Caged Q-rhodamine dextran: a new photoactivated fluorescent tracer.

An amine-reactive caged rhodamine was synthesized and conjugated to aminodextran. The resulting tracer was injected into a single cell zebrafish embryo, and a portion of the tracer was photolyzed in a single cell after development. The resulting fluorescent cell was imaged by fluorescence microscopy through a single round of cell division.

Analysis of Cx43alpha1 promoter function in the developing zebrafish embryo.

The Cx43alpha1 gap junctions play an important role in cardiovascular development. Studies using transgenic mouse models have indicated that this involves an essential role for Cx43alpha1 in modulating neural crest cell motility. We previously showed that a 6.8 kb mouse genomic sequence containing the promoter and upstream regulatory sequences of the Cx43alpha1 gene can drive lacZ reporter gene expression in all neural crest cell lineages in the mouse embryo. To obtain further insights into the sequence motifs and regulatory pathways involved in targeting Cx43alpha1 gene expression in neural crest cells, we assayed the activity of the mouse Cx43alpha1 promoter in evolutionarily distantly related zebrafish embryos. For these studies, the 6.8kb Cx43alpha1 genomic sequence and various deletion derivatives were used to generate GFP or lacZ expression vectors. The transcriptional activities of these constructs were analyzed in vivo after microinjection into one- or two- cell stage zebrafish embryos. These studies indicated that the mouse Cx43alpha1 promoter can drive lacZ expression in neural crest cells in the zebrafish embryos. Analysis by whole mount in situ hybridization showed that the endogenous zebrafish Cx43alpha1 gene is expressed maternally and zygotically, and expression is observed in regions where neural crest cells are found. To further elucidate the developmental regulation of Cx43alpha1 gene expression, we screened a zebrafish BAC library and identified a clone containing the entire zebrafish Cx43alpha1 gene and flanking upstream and downstream sequences. The upstrean Cx43alpha1 promoter sequences from zebrafish, mouse, and human were analyzed for evolutionarily conserved DNA motifs. Overall these studies suggest that the sequence motifs and transcriptional regulation involved in the targeting Cx43alpha1 expression to neural crest cells are evolutionarily conserved in zebrafish and mouse embryos.

Maternally controlled (beta)-catenin-mediated signaling is required for organizer formation in the zebrafish.

We have identified and characterized a zebrafish recessive maternal effect mutant, ichabod, that results in severe anterior and dorsal defects during early development. The ichabod mutation is almost completely penetrant, but exhibits variable expressivity. All mutant embryos fail to form a normal embryonic shield; most fail to form a head and notochord and have excessive development of ventral tail fin tissue and blood. Abnormal dorsal patterning can first be observed at 3.5 hpf by the lack of nuclear accumulation of (beta)-catenin in the dorsal yolk syncytial layer, which also fails to express bozozok/dharma/nieuwkoid and znr2/ndr1/squint. At the onset of gastrulation, deficiencies in expression of dorsal markers and expansion of expression of markers of ventral tissues indicate a dramatic alteration of dorsoventral identity. Injection of (beta)-catenin RNA markedly dorsalized ichabod embryos and often completely rescued the phenotype, but no measurable dorsalization was obtained with RNAs encoding upstream Wnt pathway components. In contrast, dorsalization was obtained when RNAs encoding either Bozozok/Dharma/Nieuwkoid or Znr2/Ndr1/Squint were injected. Moreover, injection of (beta)-catenin RNA into ichabod embryos resulted in activation of expression of these two genes, which could also activate each other. RNA injection experiments strongly suggest that the component affected by the ichabod mutation acts on a step affecting (beta)-catenin nuclear localization that is independent of regulation of (beta)-catenin stability. This work demonstrates that a maternal gene controlling localization of (beta)-catenin in dorsal nuclei is necessary for dorsal yolk syncytial layer gene activity and formation of the organizer in the zebrafish.

Regional cell movement and tissue patterning in the zebrafish embryo revealed by fate mapping with caged fluorescein.

Determination of fate maps and cell lineage tracing have previously been carried out in the zebrafish embryo by following the progeny of individual cells injected with fluorescent dyes. We review the information obtained from these experiments and then present an approach to fate mapping and cell movement tracing, utilizing the activation of caged fluorescein-dextran. This method has several advantages over single-cell injections in that it is rapid, allows cells at all depths in the embryo to be marked, can be used to follow cells starting at any time during development, and allows an appreciation of the movements of cells located in a coherent group at the time of uncaging. We demonstrate that the approach is effective in providing additional and complementary information on prospective mesoderm and brain tissues studied previously. We also present, for the first time, a fate map of placodal tissues including the otic vesicle, lateral line, cranial ganglia, lens, and olfactory epithelium. The prospective placodal cells are oriented at the 50% epiboly stage on the ventral side of the embryo with anterior structures close to the animal pole, and posterior structures nearer to the germ ring.

Characterization of a SpAN promoter sufficient to mediate correct spatial regulation along the animal-vegetal axis of the sea urchin embryo.

In order to investigate how the maternally specified animal-vegetal axis of the sea urchin embryo is established, we have examined the molecular basis of regulation of several genes transcribed differentially in nonvegetal and vegetal domains of the very early blastula. Here we present an initial characterization of the regulatory region of one of these, SpAN, which encodes a protease in the astacin family related to Drosophila tolloid and vertebrate BMP-1 (Reynolds et al., Development 114, 769-786). Tests of SpAN promoter function in vivo show that high-level activity and correct not-vegetal expression are mediated by sequences within 300 bp upstream of the basal promoter. In vitro studies have identified six protein binding sites serviced by at least five different proteins. Comparison of the structure of the SpAN promoter to that of SpHE, whose expression pattern is identical, shows that both promoters contain multiple positively acting upstream elements close to the basal promoter. We show that two elements are critical for high-level transcription of SpAN, since exact replacement of either results in 10- to 20-fold reduction in promoter strength. These shared elements are, however, not essential for spatially correct SpHE gene transcription. We conclude that the coordinate strong activities of the SpAN and SpHE promoters in the nonvegetal domain of the embryo rely primarily on different transcription factor activities.