Philpott A et al. (1999), Neuronal differentiation and patterning in Xeno...

XB-ART-13462

Dev Biol 1999 Mar 01;2071:119-32. doi: 10.1006/dbio.1998.9146.

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Neuronal differentiation and patterning in Xenopus: the role of cdk5 and a novel activator xp35.2.

Philpott A , Tsai L , Kirschner MW .

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Cdk5, a member of the cyclin-dependent kinase family, has been shown to play an important role in development of the central nervous system in mammals when partnered by its activator p35. Here we describe the cloning and characterization of a novel activator of cdk5 in Xenopus, Xp35.2. Xp35.2 is expressed during development initially in the earliest differentiating primary neurons in the neural plate and then later in differentiating neural tissue of the brain. This is in contrast to the previously described Xenopus cdk5 activator Xp35.1 which is expressed over the entire expanse of the neural plate in both proliferating and differentiating cells. Expression of both Xp35.1 and Xp35.2 and activation of cdk5 kinase occur when terminal neural differentiation is induced by neurogenin and neuro D overexpression but not when only early stages of neural differentiation are induced by noggin. Moreover, blocking cdk5 kinase activity specifically results in disruption and reduction of the embryonic eye where cdk5 and its Xp35 activators are expressed. Thus, cdk5/p35 complexes function in aspects of neural differentiation and patterning in the early embryo and particularly in formation of the eye.

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Species referenced: Xenopus
Genes referenced: cdca5 cdk5 cdk5r1 cdk5r1l eef1a2 gal.1 gchfr inhba mrc1 myc ncam1 neurod1 neurog1 nog pax6 Upk3b

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	FIG. 1. Xp35.2 is a novel member of the p35 family of cdk5 kinase activators. (A) Full nucleotide sequence and amino acid sequence of Xp35.2. (B) Sequence alignment between Xp35.2, Xp35.1, the other cloned Xenopus cdk5 kinase activator, and human p35. Note that while the N- and C-terminal domains are highly conserved there is a substantial divergence in the central domain of the Xp35.2 protein, both from human p35 and from Xp35.1.
	FIG. 2. Both Xp35.1 and Xp35.2 are able to activate cdk5 kinase activity. One nanogram RNA encoding myc-tagged Xp35.1 (lane 1) or Xp35.2 (lane 2) was injected into fertilized eggs. Uninjected embryos were used as controls (lane 3). At stage 13 extracts were produced from two injected embryos and cdk5 was specifically immunoprecipitated. These immunoprecipitates were tested for their ability to phosphorylate the model substrate histone H1.
	FIG. 3. Developmental expression of Xp35.2. Two micrograms of RNA from developmentally staged embryos was reverse transcribed in a 20-ml reaction. One microliter per lane was used for quantitative RT-PCR analysis using primers specific for Xp35.2. Ornithine decarboxylase, expressed constantly through development, was also used as a loading control.
	FIG. 4. Xp35.2 is specifically expressed in differentiating neural tissue. Albino embryos were used for in situ hybridization analysis using a digoxigenin-labeled Xp35.2 antisense probe, visualized in purple against a white/pink background. Embryonic stages are (Ai) dorsal view of neural plate stage, (Aii) dorsal view of early tailbud, and (Aiii) lateral view of the head of a late tailbud-staged embryo. Staining of the trigeminal ganglion (TG) is highlighted with an arrow. (B) A section through the head of a stage 35/36 embryo where Xp35.2 expression has been detected by whole-mount in situ hybridization and then the embryo has been sectioned. Xp35.2 staining is strong in differentiating neurons of the forebrain (thick arrow) and ganglion cells of the retina (thin arrows).
	FIG. 5. Cdk5 kinase is activated by inducers of terminal neural differentiation. (A) Embryos were left uninjected (lanes 1, 5, and 6) or injected with RNA encoding neurogenin (50 pg, lane 2), neuro D (500 pg, lane 3), or noggin (25 pg, lane 4). Animal caps were cut at stage 8.5 and incubated in 0.73 MMR without (lanes 1–4) or with (lane 5) activin at 2 ng/ml. Animal caps were aged until parallel embryos had reached stage 25 and whole embryos at this stage were also taken as a control (lane 6). Protein extracts were prepared and cdk5 was then specifically immunoprecipitated. Immunoprecipitates were tested for their ability to phosphorylate the model substrate histone H1. (B) Alternatively, six animal caps produced and aged until stage 25 exactly as described above were used for RT-PCR analysis to determine the expression of the pan-neural marker NCAM and the ubiquitously expressed EF1a message which is used as a loading control. Uninjected embryo (lane 1) or embryos injected with RNA encoding neurogenin (50 pg, lane 2), neuro D RNA, (500 pg, lane 3), or noggin (25 pg, lane 4).
	FIG. 6. Xp35.1, Xp35.2, and Xcdk5 expression is induced by neurogenic genes. Embryos were left uninjected (lanes 1–3) or injected with 25 pg noggin (lanes 4–6), 50 pg neurogenin (lanes 7–9), or 500 pg neuro D (lanes 10–12) RNA. Animal caps were cut at stage 8.5 and then aged until stage 13 (lanes 1, 4, 7, and 10), stage 19 (lanes 2, 5, 8, and 11), or stage 26 (lanes 3, 6, 9, and 12). cDNAs were prepared from caps and assayed by quantitative RT-PCR analysis for expression of Xp35.1, Xp35.2, cdk5, NCAM, and neural b-tubulin as labeled. EF1a expression was used as a loading control.
	FIG. 7. Cdk5 kinase activity is not required for expression of neural markers. (A) Embryos were either left uninjected (lane 1) or injected with 50 pg neurogenin (lanes 2–4) or 500 pg neuro D (lanes 5–7) RNA. These RNAs were either injected alone or in combination with 2 ng cdk5 WT RNA (lanes 3 and 6) or 2 ng cdk5 DN RNA (lanes 4 and 7). Animal caps were cut when embryos had reached stage 8.5 and aged until parallel embryos had reached stage 28. cDNAs were then prepared and quantitative RT-PCR analysis was used to detect expression of the neural markers NCAM and neural b-tubulin and the ubiquitous EF1a, used as a loading control. (B) Embryos were left uninjected (lane 1) or injected with 50 pg neurogenin (lane 2) or 50 pg neurogenin with 2 ng cdk5 DN (lane 3). Animal caps were cut at stage 8.5 and then aged until parallel embryos had reached stage 28. Protein extracts were prepared, endogenous cdk5 was immunoprecipitated as described under Materials and Methods, and the immunoprecipitates were tested for their ability to phosphorylate the model substrate histone H1.
	FIG. 8. (a) Blocking cdk5 kinase activity results in a specific reduction of the embryonic eye. Embryos were injected into two dorsal cells at the four-cell stage with RNA encoding cdk5 WT alone, cdk5 DN alone (2.5 ng per blastomere), or cdk5 WT and DN (5 ng per blastomere), all as labeled, along with 0.5 ng b-galactosidase RNA to act as a lineage tracer. When embryos had reached the early tailbud stage, they were fixed and stained with Xgal to detect b-galactosidase expression (in turquoise). Those embryos in which injected RNA had been correctly targeted to the head and dorsal regions were subjected to in situ hybridization to detect expression of the eye-specific gene PAX 6 (in brown/black). Note that coinjection of cdk5 WT with cdk5 DN rescues the reduced eye phenotype seen with cdk5 DN, demonstrating specificity. (b) Cdk5 DN injection leads to specific disruption of the embryonic eye. Embryos were left uninjected (i) or injected with RNA encoding cdk5 DN (5 ng, ii), cdk5 WT (5 ng, iii), or cdk5 WT with cdk5 DN (5 ng of each, iv) into two dorsal cells of four-cell embryos. Embryos were allowed to develop until stage 35/36 when they were fixed, embedded in paraffin wax, and sectioned coronally. Sections were stained with hemotoxylin and eosin to view internal tissue morphology. The lens of the eye (L), retina (R), and neural tube (NT) are indicated.
	cdk5r1 (cyclin-dependent kinase 5, regulatory subunit 1 (p35)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, dorsal view, anterior bottom left.
	cdk5r1 (cyclin-dependent kinase 5, regulatory subunit 1 (p35)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, dorsal view, anterior bottom right.
	cdk5r1 (cyclin-dependent kinase 5, regulatory subunit 1 (p35)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 36, lateral view, anterior left, dorsal up.