulted in mislocation of Rab11-family-interacting protein 3 (Rab11-FIP3) endosomes, apoptosis-linked gene 2-interacting protein X (ALIX), and endosomal sorting complex required for transport III (ESCRT-III). Furthermore, we identified FIP3 as the link between Piezo1-generated Ca2+ signals and ALIX delivery to the ICB, where ALIX recruits the ESCRT-III component charged multivesicular body protein 4B, which promotes abscission. These results provide a different view of how mechanical forces participate in cytokinesis and identify Piezo1 as a key modulator of endosome trafficking.

Yap/Taz-TEAD activity links mechanical cues to progenitor cell behavior during zebrafish hindbrain segmentation.

Cells perceive their microenvironment through chemical and physical cues. However, how the mechanical signals are interpreted during embryonic tissue deformation to result in specific cell behaviors is largely unknown. The Yap/Taz family of transcriptional co-activators has emerged as an important regulator of tissue growth and regeneration, responding to physical cues from the extracellular matrix, and to cell shape and actomyosin cytoskeletal changes. In this study, we demonstrate the role of Yap/Taz-TEAD activity as a sensor of mechanical signals in the regulation of the progenitor behavior of boundary cells during zebrafish hindbrain compartmentalization. Monitoring of in vivo Yap/Taz activity during hindbrain segmentation indicated that boundary cells responded to mechanical cues in a cell-autonomous manner through Yap/Taz-TEAD activity. Cell-lineage analysis revealed that Yap/Taz-TEAD boundary cells decreased their proliferative activity when Yap/Taz-TEAD activity ceased, which preceded changes in their cell fate from proliferating progenitors to differentiated neurons. Functional experiments demonstrated the pivotal role of Yap/Taz-TEAD signaling in maintaining progenitor features in the hindbrain boundary cell population.

A Search for Genes Mediating the Growth-Promoting Function of TGFß in the Drosophila melanogaster Wing Disc.

Transforming Growth Factor ß (TGFß) signaling has a complex influence on cell proliferation, acting to stop cell division in differentiating cells, but also promoting cell division in immature cells. The activity of the pathway in Drosophila is mostly required to stimulate the proliferation of neural and epithelial tissues. Most interestingly, this function is not absolutely required for cell division, but it is needed for these tissues to reach their correct size. It is not known how TGFß signaling promotes cell division in imaginal discs, or what the interactions between TGFß activity and other signaling pathways regulating cell proliferation are. In this work, we have explored the disc autonomous function of TGFß that promotes wing imaginal disc growth. We have studied the genetic interactions between TGFß signaling and other pathways regulating wing disc growth, such as the Insulin and Hippo/Salvador/Warts pathways, as well as cell cycle regulators. We have also identified a collection of TGFß candidate target genes affecting imaginal growth using expression profiles. These candidates correspond to genes participating in the regulation of a variety of biochemical processes, including different aspects of cell metabolism, suggesting that TGFß could affect cell proliferation by regulating the metabolic fitness of imaginal cells.

Activation and function of TGFß signalling during Drosophila wing development and its interactions with the BMP pathway.

The development of the Drosophila wing disc requires the activities of the BMP and TGFß signalling pathways. BMP signalling is critical for the correct growth and patterning of the disc, whereas the related TGFß pathway is mostly required for growth. The BMP and TGFß pathways share a common co-receptor (Punt) and a nuclear effector (Medea), and consequently it is likely that these pathways can interfere with each other during normal development. In this work we focus on the spatial activation domains and requirements for TGFß signalling during wing disc development. We found that the phosphorylation of Smad2, the specific transducer for TGFß signalling, occurs in a generalised manner in the wing disc. It appears that the expression of the four candidate TGFß ligands (Activinß, Dawdle, Maverick and Myoglianin) in the wing disc is required to obtain normal levels of TGFß signalling in this tissue. We show that Baboon, the specific receptor of the TGFß pathway, can phosphorylate Mad, the specific transducer of the BMP pathway, in vivo. However, this activation only occurs in the wing disc when the receptor is constitutively activated in a background of reduced expression of Smad2. In the presence of Smad2, the normal situation during wing disc development, high levels of activated Baboon lead to a depletion in Mad phosphorylation and to BMP loss-of-function phenotypes. Although loss of either babo or Smad2 expression reduce growth in the wing blade in a similar manner, loss of Smad2 can also cause phenotypes related to ectopic BMP signalling, suggesting a physiological role for this transducer in the regulation of Mad spatial activation.