Back and forth between cell fate specification and movement during vertebrate gastrulation

Animal body plan arises during gastrulation and organogenesis by the coordination of inductive events and cell movements. Several signaling pathways, such as BMP, FGF, Hedgehog, Nodal, and Wnt have well-recognized instructive roles in cell fate specification during vertebrate embryogenesis. Growing evidence indicates that BMP, Nodal, and FGF signaling also regulate cell movements, and that they do so through mechanisms distinct from those that specify cell fates. Moreover, pathways controlling cell movements can also indirectly influence cell fate specification by regulating dimensions and relative positions of interacting tissues. The current challenge is to delineate the molecular mechanisms via which the major signaling pathways regulate cell fate specification and movements, and how these two processes are coordinated to ensure normal development.     

The Cdc14B phosphatase contributes to ciliogenesis in zebrafish

Progression through the cell cycle relies on oscillation of cyclin-dependent kinase (Cdk) activity. One mechanism for downregulating Cdk signaling is to activate opposing phosphatases. The Cdc14 family of phosphatases counteracts Cdk1 phosphorylation in diverse organisms to allow proper exit from mitosis and cytokinesis. However, the role of the vertebrate CDC14 phosphatases, CDC14A and CDC14B, in re-setting the cell for interphase remains unclear. To understand Cdc14 function in vertebrates, we cloned the zebrafish cdc14b gene and used antisense morpholino oligonucleotides and an insertional mutation to inhibit its function during early development. Loss of Cdc14B function led to an array of phenotypes, including hydrocephaly, curved body, kidney cysts and left-right asymmetry defects, reminiscent of zebrafish mutants with defective cilia. Indeed, we report that motile and primary cilia were shorter in cdc14b-deficient embryos. We also demonstrate that Cdc14B function in ciliogenesis requires its phosphatase activity and can be dissociated from its function in cell cycle control. Finally, we propose that Cdc14B plays a role in the regulation of cilia length in a pathway independent of fibroblast growth factor (FGF). This first study of a loss of function of a Cdc14 family member in a vertebrate organism reveals a new role for Cdc14B in ciliogenesis and consequently in a number of developmental processes.     

Conserved patterns of cell movements during vertebrate gastrulation

Vertebrate embryogenesis entails an exquisitely coordinated combination of cell proliferation, fate specification and movement. After induction of the germ layers, the blastula is transformed by gastrulation movements into a multilayered embryo with head, trunk and tail rudiments. Gastrulation is heralded by formation of a blastopore, an opening in the blastula. The axial side of the blastopore is marked by the organizer, a signaling center that patterns the germ layers and regulates gastrulation movements. During internalization, endoderm and mesoderm cells move via the blastopore beneath the ectoderm. Epiboly movements expand and thin the nascent germ layers. Convergence movements narrow the germ layers from lateral to medial while extension movements elongate them from head to tail. Despite different morphology, parallels emerge with respect to the cellular and genetic mechanisms of gastrulation in different vertebrate groups. Patterns of gastrulation cell movements relative to the blastopore and the organizer are similar from fish to mammals, and conserved molecular pathways mediate gastrulation movements.     

Cyclooxygenase-1 signaling is required for vascular tube formation during development

Prostaglandin endoperoxide synthases (PTGS), commonly referred to as cyclooxygenases (COX-1 and COX-2), catalyze the key step in the synthesis of biologically active prostaglandins (PGs), the conversion of arachidonic acid (AA) into prostaglandin H2 (PGH2). Although COX and prostaglandins have been implicated in a wide variety of physiologic processes, an evaluation of the role of prostaglandins in early mammalian development has been difficult due to the maternal contribution of prostaglandins from the uterus: COX null mouse embryos develop normally during embryogenesis. Here, we verify that inhibition of COX-1 results in zebrafish gastrulation arrest and shows that COX-1 expression becomes restricted to the posterior mesoderm during somitogenesis and to posterior mesoderm organs at pharyngula stage. Inhibition of COX-1 signaling after gastrulation results in defective vascular tube formation and shortened intersomitic vessels in the posterior body region. These defects are rescued completely by PGE(2) treatment or, to a lesser extent, by PGF(2alpha), but not by other prostaglandins, such as PGI(2), TxB(2), or PGD(2). Functional knockdown of COX-1 using antisense morpholino oligonucleotide translation interference also results in posterior vessel defect in addition to enlarged posterior nephric duct, phenocopying the defects caused by inhibition of COX-1 activity. Together, we provide the first evidence that COX-1 signaling is required for development of posterior mesoderm organs, specifically in the vascular tube formation and posterior nephric duct development.     

Zebrafish Rho kinase 2 acts downstream of Wnt11 to mediate cell polarity and effective convergence and extension movements

BACKGROUND: During vertebrate gastrulation convergence and extension (CE), movements narrow and lengthen embryonic tissues. In Xenopus and zebrafish, a noncanonical Wnt signaling pathway constitutes the vertebrate counterpart to the Drosophila planar cell polarity pathway and regulates mediolateral cell polarization underlying CE. Despite the identification of several signaling molecules required for normal CE, the downstream transducers regulating individual cell behaviors driving CE are only beginning to be elucidated. Moreover, how defective mediolateral cell polarity impacts CE is not understood.RESULTS: Here, we show that overexpression of zebrafish dominant-negative Rho kinase 2 (dnRok2) disrupts CE without altering cell fates, phenocopying noncanonical Wnt signaling mutants. Moreover, Rho kinase 2 (Rok2) overexpression partially suppresses the slb/wnt11 gastrulation phenotype, and ectopic expression of noncanonical Wnts modulates Rok2 intracellular distribution. In addition, time-lapse analyses associate defective dorsal convergence movements with impaired cell elongation, mediolateral orientation, and consequently failure to migrate along straight paths. Transplantation experiments reveal that dnRok2 cells in wild-type hosts neither elongate nor orient their axes. In contrast, wild-type cells are able to elongate their cell bodies in dnRok2 hosts, even though they fail to orient their axes.CONCLUSIONS: During zebrafish gastrulation, Rok2 acts downstream of noncanonical Wnt11 signaling to mediate mediolateral cell elongation required for dorsal cell movement along straight paths. Furthermore, elongation and orientation of the cell body are independent properties that require both cell-autonomous and nonautonomous Rok2 function.     

Studies on the interactions between human serum albumin and trans-indazolium (bisindazole) tetrachlororuthenate(III)

The interactions between HInd[RuInd2Cl4] and human serum albumin have been investigated through UV-Vis, circular dichroism (CD), fluorescence spectroscopy and the inductively coupled plasma-atomic emission spectroscopy (ICP(AES)) method. Binding of Ru(III)-indazole species to albumin has strong impact on protein structure and it influences considerably albumin binding of other molecules like warfarin, heme or metal ions. The metal complex-human serum albumin (HAS) interactions cause conformational changes with loss of helical stability of the protein and local perturbation in the domain IIA binding pocket. The relative fluorescence intensity of the ruthenium-bound HSA decreased, suggesting that perturbation around the Trp 214 residue took place. This was confirmed by the destabilization of the warfarin-binding site, which includes Trp 214, observed in the metal-bound HSA.     

Studies on the interactions between human serum albumin and imidazolium [trans-tetrachlorobis(imidazol)ruthenate(III)].

The interactions between imidazolium [trans-tetrachlorobis(imidazol) ruthenate(III)] (Ru-im) and human serum albumin (HSA) have been investigated through UV-Vis, CD, fluorescence spectroscopy and by the antibody precipitation test. Binding of Ru(III)-imidazole species to albumin has a strong impact on the protein structure and influences considerably the albumin binding of other molecules such as warfarin or heme. The metal complex-HSA interactions cause conformational changes with the loss of helical stability of the protein and local perturbation in the domain IIA binding pocket. The relative fluorescence intensity of the ruthenium-bound HSA decreased, suggesting that perturbation around the Trp 214 residue took place. This was confirmed by the destabilisation of the warfarin binding site which includes Trp 214, observed in the metal-bound HSA.     

Naked1 antagonizes Wnt signaling by preventing nuclear accumulation of β-catenin

Cyto-nuclear shuttling of β-catenin is at the epicenter of the canonical Wnt pathway and mutations in genes that result in excessive nuclear accumulation of β-catenin are the driving force behind the initiation of many cancers. Recently, Naked Cuticle homolog 1 (Nkd1) has been identified as a Wnt-induced intracellular negative regulator of canonical Wnt signaling. The current model suggests that Nkd1 acts between Disheveled (Dvl) and β-catenin. Here, we employ the zebrafish embryo to characterize the cellular and biochemical role of Nkd1 in vivo. We demonstrate that Nkd1 binds to β-catenin and prevents its nuclear accumulation. We also show that this interaction is conserved in mammalian cultured cells. Further, we demonstrate that Nkd1 function is dependent on its interaction with the cell membrane. Given the conserved nature of Nkd1, our results shed light on the negative feedback regulation of Wnt signaling through the Nkd1-mediated negative control of nuclear accumulation of β-catenin.