Lack of Cyclin B1 in zebrafish causes lengthening of G2 and M phases.

An essential part of the Mitosis Promoting Factor, Cyclin B1 is indispensable for cells to enter mitosis. We report here that the zebrafish early arrest mutant specter is a loss-of-function mutation in the сyclin B1 gene. cyclin B1 is maternally transcribed in zebrafish, and the zygotic phenotype is apparent by early segmentation. Lack of zygotic Cyclin B1 does not stop cells from dividing, rather it causes an abnormal and elongated progression through the G2 and M phases of the cell cycle. Many mutant cells show signs of chromosomal instability or enter apoptosis. Using CRISPR-mediated gene editing, we produced a more severe gain-of-function mutation confirming that specter is the result of nonfunctional Cyclin B1. Although also a recessive phenotype, this new mutation produces an alternative splice-form of cyclin B1 mRNA, whose product lacks several key components for Cyclin B1, but not the Cdk1-binding domain. This mutant form of Cyclin B1 completely prevents cell division. We conclude that, although Cyclin B1 is critical for cells to enter mitosis, another cell cycle protein may be cooperating with Cdk1 at the G2/M checkpoint to sustain a partly functional Mitosis Promoting Factor.

Wilson cell origin for kupffer's vesicle in the zebrafish.

BACKGROUND: Bilaterally symmetric animals have evolved highly reproducible asymmetries between left and right. In teleosts, Kupffer's vesicle, the structure necessary for the determination of left-right asymmetry, is derived from a group of cells in the gastrula termed the dorsal forerunners. RESULTS: Wilson cells are a ring of marginal enveloping layer cells that are cytoplasmically connected to the yolk cell and thus the last blastomeres to inherit yolk cell cytoplasm. Afterward, they collapse into the yolk to form the yolk syncytial layer. Without exception, forerunner cells are the progeny of dorsal Wilson cells. At the beginning of gastrulation, these Wilson cell progeny ingress beneath the enveloping layer, transform into Kupffer's vesicle, and eventually become tail notochord and muscle. Before ingressing, the forerunner precursor cells express endodermal promoting genes and require high-levels of Nodal signaling. CONCLUSIONS: Despite a derived function of the enveloping layer as an epithelium covering the entire embryo, its dorsal margin retains many behaviors of what might be expected of the dorsal superficial layers of the ancestral fish embryo, including an early program of endodermal development, cell ingression, and an eventual contribution of cells to caudal notochord and muscle, as well as the control of laterality. Developmental Dynamics 247:1057-1069, 2018. © 2018 Wiley Periodicals, Inc.

Progressive loss of RacGAP1/ogre activity has sequential effects on cytokinesis and zebrafish development.

RacGAP1 is one of the two components of the centralspindlin complex essential for orchestrating cytokinesis in all animal cells. We report here that the early arrest mutant ogre is a maternal and zygotic loss of function mutation in the zebrafish homolog of racgap1. Like the other model organisms in which racgap1 is mutated, cells in the mutant stop dividing. In vivo cell recordings reveal that gradual loss of wild-type RacGAP1 leads progressively from a failure of abscission, then to cleavage furrow ingression, and finally complete absence of furrow formation. Despite the lack of cytokinesis, gross patterning occurs overtly normally in ogre mutants and cells continue to cycle slowly, some even attaining four or eight nuclei. Many multinucleate cells differentiate and survive, but the majority of cells enter apoptosis that we demonstrate is due to cumulative rounds of defective cytokinesis. Investigation of the cells that differentiate in the mutant indicate that RacGAP1 is also needed for long-term survival of motoneurons and the cytoskeletal organization of sensory axons. We conclude that while RacGAP1 function is crucial for cytokinesis and its activity at different levels controls different aspects of cytokinesis, these defects have occluded other critical roles of this interesting protein.

Zebrafish Tbx16 regulates intermediate mesoderm cell fate by attenuating Fgf activity.

Progenitors of the zebrafish pronephros, red blood and trunk endothelium all originate from the ventral mesoderm and often share lineage with one another, suggesting that their initial patterning is linked. Previous studies have shown that spadetail (spt) mutant embryos, defective in tbx16 gene function, fail to produce red blood cells, but retain the normal number of endothelial and pronephric cells. We report here that spt mutants are deficient in all the types of early blood, have fewer endothelial cells as well as far more pronephric cells compared to wildtype. In vivo cell tracing experiments reveal that blood and endothelium originate in spt mutants almost exclusive from the dorsal mesoderm whereas, pronephros and tail originate from both dorsal and ventral mesoderm. Together these findings suggest possible defects in posterior patterning. In accord with this, gene expression analysis shows that mesodermal derivatives within the trunk and tail of spt mutants have acquired more posterior identity. Secreted signaling molecules belonging to the Fgf, Wnt and Bmp families have been implicated as patterning factors of the posterior mesoderm. Further investigation demonstrates that Fgf and Wnt signaling are elevated throughout the nonaxial region of the spt gastrula. By manipulating Fgf signaling we show that Fgfs both promote pronephric fate and repress blood and endothelial fate. We conclude that Tbx16 plays an important role in regulating the balance of intermediate mesoderm fates by attenuating Fgf activity.

Characterization of harpy/Rca1/emi1 mutants: patterning in the absence of cell division.

We have characterized mutations in the early arrest gene, harpy (hrp), and show that they introduce premature stops in the coding region of early mitotic inhibitor1 (Rca1/emi1). In harpy mutants, cells stop dividing during early gastrulation. Lineage analysis confirms that there is little change in cell number after approximately cycle-14. Gross patterning occurs relatively normally, and many organ primordia are produced on time but with smaller numbers of cells. Despite the lack of cell division, some organ systems continue to increase in cell number, suggesting recruitment from surrounding areas. Analysis of bromodeoxyuridine incorporation shows that endoreduplication continues in many cells well past the first day of development, but cells cease endoreduplication once they begin to differentiate and express cell-type markers. Despite relatively normal gross patterning, harpy mutants show several defects in morphogenesis, cell migration and differentiation resulting directly or indirectly from the arrest of cell division.

Probing Cadherin Interactions in Zebrafish with E- and N-Cadherin Missense Mutants.

Cadherins are cell adhesion molecules that regulate numerous adhesive interactions during embryonic development and adult life. Consistent with these functions, when their expression goes astray cells lose their normal adhesive properties resulting in defective morphogenesis, disease, and even metastatic cancer. In general, classical cadherins exert their effect by homophilic interactions via their five characteristic extracellular (EC) repeats. The EC1 repeat provides the mechanism for cadherins to dimerize with each other whereas the EC2 repeat may facilitate dimerization. Less is known about the other EC repeats. Here, we show that a zebrafish missense mutation in the EC5 repeat of N-cadherin is a dominant gain-of-function mutation and demonstrate that this mutation alters cell adhesion almost to the same degree as a zebrafish missense mutation in the EC1 repeat of N-cadherin. We also show that zebrafish E- and N-cadherin dominant gain-of-function missense mutations genetically interact. Perturbation of cell adhesion in embryos that are heterozygous mutant at both loci is similar to that observed in single homozygous mutants. Introducing an E-cadherin EC5 missense allele into the homozygous N-cadherin EC1 missense mutant more radically affects morphogenesis, causing synergistic phenotypes consistent with interdependent functions being disrupted. Our studies indicate that a functional EC5 repeat is critical for cadherin-mediated cell affinity, suggesting that its role may be more important than previously thought. These results also suggest the possibility that E- and N-cadherin have heterophilic interactions during early morphogenesis of the embryo; interactions that might help balance the variety of cell affinities needed during embryonic development.

Fate mapping embryonic blood in zebrafish: multi- and unipotential lineages are segregated at gastrulation.

Vertebrate hematopoiesis first produces primitive (embryonic) lineages and ultimately generates the definitive (adult) blood. Whereas definitive hematopoiesis may produce many diverse blood types via a common multipotent progenitor, primitive hematopoiesis has been thought to produce only erythrocytes or macrophages via progenitors that are unipotent for single blood lineages. Using a variety of in vivo cell-tracing techniques, we show that primitive blood in zebrafish derives from two different progenitor types. On the dorsal gastrula, blood progenitors are unipotential cells that divide infrequently, populate the rostral blood islands, and differentiate into macrophages. In contrast, on the ventral gastrula, blood progenitors are multipotential cells with rapid cell cycles; populate the intermediate cell mass; and differentiate into erythrocytes, neutrophils, and thrombocytes. Our results demonstrate the existence of primitive hematopoietic progenitors that are segregated very early in development and that are specified to produce either a unipotent or a multipotent blood cell lineage.

A role for N-cadherin in mesodermal morphogenesis during gastrulation.

Cell adhesion molecules mediate numerous developmental processes necessary for the segregation and organization of tissues. Here we show that the zebrafish biber (bib) mutant encodes a dominant allele at the N-cadherin locus. When knocked down with antisense oligonucleotides, bib mutants phenocopy parachute (pac) null alleles, demonstrating that bib is a gain-of-function mutation. The mutant phenotype disrupts normal cell-cell contacts throughout the mesoderm as well as the ectoderm. During gastrulation stages, cells of the mesodermal germ layer converge slowly; during segmentation stages, the borders between paraxial and axial tissues are irregular and somite borders do not form; later, myotomes are fused. During neurulation, the neural tube is disorganized. Although weaker, all traits present in bib mutants were found in pac mutants. When the distribution of N-cadherin mRNA was analyzed to distinguish mesodermal from neuroectodermal expression, we found that N-cadherin is strongly expressed in the yolk cell and hypoblast in the early gastrula, just preceding the appearance of the bib mesodermal defects. Only later is N-cadherin expressed in the anlage of the CNS, where it is found as a radial gradient in the forming neural plate. Hence, besides a well-established role in neural and somite morphogenesis, N-cadherin is essential for morphogenesis of the mesodermal germ layer during gastrulation.

Origin of the zebrafish endocrine and exocrine pancreas.

Here, we report a detailed fate map of the zebrafish pancreas at the early gastrula stage of development (6 hours postfertilization; hpf). We show that, at this stage, both pancreas and liver progenitors are symmetrically localized in two broad domains relative to the dorsal organizer. We demonstrate that the dorsal and ventral pancreatic buds can derive from common progenitor pools at 6 hpf, but often derive from independent populations. Endocrine vs. exocrine pancreas show a similar pattern of progenitors, consistent with descriptions of the dorsal bud being strictly endocrine and the ventral bud primarily exocrine. In general, we find that endocrine/dorsal bud progenitors are located more dorsally than the exocrine pancreas/ventral bud progenitors. Later in gastrulation (10 hpf), pancreas progenitors have migrated to bilateral domains at the equator of the embryo. Our fate map will assist with design and interpretation of future experiments to understand early pancreas development.

Mutations in half baked/E-cadherin block cell behaviors that are necessary for teleost epiboly.

Epiboly, the spreading of the blastoderm over the large yolk cell, is the first morphogenetic movement of the teleost embryo. Examining this movement as a paradigm of vertebrate morphogenesis, we have focused on the epiboly arrest mutant half baked (hab), which segregates as a recessive lethal, including alleles expressing zygotic-maternal dominant (ZMD) effects. Here we show that hab is a mutation in the zebrafish homolog of the adhesion protein E-cadherin. Whereas exclusively recessive alleles of hab produce truncated proteins, dominant alleles all contain transversions in highly conserved amino acids of the extracellular domains, suggesting these alleles produce dominant-negative effects. Antisense oligonucleotides that create specific splicing defects in the hab mRNA phenocopy the recessive phenotypes and, surprisingly, some of the ZMD phenotypes as well. In situ analyses show that during late epiboly hab is expressed in a radial gradient in the non axial epiblast, from high concentrations in the exterior layer of the epiblast to low concentrations in the interior layer of the epiblast. During epiboly, using an asymmetric variant of radial intercalation, epiblast cells from the interior layer sequentially move into the exterior layer and become restricted to that layer; there they participate in subtle cell shape changes that further expand the blastoderm. In hab mutants, when cells intercalate into the exterior layer, they tend to neither change cell shape nor become restricted, and many of these cells 'de-intercalate' and move back into the interior layer. Cell transplantation showed all these defects to be cell-autonomous. Hence, as for the expansion of the mammalian trophoblast at a similar developmental stage, hab/E-cadherin is necessary for the cell rearrangements that spread the teleost blastoderm over the yolk.

Genetic locus half baked is necessary for morphogenesis of the ectoderm.

The zebrafish epiboly mutants partially block epiboly, the vegetalward movement of the blastoderm around the giant yolk cell. Here, we show that the epiboly mutations are located near the centromere of Linkage Group 7 in a single locus, termed the half baked locus. Nevertheless, except for the similar mutants lawine and avalanche, we find the epiboly traits of each of the alleles to be distinguishable, forming an allelic series. Using in situ analysis, we show that the specification and the formation of the germ layers is unaffected. However, during early gastrulation, convergence movements are slowed in homozygous and zygotic maternal dominant (ZMD) heterozygous mutants, especially in the epiblast layer of the blastoderm. Using triple-mutant analysis with squint and cyclops, we show that ablating involution and hypoblast formation in hab has no effect on the epiboly phenotype on the ventral and lateral sides of the embryo, suggesting that the hypoblast has no role in epiboly. Moreover, the triple mutant enhances the depletion of cells on the dorsal side of the embryo, consistent with the idea that convergence movements are defective. Double-mutant analysis with one-eyed pinhead reveals that hab is necessary in the ectodermal portion of the hatching gland. In ZMD heterozygotes, in addition to the slowing of epiboly, morphogenesis of the neural tube is abnormal, with gaps forming in the midline during segmentation stages; later, ectopic rows of neurons form in the widened spinal cord and hindbrain. Cell transplantation reveals that half baked acts both autonomously and nonautonomously in interactions among cells of the forming neural tube. Together, these results suggest that half baked is necessary within the epiblast for morphogenesis during both epiboly and neurulation and suggest that the mechanisms that drive epiboly possess common elements with those that underlie convergence and extension.

One-eyed pinhead regulates cell motility independent of Squint/Cyclops signaling.

In vertebrates, EGF-CFC factors are essential for Nodal signaling. Here, we show that the zygotic function of one-eyed pinhead, the zebrafish EGF-CFC factor, is necessary for cell movement throughout the blastoderm of the early embryo. During the blastula and gastrula stages, mutant cells are more cohesive and migrate slower than wild-type cells. Chimeric analysis reveals that these early motility defects are cell-autonomous; later, one-eyed pinhead mutant cells have a cell-autonomous tendency to acquire ectodermal rather than mesendodermal fates. Moreover, wild-type cells transplanted into the axial region of mutant hosts tend to form isolated aggregates of notochord tissue adjacent to the mutant notochord. Upon misexpressing the Nodal-like ligand Activin in whole embryos, which rescues aspects of the mutant phenotype, cell behavior retains the one-eyed pinhead motility phenotype. However, in squint;cyclops double mutants, which lack Nodal function and possess a more severe phenotype than zygotic one-eyed pinhead mutants, cells of the dorsal margin exhibit a marked tendency to widely disperse rather than cohere together. Elsewhere in the double mutants, for cells of the blastoderm and for rare cells of the gastrula that involute into the hypoblast, motility appears wild-type. Notably, cells at the animal pole, which are not under direct regulation by the Nodal pathway, behave normal in squint;cyclops mutants but exhibit defective motility in one-eyed pinhead mutants. We conclude that, in addition to a role in Nodal signaling, One-eyed pinhead is required for aspects of cell movement, possibly by regulating cell adhesion.

The guts of endoderm formation.

In this chapter, we will review the formation of the definitive endoderm, the population of cells that give rise to the lining of the digestive tract, its associated organs and the pharyngeal pouches. At the cellular level, we will describe the location and movement of endodermal cells from the onset of epiboly until the end of gastrulation. At the molecular level, we will discuss the genes associated with endoderm formation beginning with Nodal signaling. For convenience, we use the term involution, sometimes referred to as internalization; we also separate endoderm formation into the pre-involution (blastula) and post-involution (gastrula) periods although of course endoderm formation involves a continuous series of events. In addition, we refer to the cells that contribute to the endoderm as progenitors prior to their involution and precursors after their involution.

The role of the zebrafish nodal-related genes squint and cyclops in patterning of mesendoderm.

Nodal signals, a subclass of the TGFbeta superfamily of secreted factors, induce formation of mesoderm and endoderm in vertebrate embryos. We have examined the possible dorsoventral and animal-vegetal patterning roles for Nodal signals by using mutations in two zebrafish nodal-related genes, squint and cyclops, to manipulate genetically the levels and timing of Nodal activity. squint mutants lack dorsal mesendodermal gene expression at the late blastula stage, and fate mapping and gene expression studies in sqt(-/-); cyc(+/+) and sqt(-/-); cyc(+/-) mutants show that some dorsal marginal cells inappropriately form hindbrain and spinal cord instead of dorsal mesendodermal derivatives. The effects on ventrolateral mesendoderm are less severe, although the endoderm is reduced and muscle precursors are located nearer to the margin than in wild type. Our results support a role for Nodal signals in patterning the mesendoderm along the animal-vegetal axis and indicate that dorsal and ventrolateral mesoderm require different levels of squint and cyclops function. Dorsal marginal cells were not transformed toward more lateral fates in either sqt(-/-); cyc(+/-) or sqt(-/-); cyc(+/+) embryos, arguing against a role for the graded action of Nodal signals in dorsoventral patterning of the mesendoderm. Differential regulation of the cyclops gene in these cells contributes to the different requirements for nodal-related gene function in these cells. Dorsal expression of cyclops requires Nodal-dependent autoregulation, whereas other factors induce cyclops expression in ventrolateral cells. In addition, the differential timing of dorsal mesendoderm induction in squint and cyclops mutants suggests that dorsal marginal cells can respond to Nodal signals at stages ranging from the mid-blastula through the mid-gastrula.