XIer2 is required for convergent extension movements during Xenopus development

Abstract

Immediate early response 2 (Ier2) is a downstream target of fibroblast growth factor (FGF) signaling. In zebrafish, Ier2 is involved in left-right asymmetry establishment and in convergent extension movements. We isolated the Xenopus ier2 gene based on sequence similarity searches using multiple vertebrate species. Xenopus Ier2 has high homology in the N-terminal region to other vertebrate Ier2 proteins, and Xier2 transcripts were observed from oocytes through larval stages. Except for the maternal expression of xier2, the expression of this gene in the marginal region at gastrulation and in somites and the notochord at later stages is similar to the expression pattern of zebrafish ier2. XIer2 knockdown using antisense morpholinos resulted in defects of convergent extension leading to severe neural tube defects; overexpression of Ier2 showed similar, albeit milder phenotypes. Assays in animal cap explants likewise showed inhibition of elongation after blocking XIer2 expression. These results indicate that Xenopus Ier2 is essential for the execution of convergent extension movements during early Xenopus development.

Fig. 1. Alignment of vertebrate Ier2 sequences

(A) Comparison of amino acid sequences of vertebrate Ier2 proteins including human Ier2 (h) (NM_004907), mouse Ier2 (m) (NM_010499), X. laevis Ier2a (Xla) (GQ120520), X. laevis Ier2b (Xlb) (GQ120521), X. tropicalis (Xt) (predicted sequence based on Scaffold_649 of genomic sequence), and zebrafish Ier2 (Dr) (NM_001142583). Conserved sequences are highlighted in yellow and green. (B) Phylogenetic tree of vertebrate Ier2 proteins.

Fig. 2. Expression patterns of xier2

(A) RT-PCR analysis of Xenopus ier2a and b genes from early cleavage to st 40. Odc was used as control. (B-I) Whole mount in situ hybridization of xier2b, using antisense strand probe; (J-L) sense strand used as control. (B,C) Maternal expression is seen in whole mount and transverse section of two-cell stage embryo (C). (D,E) Restricted expression of xier2b in the marginal zone and involuting axial mesoderm during gastrulation at stage 11. (F,G) Hindbrain and somite expression of xier2 at st 13 (F) and st 25 (G). (H,I) Expression of xier2b in branchial arches, notochord, and somites at stage 30; (I) is a magnified view of the head region. ba, branchial arches; h, hindbrain; no, notochord, so, somites. (J-L) Embryos at st 11 (J,K) and st 29/30 (L) hybridized with sense strand as controls.

Fig. 3. Phenotypes generated by Ier2 knock-down and overexpression

(A-C) stages 25-27; (D-G) stage 35/36. (A,D) Ier2 MO, 40 ng; (B) ier2 RNA, 100 pg; (E) ier2 RNA, 200 pg; (F) ier2 RNA, 400 pg; (C,G) uninjected. (H) quantification of phenotypes: 1, normal; 2, elongated but abnormal; 3, very short axis with closed blastopore; 4, very short axis with open blastopore. Colored rectangles are placed next to embryos illustrating each phenotype in A and B. Total number of embryos scored for each injection is shown on top of the histogram, based on two independent experiments.

Fig. 4. Convergent extension defects in animal caps caused by knockdown of XIer2

Control animal caps round up (A), but elongate after addition of activin (B). Elongation is inhibited by Ier2 knockdown (C), and is rescued by co-injection of xier2 mRNA (D). The number of animal caps showing the phenotype portrayed and the number of caps tested is listed in each panel. The data are based on two independent experiments.