Enhancers can cause either periodic or non-periodic wavelike gene expression patterns to emerge from gene expression patterns driven by enhancers. Two of the enhancers discovered in this study, runB and hbA, produce gene expression waves which peak posteriorly and gradually fade as they propagate forwards.
Careful analysis of gene expression waves conforms with predictions made by the Enhancer Switching model.
Static Enhancers
Enhancers, non-coding elements that shape cell-type specific gene expression programs and responses to extracellular cues, account for most genetic variants associated with human diseases. Their activity is usually managed by multiple enhancers regulated by different transcription factors; their disruption often produces gene expression patterns with periodic or non-periodic wavelike structures.
Recent studies have demonstrated that the spatiotemporal dynamics of endogenous enhancers do not always match predictions from Speed Regulation model. More specifically, any gene has both dynamic and static enhancers which drive expression waves with different characteristics and drive expression waves asynchronously. The discovery of dynamic and static enhancers has enabled gene-regulatory experiments which probe spatiotemporal dynamics of enhancer activity at previously inaccessible timescales.
Careful analysis of these data reveals two characteristics of enhancer spatiotemporal dynamics. First, expression patterns driven by dynamic enhancer reporters often resemble total gene expression waves that move posterior to anterior; this phenomenon results from speed regulator activating and repressing effects on dynamic and static enhancers respectively.
Static enhancers exhibit a distinctive phenotype; their gene expression is independent of speed regulator presence, and does not fade with increasing speed. By identifying static enhancers we were able to study their role in Clark protocol waves by testing their effect on naive T cell polarization; deletion shifted towards pro-inflammatory T Helper 17 cells rather than regulatory T cells (TH17s).
Dynamic Enhancers
Static enhancers generate constant gene expression waves over time, while dynamic enhancers produce wavelike patterns with variable intensity and direction. Being able to recognize both types of enhancers is essential for understanding the genetic basis for both periodic and non-periodic wavelike patterning in AP morphology.
Tribolium castaneum, a short-germ insect believed to possess more ancestral methods of AP patterning than long-germ insects like Drosophila, exhibits dynamic wavelike gene expression patterns consistent with models in which morphogen gradients of “speed regulator” molecules provide periodic or nonperiodic wavelike gene expression patterns (El-Sherif et al. 2012a; Sarrazin et al. 2012; Zhu et al. 2017; Boos et al. 2018). These patterns correspond with predictions made by models in which “speed regulator” gradients drive either periodic or nonperiodic wavelike gene expression patterns (El-Sherif et al. 2012a).
The model suggests that dynamic enhancers initiate gene expression waves while static enhancers stop it at regions of stable gene expression. To test these predictions, Dr. David Huhlmann of University of Michigan developed an advanced system for tracking enhancer activity both live and fixed embryos; now used by authors to identify dynamic and static enhancers responsible for driving gene expression waves within embryos.
Careful analysis of in vivo gene expression patterns generated by dynamic and static enhancers revealed striking similarities in their spatiotemporal dynamics, in particular where expression driven by dynamic enhancers was maximum at posterior then gradually faded towards anterior as they propagated toward front. Dynamic enhancers were further distinguished by activating speed regulators while having a repressive effect on static enhancers.
Visualizing enhancer activity using in vivo methods may reveal only part of its dynamics. For example, fluorescent protein mCherry staining alone is only capable of showing aggregate enhancer activity – not individual enhancer activity – making these methods limited. To overcome this limitation of these methods and track individual enhancer activities over time more precisely. This limitation was overcome in this study using more sensitive reporter genes such as yellow fluorescent protein encoded gene to detect enhancer activity both fixed and live embryos as well as rapidly degradable fluorescent tag on mCherry reporter that allowed authors to follow individual enhancer activities over time over time.
In Situ Staining
In situ hybridization (or in situ staining) is a technique for visualizing specific DNA and RNA sequences within tissue samples, using either FISH (Fluorescent in situ hybridization) or CISH (Chromogenic in situ hybridization). Two main forms of in situ staining include FISH (Fluorescent in situ hybridization) and CISH (Chromogenic in situ hybridization). Staining involves adding nucleotide probes labelled with radioactive isotope labeling or fluorescent microscopy to detect the DNA or RNA within samples before autoradiography for radioactively labeled probes or fluorescent microscopy detect the DNA/RNA presence using either autoradiography for radioactive probes or fluorescent microscopy for non-isotope labeled probes or fluorescent microscopy. Staining also plays an integral part in Clark Protocol wave genetics as it allows detection of non-periodic wavelike patterns of gene expression which occur without the influence of morphogen gradient.
Reporter Genes
Reporter genes are genetic markers that can be added to an otherwise obscure gene to make its expression visible, such as fluorescent proteins or enzymes that convert invisible substrates into luminescent or fluorescent products. They have become widespread tools in molecular biology because of their easy-to-see signals used for studying regulatory sequences without overexpression or complex genetic manipulations; popular reporters include Luciferase, GFP and b-galactosidase which come standard plasmid constructs for cloning and expression.
The choice of reporter genes depends on whether your experiment will be qualitative or quantitative and which tissues or cells it will take place on. For instance, fluorescent protein yellow has an extended degradation delay which coincides with endogenous run expression early in Tribolium development, disrupting clarity in clark protocol wave data (see figure 4). On the other hand, GFP and mCherry both have very short degradation kinetics, so they may be better options to choose.
