H. Joseph Yost

The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.

Transgenesis is an important tool for assessing gene function. In zebrafish, transgenesis has suffered from three problems: the labor of building complex expression constructs using conventional subcloning; low transgenesis efficiency, leading to mosaicism in transient transgenics and infrequent germline incorporation; and difficulty in identifying germline integrations unless using a fluorescent marker transgene. The Tol2kit system uses site‐specific recombination‐based cloning (multisite Gateway technology) to allow quick, modular assembly of [promoter]–[coding sequence]–[3′ tag] constructs in a Tol2 transposon backbone. It includes a destination vector with a cmlc2:EGFP (enhanced green fluorescent protein) transgenesis marker and a variety of widely useful entry clones, including hsp70 and beta‐actin promoters; cytoplasmic, nuclear, and membrane‐localized fluorescent proteins; and internal ribosome entry sequence–driven EGFP cassettes for bicistronic expression. The Tol2kit greatly facilitates zebrafish transgenesis, simplifies the sharing of clones, and enables large‐scale projects testing the functions of libraries of regulatory or coding sequences. Developmental Dynamics 236:3088–3099, 2007.

Siah-1 Mediates a Novel β-Catenin Degradation Pathway Linking p53 to the Adenomatous Polyposis Coli Protein

The adenomatous polyposis coli (APC) tumor-suppressor protein, together with Axin and GSK3beta, forms a Wnt-regulated signaling complex that mediates phosphorylation-dependent degradation of beta-catenin by the proteasome. Siah-1, the human homolog of Drosophila seven in absentia, is a p53-inducible mediator of cell cycle arrest, tumor suppression, and apoptosis. We have now found that Siah-1 interacts with the carboxyl terminus of APC and promotes degradation of beta-catenin in mammalian cells. The ability of Siah-1 to downregulate beta-catenin signaling was also demonstrated by hypodorsalization of Xenopus embryos. Unexpectedly, degradation of beta-catenin by Siah-1 was independent of GSK3beta-mediated phosphorylation and did not require the F box protein beta-TrCP. These results indicate that APC and Siah-1 mediate a novel beta-catenin degradation pathway linking p53 activation to cell cycle control.

RNA splicing is interrupted by heat shock and is rescued by heat shock protein synthesis.

The transcripts of most eukaryotic genes contain intervening sequences and must be spliced to yield functional messenger RNA. We report that a brief severe heat shock blocks the processing of intervening sequences in Drosophila cells and that this block persists for at least 2 hr after cells are returned to normal temperatures. If a mild heat shock, which induces the synthesis of heat shock proteins, is administered prior to the severe heat shock, processing occurs under otherwise restrictive conditions. When heat shock protein synthesis is inhibited, this protection is not observed. We suggest that the disruption of intron processing contributes to heat-induced lethality and developmental abnormalities and that one function of the heat shock proteins is to protect processing from heat-induced disruption.

Conserved function for embryonic nodal cilia

How left–right handedness originates in the body plan of the developing vertebrate embryo is a subject of considerable debate. In mice, a left–right bias is thought to arise from a directional extracellular flow (nodal flow) that is generated by dynein-dependent rotation of monocilia on the ventral surface of the embryonic node. Here we show that the existence of node monocilia and the expression of a dynein gene that is implicated in ciliary function are conserved across a wide range of vertebrate classes, indicating that a similar ciliary mechanism may underlie the establishment of handedness in all vertebrates.

The roles of cilia in developmental disorders and disease

Cilia are highly conserved organelles that have diverse motility and sensory functions. Recent discoveries have revealed that cilia also have crucial roles in cell signaling pathways and in maintaining cellular homeostasis. As such, defects in cilia formation or function have profound effects on the development of body pattern and the physiology of multiple organ systems. By categorizing syndromes that are due to cilia dysfunction in humans and from studies in vertebrate model organisms, molecular pathways that intersect with cilia formation and function have come to light. Here, we summarize an emerging view that in order to understand some complex developmental pathways and disease etiologies, one must consider the molecular functions performed by cilia.

Mechanical force mobilizes zyxin from focal adhesions to actin filaments and regulates cytoskeletal reinforcement

Organs and tissues adapt to acute or chronic mechanical stress by remodeling their actin cytoskeletons. Cells that are stimulated by cyclic stretch or shear stress in vitro undergo bimodal cytoskeletal responses that include rapid reinforcement and gradual reorientation of actin stress fibers; however, the mechanism by which cells respond to mechanical cues has been obscure. We report that the application of either unidirectional cyclic stretch or shear stress to cells results in robust mobilization of zyxin from focal adhesions to actin filaments, whereas many other focal adhesion proteins and zyxin family members remain at focal adhesions. Mechanical stress also induces the rapid zyxin-dependent mobilization of vasodilator-stimulated phosphoprotein from focal adhesions to actin filaments. Thickening of actin stress fibers reflects a cellular adaptation to mechanical stress; this cytoskeletal reinforcement coincides with zyxin mobilization and is abrogated in zyxin-null cells. Our findings identify zyxin as a mechanosensitive protein and provide mechanistic insight into how cells respond to mechanical cues.

Left–right development: Conserved function for embryonic nodal cilia

How left–right handedness originates in the body plan of the developing vertebrate embryo is a subject of considerable debate. In mice, a left–right bias is thought to arise from a directional extracellular flow (nodal flow) that is generated by dynein-dependent rotation of monocilia on the ventral surface of the embryonic node. Here we show that the existence of node monocilia and the expression of a dynein gene that is implicated in ciliary function are conserved across a wide range of vertebrate classes, indicating that a similar ciliary mechanism may underlie the establishment of handedness in all vertebrates.

Structure and function of the developing zebrafish heart

The combination of optical clarity and large scale of mutants makes the zebrafish vital for developmental biologists. However, there is no comprehensive reference of morphology and function for this animal. Since study of gene expression must be integrated with structure and function, we undertook a longitudinal study to define the cardiac morphology and physiology of the developing zebrafish. Our studies included 48‐hr, 5‐day, 2‐week, 4‐week, and 3‐month post‐fertilization zebrafish. We measured ventricular and body wet weights, and performed morphologic analysis on the heart with H&E and MF‐20 antibody sections. Ventricular and dorsal aortic pressures were measured with a servonull system. Ventricular and body weight increased geometrically with development, but at different rates. Ventricle‐to‐body ratio decreased from 0.11 at 48‐hr to 0.02 in adult. The heart is partitioned into sinus venosus, atrium, ventricle, and bulbus arteriosus as identified by the constriction between the segments at 48‐hr. Valves were formed at 5‐day post‐fertilization. Until maturity, the atrium showed extensive pectinate muscles, and the atrial wall increased to two to three cell layers. The ventricular wall and the compact layer increased to three to four cell layers, while the extent and complexity in trabeculation continued. Further thickening of the heart wall was mainly by increase in cell size. The bulbus arteriosus had similar characteristics to the myocardium in early stages, but lost the MF‐20 positive staining, and transitioned to smooth muscle layer. All pressures increased geometrically with development, and were linearly related to stage‐specific values for body weight (P < 0.05). These data define the parameters of normal cardiac morphology and ventricular function in the developing zebrafish. Anat Rec 260:148–157, 2000.

FGF signalling during embryo development regulates cilia length in diverse epithelia

Cilia are cell surface organelles found on most epithelia in vertebrates. Specialized groups of cilia have critical roles in embryonic development, including left–right axis formation. Recently, cilia have been implicated as recipients of cell–cell signalling. However, little is known about cell–cell signalling pathways that control the length of cilia. Here we provide several lines of evidence showing that fibroblast growth factor (FGF) signalling regulates cilia length and function in diverse epithelia during zebrafish and Xenopus development. Morpholino knockdown of FGF receptor 1 (Fgfr1) in zebrafish cell-autonomously reduces cilia length in Kupffer’s vesicle and perturbs directional fluid flow required for left–right patterning of the embryo. Expression of a dominant-negative FGF receptor (DN-Fgfr1), treatment with SU5402 (a pharmacological inhibitor of FGF signalling) or genetic and morpholino reduction of redundant FGF ligands Fgf8 and Fgf24 reproduces this cilia length phenotype. Knockdown of Fgfr1 also results in shorter tethering cilia in the otic vesicle and shorter motile cilia in the pronephric ducts. In Xenopus, expression of a dn-fgfr1 results in shorter monocilia in the gastrocoel roof plate that control left–right patterning and in shorter multicilia in external mucociliary epithelium. Together, these results indicate a fundamental and highly conserved role for FGF signalling in the regulation of cilia length in multiple tissues. Abrogation of Fgfr1 signalling downregulates expression of two ciliogenic transcription factors, foxj1 and rfx2, and of the intraflagellar transport gene ift88 (also known as polaris), indicating that FGF signalling mediates cilia length through an Fgf8/Fgf24–Fgfr1–intraflagellar transport pathway. We propose that a subset of developmental defects and diseases ascribed to FGF signalling are due in part to loss of cilia function.

The T Box Transcription Factor No Tail in Ciliated Cells Controls Zebrafish Left-Right Asymmetry

The heart, brain, and gut develop essential left-right (LR) asymmetries. Specialized groups of ciliated cells have been implicated in LR patterning in mouse, chick, frog, and zebrafish embryos. In zebrafish, these ciliated cells are found in Kupffers vesicle (KV) and are progeny of dorsal forerunner cells (DFCs). However, there is no direct evidence in any vertebrate that the genes involved in LR development are specifically required in ciliated cells. By using a novel method in zebrafish, we knocked down the function of no tail (ntl, homologous to mouse brachyury) in DFCs without affecting its expression in other cells in the embryo. We find that the Ntl transcription factor functions cell autonomously in DFCs to regulate KV morphogenesis and LR determination. This is the first evidence that loss-of-gene function exclusively in ciliated cells perturbs vertebrate LR patterning. Our results demonstrate that the ciliated KV, a transient embryonic organ of previously unknown function, is involved in the earliest known step in zebrafish LR development, suggesting that a ciliary-based mechanism establishes the LR axis in all vertebrate embryos.