A twist in Pitx2 regulation of gut looping.

Asymmetric expression of the transcription factor Pitx2 is important for correct asymmetry in organs during development. In a recent issue of Science, Sanketi et al. find Pitx2 expression directing gut tilting is independent of Nodal and acts as a "brake" to counteract BMP4 signaling on the right.

Dynamic optima in cell sizes during early development enable normal gastrulation in zebrafish embryos.

Cell migration is the main driver of the evolutionarily conserved process of gastrulation, which shapes metazoan embryo morphology. The molecular and cellular mechanisms of cell migration during gastrulation though well researched lacks an understanding of the contribution of cell sizes to collective cell migration. This is especially important during the early phase of metazoan development, which is dominated by constantly changing cell sizes in the background of which cells migrate en mass to shape the embryo. Here we investigate this phenomenon in zebrafish embryos, a model system in which early cell divisions causes cell sizes to decrease naturally over time as cells migrate collectively to sculpt the embryonic body plan. Because mutations that can perturb cell sizes so early in development do not exist, we generate haploid and tetraploid zebrafish embryos and show that cell sizes in such embryos are smaller and larger than the diploid norm, respectively. Cells in embryos made of smaller or larger than normal cells migrate sub-optimally, leading to gastrulation defects. Gene expression analysis suggests that the observed defects originate from altered cell size, and not from pleiotropic effects of altered ploidy. This interpretation is strengthened when gastrulation defects are rescued by increasing cell sizes in embryos wherein cell sizes are smaller than normal. We show that the migration defects are cell-autonomous by live imaging migrating haploid and tetraploid cells during gastrulation in chimeric diploid embryos. Analysis of membrane protrusion dynamics in single cells shows that cells normally extend protrusions non-uniformly during migration, a phenomenon which is perturbed when cell sizes deviate from the norm. Thus, an optimal range of developmental stage-specific cell sizes appears necessary for collective cell migration to correctly position cells in space and time to shape an amorphous ball of blastoderm into an embryo.

Experimental Manipulation of Ploidy in Zebrafish Embryos and Its Application in Genetic Screens.

Metazoan animals are typically diploid, possessing two sets of a chromosome in the somatic cells of an organism. In naturally diploid species, alteration from the endogenous diploid state is usually embryonic lethal. However, the ability to experimentally manipulate ploidy of animal embryos has fundamental as well as applied biology advantages. In this chapter we describe experimental procedures to convert normally diploid zebrafish embryos into haploid or tetraploid states. We also describe methodologies to verify the ploidy of embryos and the utility of ploidy manipulation in expediting the isolation of mutations using both forward and reverse genetic strategies in zebrafish.

Transient window of resilience during early development minimizes teratogenic effects of heat in zebrafish embryos.

BACKGROUND: Transient heat shock during early development is an established experimental paradigm for doubling the genome of the zebrafish zygote, which has practical applications in expedited identification of recessive mutations in genetic screens. Despite the simplicity of the strategy and the genetic tractability of zebrafish, heat shock has not been used for genome doubling since the proof-of-principle experiments done in the 1980s. This is because of poor survival of embryos that ensue from transient heat shocks and gross developmental abnormalities in the few survivors, which is incompatible with phenotype driven screens. RESULTS: We show that heat shocks during early zebrafish development uncouple the second cycle of DNA and centrosome duplication. Interestingly, the developmental time of the heat shock that triggers the dissociation between DNA and centrosome duplication cycles significantly affect the potential of embryos to survive and attain normal morphology. The potential to develop normally after a heat shock alters in a developmental time span of 2 min in zebrafish embryos, a phenomenon that has not been reported in any species. CONCLUSIONS: The existence of heat resilient developmental windows and reduced heat teratogenicity during these windows could be an effective step forward in practical application of transient heat for experimental manipulation of ploidy in zebrafish. More broadly, heat resilience before zygotic genome activation suggests that metazoan embryos may possess innate protective features against heat beyond the canonical heat shock response. Developmental Dynamics 247:992-1004, 2018. © 2018 Wiley Periodicals, Inc.

Effective Generation of Gynogenic Haploid Zebrafish Embryos Using Low Dosage of UV Rays.

The phenomenon of phenotype manifestation when the single allele in a haploid is affected is desirable for uncovering recessive mutations expeditiously in a diploid organism. However, experimentally generated haploids manifest extensive lethality and a cluster of non-specific developmental defects known as the haploid syndrome. This precludes the use of experimentally generated haploids for genetic screens due to an insufficient number of embryos for screening and the possibility of phenotypes due to the affected gene being masked by the haploid syndrome. We show here that gynogenic haploid zebrafish can be generated by irradiation of spermatozoa with a lower UV dosage than is currently used. This strategy results in reduced haploid lethality, incidence and severity of haploid syndrome. When viewed in the context of zebrafish as a genetically tractable model organism for forward and reverse genetic strategies, these results place zebrafish in a unique niche as a vertebrate in which haploid genetic screens for developmental phenotypes could be successfully attempted.