The Journal of Neuroscience, May 24, 2006, ():
Motor Axon Guidance of the Mammalian Trochlear and Phrenic Nerves: Dependence on the Netrin Receptor Unc5c and Modifier Loci
J. Neurosci. Burgess et al.
26: 5756
Supplemental data
Files in this Data Supplement:
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Figure S1. Diaphragm innervation. (A) In wild-type mice, the phrenic nerve arises from motor neurons in the ventral horn of the cervical spinal cord (C3 through C6). Motor axons exit the spinal cord ipsilaterally through the ventral roots and migrate towards their target muscles. While most motor axons project laterally from the brachial plexus to innervate the forelimbs, axons of the phrenic continue caudally through the thoracic cavity to contact the diaphragm. After making contact with the muscle near the dorsal/ventral midpoint on E12, three main branches form: one branch projects ventrally and one branch projects dorsally to innervate most of the muscle, and another branch moves dorsal/medially to innervate the crus of the diaphragm. Like most muscles, the endplate band of neuromuscular junctions is found near the middle of the muscle fibers. (B) In B6.Unc5c-/- mice, the innervation of the diaphragm is disrupted. The phrenic nerve is reduced in diameter and contains fewer axons than in control mice. As a consequence, the nerve is unable to elaborate endings over the entire surface of the diaphragm, resulting in uninnervated muscle domains. The nerve still contacts the muscle at the correct point, and uninnervated regions of the muscle tend to be in the ventral or dorsal portions of the muscle. The phenotype is often asymmetric, with the left side and right side varying in their severity. In some cases, one nerve is completely missing, resulting in a complete lack of innervation unilaterally (represented by dashed lines), while the other side of the muscle is similar to control. In other cases, both sides are affected to varying degrees.
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Figure S2. Intercostal innervation in B6.Unc5c mice. The intercostal nerves of E18.5 control (A, C) and Unc5c-/- (B, D) animals show only normal animal-to-animal variability when visualized by either Thy1-YFP16 fluorescence (A, B) or wholemount antibody staining (C, D). There is no failure of innervation in the intercostal muscles and the branching patterns of the nerves is similar in all cases. Unlike the trochlear or phrenic nerves, which could be used to reliably predict genotype (Unc5c-/- or control), the intercostal innervation pattern was indistinguishable between these genotypes. Scale bars: 200µm (A, B); 240 µm (C, D).
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Figure S3. Projection and fasciculation of Unc5c-/- spinal motor axons. In an attempt to determine where the axons of the phrenic nerve are misprojecting in the Unc5c-/- embryos, the motor column of the spinal cord was examined using confocal microscopy. Motor neurons were visualized by breeding Thy1-eYFP transgenic mice into the Unc5c background. Cells bodies and early axonal projections in E13 control (A) and mutant mice (B) were examined. In both genotypes, occasional dorsal and medial misprojections were observed, but the bulk of the axons exit the cord ventrally and laterally (ventral is down). The misprojections were rare and difficult to characterize, and therefore could not be quantified. (A) and (B) are images from littermate animals, both are from the cervical spinal cord. Fasciculation of the ventral roots was also studied in the EYFP transgenic mice at E13.5. The lateral spinal cord was examined by confocal microscopy. The ventral roots of control (C) and Unc5c-/- (D) embryos were indistinguishable. Scale bars: 100 µm (A, B); 200 µm (C, D).
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Figure S4. Expression patterns of netrin1 receptors in the developing spinal cord. The expression patterns of Unc5c (A), Dcc (B), and Neo1 (C) were examined by fluorescent in situ hybridization at E14. While the expression patterns were distinct, particularly in dorsal spinal cord, all showed some expression in the ventral horn and overlapped the regions containing the Chat-positive motor neurons (D). The sections shown are from serial sections of cervical spinal cord, though the pattern did not change in more caudal regions (not shown). The results are summarized schematically (E). Solid bars represent regions of strong expression, and dashes represent regions of weaker expression. The color code is indicated in the legend; the pink background represents low levels of Neo1 expression. MN=motor neurons, DRG=Dorsal Root Ganglia, CC=Central Canal. Scale bar: 240µm (A-D).
