Vertebrate gastrulation involves the specification and coordinated movement of large populations of cells that give rise to the ectodermal, mesodermal and endodermal germ layers. Although many of the genes involved in the specification of cell identity during this process have been identified, little is known of the genes that coordinate cell movement. Here we show that the zebrafish silberblick (slb) locus encodes Wnt11 and that Slb/Wnt11 activity is required for cells to undergo correct convergent extension movements during gastrulation. In the absence of Slb/Wnt11 function, abnormal extension of axial tissue results in cyclopia and other midline defects in the head. The requirement for Slb/Wnt11 is cell non-autonomous, and our results indicate that the correct extension of axial tissue is at least partly dependent on medio-lateral cell intercalation in paraxial tissue. We also show that the slb phenotype is rescued by a truncated form of Dishevelled that does not signal through the canonical Wnt pathway, suggesting that, as in flies, Wnt signalling might mediate morphogenetic events through a divergent signal transduction cascade. Our results provide genetic and experimental evidence that Wnt activity in lateral tissues has a crucial role in driving the convergent extension movements underlying vertebrate gastrulation.

SUMMARYThe importance of cilia in embryonic development and adult physiology is emphasized by human ciliopathies. Despite its relevance, molecular signalling pathways behind cilia formation are poorly understood. We show that Notch signalling is a key pathway for cilia length control. In deltaD zebrafish mutants, cilia length is reduced in Kupffer´s vesicle and can be rescued by the ciliogenic factor foxj1a. Conversely, cilia length increases when Notch signalling is hyperactivated. Short cilia found in deltaD mutants reduce the fluid flow velocity inside Kupffer´s vesicle, thus compromising the asymmetric expression of the flow sensor charon. Notch signalling brings together ciliary length control and fluid flow hydrodynamics with transcriptional activation of laterality genes. In addition, our deltaD mutant analysis discloses an uncoupling between gut and heart laterality.

SUMMARYSomites are formed from the presomitic mesoderm (PSM) and give rise to the axial skeleton and skeletal muscles. The PSM is dynamic; somites are generated at the anterior end, while the posterior end is continually renewed with new cells entering from the tailbud progenitor region. Which genes control the conversion of tailbud progenitors into PSM and how is this process coordinated with cell movement? Using loss-and gain-of-function experiments and heat-shock transgenics we show in zebrafish that the transcription factor Mesogenin 1 (Msgn1), acting with Spadetail (Spt), has a central role. Msgn1 allows progression of the PSM differentiation program by switching off the progenitor maintenance genes ntl, wnt3a, wnt8 and fgf8 in the future PSM cells as they exit from the tailbud, and subsequently induces expression of PSM markers such as tbx24. msgn1 is itself positively regulated by Ntl/Wnt/Fgf, creating a negative-feedback loop that might be crucial to regulate homeostasis of the progenitor population until somitogenesis ends. Msgn1 drives not only the changes in gene expression in the nascent PSM cells but also the movements by which they stream out of the tailbud into the PSM. Loss of Msgn1 reduces the flux of cells out of the tailbud, producing smaller somites and an enlarged tailbud, and, by delaying exhaustion of the progenitor population, results in supernumerary tail somites. Through its combined effects on gene expression and cell movement, Msgn1 (with Spt) plays a key role both in genesis of the paraxial mesoderm and in maintenance of the progenitor population from which it derives.

Activities of a variety of signaling proteins that regulate embryogenesis are limited by endogenous antagonists. The zebrafish Nodal-related ligands, Squint and Cyclops, and their antagonists, Lefty1 and Lefty2, belong to the TGFbeta-related protein superfamily, whose members have widespread biological activities. Among other activities, Nodals direct the formation of most mesendoderm. By inducing their own transcription and that of the Lefties, Nodal signals establish positive and negative autoregulatory loops. To investigate how these autoregulatory pathways regulate development, we depleted zebrafish embryos of Lefty1 and/or Lefty2 by using antisense morpholino oligonucleotides. Loss of Lefty1 causes aberrations during somitogenesis stages, including left-right patterning defects, whereas Lefty2 depletion has no obvious consequences. Depletion of both Lefty1 and Lefty2, by contrast, causes unchecked Nodal signaling, expansion of mesendoderm, and loss of ectoderm. The expansion of mesendoderm correlates with an extended period of rapid cellular internalization and a failure of deep-cell epiboly. The gastrulation defects of embryos depleted of Lefty1 and Lefty2 result from the deregulation of Squint signaling. In contrast, deregulation of Cyclops does not affect morphology or the transcription of Nodal target genes during gastrulation. Furthermore, we find that Cyclops is specifically required for the maintenance of lefty1 and lefty2 transcription.

BackgroundThe zebrafish has the capacity to regenerate many tissues and organs. The caudal fin is one of the most convenient tissues to approach experimentally due to its accessibility, simple structure and fast regeneration. In this work we investigate how the regenerative capacity is affected by recurrent fin amputations and by experimental manipulations that block regeneration.Methodology/Principal FindingsWe show that consecutive repeated amputations of zebrafish caudal fin do not reduce its regeneration capacity and do not compromise any of the successive regeneration steps: wound healing, blastema formation and regenerative outgrowth. Interfering with Wnt/ß-catenin signalling using heat-shock-mediated overexpression of Dickkopf1 completely blocks fin regeneration. Notably, if these fins were re-amputated at the non-inhibitory temperature, the regenerated caudal fin reached the original length, even after several rounds of consecutive Wnt/ß-catenin signalling inhibition and re-amputation.Conclusions/SignificanceWe show that the caudal fin has an almost unlimited capacity to regenerate. Even after inhibition of regeneration caused by the loss of Wnt/ß-catenin signalling, a new amputation resets the regeneration capacity within the caudal fin, suggesting that blastema formation does not depend on a pool of stem/progenitor cells that require Wnt/ß-catenin signalling for their survival.

The coatomer vesicular coat complex is essential for normal Golgi and secretory activities in eukaryotic cells. Through positional cloning of genes controlling zebrafish notochord development, we found that the sneezy, happy, and dopey loci encode the alpha, beta, and beta' subunits of the coatomer complex. Export from mutant endoplasmic reticulum is blocked, Golgi structure is disrupted, and mutant embryos eventually degenerate due to widespread apoptosis. The early embryonic phenotype, however, demonstrates that despite its "housekeeping" functions, coatomer activity is specifically and cell autonomously required for normal chordamesoderm differentiation, perinotochordal basement membrane formation, and melanophore pigmentation. Hence, differential requirements for coatomer activity among embryonic tissues lead to tissue-specific developmental defects. Moreover, we note that the mRNA encoding alpha coatomer is strikingly upregulated in notochord progenitors, and we present data suggesting that alpha coatomer transcription is tuned to activity- and cell type-specific secretory loads.

To establish the vertebrate body plan, it is fundamental to create left-right asymmetry in the lateral-plate mesoderm to correctly position the organs. However, it is also crucial to maintain symmetry between the left and the right sides of the presomitic mesoderm, ensuring the allocation of symmetrical body structures, such as the axial skeleton and skeletal muscles. Here, we show that terra is an early left-sided expressed gene that links left-right patterning with bilateral synchronization of the segmentation clock.

BackgroundZebrafish (Danio rerio) has a remarkable capacity to regenerate many organs and tissues. During larval stages the fin fold allows the possibility of performing long time-lapse imaging making this system very appealing to study the relationships between tissue movements, cell migration and proliferation necessary for the regeneration process.ResultsThrough the combined use of transgenic fluorescently-labeled animals and confocal microscopy imaging, we characterized in vivo the complete fin fold regeneration process. We show, for the first time, that there is an increase in the global rate of epidermal growth as a response to tissue loss. Also enhanced significantly is cell proliferation, which upon amputation happens in a broad area concerning the amputation level and not in a blastema-restricted way. This reveals a striking difference with regard to the adult fin regeneration system. Finally, an accumulation of migratory, shape-changing fibroblasts occurs proximally to the wound area, resembling a blastemal-like structure, which may act as a signaling center for the regeneration process to proceed.ConclusionsThese findings provide a novel in vivo description of fundamental mechanisms occurring during the fin fold regeneration process, thereby contributing to a better knowledge of this regenerative system and to reveal variations in the epimorphic regeneration field.