QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online February 18, 2004
doi: 10.1242/10.1242/dev.01013

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Guha, U. Articles by Kessler, J. A. Search for Related Content PubMed PubMed Citation Articles by Guha, U. Articles by Kessler, J. A.

Target-derived BMP signaling limits sensory neuron number and the extent of peripheral innervation in vivo

¹ Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461, USA
² Department of Neurology, Northwestern University, Feinberg Medical School, Chicago, IL 60611, USA
³ Department of Neuroscience, Albany Medical College, Albany, NY 12208, USA

View larger version (46K): [in a new window]   Fig. 1. Generation of K14-BMP4 and K14-noggin transgenic mice. (A) Transgenic constructs as diagrammed were injected into one-cell stage Fvb embryos to generate founders. (B,D) Southern blot analyses of genomic DNA from mouse tails shows the BMP4 and noggin transgenic bands in the respective transgenic founders. (C,E) Western blot analyses of lysates from back skin of one-month-old mice shows that BMP4 and noggin proteins are overexpressed in the skin compared with wild-type (wt) littermates. (F-I) Whole-mount in situ hybridization of E12.5 mouse embryos shows that the Bmp4 (G) and noggin (I) transgenes are overexpressed in the whisker pad (arrows) of the corresponding transgenic embryos compared with wild-type littermates (F,H).

View larger version (104K): [in a new window]   Fig. 2. Hyperinnervation of the skin in postnatal and adult K14-noggin animals. Innervation of the epidermis and upper dermis at comparable locations in the intervibrissal fur (A-D) and at the mouth of vibrissal follicles (E,F) in wild-type and K14-noggin overexpressing mice (K14-Nog) as shown by double-label immunofluorescence with anti-CGRP revealed with Cy3 and anti-PGP revealed with Cy2. e, epidermis; f, hair follicle; *, the mouth of a whisker follicle. The broken line indicates the border between the epidermis and dermis. Green arrowheads indicate examples of FNEs in the epidermis that are labeled only with anti-PGP. Yellow arrowheads indicate examples of epidermal FNEs that also label with anti-CGRP. Note that the epidermis of the K14-Nog mice contains far more FNEs with and without CGRP-IR. Broad arrowheads with numbers 2, 3 and 4 indicate regions of the second, third and fourth tiers of the dermal plexus respectively. In K14-Nog mice, the bundles of axons that compose the fourth tier are much larger and occupy a greater proportion of the epidermal-dermal border. In wild type, axons with CGRP-IR (yellow arrows) are mostly restricted to the second and third tiers. In K14-Nog, numerous axons with CGRP-IR are also located in the fourth tier. However, at E15 there are no detectable differences in PGP immunostaining between transgenic and control mice (G,H). Panels A-H are all mice from an Fvb background. (I-L) Quantification of the innervation to the epidermis and upper dermis in K14-Nog mice. The quantitative analyses were carried out on five sections for each mouse and the mean for each parameter obtained. Mean±s.e.m. of three mice for each genotype are shown (*P<0.05; **P<0.005; ***P<0.0005).

View larger version (137K): [in a new window]   Fig. 3. Diminished innervation of the skin in K14-BMP4 mice. Innervation of the epidermis and upper dermis at comparable locations in the intervibrissal fur in wild type and K14-BMP4 mice. Labels are similar to those in Fig. 2. (A-D) Double-label immunofluorescence with anti-CGRP (Cy3) and anti-PGP (Cy2). At 1 month, the innervation to the epidermis appears comparable in wild type and K14-BMP4 mice. At 6 months, innervation in the K14-BMP4 mice has become reduced and fragmented. However, most of the remaining innervation expresses CGRP-IR, which may be more than in wild type. Yellow arrows indicate sites where axons express CGRP IR. In contrast to wild type, in K14-BMP4 transgenics most epidermal innervation seems to be supplied directly from the third tier of the dermal plexus. Axon bundles at the epidermal-dermal border, indicative of the fourth tier, are readily encountered in wild type. The fourth tier was rarely detected in K14-BMP4 intervibrissal fur. A-D are all mice from CB6F1 background. (E-H) Comparison of the innervation to the epidermis and upper dermis at comparable locations in the intervibrissal fur and in wild-type, K14-BMP4 and K14-noggin mice as shown by double-label immunofluorescence with anti-CGRP (Cy3) and anti-NF200 (Cy2). E,F are from a CB6F1 background; G,H are from a Fvb background. Red arrows indicate axons labeled only with anti-CGRP, green arrows labeled only with anti-NF200, yellow arrows labeled with both anti-CGRP and anti-NF. Note the massive increase in CGRP-positive innervation at all levels of the dermal plexus in the K14-noggin fur. In both wild-type and K14-noggin mice, the third and fourth tiers of the dermal plexus contain anti-CGRP labeled axons that are mostly NF200 negative (red arrows). Likewise, the anti-CGRP labeled endings in the epidermis lack NF200-IR (red arrowheads). A few axons in the third tier contain NF200 but most of the CGRP-positive/NF200-positive axons are in the second tier. By contrast, in the K14-BMP4 fur, most of the CGRP positive innervation at all levels co-expresses NF200-IR. This includes an ending in the epidermis that co-expresses CGRP-IR and NF200-IR (yellow arrowhead), which is never seen in the wild-type epidermis.

View larger version (130K): [in a new window]   Fig. 4. Merkel cells are increased in K14-noggin and decreased in K14-BMP4 mice. Innervation to the mouth of whisker follicles in wild-type, K14-noggin and K14-BMP4 mice as shown by double-label immunofluorescence with anti-NF200 (Cy3) and anti-PGP (Cy2). e, epidermis at the mouth of a whisker follicle. A-D are from an Fvb background; E,F are from CB6F1 background. The broken line indicates the border between the epidermis and dermis. Green arrowheads indicate examples of FNEs in the epidermis which are labeled only with anti-PGP. Note that the epidermis of the K14-noggin mice contains far more FNEs and that the K14-BMP4 mice have fewer FNEs than their corresponding wild-type littermates. Yellow curved arrows indicate Merkel endings labeled with anti-NF200 and anti-PGP that terminate on Merkel cells (asterisks) labeled only with anti-PGP. Note that the Merkel cells and innervation are increased in K14-Nog and decreased in K14-BMP4 mice.

View larger version (49K): [in a new window]   Fig. 5. Summary of changes in the innervation within the intervibrissal fur and the mouth of the whisker follicles of the K14-noggin and K14-BMP4 mice.

View larger version (31K): [in a new window]   Fig. 6. BMP signaling decreases neuron numbers in vivo and in vitro. (A) Neuron numbers in the trigeminal ganglion and DRG of K14-noggin animals and the DRG of the K14-BMP4 animals normalized to their respective wild-type controls for each ganglion. Neuron numbers were significantly increased in the trigeminal and dorsal root ganglia of the K14-noggin animals, but significantly reduced in the DRGs of K14-BMP4 animals. The mean and s.e.m. of seven wild-type and seven transgenic ganglia are shown. *P<0.02; **P<0.01; ***P<0.015. (B) Preferential increase of the smaller size neuronal population in K14-noggin trigeminal ganglia. The means and s.e.m. of three wild-type and four transgenic ganglia are shown. *P<0.05; **P<0.005. (C) Effects of BMP4 and NGF on cultured trigeminal neurons. E10 trigeminal ganglia were dissociated, and 600 cells were plated per well in the presence of BDNF (5 ng/ml). Six hours after plating, some cultures were treated with BMP4 (30 ng/ml), NGF (10 ng/ml), or both, or with additional BDNF (10 ng/ml). Cell numbers were counted 72 hours later and are expressed as mean±s.e.m. per dish. Note that BMP4 treatment reduced cell survival. Treatment with NGF but not additional BDNF prevented the BMP4-mediated cell death (*P<0.04 using ANOVA).

View larger version (41K): [in a new window]   Fig. 7. Neuron numbers are unchanged in E12.5 K14-noggin trigeminal ganglia. (A) Total number of ß-tubulin positive neurons in the trigeminal ganglion is unchanged in K14-noggin compared with wild-type littermates. Mean and s.e.m. of four animals of each genotype are shown. (B) Total number of BrdU-positive cells is not significantly different in K14-noggin versus wild-type trigeminal ganglia. Means and s.e.m. of four animals of each genotype are shown. (C) Total number of TUNEL-positive cells is unchanged. Mean and s.e.m. of four animals of each genotype are shown. (D) Neuron numbers in the trigeminal ganglion decrease by about 33% between E12.5 and adult life in wild type but do not change in the K14-noggin animals (*P<0.02).

View larger version (23K): [in a new window]   Fig. 8. Neurotrophin levels are not increased in the skin of K14-noggin animals. NGF and BDNF proteins as measured by ELISA of lysates from the whisker pads of 3-day-old mice are unchanged in K14-noggin compared with the wild-type mice and levels of NT3 are actually reduced significantly. Mean±s.e.m. of five animals of each genotype are shown (*P<0.025).

View larger version (56K): [in a new window]   Fig. 9. Neurotrophin transcript expression in the mystacial pads of K14-noggin mice. Expression of neurotrophin mRNAs was analyzed by quantitative RT-PCR performed with mRNA extracted from the mystacial pads of E14.5 and postnatal day 2 (P2) K14-noggin and wild-type littermates. Shown are two curves each for transgenic (blue) and wild-type (red) tissues. At E14.5 there were no changes in NGF, NT3 or GDNF transcripts, while BDNF transcripts were slightly increased about twofold in the K14-noggin animals. At PN2 there were no changes in transcript levels of BDNF, NT3 or GDNF, but there was a small (fourfold) increase in NGF transcripts in the K14-noggin animals.