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Abstract
While there is convincing evidence implicating Drosophila int-1 in pattern regulation, the normal role of int-1 in vertebrate development is unclear. We have injected Xenopus eggs with mouse int-1 RNA and monitored subsequent development. Injected RNA is translated and the protein widely distributed. Embryos develop into apparently normal gastrulae, but almost all surviving neurulae have a bifurcated anterior and expanded posterior neural plate. Bifurcation of the neural plate was abolished by substitution of a single, conserved cysteine residue and was dependent on the presence of a signal peptide sequence in the int-1 protein. Histological examination indicates that underlying axial mesodermal structures were duplicated. This result suggests that ectopic int-1 expression leads to dual axis formation and points to a role for int-1 in patterning processes in vertebrate development.
Figure 1. Schematic Representation of the int- 1 cDNA Clone and Predicted Proteins Produced from Modified int-1 Transcripts
(a) The 2.3 kb mouse W-1 cDNA clone consists
of approximately 185 bp of 5’untranslated sequence and 1 kb of 3’untranslated sequence (single line). Coding sequences are represented by the open box. Plasmids were linearized at a unique 3’EcoRI (E) site and transcribed with T7 RNA polymerase. (b) Modification of the parental plasmid (~286) gives rise to RNAs encoding modified int-1 proteins.286 is a schematic representation of the 41 kd int-1 protein. Vertical lines indicate the position of the 23 cysteine residues that are absolutely conserved in Xenopus, Drosophila, and human int-1. 284 was generated by addition of a 30 bp oligonucleotide encoding 10 amino acids of human c-myc (diagonal filled line) at a unique BamHl site (B in Figure 1a). p285 was generated by cleavage at a unique Xhol site in p286 (X in Figure la), end filling, and religation. The T7-derived transcript encodes a truncated int-1-protein 285, which contains a C-terminal extension of non-int-1-coding sequence (horizontal filled line) due to the frame shift introduced at the Xhol site. p293 was generated by Accl digestion of a modified in&l construct in which a new Sall-Accl ((S) in Figure 1a) was introduced at the C terminus of the coding sequence. Accl digestion and religation removes a C-terminal coding segment of int-1. Translation of p293-derived RNA generates an almost full-length in&l protein, 293, in which 50% of the C-terminal cysteine residues are deleted. A C-terminal extension of a non-int-1 sequence (horizontal filled line) is introduced due to a frame shift on deletion of the Accl fragment
Figure 3. Phenotype of Xenopus Embryos Injected with Mouse int-1 RNAs
(a) uninjectecl neurulae; (b) 285 injected; (c) 286 injected; (d) 284-injected; (e) 293 injected; (1) 266-injected tailbud stage. Uninjected, 285- injected, and 293injected neurulae develop normal single neural plates (open arrows). Both 286 and 284-injected neurulae show broad posterior neural plates and anterior bi- furcation of the neural axis (closed arrows). This phenotype is less severe in embryos injected with the int-1 myc RNA (284, [d]). Most embryos injected with 285 or 293 RNAs were normal at the tailbud stage (see Table 1) whereas few embryos injected with 286 RNA develop to this stage. Of these, approximately 10% show a clear bifurcation of anterior structures ([fl, closed arrow; see Table 1)
Figure 4. Distribution in int-1 [wnt1] myc Protein in 284-injected Neurulae.
Whole-mount immunohistochemsitry was performed on uninjected (a) and 284-injected embryos (b) at the neurulae stage. A monoclonal antibody, 9E10, directed against the myc epitope, was used to recognized the int-1 myc protein. Immunoperoxidase detection was used to visualize the int-1 myc protein. Uninjected embryos show wides spread distribution of int-1 myc protein throughout the embryo, with some suggestion of accumulation along the neural tube (b).
Figure 5. Carboxy-Terminal Point Mutations In the in&l Protein Sequence
The C-terminal sequences of mouse, Xenopus, and Drosophila int-1l, and human and mouse int-1-related proteins (h-irp and m-irp, respectively) are shown. Each protein contains an absolutely conserved cysteine residue (open box), which in mouse int-1 is at position 369 in the 370 amino acid protein. At position 368 is a glutamic acid that is substituted by a threonine residue at this position in Drosophila int-1 and human and mouse irp. Oligonucleotide mutagenesis was used to change amino acid 368 from glutamic acid (E)->threonine (T, stippled box, 322) and amino acid 369 from cysteine (C)->tryptophan (W, open box, 323) in the mouse int-1 protein.
Figure 6. RNA Blot Analysis of 322 and 323 RNA in Injected Embryos.
Eggs were injected with l-2ng of in vitro-transcribed 322 (lanes 4, 6, and 8) or 323 (lanes 5, 7, and 9) RNA and total RNA extracted from approximately five embryo equivalents of eggs (E, lanes 4 and 5). gastrulae (G, lanes 6 and 7), and neurulae (N, lanes 8 and 9) following injection. RNA was fractionated by electrophoresis. transferred to Gene- screen, and probed with a mouse int-1probe at high stringency. This probe does not cross-react with Xenopus int-1 RNA under these conditions (lanes 10-12). Injected RNA (open arrow) was detected at all stages (only after a long exposure for neurula samples). in&l RNA lev- els were quantitated by densitometry relative to known standards of in vitro-transcribed int-1 RNA run in parallel (10 ng. lane 1; 1 ng. lane 2; 0.1 ng. lane 3). Subsequent hybridization with a ribosomal RNA probe indicates that approximately equal amounts of RNA were present in all lanes (closed arrow).
Figure 7 Ammo-Terminal Sequences of int-1 Constructs.
Construct 266, wild-type int-1, construct 337, int-1 with signal peptlde deletion: construct 351. ml-1 with modified chick lysozyme signal pep tide sequence (For further detais, see Ex-perimental Procedures.) Closed arrow. lysozyme signal peptlde cleavage site; open arrow. proposed signal peptlde cleavage site for int-1 (Brown et al 1987)
Figure 8. Transverse Sections through Normal and Bifurcated Embryos
(a) anterior; (b) posterior sectlons through normal neurula infected with 265 RNA. (c-f) antenor to posterior sections through bifurcated neurula Injected with 266 RNA. In the normal embryos (a and b). a single notochord (n) underlies a closed neural tube (arrow). Paired somites (s) lie lateral to the notochord. In anterior sections through 2664njected embryos (c and d). there is a continuous enlarged neural plate, which is rouning up into tube-like structures (arrows) above paired notochords. Paired somites lie lateral to each notochord (d). In more posterior sections (e and f), the neural plate has closed and the paired notochords ap- proach the mid-llne (e) and fuse (f)
Figure 9. Somlte FormatIon in 323-and 322-lnjected Neurulae.
Whole-mount immunohlstochemistry was performed on 323- and 322- injected neurulae using an antIbody. 12001, which detects somlic derivatives. (a) Staining In the absence of 12/101. (b) stalnmg of 323-injected neurula; (c) staining of 322-injected neurula. Somites In 323-injected embryos are arranged as normal in pairs about the midline (arrows) In 322- injected embryos, pairs of somItes surround each axis (arrows). Anterior is to the left.
