XB-ART-10769J Comp Neurol July 31, 2000; 423 (3): 512-31.
Xenopus laevis peripherin (XIF3) is expressed in radial glia and proliferating neural epithelial cells as well as in neurons.
Neuronal intermediate filament (nIF) proteins form the most abundant component of the axonal cytoskeleton. Thus, understanding their function and the regulation of their expression is essential for comprehending how axonal structure is regulated. Although most vertebrate nIF proteins are classified as type IV intermediate filament (IF) proteins, additional nIF proteins exist in frogs (Xenopus laevis), cyprinid fishes, and mammals (called XIF3, plasticin, and peripherin, respectively) that are classified as type III. Expression of a type III nIF protein is correlated strongly with the earliest phases of axonal outgrowth in fishes but less so in mammals. To understand better how the correlation between type III nIF protein expression and early phases of axonal outgrowth has changed during evolution, the authors examined XIF3 expression in Xenopus laevis. In Xenopus, the association between XIF3 expression and early axonal outgrowth was especially strong. For example, during early axonal development, XIF3 expression preceded and was more abundant and widespread than that of any of the type IV nIF proteins. As axons matured, neuronal expression of XIF3 gradually became more restricted while that of type IV nIF proteins increased. These results support the idea that type III nIF proteins play a special role during early phases of axonal outgrowth. In addition to finding XIF3 in neurons, the authors also unexpectedly found it in regions of the central nervous system that contain proliferating cells and radial glia. As a framework for interpreting variations in nIF expression in different vertebrate species, the authors built phylogenetic trees to clarify relationships among vertebrate nIF proteins. These trees supported the classification of XIF3, plasticin, and peripherin as orthologs (products of the same genetic locus, evolving separately only since the species lineages diverged). Thus, XIF3, plasticin, and peripherin probably should be referred to as Xenopus, fish, and mammalian peripherin, respectively. This finding argues that differences in expression of these three proteins in frogs, fishes, and mammals are the result of regulatory changes to the peripherin ancestral gene along each lineage. The expression of a peripherin ortholog in Xenopus glia may represent either an adaptation that arose since the divergence of Xenopus from mammals or, alternatively, a feature retained from an ancestral IF protein that was expressed originally both in neurons and in glia.
PubMed ID: 10870090
Article link: J Comp Neurol
Species referenced: Xenopus laevis
Genes referenced: drg1 grina ina nefl nif pcna prph vim
Antibodies: Prph Ab1
Article Images: [+] show captions
|Fig. 1. Early expression of Xenopus laevis peripherin (XIF3) determined by wholemount in situ hybridization of embryos hybridized with cRNA probes equivalently labeled with digoxigenin. A: Anterodorsal view of stage 18 embryos hybridized with XIF3 antisense (left) or sense (right) probe. B,C: Lateral views of stage 22 embryos hybridized with antisense probes to XIF3 (B) or Xenopus neuronal intermediate filament (XNIF; C). Principal structures containing labeled cells are indicated by labeled arrowheads. o, Olfactory pit; rh, rhombencephalon; sc, spinal cord; tg, trigeminal ganglion. Scale bars 5 500 mm in A and C (C also applies to B).|
|Fig. 2. Wholemount in situ hybridization with equivalently labeled probes for the four low-molecular-mass neuronal intermediate filament (nIF) mRNAs in newly hatched (stage 33/34–35/36) tadpoles. Embryos were hybridized with cRNA probes to XIF3 (A), XNIF (B), xefiltin (C), and low-molecular-weight neurofilament protein (NF-L; D) under parallel conditions to provide an indication of the relative abundance of each. Specimens were photographed without clearing to avoid confusion from underlying structures. The right-left axis of D was reversed to match the orientation of the other photomicrographs. m, Mesencephalon; o, olfactory pit; p, prosencephalon; rh, rhombencephalon; sc, spinal cord. Roman numerals indicate respective cranial ganglia. Scale bar 5 300 mm.|
|Fig. 3. Relative changes in XIF3 (A,C,E) and XNIF (B,D,F) mRNA expression in spinal cord during early tadpole stages (stages 28–42). A,B: Lateral view of the spinal cord of stage 32 embryos hybridized with cRNA probes to XIF3 (A) and XNIF (B). Anterior is left, and dorsal is up. C,D: Transverse sections through the spinal cords of embryos at a stage similar to that shown in A and B and hybridized with cRNA probes to XIF3 (C) and XNIF (D). Arrows indicate the perimeter of the spinal cord. Arrowheads point to examples of large, labeled cells. Yolk granules make it difficult to discern the outlines of unlabeled cell bodies. E,F: Transverse sections through a stage 42 tadpole spinal cord hybridized with cRNA probes to XIF3 (E) and XNIF (F). In C and E, XIF3 labeling surrounding the central canal is indicated by an asterisk. In F, only a small portion of the dorsal root ganglion (drg) is contained in the section, making the level of XNIF appear lower than it actually is. c, Central canal; n, notochord. Scale bars 5 300 mm in B (also applies to A); 40 mm in D (also applies to C) and F (also applies to E).|
|Fig. 4. Comparison of XIF3 (A,C,E) and XNIF (B,D,F) mRNA distributions in cell bodies contributing to the peripheral nervous system. A,B: Transverse section through a dorsal root ganglion of a juvenile frog. C,D: Transverse section through the ventral horn of the spinal cord of a juvenile frog. E,F: Transverse section through the gut of a stage 51 tadpole. Arrowheads point to examples of enteric neurons in the intestinal wall. Insets show the labeled neurons marked by asterisks in E and F at higher magnification. The dark material within the intestinal lumen is nonspecifically labeled chyme. g, Gray matter; w, white matter. Scale bars 5 100 mm in B (also applies to A,C,D), 300 mm in F (also applies to E), 30 mm in F inset (also applies to E inset).|
|Fig. 5. XIF3 (A,C) and XNIF (B,D) mRNA distribution in the eye of stage 51 tadpoles. A,B: Transverse sections through the eye. C,D: Views at higher magnification of the left retinal ciliary margins. Arrowheads in A and C mark the farthest extent of labeled retinal ganglion cells. XIF3 mRNA was restricted to the ciliary margin and adjacent retinal cells (A,C), whereas XNIF mRNA was found in retinal ganglion cells throughout the retina (B,D). c, Ciliary margin; i, inner nuclear layer; p, pigmented retinal epithelium (naturally pigmented and not labeled); r, retinal ganglion cells layer. Scale bars 5 200 mm in B (also applies to A), 50 mm in D (also applies to C).|
|Fig. 6. Distributions of XIF3 (A) and xefiltin (B) mRNA in olfactory epithelium of stage 51 tadpoles (transverse sections). Arrowheads in A point to examples of labeled cells in the basal layer. oe, Olfactory epithelium; op, olfactory pit. Scale bar 5 300 mm.|
|Fig. 7. XIF3 (A) and XNIF (B) mRNA distributions in transverse sections through juvenile frog spinal cord and dorsal root ganglion (drg). Arrowheads point to motoneurons of the ventral horn; arrows in A point to preganglionic neurons of the autonomic nervous system. cf, Central field; g, gray matter; w, white matter. Scale bar 5 300 mm.|
|Fig. 8. Distribution of XIF3 (A,C) and XNIF (B,D) mRNAs in transverse sections through the hind brains of juvenile frogs (A,B) and 7-year-old adult frogs (C,D). A,C: XIF3 labeling is visible in ventricular regions (vr) near the obex (ob) and in reticular neurons (rf). B,D: The same regions are also identified in the sections that were stained for XNIF. A–D are shown at the same magnification. Scale bar 5 300 mm.|
|Fig. 9. Proliferating cell nuclear antigen (PCNA) immunoreactivity in a stage 51 tadpole (A,B) and a juvenile frog (D). A illustrates PCNA immunoreactivity in cells of the central field (cf) of the spinal cord, whereas, for comparison, C shows a neighboring section that was labeled by using in situ hybridization for XIF3 mRNA, which was found in the central field (cf) as well as in ventral motoneurons (m). After metamorphosis, PCNA immunoreactivity greatly diminished (D). B shows PCNA immunoreactivity in the retinal ciliary margin (c) of a stage 51 tadpole (which can be compared to a neighboring section labeled by in situ hybridization for XIF3 mRNA in Fig. 5A,C). The tadpole was stained with anti-PCNA at a dilution of 1:5,000, whereas juvenile frog spinal cord was stained with antibody at a dilution of 1:1,000. p, Pigmented retinal epithelium; i, inner nuclear layer; r, retinal ganglion cell layer. Scale bars 5 50 mm in B, 150 mm in C (also applies to A), 300 mm in D.|
|Fig. 10. Western blot analysis of whole homogenates of Xenopus laevis spinal cord (a) and brain (b). The antibodies that were used are XIF3 antiserum, XIF3 preimmune serum, Xenopus NF-L (XNF-L) antiserum, XNIF antiserum, vimentin antiserum, and a monoclonal antibody (IFA) directed against an epitope common to all intermediate filament proteins. Labeled bars on the left of each group of blots indicate the positions of prestained protein molecular mass standards (molecular masses in kDa). Arrowheads on the right of each strip point to the bands that were stained by each respective antibody, except for the strip labeled IFA, in which the arrowhead points only to the band that comigrated with XIF3.|
|Fig. 11. A–G: XIF3 immunostaining in central nervous system radial glia and peripheral axons. A, C, and D show progressively more caudal transverse sections through juvenile frog spinal cord at lower magnification. XIF3 immunostaining was abundant in radially oriented processes extending from cell bodies in the central field to end feet at the pial surface. B: Examples of XIF3 immunostaining in scattered axons of the dorsal root (arrowheads) and adjacent spinal cord glial end feet at higher magnification (from A). E: XIF3 immunoreactivity in radial glia from the dorsal column in C shown at higher magnification. Arrowheads and arrows point to corresponding examples of glial cell bodies and end feet at the pial surface (p), respectively. F: XIF3-immunoreactive axons in a cranial nerve. G: XIF3-immunoreactive cell bodies (arrowheads) and proximal axonal segments from the dorsal root ganglion in D. cf, Central field; g, gray matter; w, white matter; p, pial surface; dr, dorsal root; drg, dorsal root ganglion. Scale bars 5 300 mm in D (also applies to A,C), 50 mm in F, 100 mm in G (also applies to B,E).|