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Developmental waves of mechanosensitivity acquisition in sensory neuron subtypes during embryonic development.

Lechner SG, Frenzel H, Wang R, Lewin GR - EMBO J. (2009)

Bottom Line: Sensory neurons that are mechanoreceptors or proprioceptors acquire mature mechanotransduction indistinguishable from the adult already at E13.In contrast, most nociceptive (pain sensing) sensory neurons acquire mechanosensitive competence as a result of exposure to target-derived nerve growth factor.The highly regulated process of mechanosensory acquisition unveiled here, reveals new strategies to identify molecules required for sensory neuron mechanotransduction.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Max-Delbrück Center for Molecular Medicine, Berlin-Buch, Germany.

ABSTRACT
Somatic sensation relies on the transduction of physical stimuli into electrical signals by sensory neurons of the dorsal root ganglia. Little is known about how and when during development different types of sensory neurons acquire transduction competence. We directly investigated the emergence of electrical excitability and mechanosensitivity of embryonic and postnatal mouse sensory neurons. We show that sensory neurons acquire mechanotransduction competence coincident with peripheral target innervation. Mechanotransduction competence arises in different sensory lineages in waves, coordinated by distinct developmental mechanisms. Sensory neurons that are mechanoreceptors or proprioceptors acquire mature mechanotransduction indistinguishable from the adult already at E13. This process is independent of neurotrophin-3 and may be driven by a genetic program. In contrast, most nociceptive (pain sensing) sensory neurons acquire mechanosensitive competence as a result of exposure to target-derived nerve growth factor. The highly regulated process of mechanosensory acquisition unveiled here, reveals new strategies to identify molecules required for sensory neuron mechanotransduction.

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Developmental expression of TRP channels and Deg/ENaC channels. (A) Expression levels of TRP- and Deg/ENaC family members that exhibit a >2-fold increase in expression between E11.5 and E14.5 and between E11.5 and adult. mRNA was extracted from whole DRGs and expression levels were normalized to the housekeeping gene HPRT. (B) x-fold increase of channel mRNA expression in culture prepared from E12.5 DRGs after 24 h NT-3 treatment. In such cultures, the majority of large neurons acquire RA-currents (see Figure 5b). Channel mRNAs that are up-regulated are highlighted in red. (C) Relative expression levels of known stretch-activated TRP channels. (D, E) RA-currents were measured in ASIC2- and ASIC3-deficient mice, no significant differences were found with respect to maximal RA-current amplitude (D) and inactivation time constant (E).
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f8: Developmental expression of TRP channels and Deg/ENaC channels. (A) Expression levels of TRP- and Deg/ENaC family members that exhibit a >2-fold increase in expression between E11.5 and E14.5 and between E11.5 and adult. mRNA was extracted from whole DRGs and expression levels were normalized to the housekeeping gene HPRT. (B) x-fold increase of channel mRNA expression in culture prepared from E12.5 DRGs after 24 h NT-3 treatment. In such cultures, the majority of large neurons acquire RA-currents (see Figure 5b). Channel mRNAs that are up-regulated are highlighted in red. (C) Relative expression levels of known stretch-activated TRP channels. (D, E) RA-currents were measured in ASIC2- and ASIC3-deficient mice, no significant differences were found with respect to maximal RA-current amplitude (D) and inactivation time constant (E).

Mentions: If an ion channel is alone necessary for the emergence of the mechanosensitive current, the mRNA level for the channel might be low at non-mechanosensitive stages (E11.5) and dramatically elevated at mechanosensitive stages (E14.5). We tested this hypothesis using quantitative real-time PCR for a panel of all known members (33 in total) of the transient receptor potential (TRP) family of ion channels. The members of this channel family have been implicated in mechanosensory transduction in several species (Lin and Corey, 2005; Lumpkin and Caterina, 2007). We also tested the expression of 10 members of the Degenerin/ENaC sodium channel super family, the Caenorhabditis elegans members of this family MEC-4 and MEC-10 are necessary for normal body touch transduction (Ernstrom and Chalfie, 2002; Goodman et al, 2004; O'Hagan et al, 2005). Of the 43 candidate genes tested (for complete list see Supplementary Table S2), only 8 genes fulfilled the ideal expression pattern in that mRNA was barely detectable at E11.5 but was significantly higher (>2-fold) in the E14.5 and adult DRG (Figure 8A). This group of ion channel genes includes Acid Sensitive Ion Channels (ASIC2b and ASIC3) that have been implicated in mechanosensitivity (Price et al, 2000, 2001), but also a number of TRP channels (Figure 8A). We took E12.5 DRG cultures incubated with NT-3 and asked whether the expression of the eight candidate channels is up-regulated coincident with the appearance of mechanosensitive current. The expression of each mRNA was compared between 4 and 24 h in culture, and the expression level of each gene was normalized to a panel of neuron-specific genes. Of all the tested genes, only the mRNA for ASIC2b, ASIC3, TRPV1, TRPM8 and TRPC4 were up-regulated under these conditions (Figure 8B). Several members of the TRP family of channels can be activated by membrane stretch (Clapham et al, 2005) and direct gating of channels by membrane stretch has been proposed as a possible mechanism for mechanotransduction in sensory neurons (Cho et al, 2002, 2006; Kung, 2005). However, the expression of stretch activated TRP channels was not consistent with such a model as high expression levels for these genes were already found at non-mechanosensitive stages (Figure 8C). The increased expression of ASIC2b and ASIC3 mRNA at mechanosensitive stages led us to test whether the RA-type mechanosensitive current is altered or absent in E13.5 sensory neurons taken from ASIC2−/− or ASIC3−/− mice. The incidence, amplitude and inactivation kinetics of the RA-type current were, however, found to be unchanged compared with wild-type controls (Figure 8D and E; Supplementary Table S3). The TRPC4 gene also fulfilled our expression criteria and has not been implicated earlier in sensory mechanotransduction. We asked whether TRPC4 transcripts are enriched in larger sensory neurons cultured from E13.5 embryos. To do this, we collected single neurons larger or smaller than 14 μm in diameter using a glass micropipette (500 small neurons and 500 large neurons, n=3), cDNA was synthesized for the two populations and real-time PCR used to detect TRPC4 mRNA. In 6/6 reactions on pools of large neurons, the TRPC4 transcript was always detected. However, from pools of small neurons, TRPC4 was only detectable in 2/6 experiments.


Developmental waves of mechanosensitivity acquisition in sensory neuron subtypes during embryonic development.

Lechner SG, Frenzel H, Wang R, Lewin GR - EMBO J. (2009)

Developmental expression of TRP channels and Deg/ENaC channels. (A) Expression levels of TRP- and Deg/ENaC family members that exhibit a >2-fold increase in expression between E11.5 and E14.5 and between E11.5 and adult. mRNA was extracted from whole DRGs and expression levels were normalized to the housekeeping gene HPRT. (B) x-fold increase of channel mRNA expression in culture prepared from E12.5 DRGs after 24 h NT-3 treatment. In such cultures, the majority of large neurons acquire RA-currents (see Figure 5b). Channel mRNAs that are up-regulated are highlighted in red. (C) Relative expression levels of known stretch-activated TRP channels. (D, E) RA-currents were measured in ASIC2- and ASIC3-deficient mice, no significant differences were found with respect to maximal RA-current amplitude (D) and inactivation time constant (E).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2664657&req=5

f8: Developmental expression of TRP channels and Deg/ENaC channels. (A) Expression levels of TRP- and Deg/ENaC family members that exhibit a >2-fold increase in expression between E11.5 and E14.5 and between E11.5 and adult. mRNA was extracted from whole DRGs and expression levels were normalized to the housekeeping gene HPRT. (B) x-fold increase of channel mRNA expression in culture prepared from E12.5 DRGs after 24 h NT-3 treatment. In such cultures, the majority of large neurons acquire RA-currents (see Figure 5b). Channel mRNAs that are up-regulated are highlighted in red. (C) Relative expression levels of known stretch-activated TRP channels. (D, E) RA-currents were measured in ASIC2- and ASIC3-deficient mice, no significant differences were found with respect to maximal RA-current amplitude (D) and inactivation time constant (E).
Mentions: If an ion channel is alone necessary for the emergence of the mechanosensitive current, the mRNA level for the channel might be low at non-mechanosensitive stages (E11.5) and dramatically elevated at mechanosensitive stages (E14.5). We tested this hypothesis using quantitative real-time PCR for a panel of all known members (33 in total) of the transient receptor potential (TRP) family of ion channels. The members of this channel family have been implicated in mechanosensory transduction in several species (Lin and Corey, 2005; Lumpkin and Caterina, 2007). We also tested the expression of 10 members of the Degenerin/ENaC sodium channel super family, the Caenorhabditis elegans members of this family MEC-4 and MEC-10 are necessary for normal body touch transduction (Ernstrom and Chalfie, 2002; Goodman et al, 2004; O'Hagan et al, 2005). Of the 43 candidate genes tested (for complete list see Supplementary Table S2), only 8 genes fulfilled the ideal expression pattern in that mRNA was barely detectable at E11.5 but was significantly higher (>2-fold) in the E14.5 and adult DRG (Figure 8A). This group of ion channel genes includes Acid Sensitive Ion Channels (ASIC2b and ASIC3) that have been implicated in mechanosensitivity (Price et al, 2000, 2001), but also a number of TRP channels (Figure 8A). We took E12.5 DRG cultures incubated with NT-3 and asked whether the expression of the eight candidate channels is up-regulated coincident with the appearance of mechanosensitive current. The expression of each mRNA was compared between 4 and 24 h in culture, and the expression level of each gene was normalized to a panel of neuron-specific genes. Of all the tested genes, only the mRNA for ASIC2b, ASIC3, TRPV1, TRPM8 and TRPC4 were up-regulated under these conditions (Figure 8B). Several members of the TRP family of channels can be activated by membrane stretch (Clapham et al, 2005) and direct gating of channels by membrane stretch has been proposed as a possible mechanism for mechanotransduction in sensory neurons (Cho et al, 2002, 2006; Kung, 2005). However, the expression of stretch activated TRP channels was not consistent with such a model as high expression levels for these genes were already found at non-mechanosensitive stages (Figure 8C). The increased expression of ASIC2b and ASIC3 mRNA at mechanosensitive stages led us to test whether the RA-type mechanosensitive current is altered or absent in E13.5 sensory neurons taken from ASIC2−/− or ASIC3−/− mice. The incidence, amplitude and inactivation kinetics of the RA-type current were, however, found to be unchanged compared with wild-type controls (Figure 8D and E; Supplementary Table S3). The TRPC4 gene also fulfilled our expression criteria and has not been implicated earlier in sensory mechanotransduction. We asked whether TRPC4 transcripts are enriched in larger sensory neurons cultured from E13.5 embryos. To do this, we collected single neurons larger or smaller than 14 μm in diameter using a glass micropipette (500 small neurons and 500 large neurons, n=3), cDNA was synthesized for the two populations and real-time PCR used to detect TRPC4 mRNA. In 6/6 reactions on pools of large neurons, the TRPC4 transcript was always detected. However, from pools of small neurons, TRPC4 was only detectable in 2/6 experiments.

Bottom Line: Sensory neurons that are mechanoreceptors or proprioceptors acquire mature mechanotransduction indistinguishable from the adult already at E13.In contrast, most nociceptive (pain sensing) sensory neurons acquire mechanosensitive competence as a result of exposure to target-derived nerve growth factor.The highly regulated process of mechanosensory acquisition unveiled here, reveals new strategies to identify molecules required for sensory neuron mechanotransduction.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Max-Delbrück Center for Molecular Medicine, Berlin-Buch, Germany.

ABSTRACT
Somatic sensation relies on the transduction of physical stimuli into electrical signals by sensory neurons of the dorsal root ganglia. Little is known about how and when during development different types of sensory neurons acquire transduction competence. We directly investigated the emergence of electrical excitability and mechanosensitivity of embryonic and postnatal mouse sensory neurons. We show that sensory neurons acquire mechanotransduction competence coincident with peripheral target innervation. Mechanotransduction competence arises in different sensory lineages in waves, coordinated by distinct developmental mechanisms. Sensory neurons that are mechanoreceptors or proprioceptors acquire mature mechanotransduction indistinguishable from the adult already at E13. This process is independent of neurotrophin-3 and may be driven by a genetic program. In contrast, most nociceptive (pain sensing) sensory neurons acquire mechanosensitive competence as a result of exposure to target-derived nerve growth factor. The highly regulated process of mechanosensory acquisition unveiled here, reveals new strategies to identify molecules required for sensory neuron mechanotransduction.

Show MeSH
Related in: MedlinePlus