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Phosphorylation of the neuronal cytoskeletal proteins NF-H, NF-M and tau is important for normal axonal development, and is involved in axonal injury and neurodegenerative diseases. In mammalian neurons, one kinase that phosphorylates these axonal cytoskeletal proteins is cyclin-dependent kinase 5 (cdk5). Cdk5 is a member of the family of cyclin-dependent kinases (cdks), whose other family members regulate mitosis. Unlike the other cdks, cdk5 is abundant in differentiated neurons. Embryos of the clawed frog Xenopus laevis have proved useful for studying other cyclin-dependent kinases, neurofilament proteins and tau during development. As a first step in studying the role of cdk5 and its effects on neurofilaments during Xenopus neural development, four cDNA clones were isolated by screening a frog brain cDNA library at lowered stringency with a cDNA probe to rat cdk5. The frog cdk5 clones encoded a protein of 292 amino acids that was 97% identical to rat cdk5. In situ hybridization demonstrated that the Xenopus cdk5 transcript, like that of mammals, was expressed in differentiated post-mitotic neurons. The high degree of sequence homology and shared neuronal expression suggests that the role of cdk5 in neurons is highly conserved between mammals and amphibians. Northern blot analysis indicated that during Xenopus development, cdk5 mRNA was first expressed between the midblastula transition and gastrulation, which both occur long before neuronal differentiation. These stages mark the transition from synchronous to asynchronous cell division and are the earliest stages of zygotic gene expression. This early expression of Xenopus cdk5 mRNA implies a role for cdk5 during embryogenesis that is separate from its role as an axonal cytoskeletal protein kinase. These observations provide the foundation for exploiting X. laevis embryos to study the role of cdk5 both in the early stages of axonal differentiation and also in early embryogenesis.
Fig. 1. Identification of a X. laevis transcript homologous to rat cdkf.
Samples consisting of 10 /.tg of total RNA were isolated from adult rat
brain (lane 1), juvenile ,7(. laevis liver (lane 2), brain (lane 3) and spinal
cord (lane 4), separated by agarose gel electrophoresis and transferred to
nitrocellulose paper. A eDNA probe to rat cdk5 was labeled with
[32p]dCTP through random primed synthesis, and hybridized with the
blot, w.'.,!,-h was subsequently washed at 50°C in 0.1 x SSC. The bars
labeled 18S and 28S mark the positions of rat ribosomal RNAs.
Fig. 2. Nucleotide and derived amino acid sequences of ~ ~c~'~ cd~. Dots above the nucleotide sequence indicate cve~ tenth nucleotide. This sequence
is available from GenBank under accession number U24397.
Fig. 3. Comparison of the derived amino acid sequences of X. ~ecis and rat cdk5. Vertical lines indicate identical amino acid residues. The overall amino
acid identity is 97% between rat and laevis. The nucleotide identi~v over the same coding region is 83%.
Fig, 4! nervous system of juvenile (2-3 months post:metamo~hic)Xenopus laer'is frogs, as determined by in
situ hvbridizationi Transverse sections, 16/~m thick, were hybridized with cRNA probes labeled with ot-thio-[a5S]UTe. A: Section through the trigeminal
ganglion, hybridized with an antisense probe to cdk5. A strong signal is localized over the neuronal somas, whereas axons are unlabeled. B: A section, near
that shown in panel A, hybridized with a sense probe. The bright areas enveloping the ganglia of both panel A and B are due to an artifact caused by
photographic emulsion drying onto the surrounding cartilage. The: 200/zm bar in A is valid for botl~ A and B. C: Localization of cdk5 mRNA in the spinal
cord. Cell bodies throughout the gray matter (g) are labeled, with the signal being especially intense over the large motoneurons of the ventral horn
(arrowhead at vh). Axons and glial cells in the white matter (w) fail to show any specific labeling. The apparent labeling surrounding the spinal cord
originates from an edge artifact. D: The dorsal root ganglion taken from the same section as the spinal cord of panel C, and viewed at a higher
magnification. Specific labeling is clearly visible in the somas (examples of which are shown at the arrowheads) of the sensory neurons.
Fig. 5. The developmental expression of X. lae~'is cdk5 mRNA as
assayed by Northern biot hybridization. Total RNA was isolated from
various developmental stages of Xenopus lae~is. Samples consisting
nominally of 8 p,g of RNA were separated by agarose gel electrophoresis
and transferred to nitrocellulose paper. A: The blot was hybridized first to
a cDNA probe to X. laecis cdk5 labeled with a-[ 3z P]dCTP by random
primed synthesis. The blot was then washed at high stringency (60':C,
0.1 xSSC), and used to expose X-ray film for 2 weeks. B: To confirm
that each sample contained intact RNA the blot was then stripped of
labeled probe, and rehybridized with a eDNA probe directed against a 1
kb transcript that is ubiquitously expressed at all developmental stages in
X. laecis. The actual amount of RNA loaded was somewhat greater at
stages 35 and 66 than at other stages. The Nieuwkoop and Faber [33]
developmental stages for each RNA sample are listed across the top.
They are: stage 2, 2-cell blastula; stage 3, 4-cell blastula; stage 8,
midblastula transition; stage 10, ear!,,', gastrula; stage ._'~4, tailbudlarva:
stage 35, newly hatched tadpole; stage 42, 3 day old free-swimming
tadpole; stage 66, post-metamorphic juve.,z.ilc fi-og. RNA from stages 42
and earlier was isolated from whole embryos. RNA at stage 66 was
isolated from the brain.
