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We have isolated and characterized cDNAs corresponding to the Xenopus En-2 gene. Comparison of amino acid sequences between the entire Xenopus En-2 and the Drosophila engrailed proteins confirms conservation of sequences inside as well as proximal to the homeobox and reveals a region of similarity towards the N terminus. Two transcripts encode the Xenopus En-2 protein. Both transcripts are regulated temporally in an identical fashion and are likely to be transcribed from two copies of the En-2 gene. We have also analyzed the distribution of the protein in the headtissue and in the dissected brain of tailbud stage embryos. In addition to the main band of expression at the midbrain-hindbrain boundary, we show that the protein is expressed in three novel areas: the mandibular arch, the optic tectum and the region of anterior pituitary.
Fig. 4. Characterization of En-2 transcripts during development. (A) Developmental pattern of expression of En-2. The top panel shows En-2 transcripts in poly(A) RNA from blastula to swimming tadpole. The numbers at the top of the lanes correspond to embryonic stages (Nieuwkoop and Faber, 1967). Stages 9 and 10 are late blastula, stages 10 to 12 are gastrula, stages 12 to 20 are neurula and stage 36 is tadpole. The M lane is the size marker (A DNA digested with EcoRl and Hindlll and radiolabeled). The bottom panel is the expression of the Xenopus src gene during development using the same filter, stripped of the En-2 probe, to show the amount of poly(A) RNA in each lane. The quantity of src transcripts remains fairly constant throughout early Xenopus embryogenesis (Condie Ph.D. Thesis, 1989). (B) Spatial distribution of the En-2 transcripts in a stage 28 tailbud. The top panel represents the dissection (a single cut anterior to the otic vesicle). The middle panel shows that both En-2 transcripts are present in the head (H) and absent in the tail (T) region. The bottom panel shows the hybridization of the same filter with the actin probe to show abundance of RNA in each lane. cskl=cytoskeletal actin, muscle=muscle actin.
Fig. 5. Localization of En-2 protein in anterior structures and in the whole-mount brain. (A) Expression of En-2 protein in the mandibular arch of a stage 23 embryo. Arrows point to the nuclear staining. (B) Plane of frontal sections of stage 23 and 28 embryos shown in C, D and E. (C) Frontal section of the embryo shown in A; arrows point to the nuclei expressing En-2 in the presumptive mandibular arch. (D) Frontal section of a stage 28 tailbud (differential interference contrast optics) to show pharyngeal structures. MA=Mandibular arch, PPl=Pharyngeal pouch 1 and PP2=Pharyngeal pouch 2. (E) Same section as D (bright-field optics). Arrow indicate the nuclear staining in the mandibular arch. (F) Dissected brain from a stage 35/36 tadpole stained with mAb 4D9. The two boxes delineate the optic tectum shown in G and the pituitary shown in I. (G) Expression of En-2 in the optic tectum. The arrow points to the 'salt and pepper' like expression in the midbrain. (H) Whole-mount brain from a stage 35/36 embryo with HRP-stained retinal axons. The optic fibers are seen to terminate in the optic tectum (t), of the midbrain (mb). (I) Expression of En-2 in the region of the pituitary. The arrow points to the nuclear staining. The major area of the brain are indicated, t=optic tectum, p=Pineal, ot=optic tract, tel=Telencephalon, di=Diencephalon, mb=Midbrain, hb=Hindbrain.
