Cyclin D1 belongs to the core cell cycle machinery, and it is frequently overexpressed in human cancers 1,2 . The full repertoire of cyclin D1 functions in normal development and in oncogenesis is currently unclear. Here we developed FLAG-and HA-tagged cyclin D1 knock-in mouse strains that allowed high-throughput mass spectrometry approach to search for cyclin D1-binding proteins in different mouse organs. In addition to cell cycle partners, we observed several proteins involved in transcription. Genome-wide location (ChIP-chip) analyses revealed that during mouse development cyclin D1 occupies promoters of abundantly expressed genes. In particular, we found that in developing mouse retinas -an organ that critically requires cyclin D1 function 3,4 -cyclin D1 binds the upstream regulatory region of the Notch1 gene where it serves to recruit CBP histone acetyltransferase. Genetic ablation of cyclin D1 resulted in decreased CBP recruitment, decreased histone acetylation of the Notch1 promoter region, and led to decreased levels of the Notch transcript and protein in cyclin D1-null retinas. Transduction of an activated allele of Notch1 into cyclin D1 −/− retinas increased proliferation of retinal progenitor cells, indicating that upregulating Notch1 signaling alleviates the phenotype of cyclin D1-deficiency. These studies reveal that in addition to its well-established cell cycle roles, cyclin D1 plays an in vivo transcriptional function in mouseCorrespondence and request for materials should be addressed to P.S. (Peter_Sicinski@dfci.harvard.edu). * These authors contributed equally to this work. 8 Current address: Stanford University School of Medicine, Stanford, CA 94305, USA.Supplementary Information is linked to the online version of the paper at www.nature.com/nature Author Contributions F.B. and PS designed the study, analyzed the data and wrote the manuscript. F.B. performed the experiments with the help of co-authors as detailed below. S.J. performed protein purifications. J.E.E. performed and together with S.P.G. Author InformationThe complete ChIP-chip and expression datasets have been submitted to the online data repository GEO, record GSE13636 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=zvuhbiaakuwumxa&acc=GSE13636). Reprints and permissions information is available at www.nature.com/reprints. NIH Public Access Author ManuscriptNature. Author manuscript; available in PMC 2010 September 22. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript development. Our approach, which we term "genetic-proteomic" can be used to study the in vivo function of essentially any protein.To study the molecular functions of cyclin D1 during development and in cancer formation, we generated knock-in mouse strains in which tandem (FLAG-and HA-) tags were inserted into the endogenous cyclin D1 locus through homologous recombination in embryonal stem cells. Tags were introduced into N-terminus of cyclin D1 (D1 Ntag allele) or into C-terminus (D1 Ctag ) and homozygous D1 Ntag/Ntag and D1 C...

SUMMARYSeveral models of cell fate determination can be invoked to explain how single retinal progenitor cells (RPCs) produce different cell types in a terminal division. To gain insight into this process, the effects of the removal of a cell fate regulator, Notch1, were studied in newly postmitotic cells using a conditional allele of Notch1 (N1-CKO) in mice. Almost all newly postmitotic N1-CKO cells became rod photoreceptors, whereas wild-type (

The Bone morphogenetic proteins (BMPs) mediate a wide range of diverse cellular behaviors throughout development. Previous studies implicated an important role for BMP signaling during the differentiation of the definitive mammalian kidney, the metanephros. In order to examine whether BMP signaling also plays an important role during the patterning of earlier renal systems, we examined the development of the earliest nephric system, the pronephros. Using the amphibian model system Xenopus laevis, in combination with reagents designed to inhibit BMP signaling during specific stages of nephric development, we revealed an evolutionarily conserved role for this signaling pathway during renal morphogenesis. Our results demonstrate that conditional BMP inhibition after specification of the pronephric anlagen is completed, but prior to the onset of morphogenesis and differentiation of renal tissues, results in the severe malformation of both the pronephric duct and tubules. Importantly, the effects of BMP signaling on the developing nephron during this developmental window are specific, only affecting the developing duct and tubules, but not the glomus. These data, combined with previous studies examining metanephric development in mice, provide further support that BMP functions to mediate morphogenesis of the specified renal field during vertebrate embryogenesis. Specifically, BMP signaling is required for the differentiation of two types of nephric structures, the pronephric tubules and duct. Developmental Dynamics 237:132-144, 2008.

The authors have declared no conflicts of interest for this article.

Background The vertebrate retina comprises sensory neurons, the photoreceptors, as well as many other types of neurons and one type of glial cell. These cells are generated by multipotent and restricted retinal progenitor cells (RPCs), which express Notch1. Loss of Notch1 in RPCs late during retinal development results in the overproduction of rod photoreceptors at the expense of interneurons and glia. Results To examine the molecular underpinnings of this observation, microarray analysis of single retinal cells from wild-type or Notch1 conditional knockout retinas was performed. In situ hybridization was carried out to validate some of the findings. Conclusions The majority of Notch1-mutant cells lost expression of known Notch target genes. These cells also had low levels of RPC and cell cycle genes, and robustly up-regulated rod precursor genes. In addition, single wild-type cells, in which cell cycle marker genes were down-regulated, expressed markers of both rod photoreceptors and interneurons.

During convergent differentiation, multiple developmental lineages produce a highly similar or identical cell type. However, few molecular players that drive convergent differentiation are known. Here, we show that the C. elegans Forkhead transcription factor UNC-130 is required in only one of three convergent lineages that produce the same glial cell type. UNC-130 acts transiently as a repressor in progenitors and newly-born terminal cells to allow the proper specification of cells related by lineage rather than by cell type or function. Specification defects correlate with UNC-130:DNA binding, and UNC-130 can be functionally replaced by its human homolog, the neural crest lineage determinant FoxD3. We propose that, in contrast to terminal selectors that activate cell-type specific transcriptional programs in terminally differentiating cells, UNC-130 acts early and specifically in one convergent lineage to produce a cell type that also arises from molecularly distinct progenitors in other lineages.

A central goal in developmental biology is to decipher the molecular events that govern cell fate specification in each developmental lineage. Here, we show that the C. elegans Forkhead transcription factor UNC-130 specifies two glial types that arise from one lineage, but does not affect equivalent glia that are produced in different anatomical regions from other lineages. We show that glial defects correlate with UNC-130:DNA binding, and that UNC-130 acts as a transcriptional repressor via two independent domains. UNC-130 can be functionally replaced by its human homolog, the neural crest lineage determinant FoxD3, and other neural crest factors (UNC-86/Brn3 and RNT-1/Runx) act in the same pathway. We propose that, in contrast to "terminal selectors", UNC-130 acts as a "lineage selector" to enable molecularly distinct progenitor cells to generate regionally equivalent cell types. This novel mechanism may underlie the recent observation of convergent lineages as a prevalent feature of vertebrate development.

The extracellular matrix (ECM) guides and constrains the shape of the nervous system. In C. elegans, DIG-1 is a giant ECM component that is required for fasciculation of sensory dendrites during development and for maintenance of axon positions throughout life. We identified four novel alleles of dig-1 in three independent screens for mutants affecting disparate aspects of neuronal and glial morphogenesis. First, we find that disruption of DIG-1 causes fragmentation of the amphid sheath glial cell in larvae and young adults. Second, it causes severing of the BAG sensory dendrite from its terminus at the nose tip, apparently due to breakage of the dendrite as animals reach adulthood. Third, it causes embryonic defects in dendrite fasciculation in inner labial (IL2) sensory neurons, as previously reported, as well as rare defects in IL2 dendrite extension that are enhanced by loss of the apical ECM component DYF-7, suggesting that apical and basolateral ECM contribute separately to dendrite extension. Our results highlight novel roles for DIG-1 in maintaining the cellular integrity of neurons and glia, possibly by creating a barrier between structures in the nervous system.