The segmentation clock drives oscillatory gene expression across the pre-somitic mesoderm (PSM). Dynamic Notch activity is key to this process. We use imaging and computational analyses to extract temporal dynamics from spatial expression data to demonstrate that Delta ligand and Notch receptor expression oscillate in the vertebrate PSM.
During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a strict species-specific periodicity. The periodicity of the process is regulated by a molecular oscillator, known as the “segmentation clock,” acting in the PSM cells. This clock drives the oscillatory patterns of gene expression across the PSM in a posterior-anterior direction. These so-called clock genes are key components of three signaling pathways: Wnt, Notch, and fibroblast growth factor (FGF). In addition, Notch signaling is essential for synchronizing intracellular oscillations in neighboring cells. We recently gained insight into how this may be mechanistically regulated. Upon ligand activation, the Notch receptor is cleaved, releasing the intracellular domain (NICD), which moves to the nucleus and regulates gene expression. NICD is highly labile, and its phosphorylation-dependent turnover acts to restrict Notch signaling. The profile of NICD production (and degradation) in the PSM is known to be oscillatory and to resemble that of a clock gene. We recently reported that both the Notch receptor and the Delta ligand, which mediate intercellular coupling, themselves exhibit dynamic expression at both the mRNA and protein levels. In this article, we describe the sensitive detection methods and detailed image analysis tools that we used, in combination with the computational modeling that we designed, to extract and overlay expression data from distinct points in the expression cycle. This allowed us to construct a spatio-temporal picture of the dynamic expression profile for the receptor, the ligand, and the Notch target clock genes throughout an oscillation cycle. Here, we describe the protocols used to generate and culture the PSM explants, as well as the procedure to stain for the mRNA or protein. We also explain how the confocal images were subsequently analyzed and temporally ordered computationally to generate ordered sequences of clock expression snapshots, hereafter defined as “kymographs,” for the visualization of the spatiotemporal expression of Delta-like1 (Dll1) and Notch1 throughout the PSM.
Somites are the first segments formed in the elongating body axis in developing vertebrate species and are the precursors of the spine, ribs, and dermis tissue, as well as of muscle and endothelial cells. During somitogenesis, epithelial somites form from the unsegmented presomitic mesoderm (PSM) (reviewed in Reference 1). This process is regulated by the "segmentation clock," which consists of a network of oscillatory genes and proteins, mostly belonging to the Notch signaling pathway. The segmentation clock consists of various negative feedback loops, which enable the pulsatile production of Notch activity within a single cell2 (reviewed in References 3 – 6). While the intracellular method of oscillation is well characterized, it is still largely unknown how these oscillations are coordinated across the PSM tissue. It has been recently shown, through both experimental and theoretical studies, that these oscillations are essential to the process of somitogenesis and that the Notch pathway plays a crucial role in the process of both segmentation and oscillatory gene expression7,8. However, it has been widely reported that Notch receptor 1 (Notch1) and Delta-like ligand (Dll)-1 have static gradients in the PSM9,10,11.
We hypothesized that Notch-dependent oscillations of the PSM segmentation clock depend upon the periodic activation of the main Notch pathway receptor and ligand, Notch1 and Dll1, respectively, across the mouse PSM. The conclusions of previous studies that reported a static rostral-caudal gradient of these proteins were due, we predict, to a lack of sensitivity in immunostaining techniques. They were therefore unable to detect low-level fluctuations of Dll1 and Notch1 in the caudal PSM.
We have devised a method to more closely examine these factors, combining experimental data with mathematical modeling to predict a mechanism by which the oscillations of the proteins of clock components are coordinated across the PSM12.
The overall goal of this method is to detect and quantify low-level, dynamic protein expression in the PSM and to map the expression profiles of proteins of interest according to the expression of the known clock gene, Lunatic fringe (Lfng). Since one cycle of the segmentation clock in the mouse embryo takes 2 h to complete, various samples are required to build a complete spatiotemporal profile of Dll1 and Notch1 protein expression during one Lfng oscillation in the PSM. We have thus developed this protocol to allow for the high-throughput detection of low-level protein expression in whole-mount, contralateral PSM explants. However, this technique can also be useful for studies that aim to characterize low-level protein dynamics within any embryonic tissue that can be split into contralateral halves.
All experiments were performed under project license number 6004219 in strict adherence to the Animals (Scientific Procedures) Act of 1986 and the UK Home Office Codes of Practice for the use of animals in scientific procedures.
1. PSM Explant Dissection
2. Immunohistochemistry of PSM Explants
3. Fluorescent In Situ Hybridization (FISH) of PSM Explants
4. Sample Preparation for Imaging
5. Post-acquisition Image Analysis
6. Temporal Ordering of Samples
This protocol permits the visualization of the spatiotemporal profile of a protein of interest alongside clock gene transcription in the mouse PSM12. For example, Dll1 (Figure 1A-C) and Notch1 (Figure 1D-F) protein expression are shown to oscillate out of synchrony with the nascent transcription of the Notch-regulated segmentation clock gene Lfng. Quantification of Dll1, Notch1, and Lfng(i) signal intensity in relation to the antero-posterior (AP) axis of the PSM (Figure 1G) reveals clear oscillatory expression dynamics for these targets (Figure 1H-J). The spatiotemporal profile of Dll1 and Notch1 protein expression throughout the clock cycle are clearly visualized and quantified using this protocol through the post-acquisition image analysis of high-resolution fixed tissue image data.
Figure 1: Spatio-temporal Visualization and Quantification of Dll1 and Notch1 Protein Expression Dynamics. (A-F) Pairs of explants from six E10.5 embryos (A-F) showing the spatial distribution of Dll1 protein (A-C) or Notch1 protein (D-F) in one half alongside the detection of Lfng pre-mRNA (Lfng(i)) in the corresponding contralateral half of each pair. Panels are arranged according to Phase 1 (A and D), Phase 2 (B and E), and Phase 3 (C and F) of the segmentation clock cycle, as determined by the spatial profile of Lfng(i) expression. The extent of the expression domains for Dll1 (green), Notch1 (red), and Lfng(i) (gray) along the antero-posterior axis of the PSM have been demarcated by color-coded bars. The dotted lines demarcate the positions of the most recently formed somite(s), the outer edges of the PSM, and the adjacent neural tissue (C and E). Scale bars (bottom left of each panel, A-F) represent 100 µm. (G) An example intensity plot depicting the axial variation in signal intensity across the PSM. The data is plotted from two explant pairs showing Lfng pre-mRNA (black hashed line) in one explant compared to Notch1 protein (red) in the contralateral explant (Embryo 1), as well as Lfng pre-mRNA (black solid line) in another explant compared to Dll1 protein (green) in the contralateral explant (Embryo 2). Measured signal intensity (y-axis) is plotted against axial position (x-axis; anterior PSM [A] to the right and posterior PSM [P] to the left). (H) A kymograph showing the spatial distribution of Dll1, Notch1, and Lfng(i) across numerous PSMs. Each row of the kymograph represents the signal intensity of an individual PSM explant. Rows are arranged in temporal sequence according to the spatiotemporal distribution of Lfng pre-mRNA (I) The spatiotemporal distribution of Dll1, Notch1, and Lfng(i) through multiple clock oscillations is simulated by the periodic extension of the data shown in (H), highlighting the oscillatory nature of Dll1 and Notch1 expression dynamics. (J) Pulsatile Notch1 protein expression in the caudal PSM is highlighted by magnification of the region demarcated in the virtual kymograph shown in (I). Modified from Reference 12. Please click here to view a larger version of this figure.
Figure 2: Quantification of the Spatio-temporal Dynamics of Dll1 and Notch1 Protein Expression. (A) An example intensity plot depicts axial variation in signal intensity across the PSM. Data plotted from two explant pairs showing Lfng pre-mRNA (black hashed line) in one explant compared to Notch1 protein (red) in the contralateral explant half, as well as Lfng pre-mRNA (black solid line) in a half explant from a second tail compared to Dll1 protein (green) in the contralateral explant half of the second tail. Measured intensities (y-axis) are plotted against axial position (x-axis; rostral [A] to the right and caudal [P] to the left). (B-H) Kymographs show the spatial distribution of Notch1, Dll1, NICD, and Lfng(i) across numerous PSMs. (B and C) NICD (B) and Dll1 (C) expression in PSM sections; (D and E) Lfng(i) (D) and Dll1 (E) in contralateral explant halves; (F and G) Lfng(i) (F) and Notch1 (G) in contralateral explant halves. From Reference 12. Please click here to view a larger version of this figure.
Critical Steps within the Protocol
The present protocol describes a sensitive method to perform the quantitative analysis of low-level protein expression and oscillatory dynamics in E10.5 mouse PSM explants. A robust protocol for both immunohistochemistry and fluorescent in situ hybridization (FISH) is followed by high-resolution whole-mount confocal imaging, and then by image analysis and temporal segmentation of kymographs to generate a spatiotemporal map of protein expression across the PSM. A high signal-to-noise ratio in protein and mRNA detection is essential to ensure the success of this technique. Care must be taken to thoroughly exchange all solutions effectively during the wash steps and to maintain the temperature of the 65 °C washes in the relevant stages of step 3. It is most advantageous to take the time to source efficacious antibodies and RNA probes against the targets of interest and to test these reagents thoroughly on whole-mount samples prior to starting this protocol.
Modifications and Troubleshooting
The main issues that may be encountered when performing this protocol arise from poor signal detection strength and quality. This is largely dependent upon the efficacy of the antibodies or RNA probes used for the immunohistochemistry or FISH steps in the protocol, respectively. A number of different steps may require optimization before adequate signal detection is achieved. One common cause for poor signal detection is improper fixation; it is imperative that either fresh PFA or PFA stored at 4 °C for no longer than one week is used to fix the samples. The length of fixation may also require optimization, depending on the antibody or RNA probe used. For antibodies, it is advised to follow the manufacturer's instructions where possible, while for RNA probes, we advise the consultation of the published literature.
In this study, we used an RNA probe that specifically detects the pre-mRNA of the clock gene Lfng. Due to its relative lack of abundance, detection of Lfng pre-mRNA requires a long period of incubation with the probe in hybridization mix containing 5x saline-sodium citrate (SSC) for good signal detection. The same conditions may apply to other probes that detect weakly expressed mRNAs, but in our experience, the detection of more stable mRNA targets may require a shorter probe hybridization step and lower SSC concentrations in the hybridization mix (e.g., 1.3x SSC). For both immunohistochemistry and FISH, the protocol must first be optimized on whole embryos, and the optimal concentration of antibody or probe must be determined empirically.
Limitations of the Technique
As mentioned above, the success of this technique is highly dependent upon the quality of the protein and mRNA detection. We have outlined several suggestions as to how protein and mRNA detection can be improved, but in the absence of high-quality fluorescent signal detection, there is no way the experiment can proceed. The number of protein targets that can be analyzed in each tissue sample is limited by the spectral resolution of the confocal microscope and by the epitopes of the antibodies used. In this study, we were able to use up to three epitopes for protein detection alongside a DNA stain on each sample12. This protocol only permits the detection of one mRNA target, although current alternative methods could be employed to increase this to up to three targets14.
Significance of the Technique with Respect to Existing/Alternative Methods
The method described here provides a sensitive technique to detect low-level protein fluctuations in whole-mount PSM explants. The quantification of these dynamics is possible by performing FISH for a known clock gene in corresponding contralateral explants. A library of kymographs is generated that can be organized over one segmentation clock cycle, highlighting the spatiotemporal expression dynamics of a target of interest within this time frame. A key difference in this technique over others is the use of computational automation to temporally order large data sets, which permits the spatiotemporal expression dynamics of novel clock components to be analyzed in an unbiased manner. For example, this technique provided insight into how Dll1 and Notch1 proteins and their oscillations are co-regulated across the entire PSM. Alternative methods in this context have also relied upon immunostaining, but they did not detect the small fluctuations in Dll1 and Notch1 protein levels in the caudal PSM that were evident using this method. Instead, they reported a steady gradient of expression that is strongest in the rostral region9,10,11. This could be due to the fact that this protocol has a lengthier primary antibody incubation period (3 – 5 days, as opposed to overnight), which may be required to detect lower levels of protein. As the levels of Dll1 and Notch1 expression are relatively high in the rostral PSM, this may have influenced the authors to image the samples at a lower exposure setting than would be necessary to detect the caudal protein expression. One further potential discrepancy arises from the use of unfixed tissue in the study by Chapman et al., in which the transitory expression of Dll1 and Notch1 in the caudal PSM may have been less well-preserved9.
Future Applications or Directions after Mastering the Technique
Once this protocol has been mastered, high-throughput expression analysis can be performed for any protein of interest in the PSM. PSM explants generated from several mouse litters can be processed at once to generate the high sample number necessary for analysis. Although we have only used wild-type embryos in these studies, it is possible to perform this analysis using genetically modified embryos in order to assess the importance of one or more factors on protein expression dynamics. Beyond the PSM, this protocol can be adapted to other systems that are composed of two contralateral halves and can be used to sensitively detect low-level protein expression and oscillatory dynamics. One example for which this protocol could be adapted is the study of dynamic protein expression in the mouse neural tube, since contralateral halves could be generated and cultured, and Notch activity has been shown to be both present and important for patterning15. We encourage other groups to adapt this protocol to other systems and to provide feedback for future improvement.
The authors have nothing to disclose.
This work was supported by an MRC studentship to RAB, an MRC studentship to CSLB, and a WT project grant to JKD (WT089357MA). The work was also supported by a Welcome Trust Strategic award (097945/Z/11/Z). We thank Dr. E. Kremmer for the kind gift of the Dll1 antibody and Dr. O. Pourquie for the Lfng RNA probe.
DMEM-F12 | Gibco (ThermoFisher Scientific) | 11320033 | |
GlutaMAX™-1 (100X) | Gibco (ThermoFisher Scientific) | 35050 | |
Fetal Bovine Serum, qualified, E.U.-approved, South America origin | Gibco (ThermoFisher Scientific) | 10270106 | |
Recombinant Human FGF-basic (154 a.a.) | Peprotech | 100-18B | |
Penicillin/Streptomycin | Gibco (ThermoFisher Scientific) | 15140122 | |
anti-mouse monoclonal Notch1 antibody^ | BD Pharmingen | 552466 | |
anti-rat polyclonal Dll1 antibody^* | N/A | N/A | |
Lfng intronic anti-sense RNA probe^* | N/A | N/A | |
16% paraformaldehyde | Pierce (ThermoFischer Scientific) | PI28908 | |
Proteinase K, recombinant, PCR grade | Roche (Sigma-Aldrich) | 31158 | |
Phosphate buffered saline (PBS), pH7.4 | Made in house | N/A | |
Triton-X 100 | Sigma-Aldrich | T8787 | |
Bovine serum albumin (BSA) | Sigma-Aldrich | 5470 | |
Normal goat serum (NGS) (heat-treated) | Gibco (ThermoFisher Scientific) | 16210072 | |
Hoechst 33342 | ThermoFischer Scientific | H3570 | |
Tween-20 | Sigma-Aldrich | P9416 | |
Ethanol | Sigma-Aldrich | 46139 | |
Glutaraldehyde | Sigma-Aldrich | 340855 | |
Formamide | Sigma-Aldrich | F9037 | |
Saline-sodium citrate (SSC) | Sigma-Aldrich | 93017 | |
EDTA | Sigma-Aldrich | 798681 | |
tRNA | Roche (Sigma-Aldrich) | 101095 | |
Heparin | Sigma-Aldrich | H3149 | |
Tris-buffered saline (TBS) | Made in house | N/A | |
Blocking Buffer Reagent | Roche (Sigma-Aldrich) | 11096176001 | |
anti-DIG horseradish peroxidase (HRP) conjugated antibody | Roche (Sigma-Aldrich) | 11207733910 | |
Tyramide signal amplification (TSA) kit | Perkin Elmer | NEL744001KT | |
*NOTE: The Dll1 antibody and RNA probe used in this study are not commercially available. Please see acknowledgements for sources. | |||
^Antibodies/RNA probes should be sourced which are applicable to the research interests of the reader. |