Development of mice expressing a single D-type cyclin

Abstract

D-cyclins (cyclins D1, D2, and D3) are components of the core cell cycle machinery. To directly test the ability of each D-cyclin to drive development of various lineages, we generated mice expressing only cyclin D1, or only cyclin D2, or only cyclin D3. We found that these "single-cyclin" embryos develop normally until late gestation. Our analyses revealed that in single-cyclin embryos, the tissue-specific expression pattern of D-cyclins was lost. Instead, mutant embryos ubiquitously expressed the remaining D-cyclin. These findings suggest that the functions of the three D-cyclins are largely exchangeable at this stage. Later in life, single-cyclin mice displayed focused abnormalities, resulting in premature mortality. "Cyclin D1-only" mice developed severe megaloblastic anemia, "cyclin D2-only" mice presented neurological abnormalities, and "cyclin D3-only" mice lacked normal cerebella. Analyses of the affected tissues revealed that these compartments failed to sufficiently up-regulate the remaining, intact D-cyclin. In particular, we found that in cerebellar granule neuron precursors, the N-myc transcription factor communicates with the cell cycle machinery via cyclins D1 and D2, but not D3, explaining the inability of D3-only mice to up-regulate cyclin D3 in this compartment. Hence, the requirement for a particular cyclin in a given tissue is likely caused by specific transcription factors, rather than by unique properties of cyclins.

Figure 1

Bromodeoxyuridine (BrdU) incorporation in wild-type and single-cyclin embryos at E13.5. Pregnant females were injected with a single dose of BrdU, and the embryos were collected 1 h later and processed for histology. (A) Sagittal sections of the entire embryos were stained with an anti-BrdU antibody. (B) Higher magnification showing normal BrdU incorporation rate in single-cyclin hearts (H) and livers (L). (C) Higher magnification showing normal BrdU incorporation rate in single-cyclin roofs of neopalial cortex.

Figure 2

Molecular analyses of single-cyclin embryos. (A–C) Protein lysates were prepared from indicated organs of mutant or wild-type embryos and subjected to Western blotting. (A) Comparison of the levels of particular D-cyclins between wild-type and single-cyclin embryos. In mutant embryos, the remaining, intact D-cyclin becomes up-regulated in compartments in which it is normally not expressed or is expressed at low levels. Developing livers and spleens were collected at E13.5 and E16.5, respectively. (B) Expression pattern of the D-type cyclins in wild-type and single-cyclin embryos. Wild-type, cyclin D1^−/− D2^−/−, and D1^−/− D3^−/− embryos were collected at E18.5, whereas cyclin D2^−/− D3^−/− embryos were collected at E16.5. (C) The levels of p27^Kip1 protein in organs dissected from wild-type and single-cyclin embryos. Livers were collected at E13.5, hearts and lungs at E18.5. The blots were probed with an anti-actin antibody to ensure equal loading. (D) CDK2-associated kinase activity in single-cyclin embryos. Lysates were prepared from E13.5 embryos of the indicated genotypes. CDK2 was immunoprecipitated and kinase assays were performed using histone H1 as a substrate. For control, wild-type lysates were either treated with 15 μM roscovitine (wt + roscovitine) or were subjected to immunoprecipitation with unrelated, control anti-progesterone receptor antibody (wt + control antibody).

Figure 3

Phenotype of cyclin D1-only (cyclin D2^−/− D3^−/−) mice. (A) A graph showing survival rate at particular stages of embryo development, presented as the ratio of the observed to the expected number of live embryos × 100% (for the exact numbers, see Table 1). (B) The number of immature erythroid cells in livers of wild-type and cyclin D2^−/− D3^−/− embryos. Values were obtained by counting the number of hematopoietic cells per field of view in hematoxylin–eosin-stained sections of E16.5 wild-type (n = 4) and mutant livers (n = 5). For each specimen, five independent counts were performed. Bars indicate mean values for each genotype. Error bars denote S.D. (C) Histologic appearance of wild-type and mutant livers in E16.5 embryos. Paraffin sections were stained with hematoxylin and eosin. Arrows denote examples of immature erythroid cells. Note the paucity of erythroid cells in mutant livers. (D) Smears of peripheral blood collected from E16.5 wild-type and cyclin D2^−/− D3^−/− embryos. Pictures were taken at the same magnification. Note the striking difference in size between wild-type and mutant erythrocytes.

Figure 4

Phenotype of cyclin D2-only (cyclin D1^−/− D3^−/−) mice. (A) A graph showing survival rate at particular stages of embryo (E) or postnatal (P) development calculated as the ratio of the observed to the predicted number of live embryos or pups × 100%. For the exact numbers, see Table 1. (B) Appearance of 2-week-old wild-type and cyclin D1^−/− D3^−/− littermates. (C–E) Microscopic appearance of lungs in wild-type (C) and mutant (D,E) 1-day-old pups. Paraffin sections were stained with hematoxylin and eosin. Note the presence of meconium in lungs of mutant animals (arrows in D,E), indicating an acute asphyxiation with the gastrointestinal content. (E) Lungs with collapsed alveoli in a dead mutant.

Figure 5

Phenotype of cyclin D3-only (cyclin D1^−/− D2^−/−) mice. (A) A graph showing survival rate at particular stages of embryo (E) or postnatal (P) development calculated as the ratio of the observed to the predicted number of live embryos or pups × 100%. For the exact numbers, see Table 1. (B) Appearance of 16-day-old wild-type and cyclin D1^−/− D2^−/− littermates. Mutant mice display pronounced problems with coordination of their movements. (C) Paraffin sections of cerebella from 16-day-old wild-type and cyclin D1^−/− D2^−/− mice stained with hematoxylin and eosin. Note the dramatic difference in size and the abnormal number of foliae in cyclin D1^−/− D2^−/− cerebella. The cyclin D2^−/− cerebella, which were reported to display mild abnormalities in the anatomy of the foliae (Huard et al. 1999) are shown for comparison. (D) Higher magnification of images shown in C reveals severely reduced cell numbers within the internal granule layer (IGL) and abnormal positioning of Purkinje cells (arrows) in cyclin D1^−/− D2^−/− cerebella.

Figure 6

Molecular analyses of developing cerebella. (A) Sections of cerebella from 5-day-old wild-type (wt) or cyclin D1^−/− D2^−/− mice were hybridized with riboprobes specific for cyclin D1, D2, and D3. White color represents the hybridization signal. (B) Bromodeoxyuridine (BrdU) incorporation in wild-type and D1^−/− D2^−/− cerebella derived from 5-day-old mice. The proliferating external granule layer of wild-type mice contains numerous BrdU positive cells, whereas these numbers are reduced in mutant tissue. (Lower panel) Higher magnification of the external granule layer. (C) Protein lysates prepared from 5-day-old wild-type or mutant cerebella were probed with an antibody against the retinoblastoma protein. (ppRB) Hyperphosphorylated form of the pRB.

Figure 7

N-myc in the developing cerebella and in cultured cerebellar granule neuron precursors. (A) N-myc expression in developing cerebella of 5-day-old wild-type and cyclin D1^−/− D2^−/− mice detected by in situ hybridization with a riboprobe specific for N-myc. White color represents hybridization signal. (B) In vitro cultured cerebellar granule neuron precursors were infected with a retrovirus encoding N-myc. The levels of cyclin D1, D2, D3, and N-myc were detected by Western blotting. Blots were probed with an anti-α-tubulin antibody to ensure equal loading.

Figure 8

Analyses of mutant retinas and erythroid cells. (A) BrdU incorporation and D-type cyclin expression in developing retinas at E18.5. Pregnant females were injected with BrdU, and embryonal eyes were collected after 1 h and processed for histology. Transcripts for cyclin D1 and D2 were detected by in situ hybridization with appropriate riboprobes. Specimens were photographed by double exposure using bright-field illumination and Hoechst epifluorescence optics. The blue color represents the Hoechst staining of cell nuclei, and the red represents the hybridization signal. R denotes neural part of the retina. Note that the red signal within the pigmented epithelium layer (arrowheads) represents a false positive signal that stains all probes owing to the presence of pigmented granules in these cells. (B) Protein lysates were prepared from flow-sorted Ter119⁺ erythroid cells (derived from E14.5 embryonic livers) and then subjected to Western blotting. Blots were probed with an anti-α-tubulin antibody to ensure equal loading. Protein extracts prepared from entire wild-type embryos were used as a positive control.