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The principal function of intermediate filaments is to strengthen cells. Their developmentally regulated, tissue-specific patterns of expression further suggest that they modulate cellular structural properties during development. To explore the role of intermediate filaments in development, we injected RNA encoding a truncated form of the Xenopus laevis middle-molecular-weight neurofilament protein (NF-M) into embryonic frog blastomeres at the 2-cell stage. A similar truncated form of mammalian NF-M disrupts neurofilaments (Type IV) and vimentin (Type III) intermediate filaments in transfected fibroblasts. In cultures made from dissociated neural tubes and their adjacent myotomes, the resultant protein disrupted both desmin filaments in muscle cells and neurofilaments in neurons during the first day of culture, which corresponds to stage 35/36 in the intact embryo. We next examined the effects of this truncated neurofilament protein on development of the nervous system. The greatest effects were seen on development of cranial and primary motor nerves, which were severely stunted as late as stage 37/38. In addition to these effects, ectopic neurons also appeared immediately beneath the epidermis along the flank of tadpoles expressing the truncated neurofilament protein. Whereas the former effects on peripheral nerve development were nearly identical to effects obtained with injected neurofilament antibodies, the ectopic neurons were novel, suggesting they resulted from the disruption of intermediate filaments other than the neurofilaments. These experiments thus implicate intermediate filaments in several functions important for normal neural development.
FIG. 6. Inhibition of cranial nerve development by NF-MDT. Embryos were injected unilaterally with either NF-MDT mRNA (A)or b-galactosidase mRNA (E, F) at the 2-cell stage and then immunostained with the anti-MAP1 antibody as wholemounts at stage 28 (A and B) or stage 37/38 (C ). (A, C, and E) The side of the animal derived from the injected blastomere. (B, D, and F) The respective contralateral uninjected side of the same animals. At stage 28, Vop (A, labeled arrowhead) developed to a lesser extent than on the contralateral, uninjected side (B). The unlabeled arrowheads in B show additional segments of Vop that are missing from the injected side. At stage 37/38 (C), cranial nerve development was more dramatically inhibited. (C) Inhibition of cranial nerves Vop, Vmd, VII, IX, and X. (D) Normal development of these same nerves on the contralateral side. Xb, Xv , and Xl are especially well developed on this side compared to the injected side. (E and F) Cranial nerve development was unaffected by expression of b-galactosidase. Cranial nerves on both sides of the head developed symmetrically, and the extent of nerve development was similar to that in D. (The dorsal side of the animals shown in E and F is rotated slightly forward.) Abbreviations: E, eye; O, otic vesicle; Vop , ophthalmic branch of the trigeminal nerve; Vmd, mandibular branch of the trigeminal nerve; VII, branch of the facial nerve; IX, branch of the glossopharyngeal nerve; Xb , brachial branch of the Xth cranial nerve; Xv visceral branch of Xth cranial nerve; Xl lateral line branch of the Xth cranial nerve. The scale bar applies to all panels.
FIG. 7. Inhibition of primary motor nerve development by NF-MDT. Embryos were injected with NF-MDT mRNA into a single blastomere at the 2-cell stage and later stained as wholemounts with the anti-MAP1 antibody. (A) Lateral view of mid-level myotomes on the injected side of a stage 28 larval tadpole, showing inhibition of motor axon development. (B) The uninjected contralateral side of the same animal at the same rostrocaudal level as in A, showing normal motor nerve development. (C) Lateral view of tailsomites on the injected side of a stage 37/38 tadpole. (D) The contralateral uninjected side of the same tadpole as in C. The injected side (C) had fewer motor nerves emanating from the spinal cord than normal (D). Arrowheads at S point to the spinal cord. Arrowheads at m point to an example of a motor nerve projecting to the somites. In these panels, rostral is to the right. (The negatives were reversed when printed to maintain the same orientation in all panels.) The scale bars in B and D also apply to A and C, respectively.
FIG. 8. The presence of ectopic neurons in the trunks of tadpoles expressing NF-MDT. 2-cell stage embryos were injected unilater-ally with NF-MDT mRNA and then stained as wholemounts at neustage 37/38 with the anti-NF-M tail antibody (RMO270, 1:200). Several ectopic cells with neuronal morphologies appeared on the injected side of the tadpole (arrowheads in A). These cells were absent from the uninjected side (B). The scale is the same for both panels.
Map1a (Map-1) gene expression in Xenopus laevis embryo via immunohistochemistry, NF stage (28), lateral view, anteriorleft, dorsal up.
Image copyrighted by: Academic Press and reproduced from XB-ART-17584
Map1a (Map-1) gene expression in Xenopus laevis embryo via immunohistochemistry, NF stage (37-38), lateral view, anteriorleft, dorsal up.
Image copyrighted by: Academic Press and reproduced from XB-ART-17584
Map1a (Map-1) gene expression in Xenopus laevis embryo via immunohistochemistry, NF stage (37-38), lateral view, anteriorright, dorsal up.
Image copyrighted by: Academic Press and reproduced from XB-ART-17584
