Light-assisted small-molecule screening against protein kinases.

High-throughput live-cell screens are intricate elements of systems biology studies and drug discovery pipelines. Here, we demonstrate an optogenetics-assisted method that avoids the need for chemical activators and reporters, reduces the number of operational steps and increases information content in a cell-based small-molecule screen against human protein kinases, including an orphan receptor tyrosine kinase. This blueprint for all-optical screening can be adapted to many drug targets and cellular processes.

FAM96A is a novel pro-apoptotic tumor suppressor in gastrointestinal stromal tumors.

The ability to escape apoptosis is a hallmark of cancer-initiating cells and a key factor of resistance to oncolytic therapy. Here, we identify FAM96A as a ubiquitous, evolutionarily conserved apoptosome-activating protein and investigate its potential pro-apoptotic tumor suppressor function in gastrointestinal stromal tumors (GISTs). Interaction between FAM96A and apoptotic peptidase activating factor 1 (APAF1) was identified in yeast two-hybrid screen and further studied by deletion mutants, glutathione-S-transferase pull-down, co-immunoprecipitation and immunofluorescence. Effects of FAM96A overexpression and knock-down on apoptosis sensitivity were examined in cancer cells and zebrafish embryos. Expression of FAM96A in GISTs and histogenetically related cells including interstitial cells of Cajal (ICCs), "fibroblast-like cells" (FLCs) and ICC stem cells (ICC-SCs) was investigated by Northern blotting, reverse transcription-polymerase chain reaction, immunohistochemistry and Western immunoblotting. Tumorigenicity of GIST cells and transformed murine ICC-SCs stably transduced to re-express FAM96A was studied by xeno- and allografting into immunocompromised mice. FAM96A was found to bind APAF1 and to enhance the induction of mitochondrial apoptosis. FAM96A protein or mRNA was dramatically reduced or lost in 106 of 108 GIST samples representing three independent patient cohorts. Whereas ICCs, ICC-SCs and FLCs, the presumed normal counterparts of GIST, were found to robustly express FAM96A protein and mRNA, FAM96A expression was much reduced in tumorigenic ICC-SCs. Re-expression of FAM96A in GIST cells and transformed ICC-SCs increased apoptosis sensitivity and diminished tumorigenicity. Our data suggest FAM96A is a novel pro-apoptotic tumor suppressor that is lost during GIST tumorigenesis.

ß-arrestin control of late endosomal sorting facilitates decoy receptor function and chemokine gradient formation.

A crucial regulator of Cxcl12 is the decoy receptor Cxcr7, which controls the level of the chemokine in the tissue. The molecular mechanisms that enable Cxcr7 to function as an efficient molecular sink are not known. Using zebrafish primordial germ cells as a model, we identify a novel role for ß-arrestins in controlling the intracellular trafficking of Cxcr7. ß-arrestins facilitate the recycling of Cxcr7 from late endosomal compartments back to the plasma membrane, whereas the internalized ligand undergoes lysosomal degradation. ß-arrestins thus function in regulating chemokine gradient formation, allowing responding cells to discriminate between alternative migration targets in vivo.

Interpretation of the FGF8 morphogen gradient is regulated by endocytic trafficking.

Forty years ago, it was proposed that during embryonic development and organogenesis, morphogen gradients provide positional information to the individual cells within a tissue leading to specific fate decisions. Recently, much insight has been gained into how such morphogen gradients are formed and maintained; however, which cellular mechanisms govern their interpretation within target tissues remains debated. Here we used in vivo fluorescence correlation spectroscopy and automated image analysis to assess the role of endocytic sorting dynamics on fibroblast growth factor 8 (Fgf8) morphogen gradient interpretation. By interfering with the function of the ubiquitin ligase Cbl, we found an expanded range of Fgf target gene expression and a delay of Fgf8 lysosomal transport. However, the extracellular Fgf8 morphogen gradient remained unchanged, indicating that the observed signalling changes are due to altered gradient interpretation. We propose that regulation of morphogen signalling activity through endocytic sorting allows fast feedback-induced changes in gradient interpretation during the establishment of complex patterns.

Fgf8 morphogen gradient forms by a source-sink mechanism with freely diffusing molecules.

It is widely accepted that tissue differentiation and morphogenesis in multicellular organisms are regulated by tightly controlled concentration gradients of morphogens. How exactly these gradients are formed, however, remains unclear. Here we show that Fgf8 morphogen gradients in living zebrafish embryos are established and maintained by two essential factors: fast, free diffusion of single molecules away from the source through extracellular space, and a sink function of the receiving cells, regulated by receptor-mediated endocytosis. Evidence is provided by directly examining single molecules of Fgf8 in living tissue by fluorescence correlation spectroscopy, quantifying their local mobility and concentration with high precision. By changing the degree of uptake of Fgf8 into its target cells, we are able to alter the shape of the Fgf8 gradient. Our results demonstrate that a freely diffusing morphogen can set up concentration gradients in a complex multicellular tissue by a simple source-sink mechanism.

Ubc9 regulates mitosis and cell survival during zebrafish development.

Many proteins are modified by conjugation with Sumo, a gene-encoded, ubiquitin-related peptide, which is transferred to its target proteins via an enzymatic cascade. A central component of this cascade is the E2-conjugating enzyme Ubc9, which is highly conserved across species. Loss-of-function studies in yeast, nematode, fruit fly, and mouse blastocystes point to multiple roles of Ubc9 during cell cycle regulation, maintenance of nuclear architecture, chromosome segregation, and viability. Here we show that in zebrafish embryos, reduction of Ubc9 activity by expression of a dominant negative version causes widespread apoptosis, similar to the effect described in Ubc9-deficient mice. However, antisense-based knock down of zygotic ubc9 leads to much more specific defects in late proliferating tissues, such as cranial cartilage and eyes. Affected cartilaginous elements are of relatively normal size and shape, but consist of fewer and larger cells. Stainings with mitotic markers and 5-Bromo-2'-deoxyuridine incorporation studies indicate that fewer chondrocyte precursors are in mitosis, whereas the proportion of cells in S-phase is unaltered. Consistently, FACS analyses reveal an increase in the number of cells with a DNA content of 4n or even 8n. Our data indicate an in vivo requirement of Ubc9 for G2/M transition and/or progression through mitosis during vertebrate organogenesis. Failed mitosis in the absence of Ubc9 is not necessarily coupled with cell death. Rather, cells can continue to replicate their DNA, grow to a larger size, and finish their normal developmental program.

Eya1 is required for lineage-specific differentiation, but not for cell survival in the zebrafish adenohypophysis.

The homeodomain transcription factor Six1 and its modulator, the protein phosphatase Eya1, cooperate to promote cell differentiation and survival during mouse organ development. Here, we studied the effects caused by loss of eya1 and six1 function on pituitary development in zebrafish. eya1 and six1 are co-expressed in all adenohypophyseal cells. Nevertheless, eya1 (aal, dog) mutants show lineage-specific defects, defining corticotropes, melanotropes, and gonadotropes as an Eya1-dependent lineage, which is complementary to the Pit1 lineage. Furthermore, eya1 is required for maintenance of pit1 expression, leading to subsequent loss of cognate hormone gene expression in thyrotropes and somatotropes of mutant embryos, whereas prolactin expression in lactotropes persists. In contrast to other organs, adenohypophyseal cells of eya1 mutants do not become apoptotic, and the adenohypophysis remains at rather normal size. Also, cells do not trans-differentiate, as in the case of pit1 mutants, but display morphological features characteristic for nonsecretory cells. Some of the adenohypophyseal defects of eya1 mutants are moderately enhanced in combination with antisense-mediated loss of Six1 function, which per se does not affect pituitary cell differentiation. In conclusion, this is the first report of an essential role of Eya1 during pituitary development in vertebrates. Eya1 is required for lineage-specific differentiation of adenohypophyseal cells, but not for their survival, thereby uncoupling the differentiation-promoting and anti-apoptotic effects of Eya proteins seen in other tissues.

Destabilization of DeltaNp63alpha by Nedd4-mediated ubiquitination and Ubc9-mediated sumoylation, and its implications on dorsoventral patterning of the zebrafish embryo.

We have recently identified the p53-related DeltaNp63 gene as a transcriptional target of Bmp signaling that encodes a transcriptional repressor blocking neural development in the zebrafish ectoderm. However, in contrast to Bmps, the neural-repressing effect of forced DeltaNp63alpha expression is restricted to the presumptive forebrain, while posterior regions of the brain are not affected. Here, we show that this is due to instability of DeltaNp63alpha protein on the dorsal side of the embryo. In a yeast-two-hybrid screen, we isolated two DeltaNp63alpha-modifying enzymes, the SUMO-conjugating enzyme Ubc9 and the ubiquitin ligase Nedd4. The proteins bind to distinct sites in the C-terminal region of DeltaNp63alpha, which are absent in the shorter and more stable DeltaNp63gamma isoform. Similarly, mutant versions of DeltaNp63alpha unable to bind Nedd4 or Ubc9 are stabilized. DeltaNp63alpha is sumoylated and ubiquitinated both in HEK293 cells and in zebrafish embryos, and Nedd4 promotes ubiquitination and instability of DeltaNp63alpha protein, with lysine residue 637 serving as a potential alternative sumoylation and ubiquitination site that is crucial for DeltaNp63alpha destabilization. In zebrafish, ubc9.1 and nedd4 show restricted expression on the dorsal side of the embryo, where DeltaNp63alpha instability can be overcome upon blockage of endogenous Nedd4 activity, or upon injection of mutant versions of DeltaNp63alpha that are unable to bind Nedd4 or Ubc9. This results in a more widespread neural repression, affecting the entire Bmp-sensitive neuroectoderm. In sum, our data indicate that DeltaNp63alpha is ubiquitinated in a Nedd4- and sumoylated in a Ubc9-dependent fashion, and that these modifications can regulate DeltaNp63alpha stability in the zebrafish ectoderm.

Loss of maternal Smad5 in zebrafish embryos affects patterning and morphogenesis of optic primordia.

The mechanisms of patterning and morphogenesis of vertebrate eye primordia are heavily debated. Taking advantage of the maternal effect of a zebrafish smad5 null mutation (Mm169), we investigate the effect of early signaling by members of the bone morphogenetic proteins (Bmps) on eye field patterning and optic vesicle morphogenesis. In contrast to previous Xenopus and chick studies demonstrating a late dorsalizing effect of Bmp4 expressed in the dorsal neural retina itself, we show that patterning of the eye primordia already starts during blastula and early gastrula stages. At these stages, bmps expressed on the ventrolateral side of the embryo promote dorso-distal fates in the entire neuroectoderm, including the eye primordia. Despite a normal split of the eye field in the midline, the eye primordia of Mm169 embryos fail to evaginate laterally. They display a concentric pattern with retinal cells in the center and optic stalk cells in the periphery, representing a flattened version of the topologic relationships present in the mature wild-type eye. Different interpretations of these latter findings are presented. They can be best explained with a model according to which zebrafish eye morphogenesis occurs as a telescopic extension of disc-like, concentric primordia, similar to the development of appendages from imaginal discs in Drosophila.

Maternally supplied Smad5 is required for ventral specification in zebrafish embryos prior to zygotic Bmp signaling.

We have previously shown that the maternal effect dorsalization of zebrafish embryos from sbn(dtc24) heterozygous mothers is caused by a dominant negative mutation in Smad5, a transducer of ventralizing signaling by the bone morphogenetic proteins Bmp2b and Bmp7. Since sbn(dtc24) mutant Smad5 protein not only blocks wild-type Smad5, but also other family members like Smad1, it remained open to what extent Smad5 itself is required for dorsoventral patterning. Here, we report the identification of novelsmad5 alleles: three new isolates coming from a dominant enhancer screen, and four former isolates initially assigned to the cpt and pgy complementation groups. Overexpression analyses demonstrate that three of the new alleles, m169, fr5, and tc227, are true nulls (amorphs), whereas the initial dtc24 allele is both antimorphic and hypomorphic. We rescued m169 mutant embryos by smad5 mRNA injection. Although adult mutants are smaller than their siblings, the eggs laid by m169(-/-) females are larger than normal eggs. Embryos lacking maternal Smad5 function (Mm169(-/-) embryos) are even more strongly dorsalized thanbmp2b or bmp7 null mutants. They do not respond to injected bmp2b mRNA, indicating that Smad5 is absolutely essential for ventral development and Bmp2/7 signaling. Most importantly, Mm169(-/-) embryos display reducedbmp7 mRNA levels during blastula stages, when bmp2b and bmp7 mutants are still normal. This indicates that maternally supplied Smad5 is already required to mediate ventral specification prior to zygotic Bmp2/7 signaling to establish the initial dorsoventral asymmetry.