Expression of Otx Homeodomain Proteins Induces Cell Aggregation in Developing Zebrafish Embryos

Abstract

In the zebrafish embryo, cells fated to give rise to the rostral brain move in a concerted fashion and retain tissue coherence during morphogenesis. We demonstrate here that Otx proteins have a dramatic effect on cell-cell interactions when expressed ectopically in the zebrafish embryo. Injection of zebrafish Otx1 or Drosophila otd RNAs into a single cell at the 16-cell stage results in aggregation of descendants of the injected cell. The Otx/Otd homeodomain is necessary for aggregation and appears to be sufficient for the effect when substituted for the homeodomain of an unrelated homeodomain protein. When cells containing injected zOtx1 RNA are limited to the area that is normally fated to become the anterior brain and neural retina, the induced aggregates contribute to anterior brain and retina tissues. In many other embryonic regions, which do not express endogenous zOtx1, the aggregates appear to be incompatible with normal development and do not integrate into developing tissues. By using an activatable Otx1-glutocorticoid receptor fusion protein that results in the stimulation of cell association, we demonstrate that cell aggregates can form as a result of Otx1 activity even after gastrulation is completed. Time-lapse analysis of cell movements show that cell aggregation occurs with only a slight inhibition of the rate of convergence. These results suggest that promotion of cell adhesion or mediation of cell repulsion may be one of the normal functions of the Otx proteins in the establishment of the anterior brain.

Cadherin-6 is required for zebrafish nephrogenesis during early development

“…Staged zebrafish embryos were stained by wholemount ISH as described previously (Weinberg et al, 1996;Bellipanni et al, 2000).…”

Zebrafish protocadherin 10 is involved in paraxial mesoderm development and somitogenesis

“…Fish of the golden strain were used as wild-type in some of the experiments because of their attenuated pigment production suited for microscopy and otherwise normal development (Weinberg et al, 1996;Bellipanni et al, 2000). Fish were maintained and embryos were obtained as described elsewhere (Westerfield, 2000).…”

“…Capped mRNAs were synthesized from the pCS2ϩ vector clones as described previously (Bellipanni et al, 2000). For the Flag-tagged dominantnegative construct, Pcdh10ecF, a mixture of RNAs of GFP and the construct, each at 100 g/ml, were injected into embryos by using an IM-300 microinjector (Narishige, Tokyo, Japan).…”

“…DIG-labeled RNA probes were synthesized using the cDNA clones as templates. Staged zebrafish embryos were stained by whole-mount ISH as described elsewhere (Weinberg et al, 1996;Bellipanni et al, 2000). Embryos expressing a Flag-tagged construct were stained with rabbit polyclonal anti-Flag antibody (SigmaAldrich Japan, Tokyo, Japan) and Alexa Fluor 594 -labeled anti-rabbit IgG (HϩL) antibody (Molecular Probes, Eugene, OR).…”

“…A Nikon E-1000 compound microscope (Nikon, Tokyo, Japan) was used for manually sectioned embryos and fluorescent embryos. Embryos or sections were mounted between a glass slide and a coverslip with plastic adhesive tape as a spacer (Weinberg et al, 1996;Kozlowski et al, 1997;Bellipanni et al, 2000).…”