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Pairing of segmentation clock genes drives robust pattern formation

Abstract

Gene expression is an inherently stochastic process1,2; however, organismal development and homeostasis require cells to coordinate the spatiotemporal expression of large sets of genes. In metazoans, pairs of co-expressed genes often reside in the same chromosomal neighbourhood, with gene pairs representing 10 to 50% of all genes, depending on the species3,4,5,6. Because shared upstream regulators can ensure correlated gene expression, the selective advantage of maintaining adjacent gene pairs remains unknown6. Here, using two linked zebrafish segmentation clock genes, her1 and her7, and combining single-cell transcript counting, genetic engineering, real-time imaging and computational modelling, we show that gene pairing boosts correlated transcription and provides phenotypic robustness for the formation of developmental patterns. Our results demonstrate that the prevention of gene pairing disrupts oscillations and segmentation, and the linkage of her1 and her7 is essential for the development of the body axis in zebrafish embryos. We predict that gene pairing may be similarly advantageous in other organisms, and our findings could lead to the engineering of precise synthetic clocks in embryos and organoids.

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Fig. 1: Negative feedback loop drives correlated transcription.
Fig. 2: Segmentation is robust against perturbations by chromosomal linkage of two clock genes.
Fig. 3: Impaired oscillations of the segmentation clock underlie segmentation defects.

Data availability

Datasets containing RNA counts in each cell for each embryo are provided as Excel files in Supplementary Tables 17, 9, 10. Original microscopy image files are provided at the BioStudies (https://www.ebi.ac.uk/biostudies/studies/) (accession number S-BSST434). Source data are provided with this paper.

Code availability

Matlab and Python codes are provided at GitHub (https://github.com/ozbudak/zinani_genepairing).

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Acknowledgements

We thank S. Keskin, I. Ejikeme, M. Evren, H. Seawall, L. Tweedie, Y. Y. Lee, E. Meyer, M. Kofron, and Cincinnati Children’s Imaging Core and Veterinary Services for technical assistance, M. Simsek, A. Singh, T. Zhang, C. Hong, D. Spinzak and members of Özbudak laboratory for discussions, and B. Gebelein and R. Kopan for editing the manuscript. This work was funded by an NIH grant (GM122956) to E.M.Ö.

Author information

Affiliations

Authors

Contributions

E.M.Ö. designed and supervised the project. K.K. performed real-time imaging and O.Q.H.Z. performed all other experiments. A.A. performed simulations. A.A. and O.Q.H.Z. performed statistical analysis. O.Q.H.Z., K.K., A.A. and E.M.Ö. analysed the data and wrote the manuscript.

Corresponding author

Correspondence to Ertuğrul M. Özbudak.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks Andrew Oates, Michael Levine and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Single RNA molecules are quantified in single cells in the zebrafish PSM.

a, Top, a single z-section of PSM of a wild-type embryo. her7 mRNAs and nuclei are coloured in red and blue, respectively. Scale bar, 30 μm. n = 24, N = 2. Bottom, the PSM is divided into single-cell-wide slices. Cells containing higher or lower RNA than an arbitrary threshold are plotted as red or grey circles, respectively. Left and right halves of the PSM are located at the top and bottom portions of the image, respectively. Three oscillatory waves of her7 are visible. b, her7 RNA counts are plotted along the posterior-to-anterior direction at the left half of PSM. Each dot corresponds to the average RNA number in a spatial cell population (slice). Data are mean and two s.e.m. c, All embryos are aligned from their posterior ends and slices corresponding to the same anterior-posterior positions are grouped (blue dashed lines). d, The spatial amplitudes of oscillations of total her (her1 + her7) RNA. Data are mean and s.e.m. e, The spatial amplitudes are averaged over all positions in the PSM. Comparison of new wild-type (silver, n = 24, N = 2) data obtained with a Nikon confocal microscope versus previously published data (dark grey, n = 18, N = 4) obtained by a Zeiss Apotome14. The box spans the interquartile range, line labels median, the whiskers extend to maximal and minimal observations. Difference assessed by two-sided independent t-test with Bonferroni correction, her1 P = 0.48; her7 P = 0.106. f, Comparison of histograms of total her RNA obtained by two different microscopes. g, Spatial Spearman correlation scores for wild-type embryos. Thick line denotes the median; thin black lines denote the 25th and 75th percentiles. h, Sequencing showing two base pairs deletion in the her1 coding sequence in her1ci301 her7hu2526 and her1ci302 fish. n is the number of embryos; N is the number of independent experiments.

Source data

Extended Data Fig. 2 Gene pairing boosts correlated transcription.

a, A her1b567/+ her7b567/+ embryo with oscillatory waves of her7 transcription. Scale bar, 30 μm. b, One of the chromosomes has a large deletion including the her1her7 locus. c, The boundaries of somite segments are marked by xirp2a in situ hybridization staining in sibling wild-type or heterozygous her1b567/+ her7b567/+ (top) and homozygous her1b567her7b567 (bottom) embryos. Scale bar, 100 μm. d, her1b567/+her7b567/+ embryos (n = 24, N = 2) have reduced spatial amplitude from wild-type (n = 14, N = 2) as assessed by two-sided Welch’s t-test with Bonferroni correction for her1 and the independent samples two-sided t-test for her7 (28% her1 amplitude t(13.6) = 2.6, *P = 0.04, 28% her7 amplitude t(18) = 5.3, ***P = 9.800 × 10−5). The box spans the interquartile range, line labels median, the whiskers extend to maximal and minimal observations. e, The histogram of total her (her1+her7) RNA per cell is plotted in wild-type (grey) and her1b567/+her7b567/+ (blue) embryos. her1b567/+her7b567/+embryos have 38% less total her mRNA than wild-type. f, Spatial Spearman correlation scores reflecting correlated expression of her1 and her7 in wild-type (grey) and her1b567/+her7b567/+ (blue) embryos as assessed by the two-sided Mann–Whitney U test (U = 392.5, z = −3.0, **P = 0.003). Median is the thick line, and 25% and 75% are thin black lines. g, The nascent transcription loci (dots) are detected in nuclei (blue) of cells located in a stripe-region in the anterior PSM of a her1b567/+her7b567/+ embryo. Scale bar, 5 μm. h, The histogram of the distance between two-closest loci in wild-type embryos. i, The histogram of the distance between two-closest loci in her1b567/+her7b567/+ embryos. n is the number of embryos; N is the number of independent experiments.

Source data

Extended Data Fig. 3 Spatial Pearson correlation scores for all genotypes.

a, Spatial Pearson correlation scores of her1 and her7 in wild-type (dark grey), her1ci301/+her7hu2526/+ (silver), her1ci301her7hu2526 (red) embryos differences are assessed by the two-sided Mann–Whitney U test with Bonferroni correction (wild-type, her1ci301/+her7hu2526/+, U = 615, z = −2.3, P = 0.072; wild-type, her1ci301 her7hu2526, U = 576, z = −5.0, ***P = 1.652 × 10−6; her1ci301/+her7hu2526/+, her1ci301her7hu2526, U = 841, z = −1.3, P = 0.546). b, Spatial Pearson correlation scores of her1 and her7 in wild-type (grey) and her1b567/+ her7b567/+ (blue) embryos (U = 410, z = −2.8, **P = 0.005). c, Spatial Pearson correlation scores of her1 and her7 in wild-type embryos raised at 21.5 °C or 28 °C (U = 772, z = −2.23, *P = 0.026). d, Spatial Pearson correlation scores for gene-paired and gene-unpaired embryos raised at 21.5 °C (U = 1039, z = −4.6, ***P = 4.000 × 10−6). ad, Differences in Pearson correlation scores are assessed by the two-sided Mann–Whitney U test. Thick line denotes the median; thin black lines denote the 25th and 75th percentiles.

Source data

Extended Data Fig. 4 Simulation of alternative scenarios.

a, b, Scenario 2. a, Average transcription firing rates of her1 and her7 were kept constant. But, at each incidence of firing, the firing rate for each gene was separately and randomly chosen from a distribution that has the same average rate. b, Spatial Spearman correlation score of her1 and her7 expression over time (U = 3, z = −6.6, ***P = 3.879 × 10−11). Differences in Spearman correlation scores are assessed by the two-sided Mann–Whitney U test. c, d, Scenario 3. c Transcription and RNA degradation rates of her7 were set to 50% higher than those of her1 which led to similar average RNA numbers of two genes. d, Spatial Spearman correlation score of her1 and her7 expression over time (U = 0, z = −6.7, ***P = 2.872 × 10−11). Differences in Spearman correlation scores are assessed by the two-sided Mann–Whitney U test. Thick line denotes the median; thin black lines denote the 25th and 75th percentiles.

Source data

Supplementary information

Reporting Summary

Supplementary Table

Supplementary Table 1: Excel file of single molecule FISH (smFISH) data for wild-type embryos. The file includes cell positions, her1 and her7 expression spot count in that cell, cell volume. Each sheet is one embryo, there are 24 sheets in total.

Supplementary Table

Supplementary Table 2: Excel file of smFISH data for her1ci301/+ her7 hu2526/+ embryos. The file includes cell position, her1 and her7 expression spot count in that cell, cell volume. Each sheet is one embryo, there are 18 sheets in total.

Supplementary Table

Supplementary Table 3: Excel file of smFISH data for her1ci301 her7 hu2526 embryos. The file includes cell position, her1 and her7 expression spot count in that cell, cell volume. Each sheet is one embryo, there are 28 sheets in total.

Supplementary Table

Supplementary Table 4: Excel file of smFISH data for wild-type sibling of her1b567/+ her7b567/+ embryos. The file includes cell position, her1 and her7 expression spot count in that cell, cell volume. Each sheet is one embryo, there are 14 sheets in total.

Supplementary Table

Supplementary Table 5: Excel file of smFISH data for her1b567/+ her7b567/+ embryos. The file includes cell position, her1 and her7 expression spot count in that cell, cell volume. Each sheet is one embryo, there are 24 sheets in total.

Supplementary Table

Supplementary Table 6: Excel file of smFISH data for wild-type embryos grown at 28 °C. The file includes cell position, her1 and her7 expression spot count in that cell, cell volume. Each sheet is one embryo, there are 23 sheets in total.

Supplementary Table

Supplementary Table 7: Excel file of smFISH data for wild-type embryos grown at 21.5 °C. The file includes cell position, her1 and her7 expression spot count in that cell, cell volume. Each sheet is one embryo, there are 23 sheets in total.

Supplementary Table

Supplementary Table 8: The scores of segmentation defects.

Supplementary Table

Supplementary Table 9: Excel file of smFISH data for gene-paired embryos grown at 21.5 °C. The file includes cell position, her1 and her7 expression spot count in that cell, cell volume. Each sheet is one embryo, there are 27 sheets in total.

Supplementary Table

Supplementary Table 10: Excel file of smFISH data for gene-unpaired embryos grown at 21.5 °C. The file includes cell position, her1 and her7 expression spot count in that cell, cell volume. Each sheet is one embryo, there are 37 sheets in total.

Supplementary Table

Supplementary Table 11: The list of primers and smFISH probes.

Supplementary Table

Supplementary Table 12: The list of molecules simulated in the model.

Supplementary Table

Supplementary Table 13: The list of the reaction rates used in the model. The mRNA degradation rates mdh1 and mdh7 were changed in different model scenarios.

Supplementary Table

Supplementary Table 14: The list of the reactions simulated in the model. The propensity for each reaction is calculated as shown in the last column. For the gene-paired mutant, we changed the first and second reactions to c1->c1+mh1+mh7 and c2->c2, respectively. For model scenario 1, her1/her7 transcription led to one her1 and one her7 mRNA molecules. For scenario 2, her1/her7 transcription led to 1-2 (randomly chosen for each gene in each firing) her1 and her7 mRNA molecules. For scenario 3, her1 and her7 transcription led to 2 and 3 mRNA molecules, respectively.

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Zinani, O.Q.H., Keseroğlu, K., Ay, A. et al. Pairing of segmentation clock genes drives robust pattern formation. Nature 589, 431–436 (2021). https://doi.org/10.1038/s41586-020-03055-0

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