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D-type cyclins serve as cell cycle recipients of several oncogenic pathways. The specific sequences of the promoters of the cyclin D genes are thought to render particular D-cyclins responsive to specific oncogenic pathways. For instance, the Ras oncogene was postulated to signal through cyclin D1, while Myc can impact the cell cycle machinery by transcriptionally upregulating cyclin D2. In the current study we engineered mouse fibroblasts to express only cyclin D1, only D2, or only D3. These 'single-cyclin' cells allowed us to rigorously test the ability of cyclin D1, D2, or D3, when expressed on their own, to serve as recipients of the Ras- and Myc-driven oncogenic pathways. We found that each of the D-cyclins was sufficient to drive oncogenic proliferation of mouse fibroblasts. This, together with our recent observations that cells lacking all three D-cyclins show greatly reduced susceptibility to the oncogenic action of Ras and Myc, reveals that the Ras and Myc oncogenes can impact the core cell cycle machinery through all three D-cyclins.

Regulation of adult beta-cell mass in pancreatic islets is essential to preserve sufficient insulin secretion in order to appropriately regulate glucose homeostasis. In many tissues mitogens influence development by stimulating D-type cyclins (D1, D2, or D3) and activating cyclin-dependent kinases (CDK4 or CDK6), which results in progression through the G(1) phase of the cell cycle. Here we show that cyclins D2 and D1 are essential for normal postnatal islet growth. In adult murine islets basal cyclin D2 mRNA expression was easily detected, while cyclin D1 was expressed at lower levels and cyclin D3 was nearly undetectable. Prenatal islet development occurred normally in cyclin D2(-/-) or cyclin D1(+/-) D2(-/-) mice. However, beta-cell proliferation, adult mass, and glucose tolerance were decreased in adult cyclin D2(-/-) mice, causing glucose intolerance that progressed to diabetes by 12 months of age. Although cyclin D1(+/-) mice never developed diabetes, life-threatening diabetes developed in 3-month-old cyclin D1(-/+) D2(-/-) mice as beta-cell mass decreased after birth. Thus, cyclins D2 and D1 were essential for beta-cell expansion in adult mice. Strategies to tightly regulate D-type cyclin activity in beta cells could prevent or cure diabetes.

Mice likely represent the most-studied mammalian organism, except for humans. Genetic engineering in embryonic stem cells has allowed derivation of mouse strains lacking particular cell cycle proteins. Analyses of these mutant mice, and cells derived from them, facilitated the studies of the functions of cell cycle apparatus at the organismal and cellular levels. In this review, we give some background about the cell cycle progression during mouse development. We next discuss some insights about in vivo functions of the cell cycle proteins, gleaned from mouse knockout experiments. Our text is meant to provide examples of the recent experiments, rather than to supply an extensive and complete list.

A fundamental aspect of cancer is dysregulated cell cycle control. Unlike normal cells that only proliferate when compelled to do so by developmental or other mitogenic signals in response to tissue growth needs, the proliferation of cancer cells proceeds essentially unchecked. This does not mean that cancer cell cycles are necessarily different from those found in normal cycling cells, but rather implies that cancer cells proliferate because they are no longer subject to proliferation-inhibitory influences arising from the stroma or from gene expression pattern changes consequent to 'terminal' differentiation, nor do they necessarily require extrinsic growth factors to recruit them into or maintain their proliferative state. Finally, cancer cells have also often avoided normal controls linked to cell cycle progression that halt proliferation in the presence of damaged DNA or other physiological insults. The result of these alterations is the inappropriate proliferation commonly associated with cancerous tumor formation. This review will summarize the current understanding of dysregulation of the G0/G1-to-S-phase transition in cancer cells, with particular emphasis on recent in vivo studies that suggest a need to rethink existing models of cell cycle control in development and tumorigenesis.

D-type cyclins (cyclin D1, D2 and D3) and their associated cyclin-dependent kinases CDK4 and CDK6 were thought to represent important, perhaps essential components of the core cell cycle apparatus. However, recent analyses of mice lacking D-cyclins, or CDK4 and CDK6 reveal that these proteins are critically required for proliferation only in the selected cell types. Intriguingly, several compartments can develop in the absence of cyclin D-CDK4/6 activity, revealing that these cells can proliferate in a cyclin D-independent fashion.

D cyclins (D1, D2, and D3) are components of the core cell cycle machinery in mammalian cells. It is unclear whether each of the D cyclins performs unique, tissue-specific functions or the three proteins have virtually identical functions and differ mainly in their pattern of expression. We previously generated mice lacking cyclin D1, and we observed that these animals displayed hypoplastic retinas and underdeveloped mammary glands and a presented developmental neurological abnormality. We now asked whether the specific requirement for cyclin D1 in these tissues reflected a unique pattern of D cyclin expression or the presence of specialized functions for cyclin D1 in cyclin D1-dependent compartments. We generated a knock-in strain of mice expressing cyclin D2 in place of D1. Cyclin D2 was able to drive nearly normal development of retinas and mammary glands, and it partially replaced cyclin D1's function in neurological development. We conclude that the differences between these two D cyclins lie mostly in the tissue-specific pattern of their expression. However, we propose that subtle differences between the two D cyclins do exist and they may allow D cyclins to function in a highly optimized fashion. We reason that the acquisition of multiple D cyclins may allow mammalian cells to drive optimal proliferation of a diverse array of cell types.

Adult neurogenesis (i.e., proliferation and differentiation of neuronal precursors in the adult brain) is responsible for adding new neurons in the dentate gyrus of the hippocampus and in the olfactory bulb. We describe herein that adult mice mutated in the cell cycle regulatory gene Ccnd2, encoding cyclin D2, lack newly born neurons in both of these brain structures. In contrast, genetic ablation of cyclin D1 does not affect adult neurogenesis. Furthermore, we show that cyclin D2 is the only D-type cyclin (out of D1, D2, and D3) expressed in dividing cells derived from neuronal precursors present in the adult hippocampus. In contrast, all three cyclin D mRNAs are present in the cultures derived from 5-day-old hippocampi, when developmental neurogenesis in the dentate gyrus takes place. Thus, our results reveal the existence of molecular mechanisms discriminating adult versus developmental neurogeneses.

The proto-oncogene c-myc encodes a transcription factor that is implicated in the regulation of cellular proliferation, differentiation, and apoptosis and that has also been found to be deregulated in several forms of human and experimental tumors. We have shown that forced expression of c-myc in epithelial tissues of transgenic mice (K5-Myc) resulted in keratinocyte hyperproliferation and the development of spontaneous tumors in the skin and oral cavity. Although a number of genes involved in cancer development are regulated by c-myc, the actual mechanisms leading to Myc-induced neoplasia are not known. Among the genes regulated by Myc is the cyclin-dependent kinase 4 (CDK4) gene. Interestingly, previous studies from our laboratory showed that the overexpression of CDK4 led to keratinocyte hyperproliferation, although no spontaneous tumor development was observed. Thus, we tested the hypothesis that CDK4 may be one of the critical downstream genes involved in Myc carcinogenesis. Our results showed that CDK4 inhibition in K5-Myc transgenic mice resulted in the complete inhibition of tumor development, suggesting that CDK4 is a critical mediator of tumor formation induced by deregulated Myc. Furthermore, a lack of CDK4 expression resulted in marked decreases in epidermal thickness and keratinocyte proliferation compared to the results obtained for K5-Myc littermates. Biochemical analysis of the K5-Myc epidermis showed that CDK4 mediates the proliferative activities of Myc by sequestering p21Cip1 and p27Kip1 and thereby indirectly activating CDK2 kinase activity. These results show that CDK4 mediates the proliferative and oncogenic activities of Myc in vivo through a mechanism that involves the sequestration of specific CDK inhibitors., (Copyright 2004 American Society for Microbiology)

D-type cyclins (cyclins D1, D2, and D3) are regarded as essential links between cell environment and the core cell cycle machinery. We tested the requirement for D-cyclins in mouse development and in proliferation by generating mice lacking all D-cyclins. We found that these cyclin D1(-/-)D2(-/-)D3(-/-) mice develop until mid/late gestation and die due to heart abnormalities combined with a severe anemia. Our analyses revealed that the D-cyclins are critically required for the expansion of hematopoietic stem cells. In contrast, cyclin D-deficient fibroblasts proliferate nearly normally but show increased requirement for mitogenic stimulation in cell cycle re-entry. We found that the proliferation of cyclin D1(-/-)D2(-/-)D3(-/-) cells is resistant to the inhibition by p16(INK4a), but it critically depends on CDK2. Lastly, we found that cells lacking D-cyclins display reduced susceptibility to the oncogenic transformation. Our results reveal the presence of alternative mechanisms that allow cell cycle progression in a cyclin D-independent fashion.

More than 25% of head and neck squamous cell carcinomas (HNSCC) and 99% of cervical cancers (CxCa) are positive for high-risk human papillomaviruses (HPVs). Furthermore, the type I tyrosine kinase receptor ErbB-2 is overexpressed in at least 30% of HNSCC and CxCa. Recently, we demonstrated that E6/E7 of HPV type 16 cooperate with ErbB-2 to induce cell transformation of human normal oral epithelial (NOE) cells. This is accompanied by overexpression of cyclin D1 in NOE cells. To determine the role of cyclin D1 in E6/E7/ErbB-2 cooperation, we examined the independent effects of E6/E7 and ErbB-2, and the combined effect of E6/E7 and ErbB-2 in mouse normal embryonic fibroblast (NEF), wild type (wt), and knockout cyclin D1 (D1(-/-)) cells. We report that NEF-wt cells transduced with E6/E7 alone and E6/E7/ErbB-2 together form small and large tumors in nude mice, respectively, as well as different sized colonies in soft agar; whereas ErbB-2 alone elicits neither tumor formation in vivo nor colony formation in soft agar. More importantly, E6/E7, ErbB-2 and E6/E7/ErbB-2 together all fail to induce neoplastic transformation of cyclin D1(-/-) cells in vivo and in vitro. Furthermore, using antisense cyclin D1 we completely inhibited tumor and colony formation of NEF-wt-E6/E7 and wt-E6/E7-ErbB-2 as well as human NOE-E6/E7-ErbB-2-transformed cells. These analyses reveal that cyclin D1 is the downstream target of the neoplastic transformation induced by E6/E7 or E6/E7/ErbB-2 cooperation in normal cells. Our data suggest that anti-cyclin D1 therapy may be highly specific in the treatment of all human cancers expressing high-risk HPVs or HPVs/ErbB-2.

The ubiquitously expressed E4F protein was originally identified as an E1A-regulated cellular transcription factor required for adenovirus replication. The function of this protein in normal cell physiology remains largely unknown. To address this issue, we generated E4F knockout mice by gene targeting. Embryos lacking E4F die at the peri-implantation stage, while in vitro-cultured E4F(-/-) blastocysts exhibit defects in mitotic progression, chromosomal missegregation, and increased apoptosis. Consistent with these observations, we found that E4F localizes to the mitotic spindle during the M phase of early embryos. Our results establish a crucial role for E4F during early embryonic cell cycles and reveal an unexpected function for E4F in mitosis.

The family of mammalian E-type cyclins is composed of two proteins, termed cyclin E1 and E2. These two cyclins are widely expressed in proliferating cells. E-cyclins bind and activate cyclin dependent kinase CDK2. Cyclin E-CDK2 complexes were believed to play critical function in driving cell cycle progression of normal, nontransformed cells and of cancer cells. Several recent reports challenge this notion.

The D-type cyclins (cyclins D1, D2, and D3) are components of the core cell cycle machinery in mammalian cells. Cyclin D3 gene is rearranged and the protein is overexpressed in several human lymphoid malignancies. In order to determine the function of cyclin D3 in development and oncogenesis, we generated and analyzed cyclin D3-deficient mice. We found that cyclin D3(-/-) animals fail to undergo normal expansion of immature T lymphocytes and show greatly reduced susceptibility to T cell malignancies triggered by specific oncogenic pathways. The requirement for cyclin D3 also operates in human malignancies, as knock-down of cyclin D3 inhibited proliferation of acute lymphoblastic leukemias deriving from immature T lymphocytes. These studies point to cyclin D3 as a potential target for therapeutic intervention in specific human malignancies.

Inhibins are heterodimeric (alpha:betaA and alpha:betaB) endocrine, paracrine, and autocrine factors of the TGFbeta superfamily that are produced predominantly by ovarian granulosa cells in females and testicular Sertoli cells in males. Control of granulosa and Sertoli cell proliferation is lost in the inhibin alpha (Inhalpha) knockout mouse model, leading to gonadotropin-dependent gonadal tumors of the granulosa/Sertoli cell lineage in both females and males. Castrate Inhalpha knockout mice develop sex steroidogenic tumors of the adrenal cortex. Physiological control of granulosa/Sertoli cell cycle progression depends on p27Kip1 and cyclin D2, which function in the G1-->S phase transition. To study the cell cycle-regulatory factors involved in ovarian, testicular, and adrenal tumor development in vivo, we have bred Inhalpha mutant mice to mice with targeted disruptions of the p27 and cyclin D2 genes. Our previous studies demonstrated that inhibins act cooperatively with p27 to negatively regulate granulosa cell proliferation, as double mutant mice lacking inhibins and p27 develop and succumb to ovarian tumors more rapidly than Inhalpha knockout mice. Here, we report that cyclin D2 antagonizes this inhibition and is key in promoting gonadal growth and tumor development, and tumor development is markedly suppressed in double-mutant mice. We found that double-knockout females lacking cyclin D2 and Inhalpha lived longer than mice lacking inhibins alone; the majority of these double-knockout mice lived longer than 17 wk, as opposed to inhibin alpha single-knockout females with 50% survival at between 12 and 13 wk of age. Moreover, 95% of inhibin alpha knockout males succumb to testicular tumor development by 12 wk of age, whereas double knockouts were protected from early signs of tumor development and had a 50% survival of 40 wk. Interestingly, the results of these studies reflect tissue-specific consequences of loss of these cell cycle regulators. In castrate mice, loss of p27 has little effect on adrenal cortical tumor progression in the absence of inhibins, whereas loss of cyclin D2 prolongs the lifespan of cyclin D2, Inhalpha double knockouts. After gonadectomy, 50% of cyclin D2, Inhalpha double-knockout males live to more than 46 wk of age, 10 wk longer than 50% of littermates lacking only inhibins. Similarly, 50% of female cyclin D2, inhibin alpha double knockouts live to 47 wk of age before succumbing to adrenal tumor development, in contrast to the 50% survival of Inhalpha single-knockout females at between 27 and 28 wk. Thus, identification of genetic modifiers of the Inhalpha knockout tumor phenotype has led us to a better appreciation of how specific components of the cell cycle machinery contribute to tumorigenesis in the ovary, testis, and adrenal gland.

E type cyclins (E1 and E2) are believed to drive cell entry into the S phase. It is widely assumed that the two E type cyclins are critically required for proliferation of all cell types. Here, we demonstrate that E type cyclins are largely dispensable for mouse development. However, endoreplication of trophoblast giant cells and megakaryocytes is severely impaired in the absence of cyclin E. Cyclin E-deficient cells proliferate actively under conditions of continuous cell cycling but are unable to reenter the cell cycle from the quiescent G(0) state. Molecular analyses revealed that cells lacking cyclin E fail to normally incorporate MCM proteins into DNA replication origins during G(0)-->S progression. We also found that cyclin E-deficient cells are relatively resistant to oncogenic transformation. These findings define a molecular function for E type cyclins in cell cycle reentry and reveal a differential requirement for cyclin E in normal versus oncogenic proliferation.

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.

Chronic myeloid leukemia is caused by the tyrosine kinase oncoprotein BCR/ABL. Using oligonucleotide arrays to assay mRNAs at different phases of the cell cycle in BCR/ABL-transformed cells, we found that cyclin D2 mRNA was constitutively expressed at high levels throughout the cell cycle, a pattern confirmed by immunoblotting of protein lysates. Bone marrow cells from cyclin D2-deficient strains of mice failed to proliferate in response to infection with a retrovirus carrying BCR/ABL and failed to generate transformed lymphoid cell lines in vitro. These results establish that BCR/ABL promotes cell cycle progression by altering expression of cyclin D2 and that cyclin D2 induction plays a critical role in proliferation of hematopoietic cells by BCR/ABL.

Cyclin E1 (formerly called cyclin E) and the recently described cyclin E2 belong to the family of E-type cyclins that operate during the G(1)/S phase progression in mammalian cells. The two E-cyclins share a catalytic partner, cyclin-dependent kinase 2 (CDK2), and activate their associated kinase activities at similar times during cell cycle progression. Despite these similarities, it is unknown whether the two proteins perform distinct functions, or, alternatively, they control S-phase entry of different cell types in a tissue-specific fashion. To start addressing in vivo functions of E-cyclins, we determined the expression pattern of cyclins E1 and E2 during normal mouse development. We found that the two E-cyclins showed very similar patterns of expression; both were expressed within the proliferating compartment during embryo development. Analyses of cells and tissues lacking members of the retinoblastoma (pRB) family of proteins revealed that the expression of both cyclins is controlled in a pRB-dependent, but p107- and p130-independent fashion, likely through the pRB-dependent E2F transcription factors. We also found that cyclins E1 and E2 are expressed at high levels in mouse breast tumors driven by the Myc oncogene. Last, we found that cyclin E2 is overexpressed in approximately 24% of analyzed human mammary carcinomas. Collectively these findings suggest that the expression of cyclins E1 and E2 is governed by similar molecular circuitry.

Breast cancer is the most common malignancy among women. Most of these cancers overexpress cyclin D1, a component of the core cell-cycle machinery. We previously generated mice lacking cyclin D1 using gene targeting. Here we report that these cyclin D1-deficient mice are resistant to breast cancers induced by the neu and ras oncogenes. However, animals lacking cyclin D1 remain fully sensitive to other oncogenic pathways of the mammary epithelium, such as those driven by c-myc or Wnt-1. Our analyses revealed that, in mammary epithelial cells, the Neu-Ras pathway is connected to the cell-cycle machinery by cyclin D1, explaining the absolute dependency on cyclin D1 for malignant transformation in this tissue. Our results suggest that an anti-cyclin D1 therapy might be highly specific in treating human breast cancers with activated Neu-Ras pathways.

D-type cyclins (cyclins D1, D2, and D3) are key components of cell cycle machinery in mammalian cells. These proteins are believed to drive cell cycle progression by associating with their kinase partners, cyclin-dependent kinases, and by directing phosphorylation of critical cellular substrates. In addition, D-cyclins play a kinase-independent role by sequestering cell cycle inhibitors p27(Kip1) and p21(Cip1). In the past, we and others generated cyclin D1-deficient mice and have shown that these mice display developmental abnormalities, hypoplastic retinas, and pregnancy-insensitive mammary glands. To test the significance of cyclin D1-p27(Kip1) interaction within a living mouse, we crossed cyclin D1-deficient mice with mice lacking p27(Kip1), and we generated double-mutant cyclin D1(-/-)p27(-/-) animals. Here we report that ablation of p27(Kip1) restores essentially normal development in cyclin D1-deficient mice. Our results provide genetic evidence that p27(Kip1) functions downstream of cyclin D1.

Progression into G(1) in B lymphocytes is regulated by cyclins D2 and D3, components of the cell cycle machinery currently believed to have overlapping and potentially redundant roles in cell cycle control. To study the specific role of cyclin D2 in B lymphocyte proliferation, we examined B cells from cyclin D2(-/-) mice and demonstrate a specific requirement for cyclin D2 in BCR- but not CD40- or lipopolysaccharide-induced proliferation. Furthermore, conventional B cell development proceeds normally in the mutant mice; however, the CD5 B cell compartment is dramatically reduced, suggesting that cyclin D2 is important in CD5 B cell development as well as antigen-dependent B cell clonal expansion.

Sciatic nerve axons in cyclin D1 knockout mice develop normally, become properly ensheathed by Schwann cells, and appear to function normally. However, in the Wallerian degeneration model of nerve injury, the mitotic response of Schwann cells is completely inhibited. The mitotic block is Schwann cell autonomous and developmentally regulated. Rescue analysis (by "knockin" of cyclin E) indicates that D1 protein, rather than regulatory elements of the D1 gene, provides the essential Schwann cell function. Genetic inhibition of the Schwann cell cycle shows that neuronal responses to nerve injury are surprisingly independent of Schwann cell mitotic responses. Even axonal regrowth into the distal zone of a nerve crush injury is not markedly impaired in cyclin D1-/- mice.

Cyclin D2 is the only D-type cyclin expressed in mature mouse B-lymphocytes, and its expression is associated with retinoblastoma protein (pRB) and pRB-related protein phosphorylation and induction of E2F activity, as B-cells enter the cell cycle following stimulation via surface IgM and/or CD40. Cyclin D-dependent kinase activity is required for cell proliferation, yet cyclin D2(-/-) mice have normal levels of mature B-lymphocytes. Here we show that B-lymphocytes from cyclin D2(-/-) mice can proliferate in response to anti-IgM and anti-CD40, but the time taken to enter S-phase is longer than for the corresponding cyclin D2(+/+) cells. This is due to the compensatory induction of cyclin D3, but not cyclin D1, which causes pRb phosphorylation on CDK4-specific sites. This is the first demonstration that loss of a D-type cyclin causes specific expression and functional compensation by another member of the family in vivo and provides a rationale for the presence of mature B-lymphocytes in cyclin D2(-/-) mice.

Ectopic expression of Myc induces Cdk2 kinase activity in quiescent cells and antagonizes association of p27(kip1) with Cdk2. The target gene(s) by which Myc mediates this effect is largely unknown. We now show that p27 is rapidly and transiently sequestered by cyclin D2-Cdk4 complexes upon activation of Myc and that cyclin D2 is a direct target gene of Myc. The cyclin D2 promoter is repressed by Mad-Max complexes and de-repressed by Myc via a single highly conserved E-box element. Addition of trichostatin A to quiescent cells mimics activation of Myc and induces cyclin D2 expression, suggesting that cyclin D2 is repressed in a histone deacetylase-dependent manner in quiescent cells. Inhibition of cyclin D2 function in established cell lines, either by ectopic expression of p16 or by antibody injection, inhibits Myc-dependent dissociation of p27 from Cdk2 and Myc-induced cell cycle entry. Primary mouse fibroblasts that are cyclin D2-deficient undergo accelerated senescence in culture and are not immortalized by Myc; induction of apoptosis by Myc is unimpaired in such cells. Our data identify a downstream effector pathway that links Myc directly to cell cycle progression.

D-type cyclins and cyclin E represent two very distinct classes of mammalian G1 cyclins. We have generated a mouse strain in which the coding sequences of the cyclin D1 gene (Ccnd1) have been deleted and replaced by those of human cyclin E (CCNE). In the tissues and cells of these mice, the expression pattern of human cyclin E faithfully reproduces that normally associated with mouse cyclin D1. The replacement of cyclin D1 with cyclin E rescues all phenotypic manifestations of cyclin D1 deficiency and restores normal development in cyclin D1-dependent tissues. Thus, cyclin E can functionally replace cyclin D1. Our analyses suggest that cyclin E is the major downstream target of cyclin D1.

Formation of brain requires deftly balancing primary genesis of neurons and glia, detection of when sufficient cells of each type have been produced, shutdown of proliferation and removal of excess cells. The region and cell type-specific expression of cell cycle regulatory proteins, such as demonstrated for cyclin D2, may contribute to these processes. If so, regional brain development should be affected by alteration of cyclin expression. To test this hypothesis, the representation of specific cell types was examined in the cerebellum of animals lacking cyclin D2. The loss of this cyclin primarily affected two neuronal populations: granule cell number was reduced and stellate interneurons were nearly absent. Differences between null and wild-type siblings were obvious by the second postnatal week. Decreases in granule cell number arose from both reduction in primary neurogenesis and increase in apoptosis of cells that fail to differentiate. The dearth of stellate cells in the molecular layer indicates that emergence of this subpopulation requires cyclin D2 expression. Surprisingly, Golgi and basket interneurons, thought to originate from the same precursor pool as stellate cells, appear unaffected. These results suggest that cyclin D2 is required in cerebellum not only for proliferation of the granule cell precursors but also for proper differentiation of granule and stellate interneurons.

Cyclin D1 is a critical component of the core cell cycle machinery. Mice lacking cyclin D1 develop mammary glands that fail to undergo normal lobuloalveolar proliferation during pregnancy. Thus, cyclin D1 seems to play a critical role in pregnancy-induced proliferation of mammary epithelium. Cyclin D1 also participates in neoplasia, as the majority of human mammary carcinomas contain elevated levels of this cyclin.

THE D-type cyclins (D1, D2 and D3) are critical governors of the cell-cycle clock apparatus during the G1 phase of the mammalian cell cycle. These three D-type cyclins are expressed in overlapping, apparently redundant fashion in the proliferating tissues. To investigate why mammalian cells need three distinct D-type cyclins, we have generated mice bearing a disrupted cyclin D2 gene by using gene targeting in embryonic stem cells. Cyclin D2-deficient females are sterile owing to the inability of ovarian granulosa cells to proliferate normally in response to follicle-stimulating hormone (FSH), whereas mutant males display hypoplastic testes. In ovarian granulosa cells, cyclin D2 is specifically induced by FSH via a cyclic-AMP-dependent pathway, indicating that expression of the various D-type cyclins is under control of distinct intracellular signalling pathways. The hypoplasia seen in cyclin D2(-/-) ovaries and testes prompted us to examine human cancers deriving from corresponding tissues. We find that some human ovarian and testicular tumours contain high levels of cyclin D2 messenger RNA.

Mice lacking cyclin D1 have been generated by gene targeting in embryonic stem cells. Cyclin D1-deficient animals develop to term but show reduced body size, reduced viability, and symptoms of neurological impairment. Their retinas display a striking reduction in cell number due to proliferative failure during embryonic development. In situ hybridization studies of normal mouse embryos revealed an extremely high level of cyclin D1 in the retina, suggesting a special dependence of this tissue on cyclin D1. In adult mutant females, the breast epithelial compartment fails to undergo the massive proliferative changes associated with pregnancy despite normal levels of ovarian steroid hormones. Thus, steroid-induced proliferation of mammary epithelium during pregnancy may be driven through cyclin D1.

Dystrophin, the protein product of the Duchenne muscular dystrophy locus, is encoded by a 14 kb transcript of over 65 exons. A point mutation in the homologous mouse gene causes muscular dystrophy in mdx mice. We have examined the developmental regulation of transcription of this gene in skeletal mouse muscle and also the tissue specificity of the transcript in muscle and brain, by using the polymerase chain reaction to amplify overlapping segments of dystrophin mRNA spanning the entire coding sequence and 5'-untranslated region. We have characterised a specific embryonic transcript that would encode dystrophin with a different C-terminus and have shown that this persists from the earliest stages to the adult in mdx skeletal muscle. The brain transcript shows striking sequence homology to rat and human, being highly conserved at the 5'-untranslated region and is present in both wild-type and mdx mice.