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The Cip/Kip family of mammalian cyclin-dependent kinase (cdk) inhibitors plays important roles in development, particularly in cell fate determination and differentiation, in addition to their function of blocking cell cycle progression. We have identified two novel members of the Kip/Cip cdk inhibitor family, p16Xic2 and p17Xic3, from Xenopus laevis. Sequence analysis revealed that p16Xic2 and p17Xic3 are orthologues of mammalian p21Cip1 and p27Kip1, respectively. Overexpression of these inhibitors results in cell cycle arrest by inhibition of cdk2 activity. Interestingly, the expression of these inhibitors is highly developmentally regulated. p16Xic2 is highly expressed in differentiating somite, tail bud, lens, and cement gland, while p17Xic3 is expressed in the central nervous system. In a retinal cell fate determination assay, both p16Xic2 and p17Xic3 have an activity that influences cell fate determination. These observations suggest that p16Xic2 and p17Xic3 might be involved in cell fate determination in a tissue-specific manner by coordinating proliferation and differentiation as observed with p27Xic1.
Fig. 1. Sequence comparison of p16Xic2 and p17Xic3 with known cdk
inhibitors. (A) Schematic drawings of Kip/Cip family cdk inhibitors. (B) A
phylogenetic tree of Kip/Cip family cdk inhibitors of X. laevis, human,
mouse, C. elegans, and D. melanogaster. (C) Sequence alignment of the Cterminal
regions of p16Xic2 and p21Cip1. p16Xic2 and p21Cip1 each has a
PCNA binding domain at their C-terminal regions. (D) Sequence alignment
of the C-terminal regions of p17Xic3, p27Kip1, and p57Kip2.
Fig. 2. p16Xic2 and p17Xic3 inhibit the cell cycle. (A) Detection of
overexpressed p16Xic2 and p17Xic3 proteins. After introduction of myctagged
p16Xic2 or p17Xic3 mRNA into two-cell stage Xenopus embryos,
their expression was analyzed at the indicated stages using a myc antibody.
(B) Overexpression of p16Xic2 or p17Xic3 in Xenopus embryos delays the
cell cycle and causes apoptosis after the MBT. After injection of 2 ng of
p16Xic2 or p17Xic3 mRNA into a blastomere at the two-cell stage, the
embryo was analyzed at stages 6, 9, and 10. The injected side is indicated
in the figures. Apoptotic cells are indicated by arrowheads. (C) Effect of
p16Xic2 and p17Xic3 on cdk2-mediated phosphorylation of histone H1.
p16Xic2 and p17Xic3 inhibit the cdk2-mediated phosphorylation of
histone H1.
Fig. 3. Expression patterns of p16Xic2 and p17Xic3 during Xenopus development. (A) Semiquantitative RT-PCR analysis of p16Xic2 and p17Xic3 during
Xenopus development. cDNAs were made from total mRNAs isolated from the corresponding stages. After determination of the best conditions for
semiquantitative analysis, RT-PCR was performed. ODC was used as a control. (B–J) Whole mount in situ hybridization of p16Xic2. (B) stage 7; (C) anterior
view, stage 18; (D) lateral view, stage 23; (E) dorsal view, stage 23; (F) anterior view, stage 25; (G) lateral view, stage 25; (H) lateral view, stage 29/30; (I)
lateral view, stage 32; (J) a section of retina at stage 32; (K) whole mount in situ hybridization of p17Xic3 at stage 33.
Fig. 4. p16Xic2 and p17Xic3 influence retinal cell fate determination. (A) Stage 41 Xenopus retina that was colipofected with a p16Xic2 expression construct
and a GFP-expressing plasmid (pGFP) at stage 15; fluorescent image (left) and its schematic drawing (right, Mqller glial cells are indicated as gray). The
number of Mqller glial cells was increased. (B) Stage 41 Xenopus retina that was colipofected with a p17Xic3 expression construct and pGFP at stage 15;
fluorescent image (left) and its schematic drawing (right). There are an increased number of Mqller glial cells. (C) A control retina lipofected with pGFP. (D)
Percentage of retinal cell types labeled by misexpression of p16Xic2 plus pGFP, p17Xic3 plus pGFP, or pGFP alone. n=660 (p16Xic2 plus pGFP), n=519
(p17Xic3 plus pGFP), and n=714 (pGFP). Error bars represent S.E.M.; ***Pb0.001 by Student’s t-test.
cdkn1a (cyclin-dependent kinase inhibitor 1A (p21, Cip1)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 7, lateral view, anteriorleft, dorsal up.
cdkn1a (cyclin-dependent kinase inhibitor 1A (p21, Cip1)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, anterior view, dorsal up.
cdkn1a (cyclin-dependent kinase inhibitor 1A (p21, Cip1)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anteriorleft, dorsal up.
cdkn1b (cyclin-dependent kinase inhibitor 1B (p27, Kip1)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 33, lateral view, anteriorleft, dorsal up.
