During vertebrate eye development, the cells of the optic vesicle (OV)become either neuroretinal progenitors expressing the transcription factor Chx10, or retinal pigment epithelium (RPE) progenitors expressing the transcription factor Mitf. Chx10 mutations lead to microphthalmia and impaired neuroretinal proliferation. Mitf mutants have a dorsal RPE-to-neuroretinal phenotypic transformation, indicating that Mitfis a determinant of RPE identity. We report here that Mitf is expressed ectopically in the Chx10or-J/or-J neuroretina(NR), demonstrating that Chx10 normally represses the neuroretinal expression of Mitf. The ectopic expression of Mitf in the Chx10or-J/or-J NR deflects it towards an RPE-like identity; this phenotype results not from a failure of neuroretinal specification, but from a partial loss of neuroretinal maintenance. Using Chx10 and Mitf transgenic and mutant mice, we have identified an antagonistic interaction between Chx10 and Mitf in regulating retinal cell identity. FGF (fibroblast growth factor) exposure in a developing OV has also been shown to repress Mitf expression. We demonstrate that the repression of Mitfby FGF is Chx10 dependent, indicating that FGF, Chx10 and Mitf are components of a pathway that determines and maintains the identity of the NR.

Dystonia musculorum (dt) is a hereditary neurodegenerative disease in mice that leads to a sensory ataxia. We describe cloning of a candidate dt gene, dystonin, that is predominantly expressed in the dorsal root ganglia and other sites of neurodegeneration in dt mice. Dystonin encodes an N-terminal actin binding domain and a C-terminal portion comprised of the hemidesmosomal protein, bullous pemphigoid antigen 1 (bpag1). dt and bpag1 are part of the same transcription unit which is partially deleted in a transgenic strain of mice, Tg4, that harbours an insertional mutation at the dt locus, and in mice that carry a spontaneous dt mutation, dtAlb. We also demonstrate abnormal dystonin transcripts in a second dt mutant, dt24J. We conclude that mutations in the dystonin gene are the primary genetic lesion in dt mice.

Modifiers of position-effect-variegation in Drosophila encode proteins that are thought to modify chromatin, rendering it heritably changed in its expressibility. In an attempt to identify similar modifier genes in other species we have utilized a known sequence homology, termed chromo box, between a suppressor of position-effect-variegation, Heterochromatin protein 1 (HP1), and a repressor of homeotic genes, Polycomb (Pc). A PCR generated probe encompassing the HP1 chromo box was used to clone full-length murine cDNAs that contain conserved chromo box motifs. Sequence comparisons, in situ hybridization experiments, and RNA Northern blot analysis suggest that the murine and human sequences presented in this report are homologues of the Drosophila HP1 gene. Chromo box sequences can also be detected in other animal species, and in plants, predicting a strongly conserved structural role for the peptide encoded by this sequence. We propose that epigenetic (yet heritable) changes in gene expressibility, characteristic of chromosomal imprinting phenomena, can largely be explained by the action of such modifier genes. The evolutionary conservation of the chromo box motif now enables the isolation and study of putative modifier genes in those animal and plant species where chromosomal imprinting has been described.

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The site of integration of transgenes in the host genome can affect levels of expression and occasionally confer ectopic patterns of expression on otherwise tissue-specific genes. We describe here a line of mice in which an hsp68-lacZ transgene is expressed in unstressed developing neural tissue and where the transgene insertion has caused a mutation of a neural tissue-specific gene, dystonia musculorum (dt). This coincidence suggests that expression of the hsp68-lacZ construct may be controlled directly by cis-acting regulatory sequences that normally control the developmental expression of the dt gene. Such constructs may serve as useful tools for identifying new tissue-specific enhancers and their associated genes.

Objective Spinal muscular atrophy (SMA) is the number 1 genetic killer of young children. It is caused by mutation or deletion of the survival motor neuron 1 (SMN1) gene. Although SMA is primarily a motor neuron disease, metabolism abnormalities such as metabolic acidosis, abnormal fatty acid metabolism, hyperlipidemia, and hyperglycemia have been reported in SMA patients. We thus initiated an in-depth analysis of glucose metabolism in SMA. Methods Glucose metabolism and pancreas development were investigated in the Smn2B/− intermediate SMA mouse model and type I SMA patients. Results Here, we demonstrate in an SMA mouse model a dramatic cell fate imbalance within pancreatic islets, with a predominance of glucagon-producing α cells at the expense of insulin-producing β cells. These SMA mice display fasting hyperglycemia, hyperglucagonemia, and glucose resistance. We demonstrate similar abnormalities in pancreatic islets from deceased children with the severe infantile form of SMA in association with supportive evidence of glucose intolerance in at least a subset of such children. Interpretation Our results indicate that defects in glucose metabolism may play an important contributory role in SMA pathogenesis.

The homeodomain transcription factors Cdx1, Cdx2 and Cdx4 play essential roles in anteroposterior vertebral patterning through regulation of Hox gene expression. Cdx2 is also expressed in the trophectoderm commencing at E3.5 and plays an essential role in implantation, thus precluding assessment of the cognate-null phenotype at later stages. Cdx2 homozygous null embryos generated by tetraploid aggregation exhibit an axial truncation indicative of a role for Cdx2 in elaborating the posterior embryo through unknown mechanisms. To better understand such roles, we developed a conditional Cdx2 floxed allele in mice and effected temporal inactivation at post-implantation stages using a tamoxifen-inducible Cre. This approach yielded embryos that were devoid of detectable Cdx2 protein and exhibited the axial truncation phenotype predicted from previous studies. This phenotype was associated with attenuated expression of genes encoding several key players in axial elongation, including Fgf8, T, Wnt3a and Cyp26a1, and we present data suggesting that T, Wnt3a and Cyp26a1 are direct Cdx2 targets. We propose a model wherein Cdx2 functions as an integrator of caudalizing information by coordinating axial elongation and somite patterning through Hoxindependent and -dependent pathways, respectively. DEVELOPMENT 4100 system to derive a conditional null allele. In agreement with previous studies (Chawengsaksophak et al., 2004), loss of Cdx2 in the postimplantation embryo resulted in axial truncation posterior to the forelimb. This axial truncation, together with the nature of many of the genes impacted by Cdx2 loss, suggested that precocious cessation of the generation of presomitic mesoderm (PSM) is the primary basis for this phenotype. To further define the role of Cdx2 and to elucidate the mechanistic basis for this phenotype, we sought to determine whether any of the affected genes were direct Cdx2 targets. Chromatin immunoprecipitation (ChIP) demonstrated occupancy of the Wnt3a, Cyp26a1 and T promoters by Cdx2 in vivo. Moreover, all of these promoters harbor functional Cdx response elements (CDREs) as determined by transfection analysis or transgenic assays. Thus, Cdx2 directly regulates the expression of multiple players essential for the development of the posterior embryo. Taken together with previous work, these findings suggest that Cdx2 is required to couple the generation of paraxial mesoderm through multiple Hox-independent mechanisms with Hoxdependent AP vertebral patterning. MATERIALS AND METHODS Gene targeting and the generation of Cdx2 -/-mutantsA 5 kb fragment of genomic Cdx2 sequence encompassing the first intron through to the 3Ј UTR was subcloned into pBluescript II KS + . A floxed thymidine kinase/neomycin resistance cassette (loxPGK-TK-Neolox) (Iulianella and Lohnes, 2002) was cloned into the BglII site in intron 1, and a single loxP site was inserted into the NruI site in intron 2, generating the targeting vector (see Fig. S1A in the supplementary material). R1 embryonic stem cells were electroporated with...

Myogenesis is a complex and tightly regulated process, the end result of which is the formation of a multinucleated myofibre with contractile capability. Typically, this process is described as being regulated by a coordinated transcriptional hierarchy. However, like any cellular process, myogenesis is also controlled by members of the protein kinase family, which transmit and execute signals initiated by promyogenic stimuli. In this review, we describe the various kinases involved in mammalian skeletal myogenesis: which step of myogenesis a particular kinase regulates, how it is activated (if known) and what its downstream effects are. We present a scheme of protein kinase activity, similar to that which exists for the myogenic transcription factors, to better clarify the complex signalling that underlies muscle development.