zKLF4 is expressed in pre-polster mesendoderm, polster, and hatching gland. Expression of zKLF4 at 80% epiboly is initially restricted to a patch of mesendoderm cells called the pre-polster (a,b, arrowhead). From approximately 100% epiboly to 10 somites (C-E), zKLF4 expression is seen in the polster (arrowhead) and the lateral plate mesoderm (arrow) both anteriorly (D) and posteriorly (E). Expression of zKLF4 within this polster region is deep within the mesendoderm (shown in J, which is the inset of I; cross-section of zKLF4 expression at 3 somites). As development progresses, zKLF4 expression can then be seen in the ICM and hatching gland (F,H, arrow and arrowhead respectively) as well as in two bilateral patches (D, F, and H, *). Dual WISH with Krox20 (G, r3 and r5) and zKLF4 (G, *) shows the position of the bilateral patches with respect to rhombomeres 3 and 5.

zKLF4 is essential for hatching gland development. A: Nucleotide sequence of the 5' end of the KLF4 transcript. The translation initiation codon is in bold type face. The sequences targeted by the two separate antisense morpholino oligonucleotides used in this study are shown. The 4-bp mismatch morpholino was designed against KLF4-MO2. The mismatched bases are underlined. Embryos were injected with the 4-bp mismatch control morpholino (B,D,F,H (ii), I,K) or zKLF4 specific morpholinos (C,E,G,H (i), J,L,M). At 3 dpf, zKLF4 morphants (C) failed to hatch compared to control embryos (B) although they had normal hearts and anterior epidermal scale patterning (E). Control embryos have a prominent hatching gland (F, arrow) whereas zKLF4 morphants have no hatching gland (G, arrow). Expression of the hatching gland enzyme CatL is absent in zKLF4 morphants at 3dpf (H, i) compared to control injected embryos (H, ii). Analysis of the polster at 6 somites (I and J) and hatching gland at 20 somite s(K-M) shows that there is an absence of CatL expression from as early as 6 somites, in zKLF4 morphants (J, arrowhead shows where CatL expression is expected). When partial knockdown of zKLF4 occurred, CatL expression was slightly reduced and some CatL-positive cells remained under the forebrain (M, arrow).

The pre-polster/anterior mesendoderm is abnormal in zKLF4 morphants. Whole mount in situ hybridization at approximately 80% epiboly for CatL (A,B), zKLF4 (C,D), cap1 (E,F), and BMP4 (G,H) in zKLF4-MO injected embryos (A,C,E,G) versus 4-bp mismatch-injected embryos (B,D,F,H). Dorsal views shown in all cases. Although zKLF4 itself is expressed in the developing pre-polster (D), expression of other polster markers such as CatL (B), cap1 (F), and BMP4 (H) is dramatically reduced by knockdown of zKLF4.

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