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Diverse dependencies of developing Merkel innervation on the trkA and both full-length and truncated isoforms of trkC

¹ Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany, NY 12208, USA
² Neuroscience Unit, Howard Hughes Medical Center, University of California, San Francisco, CA 94143-0724, USA
³ Division of Life Sciences, University of Texas, San Antonio, TX 78249, USA
⁴ Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
⁵ Astra Zeneca R&D Södertälje, Department of Molecular Sciences, SE-141 57 Huddinge, Sweden
⁶ Department of Neurobiology, Millennium Pharmaceuticals Inc., Cambridge, MA 02139-4815, USA
⁷ Neural Development Group, NCI-FCRDC, Frederick, MD 21702, USA

View larger version (42K): [in a new window]   Fig. 1. Summary of impacts of various previous knockouts on innervation (black fibers) of mid-vibrissa follicle Merkel cells (dark gray ovals) (Fundin et al., 1997). An enlarged schematic of a Merkel ending and cell is shown in C. For illustrative purposes, the follicles are all shown at the same size and maturity for all ages. (A) In newborn Ntf3–/–, only a few Merkel cells are present and only a few of these are innervated (Fig. 6A,B). Only a few uninnervated Merkel cells remain on P7 and these disappear by P14. Although reduced compared with normal, more Merkel cells and innervation are present in newborn trkCK–/– than in Ntf3–/– (Fig. 6C,D, Fig. 7E). These Merkel cells and innervation are further reduced but still present by P7. Only a few uninnervated Merkel cells remain by P14. (B) In Ngf–/– or trkA–/– animals, Merkel cells and innervation from P0 to P14 are reduced and tightly packed to a level near the ICB (Fig. 7A). The impact was slightly less severe in the Ngf–/–. (C) Merkel innervation seems normal from P0 to P14 in p75–/– and gradually declines over the next few months.

View larger version (132K): [in a new window]   Fig. 2. Immunofluorescence images of developing vibrissae (v), vibrissa follicles (f), nerve bundles (large arrows) and individual Merkel-related axons (small arrows) in rat mystacial pads on E15.5 and the caudal region of rat mystacial pads on E19.5. White arrowheads indicate labeled Merkel endings and cells. Open arrowheads indicate just labeled Merkel endings. Inserts in A,C,E,G show Merkel endings at twice the magnification as shown in white rectangles. e, epidermis. Asterisks indicate sites of non-neuronal labeling. (A,B) Development proceeds along a caudal-to-rostral gradient (right to left). Developing axons completely engulf more immature rostral follicles but caudally have become restricted to the mid-follicle level. Merkel innervation begins to penetrate caudal follicles around E14.5 (not shown). Anti-PGP labels the Merkel endings and begins to label Merkel cells a day or two later. Labeled Merkel endings and cells are shown on E19.5. (C,D) Anti-trkA labeling is clear on axons at E15.5 and is faint on axons and endings by E19.5. By comparison, axons (curved arrow) ascending to innervate the epidermis are more intensely labeled. Labeling at E19.5 has been digitally enhanced in comparison with that at E15.5. TrkA is not clearly expressed on Merkel cells. (E,F) Anti-trkC immunoreactivity is intensely expressed on the developing follicle cells as well as on axons and Merkel endings at E15.5 and is present at low levels on dermal cells immediately surrounding the follicles. Follicle labeling rapidly diminishes over the next day and is lacking on E19.5, while trkC is still evident on axons and Merkel endings. (G,H) Anti-p75 labels axons and Schwann cells within the growing nerves and is present on individual axons at the level where they form Merkel endings. On E15.5 intense p75 immunoreactivity is expressed on dermal cells completely surrounding the follicles. On E19.5, anti-p75 labeling is present on Merkel endings and cells as well as other cells both within and adjacent to the follicles. Scale bar: 50 µm.

View larger version (163K): [in a new window]   Fig. 3. Immunofluorescent images of anti-PGP, trkA, trkC and p75 labeling in and around central to caudal vibrissa follicles of E15.5 wild type, trkCK–/– and trkCE–/– mice 2 days after Merkel innervation has begun to develop. Small arrows, labeled axons; large arrows, larger axon bundles; white arrowheads, Merkel endings and cells; open arrowheads, Merkel endings only; curved arrows, epidermal innervation epidermis. Brackets span locations where Merkel endings are missing or are not revealed by the particular antibody. Asterisks indicate sites of non-neuronal labeling. Inserts contain higher magnification examples of Merkel endings and cells or sites where they should be located. (A-H) In wild type and trkCK–/– fetuses, developing Merkel axons and endings label with anti-PGP, trkA and p75. Anti-trkA labeling is relatively faint compared with 2 days earlier (see Fig. 2C,D) and has been much more digitally enhanced than labeling in other panels. At this age only anti-PGP labels Merkel cells, which will express anti-p75 immunoreactivity within another day (see Fig. 2H). TrkC labeling is also expressed on wild-type Merkel axons and endings but is lacking on axons and endings in trkCK–/–. In wild type and trkCK–/–, non-neuronal labeling occurs with anti-p75 on dermal cells surrounding the follicles and with anti-trkC on cells within the follicles. Anti-trkC labeling within follicles is far less intense than it was 2 days earlier (see Fig. 2E,F) and has been digitally enhanced. (I-L) In trkCE–/– fetuses, presumptive Merkel axons label with anti-PGP, trkA and p75; however, no endings are observed with any of these labels (see inset in L). Anti-PGP also does not reveal any Merkel cells where they are seen in wild type and trkCK–/–. Merkel axons do not label with anti-trkC as also occurred in trkCK–/–. trkCE–/– fetuses have anti-p75 labeling on dermal cells surrounding the follicles comparable with that in wild-type and trkCK–/– fetuses. By contrast, the follicle cells of the trkCE–/– have little or no detectable trkC immunoreactivity. Scale bar: 50 µm.

View larger version (112K): [in a new window]   Fig. 4. Demonstration of two types of Merkel innervation: one trkAK and the other trkCK dependent. (A,B) Double labeling with anti-trkA (green) and anti-trkC (orange) of normal developing vibrissa follicles from central portions of mystacial pads in E15.5 mice and E16.5 rats. In both cases, trkA (green arrows) and trkC (orange arrows) is expressed primarily, if not entirely, on different axons. Sites of overlapping fluorophores (yellow) are due to axon intermingling. Merkel endings (open arrowheads in inset) expressed either trkA or trkC immunoreactivity, not both. Note trkC-labeling on follicle cells. (C-F) Impact of trkAK deletion, trkCK deletion and trkAK/trkCK deletion on P0 littermates. (C) Merkel axons (arrows), endings and cells (solid arrowheads) are present in wild-type mice (see Fig. 1A). (D) TrkAK–/– have reduced Merkel innervation that becomes compacted towards the upper end of the follicles (see Fig. 1B). (E) trkCK–/– have even fewer Merkel endings and cells. Many cells are uninnervated (see Fig. 1A). (F) trkAK–/–/trkCK–/– have some detectable Merkel cells but none is innervated. Scale bar: 50 µm.

View larger version (112K): [in a new window]   Fig. 5. Dark-field images of in situ hybridization demonstrating trkCTr mRNA in developing vibrissa follicles of E14.5 mice. (A,B) In normal fetuses, extensive labeling (between arrowheads) is present in follicles with the probe recognizing all isoforms of trkC (B), but virtually no labeling is present with the probe that recognizes only full length kinase forms of trkC (A). (C) In trkCK–/– animals, labeling within the follicle is still present (between arrowheads) with the probe that recognizes all forms of trkC indicating that this mutation does not eliminate the expression of truncated trkC within the follicle. (D) Virtually no labeling occurs in the follicles of trkCE–/– mutants with the probe for all isoforms of trkC, indicating that expression of trkCTr within follicles was eliminated. Scale bar: 50 µm.

View larger version (144K): [in a new window]   Fig. 6. Immunofluorescence images of follicle innervation labeled with anti-PGP or anti-NF200 in P0 Ntf3–/–, trkCK–/– and trkCE–/– mice. In addition to axons (arrows), anti-PGP labels Merkel endings and cells (white arrowheads), and anti-NF200 labels only Merkel endings (open arrowheads). Brackets span locations where Merkel endings are missing. (A,B) Newborn Ntf3–/– have only a few Merkel cells and a few fragmented axons. Anti-NF200 labels only a few axons and rarely any endings. (C,D) Newborn trkCK–/– have fewer Merkel endings and cells than normal, but much more than Ntf3–/–. Most Merkel cells have endings. (E,F) Newborn trkCE–/– lack any detectable Merkel endings or cells labeled with either anti-PGP or anti-NF200. Both trkCK–/– and trkCE–/– have lanceolate endings (open arrows) and reticular endings (broad arrows) supplied by large caliber myelinated axons as well as epidermal endings (curved arrows) supplied by C-fibers that are not dependent upon intact kinase (Fundin et al., 1997) or trkCTr. However, these other endings are severely depleted in the absence of NT3 which, in these cases, may normally signal through trkAK or trkBK (Fundin et al., 1997; Rice et al., 1998). Scale bar: 50 µm.

View larger version (114K): [in a new window]   Fig. 7. Epifluorescence images of anti-PGP, p75, trkA or trkC labeling (red) combined with transmitted light images of lacZ reporter gene labeling (blue) in and around rostral and caudal vibrissa follicles of E14 and E15 mouse fetuses. Fetuses have either a homozygous (Ntf3lacZ/lacZ) or heterozygous (Ntf3+/lacZ) lacZ reporter gene substitution for Ntf3. Small arrows indicate labeled axons; large arrows, larger axon bundles; white arrowheads, Merkel endings and cells; open arrowheads, only Merkel endings. Asterisks indicate sites of non-neuronal p75 or trkC expression. (A-F) At E14, Ntf3/lacZ is expressed only in the dermis completely surrounding less mature rostral follicles (between curved arrows in A and D). Among more mature caudal follicles (B,C,E and F), Ntf3/lacZ is expressed in a restricted zone (between broad arrows) at mid-levels inside the follicles as well as in the dermis only surrounding the upper end of the follicles (between curved arrows). The pattern of Ntf3/lacZ expression is similar in homozygotes (B and E) and heterozygotes (C and F) but follicles of heterozygotes are larger and more mature. Only the upper two thirds of follicles are shown in C and F. Among the relatively immature follicles of homozygotes, anti-trkC labeling is present on cells throughout the follicles (A) whereas anti-p75 labeling is present on dermal cells surrounding the follicles (D). Non-neuronal expression of trkC has begun to decrease within more mature caudal follicles of homozygotes (B) and has nearly disappeared in relatively more mature caudal follicles of the heterozygote (C). In homozygotes and heterozygotes, Merkel innervation labels with anti-p75. Labeled endings are present only at the site where Ntf3/lacZ is expressed within the follicles (E,F). No innervation labels with anti-trkC in homozygotes (open arrow), but some axons (small arrow) and endings (open arrowhead) are trkC positive in heterozygotes (inset, C). The insets in C and E are enlargements of areas bounded by white rectangles but the blue lacZ label has been digitally suppressed and the red anti-trkC and anti-p75 label digitally enhanced. (G-J) In E15 Ntf3lacZ/lacZ or Ntf3+/lacZ fetuses, Ntf3/lacZ is intensely expressed primarily in the dermis surrounding upper ends of follicles and, compared to E14, is substantially reduced within the mid-level of follicles. Neuronal and non-neuronal labeling with anti-trkA (G) and anti-p75 (not shown) are comparable to normal (see Figs 2, 6). In homozygotes (H), anti-trkC labels the core of the follicle where the vibrissa is developing but does not label any innervation. Anti-PGP labels Merkel endings and cells at mid-follicle levels in homozygotes and heterozygotes (I and J). Scale bar: 50 µm.

View larger version (98K): [in a new window]   Fig. 8. Schematic compilations of immunolabeling, in situ hybridization and lacZ results of Merkel innervation development in wild type (+/+), trkCK–/–, Ntf3–/– and trkCE–/– mouse fetuses at E14.5, E15.5 and P0. For simplification, follicles are shown as the same size instead of being larger and more mature at older ages. Locations of NT3, trkAK, trkCK, trkCTr and p75 are color coded (upper left). Locations where NT3 should be produced is shown in +/+, trkCK–/– and trkCE–/– schematics at E14.5 and E15.5, and are based on lacZ expression in Ntf3+/lacZ and Ntf3lacZ/lacZ fetuses. Ntf3/lacZ was not examined on P0. Notable details are as follows. (1) Developing Merkel innervation expresses p75 along with either trkAK or trkCK. (2) TrkAK disappears before birth. (3) At onset of Merkel ending development, TrkCTR is expressed throughout the follicle, whereas NT3 is produced only by follicle cells where the endings develop. (4) Merkel endings are detected before Merkel cells. (5) TrkAK is expressed with p75 on some axons in all three knockouts but other axons only label with p75. (6) The impact of Ntf3–/– is more detrimental than trkCK–/–, although at least some Merkel endings and cells form in both. (7) No Merkel endings or Merkel cells form in the absence of trkCTR.

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