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).
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