Kristen M. Kwan

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.

Notch signaling controls multiple steps of pancreatic differentiation.

Multiple cell types of the pancreas appear asynchronously during embryogenesis, which requires that pancreatic progenitor cell potential changes over time. Loss-of-function studies have shown that Notch signaling modulates the differentiation of these progenitors, but it remains unclear how and when the Notch pathway acts. We established a modular transgenic system to heritably activate mouse Notch1 in multiple types of progenitors and differentiated cells. We find that misexpression of activated Notch in Pdx1-expressing progenitor cells prevents differentiation of both exocrine and endocrine lineages. Progenitors remain trapped in an undifferentiated state even if Notch activation occurs long after the pancreas has been specified. Furthermore, endocrine differentiation is associated with escape from this activity, because Ngn3-expressing endocrine precursors are susceptible to Notch inhibition, whereas fully differentiated endocrine cells are resistant.

Dorsoventral Patterning in Hemichordates: Insights into Early Chordate Evolution

We have compared the dorsoventral development of hemichordates and chordates to deduce the organization of their common ancestor, and hence to identify the evolutionary modifications of the chordate body axis after the lineages split. In the hemichordate embryo, genes encoding bone morphogenetic proteins (Bmp) 2/4 and 5/8, as well as several genes for modulators of Bmp activity, are expressed in a thin stripe of ectoderm on one midline, historically called “dorsal.” On the opposite midline, the genes encoding Chordin and Anti-dorsalizing morphogenetic protein (Admp) are expressed. Thus, we find a Bmp-Chordin developmental axis preceding and underlying the anatomical dorsoventral axis of hemichordates, adding to the evidence from Drosophila and chordates that this axis may be at least as ancient as the first bilateral animals. Numerous genes encoding transcription factors and signaling ligands are expressed in the three germ layers of hemichordate embryos in distinct dorsoventral domains, such as pox neuro, pituitary homeobox, distalless, and tbx2/3 on the Bmp side and netrin, mnx, mox, and single-minded on the Chordin-Admp side. When we expose the embryo to excess Bmp protein, or when we deplete endogenous Bmp by small interfering RNA injections, these expression domains expand or contract, reflecting their activation or repression by Bmp, and the embryos develop as dorsalized or ventralized limit forms. Dorsoventral patterning is independent of anterior/posterior patterning, as in Drosophila but not chordates. Unlike both chordates and Drosophila, neural gene expression in hemichordates is not repressed by high Bmp levels, consistent with their development of a diffuse rather than centralized nervous system. We suggest that the common ancestor of hemichordates and chordates did not use its Bmp-Chordin axis to segregate epidermal and neural ectoderm but to pattern many other dorsoventral aspects of the germ layers, including neural cell fates within a diffuse nervous system. Accordingly, centralization was added in the chordate line by neural-epidermal segregation, mediated by the pre-existing Bmp-Chordin axis. Finally, since hemichordates develop the mouth on the non-Bmp side, like arthropods but opposite to chordates, the mouth and Bmp-Chordin axis may have rearranged in the chordate line, one relative to the other.

A microtubule-binding Rho-GEF controls cell morphology during convergent extension of Xenopus laevis

During Xenopus development, convergent extension movements mediated by cell intercalation drive axial elongation. While many genes required for convergent extension have been identified, little is known of regulation of the cytoskeleton during these cell movements. Although microtubules are required for convergent extension, this applies only to initial stages of gastrulation, between stages 10 and 10.5. To examine the cytoskeleton more directly during convergent extension, we visualized actin and microtubules simultaneously in live explants using spinning disk confocal fluorescence microscopy. Microtubule depolymerization by nocodazole inhibits lamellipodial protrusions and cell-cell contact, thereby inhibiting convergent extension. However, neither taxol nor vinblastine, both of which block microtubule dynamics while stabilizing a polymer form of tubulin, inhibits lamellipodia or convergent extension. This suggests an unusual explanation: the mass of polymerized tubulin, not dynamics of the microtubule cytoskeleton, is crucial for convergent extension. Because microtubule depolymerization elicits striking effects on actin-based protrusions, the role of Rho-family GTPases was tested. The effects of nocodazole are partially rescued using dominant negative Rho, Rho-kinase inhibitor, or constitutively active Rac, suggesting that microtubules regulate small GTPases, possibly via a guanine-nucleotide exchange factor. We cloned full-length XLfc, a microtubule-binding Rho-GEF. Nucleotide exchange activity of XLfc is required for nocodazole-mediated inhibition of convergent extension; constitutively active XLfc recapitulates the effects of microtubule depolymerization. Morpholino knockdown of XLfc abrogates the ability of nocodazole to inhibit convergent extension. Therefore, we believe that XLfc is a crucial regulator of cell morphology during convergent extension, and microtubules limit its activity through binding to the lattice.

Xbra functions as a switch between cell migration and convergent extension in the Xenopus gastrula

During Xenopus gastrulation, the dorsal mesoderm exhibits two different cell behaviors in two different regions: active cell migration of prechordal mesoderm and convergent extension of chordamesoderm. Although many genes involved in specification and differentiation of the dorsal mesoderm have been studied, the role of these genes in controlling cell behaviors is poorly understood. To understand better the link between the development and cell behaviors of the dorsal mesoderm, we have examined these behaviors in dissociated cells and explants, where activin protein can induce both active cell migration and convergent extension. We find that Xbra, a transcription factor necessary for convergent extension, actively inhibits cell migration, both in animal cap explant assays and in the endogenous dorsal mesoderm. In addition, Xbra appears to inhibit cell migration by inhibiting adhesion to fibronectin. We propose that Xbra functions as a switch to keep cell migration and convergent extension as mutually exclusive behaviors during gastrulation.

A complex choreography of cell movements shapes the vertebrate eye

Optic cup morphogenesis (OCM) generates the basic structure of the vertebrate eye. Although it is commonly depicted as a series of epithelial sheet folding events, this does not represent an empirically supported model. Here, we combine four-dimensional imaging with custom cell tracking software and photoactivatable fluorophore labeling to determine the cellular dynamics underlying OCM in zebrafish. Although cell division contributes to growth, we find it dispensable for eye formation. OCM depends instead on a complex set of cell movements coordinated between the prospective neural retina, retinal pigmented epithelium (RPE) and lens. Optic vesicle evagination persists for longer than expected; cells move in a pinwheel pattern during optic vesicle elongation and retinal precursors involute around the rim of the invaginating optic cup. We identify unanticipated movements, particularly of central and peripheral retina, RPE and lens. From cell tracking data, we generate retina, RPE and lens subdomain fate maps, which reveal novel adjacencies that might determine corresponding developmental signaling events. Finally, we find that similar movements also occur during chick eye morphogenesis, suggesting that the underlying choreography is conserved among vertebrates.

The Drosophila MOS Ortholog Is Not Essential for Meiosis

In metazoan oocytes, a metaphase arrest coordinates the completion of meiosis with fertilization. Vertebrate mos maintains the metaphase II arrest of mature oocytes and prevents DNA replication between the meiotic divisions. We identified a Drosophila homolog of mos and showed it to be the mos ortholog by two additional criteria. The dmos transcripts are present in Drosophila oocytes but not embryos, and injection of dmos into Xenopus embryos blocks mitosis and elevates active MAPK levels. In Drosophila, MAPK is activated in oocytes, consistent with a role in meiosis. We generated deletions of dmos and found that, as in vertebrates, dmos is responsible for the majority of MAPK activation. Unexpectedly, the oocytes that do mature complete meiosis normally and produce fertilized embryos that develop, although there is a reduction in female fertility and loss of some oocytes by apoptosis. Therefore, Drosophila contains a mos ortholog that activates a MAPK cascade during oogenesis and is nonessential for meiosis. This could be because there are redundant pathways regulating meiosis, because residual, low levels of active MAPK are sufficient, or because active MAPK is dispensable for meiosis in Drosophila. These results highlight the complexity of meiotic regulation that evolved to ensure accurate control over the reproductive process.

High-resolution analysis of central nervous system expression patterns in zebrafish Gal4 enhancer-trap lines

Background: The application of the Gal4/UAS system to enhancer and gene trapping screens in zebrafish has greatly increased the ability to label and manipulate cell populations in multiple tissues, including the central nervous system (CNS). However the ability to select existing lines for specific applications has been limited by the lack of detailed expression analysis. Results: We describe a Gal4 enhancer trap screen in which we used advanced image analysis, including three‐dimensional confocal reconstructions and documentation of expression patterns at multiple developmental time points. In all, we have created and annotated 98 lines exhibiting a wide range of expression patterns, most of which include CNS expression. Expression was also observed in nonneural tissues such as muscle, skin epithelium, vasculature, and neural crest derivatives. All lines and data are publicly available from the Zebrafish International Research Center (ZIRC) from the Zebrafish Model Organism Database (ZFIN). Conclusions: Our detailed documentation of expression patterns, combined with the public availability of images and fish lines, provides a valuable resource for researchers wishing to study CNS development and function in zebrafish. Our data also suggest that many existing enhancer trap lines may have previously uncharacterized expression in multiple tissues and cell types. Developmental Dynamics 244:785–796, 2015.

Loss of laminin alpha 1 results in multiple structural defects and divergent effects on adhesion during vertebrate optic cup morphogenesis.

The vertebrate eye forms via a complex set of morphogenetic events. The optic vesicle evaginates and undergoes transformative shape changes to form the optic cup, in which neural retina and retinal pigmented epithelium enwrap the lens. It has long been known that a complex, glycoprotein-rich extracellular matrix layer surrounds the developing optic cup throughout the process, yet the functions of the matrix and its specific molecular components have remained unclear. Previous work established a role for laminin extracellular matrix in particular steps of eye development, including optic vesicle evagination, lens differentiation, and retinal ganglion cell polarization, yet it is unknown what role laminin might play in the early process of optic cup formation subsequent to the initial step of optic vesicle evagination. Here, we use the zebrafish lama1 mutant (lama1(UW1)) to determine the function of laminin during optic cup morphogenesis. Using live imaging, we find, surprisingly, that loss of laminin leads to divergent effects on focal adhesion assembly in a spatiotemporally-specific manner, and that laminin is required for multiple steps of optic cup morphogenesis, including optic stalk constriction, invagination, and formation of a spherical lens. Laminin is not required for single cell behaviors and changes in cell shape. Rather, in lama1(UW1) mutants, loss of epithelial polarity and altered adhesion lead to defective tissue architecture and formation of a disorganized retina. These results demonstrate that the laminin extracellular matrix plays multiple critical roles regulating adhesion and polarity to establish and maintain tissue structure during optic cup morphogenesis.

Coming into focus: The role of extracellular matrix in vertebrate optic cup morphogenesis

The vertebrate eye acquires its basic form during the process of optic cup morphogenesis, during which the optic vesicle emerges from the brain neuroepithelium and, through a series of cell and tissue movements, transforms itself into the multilayered optic cup, containing neural retina (comprised of retinal progenitors), retinal pigmented epithelium, and the lens, which is derived from the overlying ectoderm. While great strides have been made to understand the developmental signals controlling specification, patterning, and differentiation of the optic cup, only in recent years have the cellular and molecular bases of optic cup morphogenesis begun to be unraveled. One critical component of the morphogenetic process is the extracellular matrix: the complex, glycoprotein‐rich layer that surrounds the optic vesicle and lens. Though the extracellular matrix has long been visualized by classical histological techniques and postulated to play various roles in optic cup development, its functional role was uncertain. This is now beginning to change, as live imaging techniques, quantitative image analyses, molecular genetics and in vitro models yield new insights into the process of optic cup morphogenesis and the specific influences of particular extracellular matrix components and their associated signaling pathways. Developmental Dynamics 243:1242–1248, 2014.