Limited column formation in the embryonic growth plate implies divergent growth mechanisms during pre- and postnatal bone development
- Weizmann Institute of Science, Israel
- University of Michigan-Ann Arbor, United States
- Aix Marseille Univ, INSERM, MMG, UMR1251, France
- Queen Mary University of London, United Kingdom
Abstract
Chondrocyte columns, which are a hallmark of growth plate architecture, play a central role in bone elongation. Columns are formed by clonal expansion following rotation of the division plane, resulting in a stack of cells oriented parallel to the growth direction. In this work, we analyzed hundreds of Confetti multicolor clones in growth plates of mouse embryos using a pipeline comprising 3D imaging and algorithms for morphometric analysis. Surprisingly, analysis of the elevation angles between neighboring pairs of cells revealed that most cells did not display the typical stacking pattern associated with column formation, implying incomplete rotation of the division plane. Morphological analysis revealed that although embryonic clones were elongated, they formed clusters oriented perpendicular to the growth direction. Analysis of growth plates of postnatal mice revealed both complex columns, composed of ordered and disordered cell stacks, and small, disorganized clusters located in the outer edges. Finally, correlation between the temporal dynamics of the ratios between clusters and columns and between bone elongation and expansion suggests that clusters may promote expansion, whereas columns support elongation. Overall, our findings support the idea that modulations of division plane rotation of proliferating chondrocytes determines the formation of either clusters or columns, a multifunctional design that regulates morphogenesis throughout pre- and postnatal bone growth. Broadly, this work provides a new understanding of the cellular mechanisms underlying growth plate activity and bone elongation during development.
Data availability
The datasets generated and analyzed during the current study are available on Zenodo at the following links: https://doi.org/10.5281/zenodo.10440013, https://doi.org/10.5281/zenodo.10444731, https://doi.org/10.5281/zenodo.10446055, https://doi.org/10.5281/zenodo.10446092, https://doi.org/10.5281/zenodo.10446121, https://doi.org/10.5281/zenodo.10446131, https://doi.org/10.5281/zenodo.10446123, https://doi.org/10.5281/zenodo.10446145.
-
Col2creER;Confettihet_gE14.5_E18.5_litter154_m3Zenodo, 10.5281/zenodo.10440013.
-
Col2creER;Confettihet_gE14.5_E18.5_litter154_m4enodo, 10.5281/zenodo.10444731.
-
Col2creER;Confettihet_gE14.5_E18.5_litter153_m7enodo, 10.5281/zenodo.10446055.
-
Col2creER;Confettihet_gP30_P40_litter152_m3_DFenodo, 10.5281/zenodo.10446092.
-
Col2creER;Confettihet_gP30_P40_litter152_m3_PTenodo, 10.5281/zenodo.10446121.
-
Col2creER;Confettihet_gP30_P40_litter152_m4_DFenodo, 10.5281/zenodo.10446131.
-
Col2creER;Confettihet_gP30_P40_litter151_m2enodo, 10.5281/zenodo.10446123.
-
Col2creER;Confettihet_gP30_P40_litter152_m4_PTenodo, 10.5281/zenodo.10446145.
Article and author information
Author details
Funding
Israel Science Foundation (1387/23)
- Elazar Zelzer
Weizmann - Sagol Institute for longetivity research
- Elazar Zelzer
Julie and Eric Borman Family Research Funds
- Elazar Zelzer
University of Michigan School of Dentistry startup funds
- Tomer Stern
University of Michigan Oral Health Sciences PhD program
- Meng-Jia Lian
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All animal experiments were pre-approved by and conducted according to the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the Weizmann Institute (IACUC 01750221-1 and IACUC 05700723-2). All animals used in this study had access to food and water ad libitum and were maintained under controlled humidity and temperature (45-65%, 22 {plus minus} 2{degree sign}C, respectively).
Copyright
© 2024, Rubin et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 853
- views
-
- 151
- downloads
-
- 0
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Limited column formation in the embryonic growth plate implies divergent growth mechanisms during pre- and postnatal bone development
eLife 13:e95289.
https://doi.org/10.7554/eLife.95289
Further reading
-
- Cell Biology
TGF-β stimulates CCN2 expression which in turn amplifies TGF-β signaling. This process promotes extracellular matrix production and accelerates the pathological progression of fibrotic diseases. Alternative splicing plays an important role in multiple disease development, while U2 small nuclear RNA auxiliary factor 2 (U2AF2) is an essential factor in the early steps of pre-mRNA splicing. However, the molecular mechanism underlying abnormal CCN2 expression upon TGF-β stimulation remains unclear. This study elucidates that SIRT4 acts as a master regulator for CCN2 expression in response to TGF-β by modulating U2AF2-mediated alternative splicing. Analyses of renal biopsy specimens from patients with CKD and mouse fibrotic kidney tissues revealed marked nuclear accumulation of SIRT4. The tubulointerstitial fibrosis was alleviated by global deletion or tubular epithelial cell (TEC)-specific knockout of Sirt4, and aggravated by adeno-associated virus-mediated SIRT4 overexpression in TECs. Furthermore, SIRT4 was found to translocate from the mitochondria to the cytoplasm through the BAX/BAK pore under TGF-β stimulation. In the cytoplasm, TGF-β activated the ERK pathway and induced the phosphorylation of SIRT4 at Ser36, which further promoted its interaction with importin α1 and subsequent nuclear translocation. In the nucleus, SIRT4 was found to deacetylate U2AF2 at K413, facilitating the splicing of CCN2 pre-mRNA to promote CCN2 protein expression. Importantly, exosomes containing anti-SIRT4 antibodies were found to effectively mitigate the UUO-induced kidney fibrosis in mice. Collectively, these findings indicated that SIRT4 plays a role in kidney fibrosis by regulating CCN2 expression via the pre-mRNA splicing.
-
- Cell Biology
Aging is associated with a number of physiologic changes including perturbed circadian rhythms; however, mechanisms by which rhythms are altered remain unknown. To test the idea that circulating factors mediate age-dependent changes in peripheral rhythms, we compared the ability of human serum from young and old individuals to synchronize circadian rhythms in culture. We collected blood from apparently healthy young (age 25–30) and old (age 70–76) individuals at 14:00 and used the serum to synchronize cultured fibroblasts. We found that young and old sera are equally competent at initiating robust ~24 hr oscillations of a luciferase reporter driven by clock gene promoter. However, cyclic gene expression is affected, such that young and old sera promote cycling of different sets of genes. Genes that lose rhythmicity with old serum entrainment are associated with oxidative phosphorylation and Alzheimer’s Disease as identified by STRING and IPA analyses. Conversely, the expression of cycling genes associated with cholesterol biosynthesis increased in the cells entrained with old serum. Genes involved in the cell cycle and transcription/translation remain rhythmic in both conditions. We did not observe a global difference in the distribution of phase between groups, but found that peak expression of several clock-controlled genes (PER3, NR1D1, NR1D2, CRY1, CRY2, and TEF) lagged in the cells synchronized ex vivo with old serum. Taken together, these findings demonstrate that age-dependent blood-borne factors affect circadian rhythms in peripheral cells and have the potential to impact health and disease via maintaining or disrupting rhythms respectively.
