E-cadherin is maternally expressed in most vertebrate species, but its function during early development of the vertebrate embryo proper is unknown. To directly examine E-cadherin gene (cdh1) function in zebrafish, morpholino oligonucleotides (MOs) that inhibit E-cadherin protein (Cdh1) expression were injected into embryos. Cdh1 knockdown reduced embryo survival. In early cdh1 MO-injected embryos, the cleavage plane orientation between blastomeres was irregular and adhesion defects prevented normal compaction. Cdh1 knockdown inhibited epiboly cell movements. Epiboly delay caused yolk cell lysis and produced embryos with a bifurcated embryonic axis. Cdh1 knockdown inhibited gastrulation cell movements, causing defects in convergence and extension. Additionally, prechordal plate derivatives were absent in Cdh1 knockdown embryos even though presumptive prechordal plate markers were induced normally. E-cadherin mRNA coinjection demonstrated the specificity of cdh1 MO-induced defects. Our experiments illustrate the importance of cdh1 in regulating morphogenetic cell movements and tissue formation in the early embryo.

In zebrafish, R-cadherin (cadherin-4 or Cdh4) is expressed in the retina and in retinorecipient brain regions, suggesting that Cdh4 functions during visual system development. Cdh4 function was examined during retinogenesis and retinal axon outgrowth using antisense morpholino oligonucleotides and mutant Cdh4 construct expression. In knockdowns, Cdh4 was reduced or absent, eyes were small, and retinae lacked discrete laminae. Increased cell death produced the small eye phenotype. Zn5-, Pax6-, and zpr-1-positive cells were reduced or absent in knockdown retinas but, when present, were in the correct laminae. Cdh4 knockdowns had sparse or absent retinal ganglion cell axons. When present, axons projected contralaterally but lacked fine branching and failed to reach the tectum or arborize the entire tectum. Mutant Cdh4 construct expression during retinal ganglion cell differentiation reduced or ablated neurite formation. Cdh4 is necessary for neural retina survival and differentiation, and required for normal retinotectal projection formation and tectal arborization. Developmental Dynamics 233:930 -945, 2005.

Fimbrin, an actin-bundling protein, is a component of the osteoclast adhesion complexes called podosomes. In this study, we (1) determined the localization of fimbrin in the mature rabbit osteoclast as well as in differentiating osteoclasts using the avian monocyte-derived osteoclast differentiation model, (2) characterized the distribution and accumulation of three fimbrin isotypes (T, L, and I) in avian monocytes as they fused to form multinucleate osteoclast-like cells, and (3) report for the first time, a close spatial relationship between podosomes and microtubules using fimbrin as a marker of the podosome. Immunofluorescence using anti-T-fimbrin, anti-L-fimbrin, and pan-isotype-anti-fimbrin antibodies, showed that fimbrin is an integral component of the podosome core in the mature rabbit osteoclast and in the monocyte-derived osteoclast throughout differentiation. Anti-I-fimbrin, however, did not show immunoreactivity in these cultures. These studies also show that in the avian model of monocyte-derived osteoclast differentiation, day 2 cells (D2) are predominantly mononucleate and have few podosomes. By days 4 and 6 in culture (D4 and D6), many cells have fused and punctate rows of podosomes are commonly observed at cell margins. Analysis by Western blot of protein accumulation showed that after an initial small rise from D2 to D4, L-fimbrin levels remained relatively constant from D4 to D6. However, T-fimbrin protein levels increase steadily from D2 to D6, suggesting that it may be related to the increase in podosome formation as monocytes fuse to form osteoclasts. Finally, we examined the distribution of podosomes relative to other cytoskeletal elements such as microtubules and intermediate filaments. Double immunofluorescence labeling using anti-fimbrin and anti-tubulin showed podosomes lying adjacent to microtubules at cell margins. When osteoclasts were treated with nocodazole (1 X 10(-6) M) to disrupt microtubules, the distribution of podosomes became more random and was no longer confined to the cell periphery. These results suggest that microtubule-podosome interactions may play a role in osteoclast adhesion.