XB-ART-22643Development May 1, 1993; 118 (1): 193-202.
One of the distinguishing features of vertebrate development is the elaboration of the anterior neural plate into forebrain and midbrain, yet little is known about the early tissue interactions that regulate pattern formation in this region or the genes that mediate these interactions. As an initial step toward analyzing the process of regionalization in the anterior-most region of the brain, we have screened an anterior neural cDNA library for homeobox clones and have identified one which we have called XeNK-2 (Xenopus NK-2) because of its homology to the NK-2 family of homeobox genes. From neurula stages, when XeNK-2 is first detectable, through hatching stages, XeNK-2 mRNA is expressed primarily in the anterior region of the brain. By swimming tadpole stages, XeNK-2 expression resolves into a set of bands positioned at the forebrain-midbrain and the midbrain-hindbrain boundaries, after which XeNK-2 transcripts are no longer detectable. In addition to localized expression along the anterior-posterior axis, XeNK-2 may also play a role in the process of regionalization along the dorsal-ventral axis of the developing brain. At all stages examined, XeNK-2 mRNA is restricted to a pair of stripes that are bilaterally symmetrical in the ventral-lateral region of the brain. To begin to identify the tissue interactions that are required for the proper spatial and temporal localization of XeNK-2, we have performed a series of explant experiments. Consistent with earlier work showing that the A/P axis is not fixed at mid-gastrula stages, we show that XeNK-2 expression is activated when assayed in gastrula stage explants taken from any region along the entire A/P axis and that the tissue interactions necessary to localize XeNK-2 along the A/P axis are not completed until later neurula stages.
PubMed ID: 8104140
Article link: Development
Species referenced: Xenopus
Genes referenced: acta4 nkx2-2 tac1 tbx2
Article Images: [+] show captions
|Fig. 1. Northern blot analysis. Filters were hybridized with a radiolabeled XeNK2 DNA probe. A single 2.0 kb hybridizing band is present in the lane that contains 5 mg of neurula stage poly(A)+ RNA but absent in the lane that contains 5 mg of poly(A)- RNA.|
|Fig. 2. Nucleotide sequence of XeNK2 and the deduced amino acid sequence.The nucleotide sequence of the XeNK2 cDNA clone is presented along with a conceptual translation. The homeobox region is designated by hatching while the NK-2 box is underlined. The consensus polyadenylation signal (AATAAA) at the 3¢ end of the clone is also underlined.|
|Fig. 3. Amino acid sequence comparisons among NK2 family members. Comparisons of amino acid residues within the homeobox region (A) and the NK2 box (B) between XeNK2 and the murine Nkx2.2 gene (Price et al., 1992), the Drosophila NK2 gene (Kim and Nirenberg, 1989), the rat thyroid factor, TTF-1 (Lazzaro et al., 1991) and the planarian Dth-1 gene (Garcia-Fernandez et al., 1991). Dashes indicate identical residues. The percentage identity between XeNK2 and other family members is indicated to the right of each sequence.|
|Fig. 4. Expression pattern of XeNK2 mRNA. (A) XeNK2 mRNA is readily detectable on RNase protection assays at late neurula stages (stage 19); on long exposures XeNK2, mRNA is observed at neural plate stages (stage 14; not visible on this assay). Steady-state mRNA levels increase during neurula stages and remain constant until swimming tadpole stages when XeNK2 transcripts are no longer detectable. Approximately 20 mg of total RNA for each stage was hybridized with the RNase protection assay probe described in the Materials and Methods section. For dissected material, 20 pieces were used for each region. Arrows indicate the protected fragment. (B) The spatial distribution of XeNK2 transcripts was initially determined by dissecting embryos into anterior, middle and posterior pieces (ten pieces were used in each lane) and employing RNase protection analysis to assay for the presence of XeNK2 mRNA; at all stages examined, XeNK2 mRNA was enriched in anterior third of the embryo. ‘Forebrains’ were dissected by making a transverse cut midway through the optic vesicle and pieces of tissue anterior to this cut were collected.|
|Fig. 5. Whole-mount in situ hybridization with XeNK2. Wholemount in situ hybridization was performed on Xenopus embryos at different developmental stages. (A) Neurula (stage 20), lateral view; (B) neurula (stage 20), dorsal view; (C) tailbud (stage 23), lateral view; (D) tailbud (stage 25), lateral view; (E) tailbud (stage 29/30), lateral view; (F) tailbud (stage 29/30), dorsal view; (G) hatching (stage 33/34), lateral view; (H) swimming tadpole (stage 40), dorsal-lateral view. Anterior is at the right. Arrows indicate hybridization signals. Magnifications are as follows: A, B and F, 30´; C, 20´; D, 25´; E, G and H, 50´.|
|Fig. 6. Histological analysis of XeNK2 mRNA expression. Following completion of the in situ hybridization procedure, embryos were sectioned (approximately) transversely and resulting sections photographed with bright-field optics. (A-C) A neurula (stage 20) embryo; (D-F) a hatching stage (stage 33-34) embryo; (G-I) a swimming tadpole (stage 40) embryo. A, D and G represent sections anterior to the presumptive eye level; B, E and H depict sections at the presumptive eye level; C, F and I show sections cut through the presumptive ear region. Diagrams at the left indicate the approximate plane of section. The sections in A-C and G-I are somewhat oblique; the right side of the section is therefore more anterior than the left side. Section G-I are somewhat longitudinal, making both pairs of XeNK2 bands visible in H. In all cases, XeNK2 hybridization signals are denoted by arrows. In all panels, dorsal is up, and ventral down. The magnification in panels A through F is 75´, while the magnification in panels G through I is 50´. l, lens; nr, neural retina; ov, otic vesicle; pr, pigmented retina; r, retina.|
|Fig. 7. XeNK2 expression in neural regionalization experiments. RNAse protection analysis was used to assay XeNK2 mRNA levels in response to a variety of axis-perturbing reagents as well as in neural induction experiments. (A) XeNK2 mRNA is not detectable in embryos treated with high doses of UV irradiation or retinoic acid, while XeNK2 mRNA levels are enhanced in lithium-treated embryos. (B) The presence of a signal in the lane containing recombinants of gastrula animal cap ectoderm with dorsal mesoderm and not in the lane with animal caps indicates that XeNK2 is a response to neural induction. (C) When pieces of mes-ectoderm are dissected as shown, cultured in vitro until stage 33/34 and then assayed for XeNK2 expression, appropriate restriction of spatial expression is not observed until late neurula stages. Twenty pieces of tissue were used in each lane. See text for details. Protected fragment is indicated by an arrow. UV, ultraviolet radiation; RA, retinoic acid; Li, lithium; A, anterior; MA, middle-anterior; M, middle; MP, middle-posterior; P, posterior.|