Microtubule polarity determines the lineage of embryonic neural precursor in zebrafish spinal cord.

The phenomenal diversity of neuronal types in the central nervous system is achieved in part by the asymmetric division of neural precursors. In zebrafish neural precursors, asymmetric dispatch of Sara endosomes (with its Notch signaling cargo) functions as fate determinant which mediates asymmetric division. Here, we found two distinct pools of neural precursors based on Sara endosome inheritance and spindle-microtubule enrichment. Symmetric or asymmetric levels of spindle-microtubules drive differently Sara endosomes inheritance and predict neural precursor lineage. We uncover that CAMSAP2a/CAMSAP3a and KIF16Ba govern microtubule asymmetry and endosome motility, unveiling the heterogeneity of neural precursors. Using a plethora of physical and cell biological assays, we determined the physical parameters and molecular mechanisms behind microtubule asymmetries and biased endosome motility. Evolutionarily, the values of those parameters explain why all sensory organ precursor cells are asymmetric in flies while, in zebrafish spinal cord, two populations of neural precursors (symmetric vs asymmetric) are possible.

Polarized branched Actin modulates cortical mechanics to produce unequal-size daughters during asymmetric division.

The control of cell shape during cytokinesis requires a precise regulation of mechanical properties of the cell cortex. Only few studies have addressed the mechanisms underlying the robust production of unequal-sized daughters during asymmetric cell division. Here we report that unequal daughter-cell sizes resulting from asymmetric sensory organ precursor divisions in Drosophila are controlled by the relative amount of cortical branched Actin between the two cell poles. We demonstrate this by mistargeting the machinery for branched Actin dynamics using nanobodies and optogenetics. We can thereby engineer the cell shape with temporal precision and thus the daughter-cell size at different stages of cytokinesis. Most strikingly, inverting cortical Actin asymmetry causes an inversion of daughter-cell sizes. Our findings uncover the physical mechanism by which the sensory organ precursor mother cell controls relative daughter-cell size: polarized cortical Actin modulates the cortical bending rigidity to set the cell surface curvature, stabilize the division and ultimately lead to unequal daughter-cell size.

Elongator stabilizes microtubules to control central spindle asymmetry and polarized trafficking of cell fate determinants.

Asymmetric cell division gives rise to two daughter cells that inherit different determinants, thereby acquiring different fates. Polarized trafficking of endosomes containing fate determinants recently emerged as an evolutionarily conserved feature of asymmetric cell division to enhance the robustness of asymmetric cell fate determination in flies, fish and mammals. In particular, polarized sorting of signalling endosomes by an asymmetric central spindle contributes to asymmetric cell division in Drosophila melanogaster. However, how central spindle asymmetry arises remains elusive. Here we identify a moonlighting function of the Elongator complex-an established protein acetylase and tRNA methylase involved in the fidelity of protein translation-as a key factor for central spindle asymmetry. Elongator controls spindle asymmetry by stabilizing microtubules differentially on the anterior side of the central spindle. Accordingly, lowering the activity of Elongator on the anterior side using nanobodies mistargets endosomes to the wrong cell. Molecularly, Elongator regulates microtubule dynamics independently of its acetylation and methylation enzymatic activities. Instead, Elongator directly binds to microtubules and increases their polymerization speed while decreasing their catastrophe frequency. Our data establish a non-canonical role of Elongator at the core of cytoskeleton polarity and asymmetric signalling.

A role for Flower and cell death in controlling morphogen gradient scaling.

During development, morphogen gradients encode positional information to pattern morphological structures during organogenesis1. Some gradients, like that of Dpp in the fly wing, remain proportional to the size of growing organs-that is, they scale. Gradient scaling keeps morphological patterns proportioned in organs of different sizes2,3. Here we show a mechanism of scaling that ensures that, when the gradient is smaller than the organ, cell death trims the developing tissue to match the size of the gradient. Scaling is controlled by molecular associations between Dally and Pentagone, known factors involved in scaling, and a key factor that mediates cell death, Flower4-6. We show that Flower activity in gradient expansion is not dominated by cell death, but by the activity of Dally/Pentagone on scaling. Here we show a potential connection between scaling and cell death that may uncover a molecular toolbox hijacked by tumours.

Morphogen gradient scaling by recycling of intracellular Dpp.

Morphogen gradients are fundamental to establish morphological patterns in developing tissues1. During development, gradients scale to remain proportional to the size of growing organs2,3. Scaling is a universal gear that adjusts patterns to size in living organisms3-8, but its mechanisms remain unclear. Here, focusing on the Decapentaplegic (Dpp) gradient in the Drosophila wing disc, we uncover a cell biological basis behind scaling. From small to large discs, scaling of the Dpp gradient is achieved by increasing the contribution of the internalized Dpp molecules to Dpp transport: to expand the gradient, endocytosed molecules are re-exocytosed to spread extracellularly. To regulate the contribution of endocytosed Dpp to the spreading extracellular pool during tissue growth, it is the Dpp binding rates that are progressively modulated by the extracellular factor Pentagone, which drives scaling. Thus, for some morphogens, evolution may act on endocytic trafficking to regulate the range of the gradient and its scaling, which could allow the adaptation of shape and pattern to different sizes of organs in different species.

BMP Signaling Gradient Scaling in the Zebrafish Pectoral Fin.

Secreted growth factors can act as morphogens that form spatial concentration gradients in developing organs, thereby controlling growth and patterning. For some morphogens, adaptation of the gradients to tissue size allows morphological patterns to remain proportioned as the organs grow. In the zebrafish pectoral fin, we found that BMP signaling forms a two-dimensional gradient. The length of the gradient scales with tissue length and its amplitude increases with fin size according to a power-law. Gradient scaling and amplitude power-laws are signatures of growth control by time derivatives of morphogenetic signaling: cell division correlates with the fold change over time of the cellular signaling levels. We show that Smoc1 regulates BMP gradient scaling and growth in the fin. Smoc1 scales the gradient by means of a feedback loop: Smoc1 is a BMP agonist and BMP signaling represses Smoc1 expression. Our work uncovers a layer of morphogen regulation during vertebrate appendage development.

Sara phosphorylation state controls the dispatch of endosomes from the central spindle during asymmetric division.

During asymmetric division, fate assignation in daughter cells is mediated by the partition of determinants from the mother. In the fly sensory organ precursor cell, Notch signalling partitions into the pIIa daughter. Notch and its ligand Delta are endocytosed into Sara endosomes in the mother cell and they are first targeted to the central spindle, where they get distributed asymmetrically to finally be dispatched to pIIa. While the processes of endosomal targeting and asymmetry are starting to be understood, the machineries implicated in the final dispatch to pIIa are unknown. We show that Sara binds the PP1c phosphatase and its regulator Sds22. Sara phosphorylation on three specific sites functions as a switch for the dispatch: if not phosphorylated, endosomes are targeted to the spindle and upon phosphorylation of Sara, endosomes detach from the spindle during pIIa targeting.

Polarized endosome dynamics by spindle asymmetry during asymmetric cell division.

During asymmetric division, fate determinants at the cell cortex segregate unequally into the two daughter cells. It has recently been shown that Sara (Smad anchor for receptor activation) signalling endosomes in the cytoplasm also segregate asymmetrically during asymmetric division. Biased dispatch of Sara endosomes mediates asymmetric Notch/Delta signalling during the asymmetric division of sensory organ precursors in Drosophila. In flies, this has been generalized to stem cells in the gut and the central nervous system, and, in zebrafish, to neural precursors of the spinal cord. However, the mechanism of asymmetric endosome segregation is not understood. Here we show that the plus-end kinesin motor Klp98A targets Sara endosomes to the central spindle, where they move bidirectionally on an antiparallel array of microtubules. The microtubule depolymerizing kinesin Klp10A and its antagonist Patronin generate central spindle asymmetry. This asymmetric spindle, in turn, polarizes endosome motility, ultimately causing asymmetric endosome dispatch into one daughter cell. We demonstrate this mechanism by inverting the polarity of the central spindle by polar targeting of Patronin using nanobodies (single-domain antibodies). This spindle inversion targets the endosomes to the wrong cell. Our data uncover the molecular and physical mechanism by which organelles localized away from the cellular cortex can be dispatched asymmetrically during asymmetric division.

Uninflatable and Notch control the targeting of Sara endosomes during asymmetric division.

Cell fate decision during asymmetric division is mediated by the biased partition of cell fate determinants during mitosis [1-6]. In the case of the asymmetric division of the fly sensory organ precursor cells, directed Notch signaling from pIIb to the pIIa daughter endows pIIa with its distinct fate [1-6]. We have previously shown that Notch/Delta molecules internalized in the mother cell traffic through Sara endosomes and are directed to the pIIa daughter [6]. Here we show that the receptor Notch itself is required during the asymmetric targeting of the Sara endosomes to pIIa. Notch binds Uninflatable, and both traffic together through Sara endosomes, which is essential to direct asymmetric endosomes motility and Notch-dependent cell fate assignation. Our data uncover a part of the core machinery required for the asymmetric motility of a vesicular structure that is essential for the directed dispatch of Notch signaling molecules during asymmetric mitosis.

Characterization of Neurospora crassa α-actinin.

α-Actinin, an actin-binding protein of the spectrin superfamily, is present in most eukaryotes except plants. It is composed of three domains: N-terminal CH-domains, C-terminal calcium-binding domain (with EF-hand motifs), and a central rod domain. We have cloned and expressed Neurospora crassa α-actinin as GST and GFP fusion proteins for biochemical characterization and in vivo localization, respectively. The intracellular localization pattern of α-actinin suggests that this protein is intimately associated with actin filaments and plays an important role in the processes of germination, hyphal elongation, septum formation, and conidiation. These functions were confirmed by the experiments on the effect of α-actinin gene deletion in N. crassa.

Sumoylation of Drosophila SU(VAR)3-7 is required for its heterochromatic function.

In Drosophila, SU(VAR)3-7 is an essential heterochromatin component. It is required for proper chromatin condensation, and changing its dose modifies position-effect variegation. Sumoylation is a post-translational modification shown to play a role in diverse biological processes. Here, we demonstrate that sumoylation is essential for proper heterochromatin function in Drosophila through modification of SU(VAR)3-7. Indeed, SU(VAR)3-7 is sumoylated at lysine K839; this modification is required for localization of SU(VAR)3-7 at pericentric heterochromatin, chromosome 4, and telomeres. In addition, sumoylation of SU(VAR)3-7 is a prerequisite for its ability to enhance position-effect variegation. Thus, these results show that the heterochromatic function of SU(VAR)3-7 depends on its own sumoylation, and unveil a role for sumoylation in Drosophila heterochromatin.

Transcriptional activation by GAGA factor is through its direct interaction with dmTAF3.

The GAGA factor (GAF), encoded by the Trithorax like gene (Trl) is a multifunctional protein involved in gene activation, Polycomb-dependent repression, chromatin remodeling and is a component of chromatin domain boundaries. Although first isolated as transcriptional activator of the Drosophila homeotic gene Ultrabithorax (Ubx), the molecular basis of this GAF activity is unknown. Here we show that dmTAF3 (also known as BIP2 and dTAF(II)155), a component of TFIID, interacts directly with GAF. We generated mutations in dmTAF3 and show that, in Trl mutant background, they affect transcription of Ubx leading to enhancement of Ubx phenotype. These results reveal that the gene activation pathway involving GAF is through its direct interaction with dmTAF3.

Drosophila G9a is a nonessential gene.

Mammalian G9a is a euchromatic histone H3 lysine 9 (H3K9) methyltransferase essential for development. Here, we characterize the Drosophila homolog of G9a, dG9a. We generated a dG9a deletion allele by homologous recombination. Analysis of this allele revealed that, in contrast to recent findings, dG9a is not required for fly viability.

Drosophila SETDB1 is required for chromosome 4 silencing.

Histone H3 lysine 9 (H3K9) methylation is associated with gene repression and heterochromatin formation. In Drosophila, SU(VAR)3-9 is responsible for H3K9 methylation mainly at pericentric heterochromatin. However, the histone methyltransferases responsible for H3K9 methylation at euchromatic sites, telomeres, and at the peculiar Chromosome 4 have not yet been identified. Here, we show that DmSETDB1 is involved in nonpericentric H3K9 methylation. Analysis of two DmSetdb1 alleles generated by homologous recombination, a deletion, and an allele where the 3HA tag is fused to the endogenous DmSetdb1, reveals that this gene is essential for fly viability and that DmSETDB1 localizes mainly at Chromosome 4. It also shows that DmSETDB1 is responsible for some of the H3K9 mono- and dimethyl marks in euchromatin and for H3K9 dimethylation on Chromosome 4. Moreover, DmSETDB1 is required for variegated repression of transgenes inserted on Chromosome 4. This study defines DmSETDB1 as a H3K9 methyltransferase that specifically targets euchromatin and the autosomal Chromosome 4 and shows that it is an essential factor for Chromosome 4 silencing.

Loss of the modifiers of variegation Su(var)3-7 or HP1 impacts male X polytene chromosome morphology and dosage compensation.

Loss of Su(var)3-7 or HP1 suppresses the genomic silencing of position-effect variegation, whereas over-expression enhances it. In addition, loss of Su(var)3-7 results in preferential male lethality. In polytene chromosomes deprived of Su(var)3-7, we observe a specific bloating of the male X chromosome, leading to shortening of the chromosome and to blurring of its banding pattern. In addition, the chromocenter, where heterochromatin from all polytene chromosomes fuses, appears decondensed. The same chromosomal phenotypes are observed as a result of loss of HP1. Mutations of Su(var)3-7 or of Su(var)2-5, the gene encoding HP1, also cause developmental defects, including a spectacular increase in size of the prothoracic gland and its polytene chromosomes. Thus, although structurally very different, the two proteins cooperate closely in chromosome organization and development. Finally, bloating of the male X chromosome in the Su(var)3-7 mutant depends on the presence of a functional dosage compensation complex on this chromosome. This observation reveals a new and intriguing genetic interaction between epigenetic silencing and compensation of dose.

Isolation of Su(var)3-7 mutations by homologous recombination in Drosophila melanogaster.

The Su(var)3-7 gene, a haplo-suppressor and triplo-enhancer of position-effect variegation (PEV), encodes a zinc finger heterochromatin-associated protein. To understand the role of this protein in heterochromatin and genomic silencing, mutations were generated by homologous recombination. The donor fragment contained a yellow(+) gene and 7.6 kb of the Su(var)3-7 gene inserted between two FRTs. The Su(var)3-7 sequence contained three stop codons flanking an I-SceI cut site located in the 5' half of the gene. Using two different screening approaches, we obtained an allelic series composed of three mutant alleles. The three mutations are dominant suppressors of PEV. One behaves as a null mutation and results in a maternal-effect recessive lethal phenotype that can be rescued by a zygotic paternal wild-type gene. A P transposon zygotically expressing a Su(var)3-7 full-length cDNA also rescues the mutant phenotype. One hypomorphic allele is viable and the pleiotropic phenotype showed by adult flies indicates that rapidly and late dividing cells seem the most affected by reduced amounts of Su(var)3-7 protein. All three mutants were characterized at the molecular level. Each expresses a portion of the Su(var)3-7 protein that is unable to enter the nucleus and bind chromatin.