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BACKGROUND: One prominent example of segmentation in vertebrate embryos is the subdivision of the paraxial mesoderm into repeating, metameric structures called somites. During this process, cells in the presomitic mesoderm (PSM) are first patterned into segments leading secondarily to differences required for somite morphogenesis such as the formation of segmental boundaries. Recent studies have shown that a segmental pattern is generated in the PSM of Xenopus embryos by genes encoding a Mesp-like bHLH protein called Thylacine 1 and components of the Notch signaling pathway. These genes establish a repeating pattern of gene expression that subdivides cells in the PSM into anterior and posterior half segments, but how this pattern of gene expression leads to segmental boundaries is unknown. Recently, a member of the protocadherin family of cell adhesion molecules, called PAPC, has been shown to be expressed in the PSM of Xenopus embryos in a half segment pattern, suggesting that it could play a role in restricting cell mixing at the anterior segmental boundary.
RESULTS: Here, we examine the expression and function of PAPC during segmentation of the paraxial mesoderm in Xenopus embryos. We show that Thylacine 1 and the Notch pathway establish segment identity one segment prior to the segmental expression of PAPC. Altering segmental identity in embryos by perturbing the activity of Thylacine 1 and the Notch pathway, or by treatment with a protein synthesis inhibitor, cycloheximide, leads to the predicted changes in the segmental expression of PAPC. By disrupting PAPC function in embryos using a putative dominant-negative or an activated form of PAPC, we show that segmental PAPC activity is required for proper somite formation as well as for maintaining segmental gene expression within the PSM.
CONCLUSIONS: Segmental expression of PAPC is established in the PSM as a downstream consequence of segmental patterning by Thylacine 1 and the Notch pathway. We propose that PAPC is part of the mechanism that establishes the segmental boundaries between posterior and anterior cells in adjacent segments.
Figure 1. PAPC expression in the PSM. (a) Schematic diagram showing gene expression patterns in the PSM of Xenopus embryos, in relation to somite formation. The gray scale used reflects the intensity of staining. (b) Expression analysis of PAPC. Xenopus embryos (stage 20) were bisected sagitally and each half embryo stained for PAPC expression or for the expression of Thylacine1, X-Delta-2, and Mespo RNA using whole-mount in situ hybridization. In all panels, the two halves are oriented with anterior to the top and the half embryo stained for PAPC expression is shown on the left. Arrows denote expression in somitomere 1. (b) Comparison of PAPC and Thylacine1 expression, dorsal surface view. (c) Comparison of PAPC and Thylacine1 expression, inside view after surgical removal of axial mesoderm. (d) Comparison of PAPC and X-Delta-2 expression, dorsal surface view. (e) Comparison of PAPC and Mespo expression, lateral view.
Figure 2.
PAPC expression in CHX-treated embryos. Embryos were treated with CHX for the indicated times and then stained for the expression of (a,c,e,g)PAPC or (b,d,f,h)X-Delta-2. Note that after 1 h of CHX treatment, the striped expression pattern of X-Delta-2 in somitomere 1 (s1) is lost (compare (d) with (b)). By contrast, the expression pattern of PAPC (c) does not change after 1 h of treatment, but the striped pattern is lost in somitomere 2 after 2 h of treatment (compare (e) with (a,c)). After 3 h of CHX treatment, the striped expression pattern of both X-Delta-2 and PAPC is obliterated.
Figure 3.
PAPC expression in embryos recovered from CHX treatment. Embryos were (aâd) left untreated or (eâh) treated for 1.0 h with CHX, extensively washed and allowed to recover for 4 h, when they were stained for the expression of X-Delta-2, Thylacine1, Hairy2A, and PAPC as indicated. (b,f) Note that Thylacine1 expression in the PSM of these embryos comes on and goes off in approximately the same region, but that the gaps in expression corresponding to posterior half segments are filled in. The loss of posterior segmental identity in the PSM of these embryos is further suggested by (g) the loss of Hairy2A expression and the continuous expression of (h) PAPC and (e) X-Delta-2.
Figure 4.
Regulation of PAPC expression by Notch signaling and the Mesp family of bHLH proteins. Embryos were injected unilaterally at the two-cell stage with RNA encoding (a) X-Su(H)DBM (DBM), (b) the intracellular domain (ICD) of the X-Notch-1 receptor (ICD), (c) Mespo, or (d) Thylacine1 along with RNA encoding a β-galactosidase tracer. At early tadpole stages, embryos were fixed and stained for PAPC RNA expression using whole-mount in situ hybridization. Dorsal views are shown with the injected side up and the anterior to the left.
Figure 5.
PAPC activity is required for somite segmentation. Embryos were injected unilaterally at the two-cell stage with RNA encoding a secreted form of PAPC (DN-PAPC) or a form lacking the cytoplasmic domain (M-PAPC). At tadpole stages, the embryos were fixed and stained with (a,b,d,e,g,i,k) 12/101 or with (c,f,h,j,l) Hoechst. Anterior is to the left in all panels. The myotomal array was photographed (a,b,g,i,k) as a lateral view in whole-mount or (c–f,h,j,l) as a dorsal view in tissue section. In (c,f,h,j,l), nuclei associated with somitic tissue are pseudo-colored using Photoshop. Note that the injection of (b,c) DN-PAPC and (e,f) M-PAPC RNA causes a disorganization of the segmental pattern of somites (based on (b,e) myotomal morphology and (c,f) the arrangement of somitic nuclei; arrows mark the normal array of somitic nuclei on the uninjected side). Section shown in (c) is the same embryo as in (b). (g–l) Embryos at neurula stages were treated for 1 h with CHX, washed and allowed to develop for an additional 12 h. Note that the recovered, CHX-treated embryos are arrested in terms of morphological segmentation, based on myotomal morphology (compare (i) with (g)) or by the arrangement of somitic nuclei (compare (j) with (h); arrowheads mark the same A–P level). In DN-PAPC RNA-injected embryos, the subdivision of somitic tissue in CHX-treated embryos recovers to some extent (compare (k) with (i) and (l) with (j)).
Figure 6.
PAPC activity maintains segmental gene expression in the PSM. Embryos were injected unilaterally at the two-cell stage with RNA encoding DN-PAPC, M-PAPC or full-length PAPC (FL-PAPC). At early tadpole stages, the embryos were fixed and stained for X-Delta-2 or for Hairy2A, whose expression marks anterior and posterior cells, respectively, as indicated. Anterior to the left in all panels. (a–d) Lateral views of the injected and uninjected sides; (e–g) dorsal views. Note the misposition of cells expressing X-Delta-2 and Hairy2A in response to DN-PAPC and M-PAPC.
Figure 7.
Model linking AâP patterning to AâP boundary formation during Xenopus segmentation. Cells leaving the tailbud domain, where they express Mespo (orange), undergo segmental patterning in the transition zone (TZ). A half-segmental pattern of Thylacine1 expression (gray), in which anterior cells express Thylacine1 whereas posterior cells do not, is thus established. Thylacine1 acts as a selector gene to regulate the expression of PAPC (blue color), thus producing a sharp cutoff in adhesion between anterior and posterior cells in adjacent segments. This differential cell adhesion maintains the integrity of an AâP boundary, which is subsequently required for segmental morphogenesis.
