Degenerative disorders of motor neurons include a range of progressive fatal diseases such as amyotrophic lateral sclerosis (ALS), spinal-bulbar muscular atrophy (SBMA), and spinal muscular atrophy (SMA). Although the causative genetic alterations are known for some cases, the molecular basis of many SMA and SBMA-like syndromes and most ALS cases is unknown. Here we show that missense point mutations in the cytoplasmic dynein heavy chain result in progressive motor neuron degeneration in heterozygous mice, and in homozygotes this is accompanied by the formation of Lewy-like inclusion bodies, thus resembling key features of human pathology. These mutations exclusively perturb neuron-specific functions of dynein.

For an understanding of the aberrant biology seen in mouse mutations and identification of more subtle phenotype variation, there is a need for a full clinical and pathological characterization of the animals. Although there has been some use of sophisticated techniques, the majority of behavioral and functional analyses in mice have been qualitative rather than quantitative in nature. There is, however, no comprehensive routine screening and testing protocol designed to identify and characterize phenotype variation or disorders associated with the mouse genome. We have developed the SHIRPA procedure to characterize the phenotype of mice in three stages. The primary screen utilizes standard methods to provide a behavioral and functional profile by observational assessment. The secondary screen involves a comprehensive behavioral assessment battery and pathological analysis. These protocols provide the framework for a general phenotype assessment that is suitable for a wide range of applications, including the characterization of spontaneous and induced mutants, the analysis of transgenic and gene-targeted phenotypes, and the definition of variation between strains. The tertiary screening stage described is tailored to the assessment of existing or potential models of neurological disease, as well as the assessment of phenotypic variability that may be the result of unknown genetic influences. SHIRPA utilizes standardized protocols for behavioral and functional assessment that provide a sensitive measure for quantifying phenotype expression in the mouse. These paradigms can be refined to test the function of specific neural pathways, which will, in turn, contribute to a greater understanding of neurological disorders.

Genomic imprinting is an epigenetic phenomenon that results in monoallelic gene expression according to parental origin. It has long been established that imprinted genes have major effects on development and placental biology before birth. More recently, it has become evident that imprinted genes also have important roles after birth. In this Review, I bring together studies of the effects of imprinted genes from the prenatal period onwards. Recent work on postnatal stages shows that imprinted genes influence an extraordinarily wide-ranging array of biological processes, the effects of which extend into adulthood, and play important parts in common diseases that range from obesity to psychiatric disorders.

Genomic imprinting, by which maternal and paternal alleles of some genes have different levels of activity, has profound effects on growth and development of the mammalian fetus. The action of imprinted genes after birth, in particular while the infant is dependent on maternal provision of nutrients, is far less well understood. We disrupted a paternally expressed transcript at the Gnas locus, Gnasxl, which encodes the unusual Gs alpha isoform XL alpha s. Mice with mutations in Gnasxl have poor postnatal growth and survival and a spectrum of phenotypic effects that indicate that XL alpha s controls a number of key postnatal physiological adaptations, including suckling, blood glucose and energy homeostasis. Increased cAMP levels in brown adipose tissue of Gnasxl mutants and phenotypic comparison with Gnas mutants suggest that XL alpha s can antagonize Gs alpha-dependent signaling pathways. The opposing effects of maternally and paternally expressed products of the Gnas locus provide tangible molecular support for the parental-conflict hypothesis of imprinting.

Imprinted genes tend to occur in clusters. We have identified a cluster in distal mouse chromosome (Chr) 2, known from early genetic studies to contain both maternally and paternally imprinted, but unspecified, genes. Subsequently, one was identified as Gnas, which encodes a G protein ␣ subunit, and there is clinical and biochemical evidence that the human homologue GNAS1, mutated in patients with Albright hereditary osteodystrophy, is also imprinted. We have used representational difference analysis, based on parent-of-origin methylation differences, to isolate candidate imprinted genes in distal Chr 2 and found two oppositely imprinted genes, Gnasxl and Nesp. Gnasxl determines a variant G protein ␣ subunit associated with the trans-Golgi network and Nesp encodes a secreted protein of neuroendocrine tissues. Gnasxl is maternally methylated in genomic DNA and encodes a paternal-specific transcript, whereas Nesp is paternally methylated with maternal-specific expression. Their reciprocal imprinting may offer insight into the distal Chr 2 imprinting phenotypes. Remarkably, Gnasxl, Nesp, and Gnas are all part of the same transcription unit; transcripts for Gnasxl and Nesp are alternatively spliced onto exon 2 of Gnas. This demonstrates an imprinting mechanism in which two oppositely imprinted genes share the same downstream exons.

Genomic imprinting results in allele-specific silencing according to parental origin. Silencing is brought about by imprinting control regions (ICRs) that are differentially marked in gametogenesis. The group of imprinted transcripts in the mouse Gnas cluster (Nesp, Nespas, Gnasxl, Exon 1A and Gnas) provides a model for analyzing the mechanisms of imprint regulation. We previously identified an ICR that specifically regulates the tissue-specific imprinted expression of the Gnas gene. Here we identify a second ICR at the Gnas cluster. We show that a paternally derived targeted deletion of the germline differentially methylated region (DMR) associated with the antisense Nespas transcript unexpectedly affects both the expression of all transcripts in the cluster and methylation of two DMRs. Our results establish that the Nespas DMR is the principal ICR at the Gnas cluster and functions bidirectionally as a switch for modulating expression of the antagonistically acting genes Gnasxl and Gnas. Uniquely, the Nespas DMR acts on the downstream ICR at exon 1A to regulate tissue-specific imprinting of the Gnas gene.

Genomic imprinting brings about allele-specific silencing according to parental origin. Silencing is controlled by cis-acting regulatory regions that are differentially marked during gametogenesis and can act over hundreds of kilobases to silence many genes. Two candidate imprinting control regions (ICRs) have been identified at the compact imprinted Gnas cluster on distal mouse chromosome 2, one at exon 1A upstream of Gnas itself and one covering the promoters for Gnasxl and the antisense Nespas (ref. 8). This imprinted cluster is complex, containing biallelic, maternally and paternally expressed transcripts that share exons. Gnas itself is mainly biallelically expressed but is weakly paternally repressed in specific tissues. Here we show that a paternally derived targeted deletion of the germline differentially methylated region at exon 1A abolishes tissue-specific imprinting of Gnas. This rescues the abnormal phenotype of mice with a maternally derived Gnas mutation. Imprinting of alternative transcripts, Nesp, Gnasxl and Nespas (ref. 13), in the cluster is unaffected. The results establish that the differentially methylated region at exon 1A contains an imprinting control element that specifically regulates Gnas and comprises a characterized ICR for a gene that is only weakly imprinted in a minority of tissues. There must be a second ICR regulating the alternative transcripts.

Maternally and paternally derived alleles can utilize different promoters, but allele-specific differences in cotranscriptional processes have not been reported. We show that alternative polyadenylation sites at a novel murine imprinted gene (H13) are utilized in an allele-specific manner. A differentially methylated CpG island separates polyA sites utilized on maternal and paternal alleles, and contains an internal promoter. Two genetic systems show that alleles lacking methylation generate truncated H13 transcripts that undergo internal polyadenylation. On methylated alleles, the internal promoter is inactive and elongation proceeds to downstream polyadenylation sites. This demonstrates that epigenetic modifications can influence utilization of alternative polyadenylation sites.Supplemental material is available at http://www.genesdev.org.Received January 28, 2008; revised version accepted March 10, 2008. Transcription by RNA polymerase II (Pol II) requires multiple linked steps, including the assembly of an initiation complex, promoter release, elongation followed by splicing, polyadenylation (polyA), and dissociation of the polymerase complex from the template DNA. Each step provides an opportunity for the cell to regulate gene expression, either by changing the abundance or availability of mRNAs or by generating variant gene products. Epigenetic modifications to DNA and histones can influence transcription at the initiation stage by altering the accessibility of promoter sequences to initiation complex components (Kass et al. 1997a,b). Following initiation, regions of heterochromatin can subtly impede the progress of an elongating polymerase complex, reducing transcriptional output (Lorincz et al. 2004). However, it is not clear whether epigenetic modifications downstream from a promoter can lead to variant gene products through alternative polyadenylation or splicing.Polyadenylation, the addition of multiple adenyl residues to the 3Ј end of a newly synthesized transcript, confers stability and is required for nuclear export (Huang and Carmichael 1996;Jacobson and Peltz 1996). This occurs following cleavage of the newly synthesized pre-mRNA, typically 15-30 nucleotides downstream from a conserved hexamer motif (usually AAUAAA or AUUAAA) (Proudfoot and Brownlee 1976;Zhao et al. 1999). A large proportion of human genes utilize more than one polyadenylation (polyA) site (Tian et al. 2005), making alternative polyadenylation a major source of transcriptional diversity. The mechanisms governing alternative polyA site selection in mammalian cells have been studied in detail at only a small number of loci; notably the immunoglobulin heavy chain (Takagaki and Manley 1998) and calcitonin/CGRP genes (Lou et al. 1998). In both cases, polyA site selection is cell typespecific and is regulated by changes in the concentration of diffusible RNA processing factors (Lou et al. 1998;Takagaki and Manley 1998).Imprinted genes are differentially expressed on maternally and paternally derived alleles and are estimated to com...