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The mucus secreting cement gland is the anterior-most ectodermal organ of the Xenopus embryo. The homeobox genes Pltx1 and Pitx2c are expressed in the cement gland primordium. Misexpression of both genes induced ectopic cement gland tissue in whole embryos and transcription of the marker genes Xag1 and Xag2 in animal cap explant cultures. Antisense morpholino oligonucleotides against Pitx1 and Pitx2c inhibited ectopic cement gland formation induced by otx2. Gene knock downs generated by morpholino oligonucleotides were specific and could be rescued by coinjection of Pitx mRNAs. These data demonstrate for the first time the requirement of specific genes for cement gland formation by loss-of-function experiments. genesis 30:144--148, 2001.
FIG. 1. Expression of Pitx1 and Pitx2c in the cement gland primordium. Frontal views of neurula stage Xenopus embryos (stage 19) hybridyzed with probes specific for Pitx1 (A) and Pitx2c (B). Both mRNAs were found in identical domains comprising the cement gland anlage at stage 19. Whole-mount in situ hybridization for Pitx2c was as described (Schweickert et al., 2000). The Pitx1 probe comprised nucleotides 137 (accession number AJ278330).
FIG. 2. Misexpression of Pitx1 and Pitx2c induces ectopic cement glands in embryos and animal cap explants. (A) Induction of cement glands in whole embryos. Synthetic mRNAs were injected into ventral-animal regions of one blastomere at the four-cell stage. The injected amounts were 100 pg per blastomere for Pitx1 and 250 pg for Pitx2c. Injected embryos were cultured until stage 261, assayed for morphological phenotypic changes, and processed for whole-mount in situ hybridization with a probe specific for the cement gland marker Xag1. (A) Pitx1 injected embryo. Plane of sections are indicated. (B, C) Transverse histological sections at the level of the endogenous (B) and induced (C) cement glands. (D) Pitx2c injected embryo. Plane of sections are indicated. (E, F) Transverse histological sections at the level of the endogenous (E) and induced (F) cement glands. Embryos were embedded in gelatine-albumin and sectioned (30 m) using a vibratome. (G) Induction of cement gland marker gene transcription in animal cap explants. Embryos were injected with synthetic mRNA encoding Pitx1 (100 pg) or Pitx2c (50 pg) into animal blastomeres at the 4 cell stage, animal caps were cut at stage 8, cultured until gastrula (stage 10.5), and assayed for marker gene expression by radioactive RT-PCR as described (Ding et al., 1998). RNA from stage 10.5 whole embryos served as positive control of RT-PCR reactions, EF1 transcription as loading control. PCR primers and conditions: Xag1 forward primer: CTGACTGTCCGATCAGAC, reverse primer: GAGTTGCTTCTCTGGCAT, 26 cycles at 3095, 3053, 3072 Xag2 forward primer: TTTGCATGCCCTGGAACTACTCTT, reverse primer: AGCGCTTGTGCCACCTTGAAACTC, 26 cycles at 3095, 3055, 30 72; EF1 forward primer CAGATTGGTGCTGGATATGC, reverse primer ACTGCCTTGATGACTCCTAG, 24 cycles at 3095, 3057, 3072; Xbra forward primer CACAGTTCATAGCAGTGACCG, reverse primer TTCTGTGAGTGTACGGACTGG, 26 cycles at 3095, 3057, 3072.
FIG. 3. Pitx1 and Pitx2c are required for otx2-induced ectopic ce- ment glands. (A) Classification of inductive events in (A) strong, (B) intermediate and (C) weak induction of ectopic cement gland tissue. For details see text. (D,E,G,H) Inhibition of cement gland formation by gene knock down of Pitx1 and Pitx2c. Synthetic otx2 mRNA (130 pg/embryo) was coinjected with control (D,G), Pitx1 (E) or Pitx2c (H) antisense morpholino oligonucleotides into ventral- animal regions of one blastomere at the four-cell stage. The injected amounts of morpholino oligos were 5 nl of a 660 ,000 M solution per blastomere. Embryos were cultured until stage 261, assayed for morphological phenotypic changes and processed for whole- mount in situ hybridization with a probe specific for the cement gland marker Xag1. (F and I) Specificity of Pitx gene knock down. Coinjection of Pitx1 (F, 40 pg) or Pitx2c (I, 40 0 pg) mRNA rescued cement gland induction by otx2. (K) Quantification of results.[see xb-img-]
FIG. 3. Pitx1 and Pitx2c are required for otx2-induced ectopic ce- ment glands. (K) Quantification of results.
