We identified a zebrafish caudal-related homeobox gene, cdx1b, which shares syntenic conservation with both human and mouse cdx1. Zebrafish cdx1b transcripts are deposited maternally. cdx1b is uniformly expressed in both epiblast and hypoblast cells from late gastrulation to 1-2s stages and can be identified in the retina, brain, somite, and anus during 18 to 22 hpf stages. After 28 h of development, cdx1b is exclusively expressed in the developing intestine. Both antisense morpholino-mediated knockdown and overexpression experiments were conducted to analyze cdx1b function during digestive tract development. Hypoplastic development of liver and exocrine pancreas as well as intestinal abnormalities was observed in cdx1b 96-hpf morphants. In cdx1b 85% epiboly morphants, two-fold decrease in respective number of gata5-, cas-, foxa2-, and sox17-expressing endodermal precursors were identified. Furthermore, ectopic cdx1b expression caused substantial increase in respective number of gata5-, cas-, foxa2-, and sox17-expressing endodermal precursors and altered their distribution patterns in 85% epiboly injected embryos. Conserved Cdx1 binding motifs have been identified in both gata5 and foxa2 genes by interspecific sequence comparison. Cdx1b can bind to Cdx1-binding motif located in the intron 1 of the foxa2 gene by electrophoretic mobility shift assay. Co-injection of either zebrafish or mouse foxa2 mRNA with cdx1b MO can rescue expression domains of ceruloplasmin in the liver of 54-hpf injected embryos. These results indicate that zebrafish cdx1b regulates foxa2 expression and may also modulate gata5 expression to affect early endoderm formation. This study underscores a novel role of zebrafish cdx1b in the development of different digestive organs as compared with its mammalian homologues.

Analyses of relationships between cdx1b and downstream factors of Nodal signaling. Reductions in respective gata5-, cas-, sox17-, and foxa2-expressing endodermal cell numbers were observed in 85% epiboly morphants (C, G, H, K, L, O) compared with respective cdx1b-4mm-MO-injected (A, E, F, I, J, M) and wild-type (B, N) embryos. Embryos co-injected with either cdx1b mRNA or protein showed restoration of respective gata5- (D) and foxa2-expressing (P) endodermal cell numbers. Percentages of morphants showing decreases in the respective numbers of gata5-, cas-, sox17-, and foxa2-expressing endodermal cells (Q). Comparison of the respective gata5-, cas-, sox17-, and foxa2-expressing endodermal cell numbers in morphants (yellow bars), and wild-type (orange bars) and cdx1b-4mm-MO-injected (brown bars) embryos (n=10 each) (R). Comparison of percentages of embryos showing decreased foxa2- or gata5-expressing endodermal cell numbers in respective embryos injected with either cdx1b MO (brown bars), cdx1b MO and cdx1b mRNA (orange bars), or cdx1b MO and different amounts of Cdx1b protein (1/40 x, yellow bars; 1/2x, green bars) (S). Comparison of the respective foxa2- and gata5-expressing endodermal cell numbers in morphants (brown bars) and embryos co-injected with cdx1b MO and either cdx1b mRNA (orange bars) or protein (yellow bars) (T). Error bars represent the standard error. Dfc, dorsal forerunner cell.