Visualisation and Quantification of Morphogen Gradient Formation in the Zebrafish

Abstract

During embryonic development, signalling molecules known as morphogens act in a concentration-dependent manner to provide positional information to responding tissues. In the early zebrafish embryo, graded signalling by members of the nodal family induces the formation of mesoderm and endoderm, thereby patterning the embryo into three germ layers. Nodal signalling has also been implicated in the establishment of the dorso-ventral axis of the embryo. Although one can infer the existence of nodal gradients by comparing gene expression patterns in wild-type embryos and embryos in which nodal signalling is diminished or augmented, real understanding can only come from directly observing the gradients. One approach is to determine local ligand concentrations in the embryo, but this is technically challenging, and the presence of inhibitors might cause the effective concentration of a ligand to differ from its actual concentration. We have therefore taken two approaches to visualise a direct response to nodal signalling. In the first, we have used transgenic embryos to study the nuclear accumulation of a Smad2-Venus fusion protein, and in the second we have used bimolecular fluorescence complementation to visualise the formation of a complex between Smad2 and Smad4. This has allowed us to visualise, in living embryos, the formation of a graded distribution of nodal signalling activity. We have quantified the formation of the gradient in time and space, and our results not only confirm that nodal signalling patterns the embryo into three germ layers, but also shed light on its role in patterning the dorso-ventral axis and highlight unexpected complexities of mesodermal patterning.

no tail integrates two modes of mesoderm induction

“…Consistent with this observation, some somites form in the tail region of embryos lacking nodal signalling (Feldman et al, 1998;Gritsman et al, 1999). Further evidence pointing to the complexity of ntl transcriptional regulation comes from the observation that nodal signalling is higher, and extends further towards the animal pole, in the dorsal region of the embryo compared with lateral and ventral positions (Harvey and Smith, 2009). This would predict that ntl should be expressed in more cell tiers on the dorsal side of the embryo than elsewhere, but in fact, the gene is expressed in approximately the same number of tiers throughout the margin.…”

Integration of Hedgehog and BMP signalling by the engrailed2a gene in the zebrafish myotome

“…Gli1 cDNA (Karlstrom et al, 2003) was obtained from S. Roy (IMCB, Singapore). PCR products were purified, digested and cloned into the pCS2+ Vn, pCS2+ Vc constructs (Harvey and Smith, 2009). …”

“…BiFC exploits the ability to reconstitute a fluorescent protein (modified non-aggregating forms of Venus) from its two halves (Vn-N terminal fragment and Vc-C terminal fragment), neither of which fluoresces in isolation Saka et al, 2007). We first established the interaction assay in myotomal cells using our Vn-Smad1 along with previously described VcSmad4 constructs (Harvey and Smith, 2009). We cloned both constructs into a bi-cistronic UAS vector and targeted their expression to myoblasts using the actin::GAL4 line; this resulted in a robust and characteristically punctate fluorescence signal, both in the cytoplasm and around the nuclei of muscle cells (Fig.…”

Forming and Interpreting Gradients in the Early Xenopus Embryo

“…Importantly, this provides a mechanism for "steepening" a gradient, for cells closer to the source will not only be exposed to a higher level of ligand than cells more distant, they will have been exposed to it for longer. This "integration" of time and concentration may underlie the spatial pattern of the Smad2 signal transduction pathway in the zebrafish embryo as revealed by use of a Smad2-Venus fusion protein and by Smad2/Smad4 BiFC (Harvey and Smith 2009 in the zebrafish embryo, as in Xenopus, is higher dorsally, and this may occur because expression of the nodal ligand sqt begins on the dorsal side of the embryo and persists for longer in this region (Dougan et al 2003).…”