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The structural similarity between Drosophila and vertebrate homeobox genes begs the question of whether the vertebrate gene products affect cell fate and pattern formation. To study the function of the Xenopus homeobox protein, Xhox-1A, we microinjected fertilized Xenopus eggs with an excess of synthetic Xhox-RNA and assayed for effects on development. The predominant phenotype is a disturbance in somite formation. When embryos are injected with Xhox-1A mRNA, but not with control mRNAs, morphogenesis of somites occurs chaotically and individual segments are lost. Histological staining, in situ hybridization, and immunohistochemistry indicate that the disorganized somitic tissue has differentiated into muscle cells. Overall, these results suggest that correct regulation of the Xhox-1A gene may be important for the normal development of the segmented somite pattern in early embryos. Moreover, the inferred role of Xhox-1A in somite formation indicates that there may be molecular parallels between mechanisms of segmentation in flies and vertebrates.
Figure 1. The Structure and Stability of SPG-Generated mRNAs In- jected into Xenopus Embryos (A) Diagramatic representation of the SP6 mRNAs used in injection experiments. 64T-CIA encodes the complete Xhox-1A protein. 64T- clAABgl2 encodes the same protein except that 13 C-terminal amino acids of the homeobox and the remaining 32 C-terminal amino acid of the protein are deleted. A 23 amino acid extension to the homeobox has been created from a reading frame other than that specifying Xhox-1A. XBm codes for Xenopus 8-globin. Details of construction of the three RNAs are found in Harvey et al. (1986). The open box is the coding region of the Xhox-IA mRNAs, the cross-hatched box is the globin coding region, and the striped box is the homeobox. The elipse at the 5nd of each mRNA indicates that a 5ap has been added during synthesis (Krieg and Melton, 1988).
(B) Northern blot analysis of RNA from embryos injected with either 64T-clA or XBm RNA extracted at different stages of development. The injected RNAs were detected on Northern filters with nick-translated Xhox-IA or globin DNA probes. Stages are those specified by Nieuwkoop and Faber (1956).
(C) Quantitation of the results seen for 64T-CIA in (8). Quantities of in- jected RNA were determined relative to known amounts of the syn- thetic RNA run on the same gel. The endogenous levels of the Xhox-1A mRNA were previously measured (Harvey et al., 1986). Note that the scale for injected and endogenous RNAs are in ng and pg, respec- tively.
Figure 2. Diffusion of SPG-mRNA Injected into One Blastomere of the Two-Cell Embryo
(A) 32P-labeled Xhox-1A mRNA was injected into the marginal region of one blastomere at the two-cell stage. Embryos were allowed to develop to the gastrula stage and were then fixed, horizontally sectioned, and autoradiographed. The grains detected on the uninjected side of the embryo are the result of intercellular movement and reincorporation of radioactive nucleotides produced by degradation of injected RNA (see Figure 1). This is insignificant in the highly cellularizad animal (An) and medial (Med) regions. Autoradiography can therefore be used to visualize injected RNA in these areas of the gastrula, despite the fact that 50% has been degraded by this stage (Figure 1). There is some movement of degradation products in the less cellular vegetal (Veg) regions. In some cases, as shown here. diffusion of RNA throughout the injected blastomere is extensive, resulting in half of the gastrula being labeled. bl is the blastocoel.
(B) Cartoon to indicate that the first cleavage furrow of the developing egg can bisect the embryo into two lateral halves (Klein, 1987). In these cases, injection into one of the two blastomeres results in the left or the right side of the embryo receiving the RNA. (L) indicates the left, and (R) indicates the right lateral side
Figure 3. Normal and Xhox-1A Injected Embryos at the Tailbud Stage
(A) Normal embryo. (f3) Dorsal view of a group of Xhox-1A injected embryos showing various examples of the kinked external phenotype. (C) Lateral view of a Xhox-IA injected and kinked embryo.
Figure 4. Histological Examination of Normal and Xhox-IA Injected Embryos at the Tailbud Stage (A) Horizontal section of a normal embryo at the level of the notochord and spinal chord. (8) Horizontal section of a Xhox-IA injected embryo at the level of the spinal chord and brain. (C) Horizontal section of a severely kinked and stunted Xhox-IA injected embryo at the level of the brain and notochord. (0) A 2.5x enlargement of a part of the normal embryo shown in (A). (E) A 2.5x enlargement of a part of the Xhox-IA injected embryo in (8) showing the loss of somite units in the lower or injected side. (F) A 2.5x enlargement of a part of the Xhox-1A injected embryo in (C) showing the somite-like units interdigitated between the notochord-proximalsomites on the lower or injected side (see text). (G) Diagram of the differences in the severity of the Xhox-1A phenotypes from normal on the left to totally chaotic on the right. B is the brain, E is the eye, EV is the ear vesicle, NC is the notochord, S is the somitic mesoderm, and SC IS the spinal cord.
Figure 5. In Situ Hybridization to Xhox-IA Injected Embryos Using a Muscle-Specific a-Actin Probe (A) Horizontal sectron of Xhox-1A embryos viewed with dark field op- tics. Hybridization grains are seen as white. The embryo is outlined in pigment granules which also appear as white grains in these photo- graphs The normal or uninjected side of the embryo is uppermost. Note the two patches of hybridization-negative cells on the lower side of the disturbed tissue. Br is the brain, NC is the notochord, and S is the somitic mesoderm. (8) and (C). Transverse sections of Xhox-IA in- jected embryos viewed with dark field optics. Disturbed tissue is on the left side of each case. Note the asymmetry in the shape of each pair.
Figure 6. lmmunohistochemrstry on a Xhox-1A Injected Embryo Usrng the Muscle-Specific Antibody 12/101
(A) Horizontal section of a Xhox-1A injected embryo stained with DAPI nuclear stain. Note the aligned nuclei in each normal somite in the upper, umnjected side of the embryo, and in the posterior region of the lower, injected side. Nuclei in the disturbed anterior portion of the lower injected side are not aligned. (6) The same section as in (A) stained by indirect immunofluorescence with the muscle-specific monoclonal antibody f2/tOt. This antibody highlights the membranes of myocytes. (C) and (D) 2.5x enlargements of different parts of the immunofluorescence shown in (B). The arrow in (C) indicates tissue not stained by the antibody. The arrow in (D) indicates a patch of myocytas in cross section surrounded by cells elongated in the plane of the tissue section. B is brain, E is eye, EV is ear vesicle, NC is notochord, and S is somitic mesoderm.
Figure 7. Localization of the Endogenous Xhox-IA Transcript.
Embryos were separated into the sections indicated, total RNA was extracted, and then analyzed for the presence of endogenous Xhox-IA mRNA by RNAase protection. Probes for EFla (a translation elongation factor; see Kintner and Melton, 1987) and cytoskeletal actin were used to control for RNA recovery. Similarly, a probe for muscle-specific actin was used to control for contamination of sections with muscle. (a) Examination of Xhox- 1A localization in the principal axes of the early embryo. A is anterior, P is posterior, NP is the neural plate (containing both the neural ectoderm and the underlying mesoderm), H is the head, 0 is dorsal, and V is ventral. Note that Same muscle contaminates the R, H, and V samples. Higher resolution dissections were performed in (b). (b) Dissections to separate the axial structures from the lateral mesoderm and endoderm. T is the total embryo, H is the head, Ax is the dorsal axial structures, En is the endoderm, and LP is the lateral plate mesoderm and ventral ectoderm. The poor recovery of RNA in the endoderm sample is due to these cells being large and filled with yolk granules. They contain less cytoplasm and RNA.
