Article Figures & Data
Figures
- Figure 1.
Advillin mRNA expression in the developing sensory ganglia. A, Transverse section of E14.5 embryonic head. Advillin is detected in all sensory ganglia, including trigeminal (V), vestibulocochlear (VIII), and vagus (X) ganglia but not in other areas such as cerebral cortex, thalamus, and cerebellum. Ba–Cc, Transverse sections of E11.5 (Ba, Ca), E12.5 (Bb, Ca), and E14.5 (Bc, Ca) TG (Ba–Bc) and DRG (Ca–Cc). The expression level of Advillin increases during development. Arrows in Bb and Bc indicate the expression in facial (VII) ganglia. Da, Db, Coronal sections of P0 neonatal TG (Da) and DRG (Db). Note that no expression was detected in the spinal cord. Ea–Ef, Coronal sections of P0 neonatal TG (Ea–Ec) and DRG (Ed–Ef). Advillin mRNA was detected by in situ hybridization, followed by immunostaining with pan-neuronal anti-NeuN antibody. All Advillin-positive cells were also NeuN positive, and all NeuN-positive neurons were Advillin positive. Scale bars: A, Bc, Dc, 200 μm; E, 100 μm.
- Figure 2.
Targeted insertion of hPLAP gene into the Advillin locus. A, Schematic representation of the gene insertion. hPLAP gene with ACN cassette was inserted at the translational start codon of Advillin gene. Exons are represented as white boxes. The thymidine kinase cassette is designated as TK. Arrows indicate the position of primers used for genomic PCR analysis. B, Southern blot analysis of genomic DNA from wild-type, heterozygous, and homozygous mice. The 9.9 and 5.3 kb PstI restriction fragments indicate wild-type and mutant allele, respectively. C, Genomic PCR analysis of the progeny. Wild-type and mutant alleles produced 500 and 350 bp fragments, respectively.
- Figure 3.
Trigeminal sensory innervation of whiskers visualized by AP staining in Avil-hPLAP mice. A, Coronal sections of E12.5, E14.5, E16.5, and P0 neonatal TG. hPLAP activity was detected at E12.5 onward, and the intensity reached the maximum at E16.5. B, Coronal sections of large vibrissa on mystacial pads from P7 Avil-hPLAP mice were costained for anti-PGP9.5 reactivity and for hPLAP activity using fast red as a substrate. All types of mechanosensory nerve endings, including transverse (Ba–d, arrows) and longitudinal (Ba–d, arrowheads) lanceolate endings, Merkel endings (Ba–h, asterisks), reticular endings (Bi–l), and Ruffini endings (Bm–p), are costained. C, Coronal sections containing muscles at the bottom of the whiskers were stained for hPLAP activity, followed by immunostaining with anti-PGP9.5 antibody. Facial motor axons innervating muscles were negative for hPLAP activity (arrow). Scale bars, 100 μm.
- Figure 4.
Diverse sensory endings in various tissues revealed by AP staining in Avil-hPLAP mice. A, Cutaneous skin innervation. Various types of nociceptive and mechanosensory endings are clearly visualized in glabrous skin on the forepaw from adult mice (Aa–f). Aa, Ab, Lower-magnification pictures. Ac–f, Higher-magnification pictures from Aa and Ab (boxes in Aa and Ab) show hPLAP-positive Meissner's corpuscles (Ac, arrow), Merkel endings (Ad, arrowheads), Krause's end bulbs (Ae, open arrows), free nerve endings (Ae, arrowheads), and Pacinian corpuscles (Af). In hairy skin, hPLAP-positive axons densely innervate guard hair follicles (closed arrows) but not in a subset of vellus hairs (open arrows) (Ag). B, Proprioceptive innervation of muscle spindles (Ba) and Golgi tendons (Bb) are visualized in P0 neonatal skeletal muscles. C, Sensory innervation of cornea. hPLAP-positive axons are found in cornea at P0 (Ca, inset) but not in control mice (Cb). A boundary between the upper and lower lids is shown by an arrow, where there is dense innervation by hPLAP-positive axons. Inset, Higher-magnification of Ca. D, All types of taste buds are innervated by the Advillin-positive axons. Facial, glossopharyngeal, and vagus nerves innervating the taste buds in upper soft palate (Da), filliate (Db, closed arrow), fungiform (Dc), and circumvallate papillae (Dd) are positive for hPLAP. Trigeminal mandibular axons innervating temporomandibular joint form Ruffini endings (arrowheads) and Golgi-Mazzoni corpuscles (open arrows), which are also clearly visualized. Scale bars, 100 μm.
- Figure 5.
Sequential development of whisker- and skin-innervating somatosensory nerve endings. A, Coronal sections of developing mystacial pads. At E12.5 (Aa) and E14.5 (Ab), most axons were found at the developing FSC and form a plexus, which are presumptive DVN axons (arrows), whereas the nociceptive axon tips are first found at E14.5 at the surface skin between each whisker (arrowheads). At E16.5, Merkel endings at the ring sinus level are first observed (arrows). SVN axons are not yet found at this stage (Ac, arrowheads). At P0, Merkel endings and transverse lanceolate endings, but not other type of endings, are observed (Ad, arrows). B, Trigeminal axons innervating the small whiskers are detected at P0. Mechanosensory Merkel endings, transverse and longitudinal lanceolate endings (arrows), as well as nociceptive circumferential free endings (arrowheads) are observed. C, Transverse sections of developing forelimbs. By E14.5, the sensory axons reach their targets and form a plexus underneath the presumptive dermal ridges (Ca). At P0, the innervations are distributed to cover the entire skin but have not formed specialized nerve endings (Cb). Scale bars, 100 μm.
- Figure 6.
Central innervation of TG and DRG neurons. A, Coronal brainstem sections at Pr5 (a, c, e, g) and Sp5 (b, d, f, h, i) level were stained by hPLAP activity. Very few trigeminal axons innervate the central trigeminal nuclei until E14.5. Extensive trigeminal collateral projections can be seen at E16.5. The pioneer innervation of facial, glossopharyngeal, and vagus nerves into the solitary tract nucleus are found at E14.5 onward (arrowheads). Arrows indicate the hPLAP signal in Mo5 motoneurons, which express Advillin mRNA. B, Coronal spinal cord sections. Proprioceptive Ia fibers (arrows) and cutaneous mechanosensory axons innervating layer III/IV (arrowhead) are first found at E14.5 and precede the development of nociceptive innervations (Bb). By E16.5, Ia fibers form a branch to project to the medial part of spinal cord (Bc, arrowhead). Asterisks indicate nonspecific heat-stable alkaline phosphatase activity observed in cartilages. The mature pattern of DRG central projection is established at P0 (Bd). Scale bars: A, 100 μm; B, 200 μm.
- Figure 7.
Regenerative axon growth is impaired in Avil-hPLAP homozygous trigeminal neurons. Trigeminal ganglia from P1 neonatal embryos were dissociated and cultured for 24 h. A, B, Representatives of wild-type (A) and homozygous (B) neurons stained with anti-neurofilament antibody are shown. C, D, Total axon length per cell (C) and the length of the longest axon in each cell (D) were calculated by MetaMorph software. The error bars represent ± SEM. *p < 0.03, **p < 0.001. Scale bar, 100 μm.
- Figure 8.
Reduced structural plasticity of whisker-related patterns in the hindbrain of the Avil-hPLAP homozygous mice. The row C of whiskers was electrocauterized at P1. A–P, The PrV and Sp5i levels of hindbrain at P7 were sectioned at 20 μm and stained for CO (A, D, E, H, I, L, M, P) and AP activity (B, C, F, G, J, K, N, O). Arrows indicate the position of row C. Scale bar, 100 μm. Q, R, Quantification of the lesion-induced plasticity of whisker-related patterns at the PrV (Q) and Sp5i (R) levels. The areas of row C and D were measured using MetaMorph software, and the D/C ratio was calculated as described in Materials and Methods. *p < 0.01, **p < 0.005, compared with electrocauterized heterozygous data. The error bars represent ± SEM.
Additional Files
Supplemental Data
Files in this Data Supplement:
- supplemental material - Figure Legends
- supplemental material - Figure 1
- supplemental material - Figure 2
- supplemental material - Figure 3
- supplemental material - Figure 4
