Depletion of gata5 and gata6 from the YSL causes cardia bifida but does not affect myelopoiesis. (A) The YSL expression of gata5 and gata6 was depleted by injection of gata5+gata6 morpholinos into the YSL at the 1000-cell stage. To trace the correctly targeted embryos, a fluorescent control morpholino was co-injected. Embryos positive for fluorescence in the YSL alone (+YSL) were selected at several time points and harvested as YSL gata5+gata6-depleted embryos. The embryos showing no fluorescence (-YSL) were collected as negative injection controls and should express wild-type levels of gata5 and gata6. To ensure the efficiency of the morpholinos, positive control embryos were injected with gata5+gata6 morpholinos at the one-cell stage and were fluorescent throughout the embryo (+embryos). To assess heart formation, cmlc2 and nkx2.5 expression was analysed. Depletion of gata5 and gata6 in the YSL (+YSL) showed normal levels of expression of both cmlc2 and nkx2.5, indicating that specification occurs normally in these embryos. However, cardia bifida was observed in the +YSL embryos, demonstrating that gata5 and gata6 are required in the YSL for the correct migration of the cardiac precursors to the midline. By contrast, l-plastin expression was the same as in the wild-type embryos, indicating that gata5 and gata6 expression in the YSL is dispensable for myelopoiesis. Embryos injected at the one-cell stage (+embryos) showed complete absence of cmlc2, nkx2.5 and l-plastin expression, thereby validating morpholino effectiveness. Views are anterior. For each gene and type (±YSL, +embryo) analysed, n=28-38, and the images shown depict the findings for >95% of the embryos. (B,C) Loss of endoderm in casanova morphant embryos but no defects in myelopoiesis. To establish whether endoderm plays a role in myelopoiesis, endodermless embryos were created by injection of casanova morpholinos. Casanova morphants (casmo) were assessed for endoderm formation and myelopoiesis. Myelopoiesis was not affected in cas morphants as l-plastin and mpx remained unaffected (B). Gene expression in the ALM of cas morphants was examined at 5 and 10 somites (C). The formation of myeloid precursors occurred as normal in cas morphants. Expression of pu1 at 5 somites, and runx1, ikaros and cmyb at 10 somites, was unaffected in cas morphants. Views are anterior-dorsal. For each gene analysed n=38-67, and the images shown depict the findings for >95% of the embryos.