A spontaneous semidominant mutation (Ironside, Irn) was isolated in mice, leading to severe hindlimb paralysis following multiple deletions in cis at the HoxD locus. To understand its cellular and molecular etiology, we embarked on a comparative analysis using systematic HoxD cluster deletions, produced via targeted meiotic recombination (TAMERE). Different lines of mice were classified according to the severity of their paralyses, and subsequent analyses revealed that multiple causative factors were involved, alone or in combination, in the occurrence of this pathology. Among them are the loss of Hoxd10 function, the sum of remaining Hoxd gene activity, and the ectopic gain of function of the neighboring gene Evx2, all contributing to the mispositioning, the absence, or misidentification of specific lumbo-sacral pools of motoneurons, nerve root homeosis, and hindlimb innervation defects. These results highlight the importance of a systematic approach when studying such clustered gene families, and give insights into the function and regulation of Hox and Evx2 genes during early spinal cord development.[Keywords: Homeosis; peroneal nerve; chromosome engineering; motoneuron] Supplemental material is available at http://www.genesdev.org.

Utrophin is a 400 kDa autosomal homolog of dystrophin and a component of the submembranous cytoskeleton. While multiple dystrophin isoforms have been identified along with alternatively spliced products, to date only two different mRNA species of utrophin have been identified. To determine the degree of evolutionary conservation between dystrophin and utrophin isoforms, we have compared their expression patterns in adult mice. Northern blot analysis of multiple adult tissues confirmed that only two major sizes of transcripts are produced from each gene: 13 and 5.5 kb from utrophin and 14 and 4.8 kb from dystrophin. However, western blot analysis detected several putative short utrophin isoforms that may be homologs of the dystrophin isoforms Dp140, Dp116 and Dp71. We also identified an alternatively spliced utrophin transcript that lacks the equivalent of the alternatively spliced dystrophin exon 71. Finally, we demonstrated that the C-terminal domain of utrophin targeted to neuromuscular junctions in normal mice, but localized to the sarcolemma efficiently only in the absence of dystrophin. Our results provide further evidence for a common evolutionary origin of the utrophin and dystrophin genes.

Duchenne muscular dystrophy (DMD) is caused by mutations that impair normal production of dystrophin in muscle and brain tissues. The dystrophin gene is expressed at extremely low levels in both humans and mice, which makes analysis of the 14kb mRNA a difficult task. In addition, 30% of all cases of DMD (and the genetic lesion in all three known mdx mouse models for DMD) are thought to arise from single base mutations, yet methods are not available to routinely identify and analyze these mutations and their effects on disease progression. We have been using the polymerase chain reaction (PCR) to analyze the expression of the murine dystrophin gene. A simple assay is described that distinguishes the murine dystrophin transcripts expressed from either the muscle or brain promoter. In addition, amplification of overlapping segments from the 5' end of the murine transcript has enabled the identification of DNA sequence variations between wild-type and mdx mice. These results demonstrate that the mutation in the original strain of mdx mice is distinct from those in two newer mdx isolates and that three independently isolated mdx mutants are available for study of DMD.

Embryonic development of the spinal cord proceeds through a carefully orchestrated temporal and spatial sequence of chemical cues to provide precise patterning of adult cell types. Recreating this complex microenvironment in a standard cell culture dish is difficult, if not impossible. In this paper, a microfluidic device is used to recapitulate, in vitro, the graded patterning events which occur during early spinal cord development. The microdevice design is developed using COMSOL modeling, with which the spatiotemporal profiles of multiple, diffusible morphogens are simulated. Four independently addressed source/sinks are employed to generate two overlapping orthogonal gradients within a cell culture chamber, mimicking the dorsoventral and anteroposterior axes of the developing embryo. Mouse embryonic stem cells are directed therein to differentiate into motor neurons in a spatially organized manner, reminiscent of a neural tube.

Note: Transcript has been edited for clarity and accuracy.Brian Johnstone: Thanks to the organizers for providing this forum and the speaker for his stimulating talk. Question 1 -what is your opinion on the timing of the dosing regimen with regard to ALS mouse model study design? Specifically, please provide a rationale for presymptomatic dosing as opposed to beginning treatment following onset of symptoms. Should not all preclinical testing be performed with agent given after onset of symptoms to more accurately reflect the clinical scenario? This is especially relevant given the elegant studies by Dr. Cleveland which show that the mechanism underlying disease onset fundamentally differs from that driving disease progression.Sean Scott: Brian, we do not actually believe this animal is ever presymptomatic. With that said, we start dosing at day 50 as a uniform start.Brian Johnstone: Sean, thank you for your answer. The mice may not ever be without disease, but they do not display differences in symptoms compared to control until much later in life. Certainly ALS patients are the same, but they never are treated before manifestation of disease symptoms (e.g., post-diagnosis). Peak weight or rotarod tests are reliable measures of onset (diagnosis) in mouse.Sean Scott: Brian, my concern is the subtlety of symptom detection. If mice played golf, would they start noticing errant shots around day 30? Brian Johnstone: Question 2 -the use of total lifespan as a measure of efficacy in preclinical studies is artificial and not relevant to clinical scenario, where post-onset survival time is relevant. Has ALS-TDI applied its statistical methods toward analysis of post-onset survival time in SOD1-G93A mice?Sean Scott: We are doing that analysis now. It is very evidently much noisier than survival. In the end, simply