XB-ART-24478J Cell Biol October 1, 1991; 115 (2): 565-75.
Expression of two nonallelic type II procollagen genes during Xenopus laevis embryogenesis is characterized by stage-specific production of alternatively spliced transcripts.
The pattern of type II collagen expression during Xenopus laevis embryogenesis has been established after isolating specific cDNA and genomic clones. Evidence is presented suggesting that in X. laevis there are two transcriptionally active copies of the type II procollagen gene. Both genes are activated at the beginning of neurula stage and steady-state mRNA levels progressively increase thereafter. Initially, the transcripts are localized to notochord, somites, and the dorsal region of the lateral plate mesoderm. At later stages of development and parallel to increased mRNA accumulation, collagen expression becomes progressively more confined to chondrogenic regions of the tadpole. During the early period of mRNA accumulation, there is also a transient pattern of expression in localized sites that will later not undergo chondrogenesis, such as the floor plate in the ventral neural tube. At later times and coincident with the appearance of chondrogenic tissues in the developing embryo, expression of the procollagen genes is characterized by the production of an additional, alternatively spliced transcript. The alternatively spliced sequences encode the cysteine-rich globular domain in the NH2-propeptide of the type II procollagen chain. Immunohistochemical analyses with a type II collagen monoclonal antibody documented the deposition of the protein in the extracellular matrix of the developing embryo. Type II collagen expression is therefore temporally regulated by tissue-specific transcription and splicing factors directing the synthesis of distinct molecular forms of the precursor protein in the developing Xenopus embryo.
PubMed ID: 1918153
PMC ID: PMC2289160
Article link: J Cell Biol
Grant support: AR-38648 NIAMS NIH HHS , DE-05837 NIDCR NIH HHS, HD-23250 NICHD NIH HHS
Genes referenced: col2a1
Article Images: [+] show captions
|Figure 7. Immunohistochemical localization of type II collagen in stage 20 and 31 embryos . The embryos were processed by a whole mount procedure and bound antibody to type II collagen was local- ized with a peroxidase tag. At stage20 (a),antigen can beobserved in the peri-notochordal region which already appears segmentally organized . The embryo in b was treated identically to that in (a) except it was incubated with culture supernatant from the nonimmuno-globulin secreting parental myeloma cell line to serve as a negative control. Immunolocalization of type II collagen at stage31(c);d shows a control embryo. Note the immunoreactivity localized to the vertebrae. Bar,500um.|
|Figure 8 bright-field micrograph of a cross section through a stage 21 embryo that has been stained for type II collagen, demonstrating the localization of reaction product in the peri-notochordal region (arrow) . b is a bright-field micrograph of a section through the embryo shown in Fig. 7 c, without any additional staining in order to localize the immunoreactivity further. Note the presence of immunoreactivity in the basement membranes around the notochord (n) and on the ventral side of the neural tube, as well as in the floor plate, the sub-notochordal rod and in the developing vertebra. Bars, 250 um|
|Figure 9 Bright-field (a, c, and e) and dark-field (b, d, and f) micrographs of cross sections at three different levels through a stage 21 embryo probed for type II procollagen mRNA with the general probe. (a) and (b) are cut at a posterior level showing a high level of mRNA in the notochord. In addition, mRNA is detected throughout the somites and in the more dorsal half of the lateral plate mesoderm. c and d are cut at the level of the mid-gut showing that the signal is no longer detected in the lateral plate mesoderm but is restricted to the notochord and somites. e and f are cut at a more anterior level of the same embryo in which the intense signal in the notochord and sclerotome can be noted. The level of label in the remainder of the somite is relatively weaker than in the peri-notochordal region (n, notochord; s, somites). Bar, 250um.|
|Figure 10. Bright-field (a) and dark-field (b) micrographs of a section through the same embryo as that shown in Fig. 9 at a level between c and e, shown at higher magnification . Label can be clearly localized in the floor plate (arrow), notochord (n), and somites (s), Bar, 100 um.|
|Figure 11. Bright-field (a and c) and dark-field (b and d) micrographs through the head (oblique cross section) and trunk (cross section) of a stage 46 embryo probed for type 11 procollagen mRNA with the general probe . In the head, mRNA is detected in the oto- cysts (o), chondrocranium, and periocular (p) tissue. In the section through the trunk, mRNA is detected in cells of the vertebra. In both the head and trunk regions, mRNA is detected in ectodermal cells (arrow). Bar, 250 um.|
|Figure 12. Immunolocalization of type II collagen at stage 46 in the head (a), as seen in frontal section, and the trunk (b), shown in cross section. Antigen is detected in the chondrocranium, eye (e), vertebra, and other regions expressing the mRNA, with the exception of the ectoderm Bar, 250 um|
|Figure 13. Bright-field (a) and dark-field (b) micrographs of a cross section similar to that shown in Fig. II (a and b), but showing the pattern of hybridization with the exon 2 probe. The signal is generally weaker than that obtained with the general probe, but as far as the limit of resolution permits, is otherwise indistinguishable.(o,otocyst).Bar, 250 um|
|Figure 6. Dark field micrograph of a cross section through a stage 14 embryo probed for type II procollagen mRNA with an exon 2 sequence . Essentially identical results are obtained with the general probe (not shown) . There is significant signal in the notochord (ar- row), sub-notochordal rod, and peri-notochordal regions of the somites, as well as in the floor plate (arrowhead). Bar, 250 um.|