Micro RNAs (miRNAs) are ∼22 nucleotide single-stranded noncoding RNA molecules that bind to target messenger RNAs (mRNAs) and silence their expression. This Essay explores the importance of miRNAs in animal development and their possible roles in disease and evolution.
Regulatory DNAs serve as templates to bring weakly interacting transcription factors into close proximity so they can work synergistically to switch genes on and off in time and space. Most of these regulatory DNAs are enhancers that can work over long distances — a million base pairs or more in mammals — to control gene expression. Critical enhancers are sometimes even found within the introns of neighboring genes. This review summarizes well-defined examples of enhancers controlling key processes in animal development. Potential mechanisms of transcriptional synergy are discussed with regard to enhancer structure and contemporary ChIP-sequencing assays, whereby just a small fraction of the observed binding sites represent regulatory DNAs. Finally, there is a discussion of how enhancer evolution can produce novelty in animal morphology and of the prospects for reconstructing transitions in animal evolution by introducing derived enhancers in basal ancestors.
The synthesis of gene expression data and cis-regulatory analysis permits the elucidation of genomic regulatory networks. These networks provide a direct visualization of the functional interconnections among the regulatory genes and signaling components leading to cell-specific patterns of gene activity. Complex developmental processes are thereby illuminated in ways not revealed by the conventional analysis of individual genes. In this review, we describe emerging networks in several different model systems, and compare them with the gene regulatory network that controls dorsoventral patterning of the Drosophila embryo.
Recording light-microscopy images of large, nontransparent specimens, such as developing multicellular organisms, is complicated by decreased contrast resulting from light scattering. Early zebrafish development can be captured by standard light-sheet microscopy, but new imaging strategies are required to obtain high-quality data of late development or of less transparent organisms. We combined digital scanned laser light-sheet fluorescence microscopy with incoherent structured-illumination microscopy (DSLM-SI) and created structured-illumination patterns with continuously adjustable frequencies. Our method discriminates the specimen-related scattered background from signal fluorescence, thereby removing out-of-focus light and optimizing the contrast of in-focus structures. DSLM-SI provides rapid control of the illumination pattern, exceptional imaging quality and high imaging speeds. We performed long-term imaging of zebrafish development for 58 h and fast multiple-view imaging of early Drosophila melanogaster development. We reconstructed cell positions over time from the Drosophila DSLM-SI data and created a fly digital embryo.
Channelrhodopsin-2 (ChR2) is widely used for rapid photodepolarization of neurons, yet, as it requires high-intensity blue light for activation, it is not suited for long-term in vivo applications, e. g. for manipulations of behavior, or photoactivation of neurons during development. We used "slow" ChR2 variants with mutations in the C128 residue, that exhibit delayed off-kinetics and increased light sensitivity in Caenorhabditis elegans. Following a 1 s light pulse, we could photodepolarize neurons and muscles for minutes (and with repeated brief stimulation, up to days) with low-intensity light. Photoactivation of ChR2(C128S) in command interneurons elicited long-lasting alterations in locomotion. Finally, we could optically induce profound changes in animal development: Long-term photoactivation of ASJ neurons, which regulate larval growth, bypassed the constitutive entry into the "dauer" larval state in daf-11 mutants. These lack a guanylyl cyclase, which possibly renders ASJ neurons hyperpolarized. Furthermore, photostimulated ASJ neurons could acutely trigger dauer-exit. Thus, slow ChR2s can be employed to long-term photoactivate behavior and to trigger alternative animal development.