Delaying Gal4-driven gene expression in the zebrafish with morpholinos and Gal80

The modular Gal4/UAS gene expression system has become an indispensable tool in modern biology. Several large-scale gene- and enhancer-trap screens in the zebrafish have generated hundreds of transgenic lines expressing Gal4 in unique patterns. However, the early embryonic expression of the Gal4 severely limits their use for studies on regeneration or behavior because UAS-driven effectors could disrupt normal organogenesis. To overcome this limitation, we explored the use of the Gal4 repressor Gal80 in transient assays and with stable transgenes to temporally control Gal4 activity. We also validated a strategy to delay Gal4-driven gene expression using a morpholino targeted to Gal4. The first approach is limited to transgenes expressing the native Gal4. The morphant approach can also be applied to transgenic lines expressing the Gal4-VP16 fusion protein. It promises to become a standard approach to delay Gal4-driven transgene expression and enhance the genetic toolkit for the zebrafish.     

Nodal signaling patterns the organizer

Spemann's organizer plays an essential role in patterning the vertebrate embryo. During gastrulation, organizer cells involute and form the prechordal plate anteriorly and the notochord more posteriorly. The fate mapping and gene expression analyses in zebrafish presented in this study reveal that this anteroposterior polarity is already initiated in the organizer before gastrulation. Prechordal plate progenitors reside close to the blastoderm margin and express the homeobox gene goosecoid, whereas notochord precursors are located further from the margin and express the homeobox gene floating head. The nodal-related genes cyclops and squint are expressed at the blastoderm margin and are required for prechordal plate and notochord formation. We show that differential activation of the Nodal signaling pathway is essential in establishing anteroposterior pattern in the organizer. First, overexpression of cyclops and squint at different doses leads to the induction of floating head at low doses and the induction of both goosecoid and floating head at higher doses. Second, decreasing Nodal signaling using different concentrations of the antagonist Antivin inhibits goosecoid expression at low doses and blocks expression of both goosecoid and floating head at higher doses. Third, attenuation of Nodal signaling in zygotic mutants for the EGF-CFC gene one-eyed pinhead, an essential cofactor for Nodal signaling, leads to the loss of goosecoid expression and expansion of floating head expression in the organizer. Concomitantly, cells normally fated to become prechordal plate are transformed into notochord progenitors. Finally, activation of Nodal signaling at different times suggests that prechordal plate specification requires sustained Nodal signaling, whereas transient signaling is sufficient for notochord development. Together, these results indicate that differential Nodal signaling patterns the organizer before gastrulation, with the highest level of activity required for anterior fates and lower activity essential for posterior fates.     

Targeted gene expression by the Gal4-UAS system in zebrafish

Targeted gene expression by the Gal4-UAS system is a powerful methodology for analyzing function of genes and cells in vivo and has been extensively used in genetic studies in Drosophila . On the other hand, the Gal4-UAS system had not been applied effectively to vertebrate systems for a long time mainly due to the lack of an efficient transgenesis method. Recently, a highly efficient transgenesis method using the medaka fish Tol2 transposable element was developed in zebrafish. Taking advantage of the Tol2 transposon system, we and other groups developed the Gal4 gene trap and enhancer trap methods and established various transgenic fish expressing Gal4 in specific cells. By crossing such Gal4 lines with transgenic fish lines harboring various reporter genes and effector genes downstream of UAS (upstream activating sequence), specific cells can be visualized and manipulated in vivo by targeted gene expression. Thus, the Gal4 gene trap and enhancer trap approaches together with various UAS lines should be important tools for investigating roles of genes and cells in vertebrates.     

Tailbud-derived Bmp4 drives proliferation and inhibits maturation of zebrafish chordamesoderm

In zebrafish, BMP signaling establishes cell identity along the dorsoventral (DV) axis during gastrulation. Owing to the early requirements of BMP activity in DV patterning, it has been difficult to assign later roles in cell fate specification to specific BMP ligands. In this study, we have taken advantage of two follistatin-like genes (fstl1 and fstl2), as well as a transgenic zebrafish line carrying an inducible truncated form of the BMP-type 1 receptor to study the role of Bmp4 outside of the context of DV specification. Characterization of fstl1/2 suggests that they exert a redundant role as BMP antagonists during late gastrulation, regulating BMP activity in axial mesoderm. Maintenance of appropriate levels of BMP signaling is crucial for the proper development of chordamesoderm, a subset of axial mesoderm that gives rise to the notochord, but not prechordal mesoderm, which gives rise to the prechordal plate. Bmp4 activity in particular is required during a crucial window beginning at late gastrulation and lasting through early somitogenesis to promote chordamesoderm proliferation. In the absence of Bmp4, the notochord precursor pool is depleted, and the notochord differentiates prematurely. Our results illustrate a role for Bmp4 in the proliferation and timely differentiation of axial tissue after DV axis specification.     

Morphogenesis of prechordal plate and notochord requires intact Eph/ephrin B signaling

Eph receptors and their ligands, the ephrins, mediate cell-to-cell signals implicated in the regulation of cell migration processes during development. We report the molecular cloning and tissue distribution of zebrafish transmembrane ephrins that represent all known members of the mammalian class B ephrin family. The degree of homology among predicted ephrin B sequences suggests that, similar to their mammalian counterparts, zebrafish B-ephrins can also bind promiscuously to several Eph receptors. The dynamic expression patterns for each zebrafish B-ephrin support the idea that these ligands are confined to interact with their receptors at the borders of their complementary expression domains. Zebrafish B-ephrins are expressed as early as 30% epiboly and during gastrula stages: in the germ ring, shield, prechordal plate, and notochord. Ectopic overexpression of dominant-negative soluble ephrin B constructs yields reproducible defects in the morphology of the notochord and prechordal plate by the end of gastrulation. Notably disruption of Eph/ephrin B signaling does not completely destroy structures examined, suggesting that cell fate specification is not altered. Thus abnormal morphogenesis of the prechordal plate and the notochord is likely a consequence of a cell movement defect. Our observations suggest Eph/ephrin B signaling plays an essential role in regulating cell movements during gastrulation.     

Transgenic line with gal4 insertion useful to study morphogenesis of craniofacial perichondrium, vascular endothelium-associated cells, floor plate, and dorsal midline radial glia during zebrafish development

Zebrafish is a good model for studying vertebrate development because of the availability of powerful genetic tools. We are interested in the study of the craniofacial skeletal structure of the zebrafish. For this purpose, we performed a gene trap screen and identified a Gal4 gene trap line, SAGFF(LF)134A. We then analyzed the expression pattern of SAGFF(LF)134A;Tg(UAS:GFP) and found that green fluorescent protein (GFP) was expressed not only in craniofacial skeletal elements but also in the vascular system, as well as in the nervous system. In craniofacial skeletal elements, strong GFP expression was detected not only in chondrocytes but also in the perichondrium. In the vascular system, GFP was expressed in endothelium-associated cells. In the spinal cord, strong GFP expression was found in the floor plate, and later in the dorsal radial glia located on the midline. Taking advantage of this transgenic line, which drives Gal4 expression in specific tissues, we crossed SAGFF(LF)134A with several UAS reporter lines. In particular, time-lapse imaging of photoconverted floor-plate cells of SAGFF(LF)134A;Tg(UAS:KikGR) revealed that the floor-plate cells changed their shape within 36 h from cuboidal/trapezoidal to wine glass shaped. Moreover, we identified a novel mode of association between axons and glia. The putative paths for the commissural axons, including pax8-positive CoBL interneurons, were identified as small openings in the basal endfoot of each floor plate. Our results indicate that the transgenic line would be useful for studying the morphogenesis of less-well-characterized tissues of interest, including the perichondrium, dorsal midline radial glia, late-stage floor plate, and vascular endothelium-associated cells.     

Goosecoid and cerberus-like do not interact during mouse embryogenesis

Mouse Cerberus-like (Cer-l) is a neural inducer molecule, capable of inhibiting Nodal and BMP-4 signals in the extracelular space. The cer-l expression domain in the Anterior Visceral Endoderm (AVE) and prechordal plate, tissues involved in head induction and patterning, respectively, suggested a role for this gene in head formation. However, animals homozygous for the cer-l null allele failed to show any abnormality, leading us to propose the existence of other factor(s) that might compensate for cer-l loss-of-function. Since goosecoid (gsc) shares some domains of expression with cer-l and was shown to be essential for head morphogenesis, we tested its ability to interact genetically with cer-l. With this aim we generated cer-l;gsc double mutants. These animals were analyzed at birth for skeletal defects and revealed the same phenotype as gsc-/- single mutants. We also investigated the proper patterning of structures adjacent to the prechordal plate by performing in situ hybridization of HNF-3beta, Six-3 and BF-1, genes whose expression domains remained unchanged. In conclusion, the analysis carried out indicated that gsc does not compensate for cer-l loss-of-function and that these genes do not interact genetically.