L-type voltage-gated Ca2+ channel CaV1.2 regulates chondrogenesis during limb development
Contributed by Clifford J. Tabin, September 3, 2019 (sent for review May 24, 2019; reviewed by Cheng-Ming Chuong and Min Zhao)
Significance
Membrane potential is the difference in electric potential between the inside and outside of a cell. Cells can alter their membrane potential through modulating ion channels and pumps. The role of membrane potential has been well studied in a variety of settings, including as a mechanism of coding and transmitting information in neurons. However, far less is known about the role of regulated membrane potential in other contexts, such as in cell fate decisions during embryonic morphogenesis. We show a critical role for membrane depolarization in triggering chondrogenesis during limb development, mediated through the opening of a voltage-gated Ca2+ channel, CaV1.2, which in turn activates expression of downstream genes, expanding our knowledge of the role bioelectric signals play in development.
Abstract
All cells, including nonexcitable cells, maintain a discrete transmembrane potential (Vmem), and have the capacity to modulate Vmem and respond to their own and neighbors’ changes in Vmem. Spatiotemporal variations have been described in developing embryonic tissues and in some cases have been implicated in influencing developmental processes. Yet, how such changes in Vmem are converted into intracellular inputs that in turn regulate developmental gene expression and coordinate patterned tissue formation, has remained elusive. Here we document that the Vmem of limb mesenchyme switches from a hyperpolarized to depolarized state during early chondrocyte differentiation. This change in Vmem increases intracellular Ca2+ signaling through Ca2+ influx, via CaV1.2, 1 of L-type voltage-gated Ca2+ channels (VGCCs). We find that CaV1.2 activity is essential for chondrogenesis in the developing limbs. Pharmacological inhibition by an L-type VGCC specific blocker, or limb-specific deletion of CaV1.2, down-regulates expression of genes essential for chondrocyte differentiation, including Sox9, Col2a1, and Agc1, and thus disturbs proper cartilage formation. The Ca2+-dependent transcription factor NFATc1, which is a known major transducer of intracellular Ca2+ signaling, partly rescues Sox9 expression. These data reveal instructive roles of CaV1.2 in limb development, and more generally expand our understanding of how modulation of membrane potential is used as a mechanism of developmental regulation.
Data Availability
Data deposition: The raw data for quantifications including Alicia blue staining, quantitative PCR data, cell counting, and length measurements of mouse limbs have been deposited at https://bitbucket.org/tabinlab/pnas-01908981/.
Acknowledgments
We thank Drs. Dany S. Adams, Patrick McMillen, Juanita Mathews, and Joshua Finkelstein (Tufts University) for useful technical comments; members of the C.J.T. and Constance Cepko laboratories, especially Drs. Tyler Huycke, Changhee Lee, John J. Young, and Rose G. Long for helpful discussions. The RCAS-GCaMP6s-2A-mCherry plasmid was a kind gift from Dr. Ang Li (University of Southern California). This work was supported by the Allen Discovery Center program through The Paul G. Allen Frontiers Group (to M.L. and C.J.T.), and NIH Grant HD03443 (to C.J.T.). Y.A. is a recipient of fellowships of the Naito Foundation and Japan Society for the Promotion of Science (JSPS).
Supporting Information
Appendix (PDF)
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Movie S1.
Time-lapse analysis using GCaMP/mCherry-infected chicken limb bud cells (Day0). Frames were taken with a 20x objective lens every minute for 20 min. This movie is related to Fig. 2A, B, E.
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- 1.10 MB
Movie S2.
Time-lapse analysis using GCaMP/mCherry-infected chicken limb bud cells cultured in the presence of K-gluconate (Day0). Frames were taken with a 20x objective lens every minute for 20 min. This movie is related to Fig. 2C, D, E.
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- 1.59 MB
Movie S3.
Time-lapse analysis using GCaMP/mCherry-infected chicken limb bud cells (Day0). Frames were taken with a 20x objective lens every minute for 20 min. This movie is related to Fig. 2F, G, J.
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- 1.85 MB
Movie S4.
Time-lapse analysis using GCaMP/mCherry-infected chicken limb bud cells cultured in the presence of Nifedipine (Day0). Frames were taken with a 20x objective lens every minute for 20 min. This movie is related to Fig. 2H, I, J.
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- 1.91 MB
Movie S5.
Time-lapse analysis using GCaMP/mCherry-infected chicken limb bud cells (Day0). Frames were taken with a 20x objective lens every minute for 20 min. This movie is related to Fig. 2K, L, O.
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- 2.01 MB
Movie S6.
Time-lapse analysis using GCaMP/mCherry-infected chicken limb bud cells cultured in the presence of both K-gluconate and Nifedipine (Day0). Frames were taken with a 20x objective lens every minute for 20 min. This movie is related to Fig. 2M, N, O.
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- 1.13 MB
Movie S7.
Time-lapse analysis using GCaMP/mCherry-infected chicken limb bud cells (Day2). Frames were taken with a 20x objective lens every minute for 20 min. This movie is related to Fig. S3A, B, E.
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- 2.47 MB
Movie S8.
Time-lapse analysis using GCaMP/mCherry-infected chicken limb bud cells cultured in the presence of Nifedipine (Day2). Frames were taken with a 20x objective lens every minute for 20 min. This movie is related to Fig. S3C, D., E
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- 2.53 MB
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Cover image: Pictured is a flooded farm in northeastern Arkansas. Matteo Coronese et al. examined the impacts of natural disasters between 1960 and 2015 and found evidence of mounting economic damages tied to severe events, including those that extended beyond tropical regions into temperate areas of the globe. The authors also found increased death tolls from extreme temperature events in both rich and poor countries. The findings highlight hazards posed by severe events associated with changing climate conditions. See the article by Coronese et al. on pages 21450–21455. Image courtesy of the Federal Emergency Management Agency/Samir Valeja.
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© 2019. Published under the PNAS license.
Data Availability
Data deposition: The raw data for quantifications including Alicia blue staining, quantitative PCR data, cell counting, and length measurements of mouse limbs have been deposited at https://bitbucket.org/tabinlab/pnas-01908981/.
Submission history
Published online: October 7, 2019
Published in issue: October 22, 2019
Keywords
Acknowledgments
We thank Drs. Dany S. Adams, Patrick McMillen, Juanita Mathews, and Joshua Finkelstein (Tufts University) for useful technical comments; members of the C.J.T. and Constance Cepko laboratories, especially Drs. Tyler Huycke, Changhee Lee, John J. Young, and Rose G. Long for helpful discussions. The RCAS-GCaMP6s-2A-mCherry plasmid was a kind gift from Dr. Ang Li (University of Southern California). This work was supported by the Allen Discovery Center program through The Paul G. Allen Frontiers Group (to M.L. and C.J.T.), and NIH Grant HD03443 (to C.J.T.). Y.A. is a recipient of fellowships of the Naito Foundation and Japan Society for the Promotion of Science (JSPS).
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The authors declare no competing interest.
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