Single-molecule imaging correlates decreasing nuclear volume with increasing TF-chromatin associations during zebrafish development

Abstract

Zygotic genome activation (ZGA), the onset of transcription after initial quiescence, is a major developmental step in many species, which occurs after ten cell divisions in zebrafish embryos. How transcription factor (TF)-chromatin interactions evolve during early development to support ZGA is largely unknown. We establish single molecule tracking in live developing zebrafish embryos using reflected light-sheet microscopy to visualize two fluorescently labeled TF species, mEos2-TBP and mEos2-Sox19b. We further develop a data acquisition and analysis scheme to extract quantitative information on binding kinetics and bound fractions during fast cell cycles. The chromatin-bound fraction of both TFs increases during early development, as expected from a physical model of TF-chromatin interactions including a decreasing nuclear volume and increasing DNA accessibility. For Sox19b, data suggests the increase is mainly due to the shrinking nucleus. Our single molecule approach provides quantitative insight into changes of TF-chromatin associations during the developmental period embracing ZGA.

Fig. 1

TF binding and zygotic genome activation. a Sketch of zygotic mRNA levels as a function of embryo stage. Inset: schemes of embryos at different developmental stages. b Scheme of TF binding to chromatin: free TFs associate with rate constant k_on to and dissociate with rate constant k_off from an apparent number of chromatin binding sites D₀. The concentrations of all species involved depend on their quantities and the nuclear volume V_n. Inset: formula describing the chromatin-bound fraction of TFs that does not depend on the concentration of TFs

Fig. 2

Single molecule imaging in live zebrafish embryos by RLSM. a Sketch of reflected light-sheet microscopy of a zebrafish embryo. Inset: close-up view of a single cell within the embryo. b Sketch of mEos2-TBP and mEos2-Sox19b fusion proteins. mRNA coding for the constructs was injected into the 1-cell stage embryo. c Western blot analysis of TBP in wild type (wt) embryos (left lane) and injected with mEos2-TBP (right lane). γ-tubulin was used as loading control. Expected sizes are 50 kDa (γ-tubulin), 33 kDa (wt-TBP) and 59.4 kDa (mEos2-TBP). d ChIP-qPCR analysis of 2xHA-mEos2-TBP (blue, 3 technical replicates) binding to chromatin in the promoter region of genes hmga1a, brd2a and a genomic control and corresponding IgG controls. Values are given in % of input (mean ± s.e.m). e ChIP-qPCR analysis of 2xHA-mEos2-Sox19b (red, 3 technical replicates) binding to chromatin in the promoter region of genes dusp6, apoeb, pcdh18a and a genomic control and corresponding IgG controls. Values are given in % of input (mean ± s.e.m.). f Effect of mEos2-TBP and mEos2-Sox19b expression in embryos grown at 22 °C (up to 6-somite stage) or at 25 °C until 24 hpf. The scale bar is 100 µm up to the 6-somite stage and 1 mm at 24 hpf. g Fluorescence images of a 64-cell stage embryo and an oblong stage embryo expressing mEos2-TBP. The surface of the animal cap and the outline of the nucleus are indicated (white lines). Red arrows point to single mEos2-TBP molecules. The scale bar is 10 µm. h Time traces of mEos2-fluorescence of the molecules indicated in g. i Signal-to-noise ratio of single nuclear mEos2-TBP molecules as a function of nucleus distance from the surface of the animal cap. Values are calculated from >200 detected molecules within each nucleus (data from 3 embryos, mean ± s.e.m.)

Fig. 3

mEos2-TFs exhibit two dissociation rate constants. a Scheme of time-lapse microscopy. Red spheres indicate a detected mEos2-TF molecule. τ_on : time laser is on, τ_off : time laser is off, τ_tl : time-lapse time. b Schematic decay of visible fluorescence over time under short (blue), intermediate (magenta) and long (red) time-lapse intervals indicated by the horizontal lines. Inset: Measured mEos2-TBP molecules still bound after 4 short (left), 2 intermediate (middle) and 2 long (right) time-lapse intervals demonstrating effects of photobleaching (left) and dissociation (right). Starting number were 100 molecules. The scale bar is 5 µm. c, d Histograms of fluorescent “on” times at different time-lapse conditions of c mEos2-TBP and d mEos2-Sox19b in nuclei of oblong embryos (mean ± s.d.). Time-lapse intervals are written on top of the data points. Lines: global fit of a bi-exponential decay model (Equation 1 in Methods). Data includes 4780 molecules from 15 embryos for mEos2-TBP and 3330 molecules from 4 embryos for mEos2-Sox19b. For clarity, only the first six points of histograms are shown. e, f Residence times of e mEos2-TBP and f mEos2-Sox19b. Errors are the s.d. from the fits in c and d

Fig. 4

mEos2-TFs bind according to the law of mass action. a Scheme of interlaced time-lapse microscopy. Red spheres indicate a detected mEos2-TF molecule. τ_on : time laser is on, τ_off : time laser is off. b Schematic decay of visible fluorescence over time for the short (blue) and the long (red) bound fraction of molecules. c, d Number of molecules classified as bound compared to all detected molecules at the indicated stages. Every dot represents one embryo injected with c mEos2-TBP and d mEos2-Sox19b. The panel includes in total 53504 molecules from 5 embryos (64-cell stage) and 6 embryos (later stages) for mEos2-TBP and 76407 molecules from 5 embryos for mEos2-Sox19b. Insets: time-projection of all molecules within a nucleus classified as unbound (blue), short bound (magenta) and long bound (red). The scale bar is 5 µm

Fig. 5

The chromatin-bound fractions of mEos2-TFs increase during early development. a Chromatin-bound fraction of mEos2-TBP (blue) and mEos2-Sox19b (red) during development (calculated from data presented in Fig. 4 and Supplementary Figure 11, mean ± s.e.m). Lines represent the chromatin-bound fraction as calculated from the law of mass action using data from b and c and extracting the apparent number of chromatin binding sites as free parameter. Shades represent error intervals propagated from the errors in stable bound proportion and nuclear volume. The panel includes in total 120572 molecules from 5 embryos (64-cell stage) and 6 embryos (later stages) for mEos2-TBP and 158912 molecules from 5 embryos for mEos2-Sox19b. b Stable bound proportion of chromatin-bound mEos2-TBP (blue) and mEos2-Sox19b (red) molecules during development (calculated from data presented in Fig. 4 and Supplementary Figure 11, mean ± s.e.m.). The panel includes in total 5677 molecules from 3 embryos (64-cell stage), 4 embryos (128-cell stage), 5 embryos (256-cell stage) and 6 embryos (later stages) for mEos2-TBP and 13103 molecules from 5 embryos for mEos2-Sox19b. The dashed line is given as guide to the eye. c Nuclear volume of mEos2-TBP (blue spheres) and mEos2-Sox19b (red triangles) injected embryos during development. Data includes 5, 16, 22, 25, 32, 37, 84 measurements from 3 embryos (64-cell stage) and 4 embryos (later stages) for mEos2-TBP and 17, 21, 33, 60, 42, 90, 98 measurements from 5 embryos for mEos2-Sox19b (mean ± s.e.m. mean values for single embryos shown as open circles for TBP and open triangles for Sox19b). The dashed line is given as guide to the eye. d Apparent number of chromatin binding sites of mEos2-TBP (blue) and mEos2-Sox19b (red) as calculated from the law of mass action using data from a, b, and c. Shades represent error intervals propagated from the errors in chromatin-bound fraction, stable bound proportion and nuclear volume. e Scheme of the concentration model. During early embryo development the size of individual nuclei decreases, thereby increasing the concentration of DNA and increasing the chromatin-bound fraction of TFs