The Hubble Deep Field South Flanking Fields

As part of the Hubble Deep Field South program, a set of shorter 2-orbit observations were obtained of the area adjacent to the deep fields. The WFPC2 flanking fields cover a contiguous solid angle of 48 square arcminutes. Parallel observations with the STIS and NICMOS instruments produce a patchwork of additional fields with optical and near-infrared (1.6 micron) response. Deeper parallel exposures with WFPC2 and NICMOS were obtained when STIS observed the NICMOS deep field. These deeper fields are offset from the rest, and an extended low surface brightness object is visible in the deeper WFPC2 flanking field. In this data paper, which serves as an archival record of the project, we discuss the observations and data reduction, and present SExtractor source catalogs and number counts derived from the data. Number counts are broadly consistent with previous surveys from both ground and space. Among other things, these flanking field observations are useful for defining slit masks for spectroscopic follow-up over a wider area around the deep fields, for studying large-scale structure that extends beyond the deep fields, for future supernova searches, and for number counts and morphological studies, but their ultimate utility will be defined by the astronomical community.Comment: 46 pages, 15 figures. Images and full catalogs available via the HDF-S at http://www.stsci.edu/ftp/science/hdfsouth/hdfs.html at present. The paper is accepted for the February 2003 Astronomical Journal. Full versions of the catalogs will also be available on-line from AJ after publicatio

The Role of Muscle microRNAs in Repairing the Neuromuscular Junction

microRNAs have been implicated in mediating key aspects of skeletal muscle development and responses to diseases and injury. Recently, we demonstrated that a synaptically enriched microRNA, miR-206, functions to promote maintenance and repair of the neuromuscular junction (NMJ); in mutant mice lacking miR-206, reinnervation is impaired following nerve injury and loss of NMJs is accelerated in a mouse model of amyotrophic lateral sclerosis (ALS). Here, we asked whether other microRNAs play similar roles. One attractive candidate is miR-133b because it is in the same transcript that encodes miR-206. Like miR-206, miR-133b is concentrated near NMJs and induced after denervation. In miR-133b null mice, however, NMJ development is unaltered, reinnervation proceeds normally following nerve injury, and disease progression is unaffected in the SOD1(G93A) mouse model of ALS. To determine if miR-206 compensates for the loss of miR-133b, we generated mice lacking both microRNAs. The phenotype of these double mutants resembled that of miR-206 single mutants. Finally, we used conditional mutants of Dicer, an enzyme required for the maturation of most microRNAs, to generate mice in which microRNAs were depleted from skeletal muscle fibers postnatally, thus circumventing a requirement for microRNAs in embryonic muscle development. Reinnervation of muscle fibers following injury was impaired in these mice, but the defect was similar in magnitude to that observed in miR-206 mutants. Together, these results suggest that miR-206 is the major microRNA that regulates repair of the NMJ following nerve injury.National Institutes of Health (U.S.) (NIH grant R01AG032322)National Institute of Neurological Disorders and Stroke (U.S.) (NRSA Postdoctoral Fellowship from NINDS/NIH)Ruth K. Broad Biomedical Research Foundation (Fellowship)McGovern Institute for Brain Research at MIT (Poitras Center for Affective Disorders Research

Normal Midbrain Dopaminergic Neuron Development and Function in miR-133b Mutant Mice

Midbrain dopaminergic (mDA) neurons control movement and emotion, and their degeneration leads to motor and cognitive defects in Parkinson's disease (PD). miR-133b is a conserved microRNA that is thought to regulate mDA neuron differentiation by targeting Pitx3, a transcription factor required for appropriate development of mDA substantia nigra neurons. Moreover, miR-133b has been found to be depleted in the midbrain of PD patients. However, the function of miR-133b in the intact midbrain has not been determined. Here we show that miR-133b null mice have normal numbers of mDA neurons during development and aging. Dopamine levels are unchanged in the striatum, while expression of dopaminergic genes, including Pitx3, is also unaffected. Finally, motor coordination and both spontaneous and psychostimulant-induced locomotion are unaltered in miR-133b null mice, suggesting that miR-133b does not play a significant role in mDA neuron development and maintenance in vivo.National Institute of Neurological Disorders and Stroke (U.S.)Poitras Center for Affective Disorders Researc

MiR-133b does not regulate muscle reinnervation or ALS disease progression.

<p>(<b>A</b>) Semi-quantitative RT-PCR of cDNA from control or denervated hindlimb muscle 2 and 4 days after unilateral sciatic nerve cut. Levels of pre-miR-133b and AChRγ increase dramatically in denervated muscle, while levels of pre-miR-133a-1, pre-miR-133a-2 and GAPDH are unchanged, suggesting differential regulation of miR-133a and miR-133b. (<b>B–E</b>) Analysis of muscle reinnervation in tibialis anterior muscle from control (B) and miR-133b null mice (C) 3 weeks following nerve cut. (<b>D</b>) Percentage of tibialis anterior NMJs that were reinnervated. (<b>E</b>) Percentage of NMJs that were denerverated, partially reinnervated, or fully reinnervated. (<b>F,G</b>) Analysis of sternomastoid muscle reinnervation 9 days following accessory nerve crush. At least 6 mice were analyzed and 200 NMJs were examined per animal. Error bars indicate SEM. Scale bar  = 20 μm. (<b>H–J</b>) In the SOD1-G93A mouse model for ALS, loss of miR-133b does not exacerbate symptoms; disease onset (H), survival rate (I), and disease progression (J) are unchanged in the absence of miR-133b. Data were obtained from: 8 female, 8 male SOD1G93A; 10 female, 8 male miR-133b+/−;SOD1G93A; and 6 female, 9 male R-133b−/−;SOD1G93A mice. Error bars indicate SEM.</p

Lack of both miR-206 and miR-133b delays NMJ regeneration.

<p>(<b>A–D</b>) To determine whether both miRNAs, miR-206 and miR-133b (7H4), act in concert to affect muscle reinnervation, the peroneal nerve was crushed in control (A) and 7H4 knockout mice (B) and reinnervation of the extensor digitorium longus was examined 9 days post injury. In 7H4 muscles, the incidence of partially and completely denervated NMJs is higher than that in muscles from control animals (C, D). At least 6 mice were examined per genotype and 50 NMJs per mouse visualized. FI, fully innervated; PI, partially innervated; FD, fully denervated NMJs. Error bar  =  SEM. P-value (*) <0.02. Scale bar  = 50 μm. (<b>E, F</b>) Quantitative mRNA expression of pre-miR-1-1, pre-miR-1-2, pre-miR-133a-1, and pre-miR-133a-2 in EDL (E) and soleus (F) muscle of adult WT (black circles represent individual values and black line the mean) and 7H4 knockout (red circles represent individual values and red line the mean) mice. Gene expression is normalized to Gapdh and results are scaled to the average value of the WT samples.</p

Development of NMJs in 7H4 mice.

<p>(<b>A–D</b>) Both miR-206 and miR-133b are dispensable for development of the NMJ. There is no obvious difference in the transformation of the postsynapse (stained using f-BTX, red) from a small plaque into a large pretzel between 7H4 knockout (B and D) and control mice of the same age (A and C). The formation of the presynaptic apparatus is also indistinguishable between 7H4 knockout mice and control mice of the same age, visualized using antibodies against synaptotagmin-2, green, and neurofilament, blue, in young animals (A and B) and YFP expressed in motor axons (C and D). Scale bar  = 10 μm for P9 and 20 μm for adult NMJs.</p

Normal NMJ development in miR-133b knockout mice.

<p>(<b>A</b>) Immunofluorescence staining of axonal neurofilaments and vesicular synaptophysin (green) and BTX staining of postsynaptic nAChRs (red) to visualize axons innervating synaptic sites. Filled white arrowheads, NMJs with multiple axon innervation; empty arrowheads, retraction bulbs. Scale bar  = 20 μm. (<b>B</b>) The proportion of sternomastoid NMJs with multiple innervation decreases at a similar rate in control and knockout mice. (<b>C</b>) Proportion of developing sternomastoid NMJs with single, double or triple innervation is similar in control and knockout mice.</p

Generation and analysis of miR-206 and miR-133b double knockout mice.

<p>(<b>A</b>) <i>Trans</i>-allelic targeted meiotic recombination was used to generate mice lacking both miR-206 and miR-133b. MiR-206 and miR-133b heterozygous mice each containing a loxP site in place of the miRNA stemloop were bred together and with mice expressing Cre recombinase in the germline. Zygotes produced from sperm that underwent <i>trans</i>-allelic recombination contained one chromosome lacking miR-206 and miR-133b and one chromosome with a miR-206 and miR-133b duplication. These animals were then bred to obtain miR-206 and miR-133b double knockout mice, i.e. 7H4 knockout mice. P1, forward primer upstream of miR-206. P2, reverse primer downstream of miR-133b. (<b>B</b>) PCR using P1 and P2 primers (in A) gives a detectable product (550 bp) only in 7H4 heterozygous and knockout mice, demonstrating that the 7H4 genomic region containing the miR-206 and miR-133b stem loops is completely missing from the 7H4 null allele. (<b>C</b>) PCR using primers specific for the miR-133b allele yields a 600 bp band only when the WT allele is present, but no band for the 7H4 null allele. (<b>D</b>) Quantitative RT-PCR for the stemloop regions of miR-206 and miR-133b. As expected, miR-206 and miR-133b are absent in 7H4 knockout mice.</p