Repressive Gene Regulation Synchronizes Development with Cellular Metabolism.

Metabolic conditions affect the developmental tempo of animals. Developmental gene regulatory networks (GRNs) must therefore synchronize their dynamics with a variable timescale. We find that layered repression of genes couples GRN output with variable metabolism. When repressors of transcription or mRNA and protein stability are lost, fewer errors in Drosophila development occur when metabolism is lowered. We demonstrate the universality of this phenomenon by eliminating the entire microRNA family of repressors and find that development to maturity can be largely rescued when metabolism is reduced. Using a mathematical model that replicates GRN dynamics, we find that lowering metabolism suppresses the emergence of developmental errors by curtailing the influence of auxiliary repressors on GRN output. We experimentally show that gene expression dynamics are less affected by loss of repressors when metabolism is reduced. Thus, layered repression provides robustness through error suppression and may provide an evolutionary route to a shorter reproductive cycle.

miR-9a minimizes the phenotypic impact of genomic diversity by buffering a transcription factor.

Gene expression has to withstand stochastic, environmental, and genomic perturbations. For example, in the latter case, 0.5%-1% of the human genome is typically variable between any two unrelated individuals. Such diversity might create problematic variability in the activity of gene regulatory networks and, ultimately, in cell behaviors. Using multigenerational selection experiments, we find that for the Drosophila proneural network, the effect of genomic diversity is dampened by miR-9a-mediated regulation of senseless expression. Reducing miR-9a regulation of the Senseless transcription factor frees the genomic landscape to exert greater phenotypic influence. Whole-genome sequencing identified genomic loci that potentially exert such effects. A larger set of sequence variants, including variants within proneural network genes, exhibits these characteristics when miR-9a concentration is reduced. These findings reveal that microRNA-target interactions may be a key mechanism by which the impact of genomic diversity on cell behavior is dampened.

A microRNA imparts robustness against environmental fluctuation during development.

The microRNA miR-7 is perfectly conserved from annelids to humans, and yet some of the genes that it regulates in Drosophila are not regulated in mammals. We have explored the role of lineage restricted targets, using Drosophila, in order to better understand the evolutionary significance of microRNA-target relationships. From studies of two well characterized developmental regulatory networks, we find that miR-7 functions in several interlocking feedback and feedforward loops, and propose that its role in these networks is to buffer them against perturbation. To directly demonstrate this function for miR-7, we subjected the networks to temperature fluctuation and found that miR-7 is essential for the maintenance of regulatory stability under conditions of environmental flux. We suggest that some conserved microRNAs like miR-7 may enter into novel genetic relationships to buffer developmental programs against variation and impart robustness to diverse regulatory networks.

Hexagonal Patterning of the Insect Compound Eye: Facet Area Variation, Defects, and Disorder.

The regular hexagonal array morphology of facets (ommatidia) in the Drosophila compound eye is accomplished by regulation of cell differentiation and planar cell polarity during development. Mutations in certain genes disrupt regulation, causing a breakdown of this perfect symmetry, so that the ommatidial pattern shows onset of disorder in the form of packing defects. We analyze a variety of such mutants and compare them to normal (wild-type), finding that mutants show increased local variation in ommatidial area, which is sufficient to induce a significant number of defects. A model formalism based on Voronoi construction is developed to predict the observed correlation between ommatidium size variation and the number of defects, and to study the onset of disorder in this system with statistical tools. The model uncovers a previously unknown large-scale systematic size variation of the ommatidia across the eye of both wild-type and mutant animals. Such systematic variation of area, as well as its statistical fluctuations, are found to have distinct effects on eye disorder that can both be quantitatively modeled. Furthermore, the topological order is also influenced by the internal structure of the ommatidia, with cells of greater relative mechanical stiffness providing constraints to ommatidial deformation and thus to defect generation. Without free parameters, the simulation predicts the size-topology correlation for both wild-type and mutant eyes. This work develops formalisms of size-topology correlation that are very general and can be potentially applied to other cellular structures near the onset of disorder.

A comparative study of Pointed and Yan expression reveals new complexity to the transcriptional networks downstream of receptor tyrosine kinase signaling.

The biochemical regulatory network downstream of receptor tyrosine kinase (RTK) signaling is controlled by two opposing ETS family members: the transcriptional activator Pointed (Pnt) and the transcriptional repressor Yan. A bistable switch model has been invoked to explain how pathway activation can drive differentiation by shifting the system from a high-Yan/low-Pnt activity state to a low-Yan/high-Pnt activity state. Although the model explains yan and pnt loss-of-function phenotypes in several different cell types, how Yan and Pointed protein expression dynamics contribute to these and other developmental transitions remains poorly understood. Toward this goal we have used a functional GFP-tagged Pnt transgene (Pnt-GFP) to perform a comparative study of Yan and Pnt protein expression throughout Drosophila development. Consistent with the prevailing model of the Pnt-Yan network, we found numerous instances where Pnt-GFP and Yan adopt a mutually exclusive pattern of expression. However we also observed many examples of co-expression. While some co-expression occurred in cells where RTK signaling is presumed low, other co-expression occurred in cells with high RTK signaling. The instances of co-expressed Yan and Pnt-GFP in tissues with high RTK signaling cannot be explained by the current model, and thus they provide important contexts for future investigation of how context-specific differences in RTK signaling, network topology, or responsiveness to other signaling inputs, affect the transcriptional response.

MicroRNA miR-7 Regulates Secretion of Insulin-Like Peptides.

The insulin/insulin-like growth factor (IGF) pathway is essential for linking nutritional status to growth and metabolism. MicroRNAs (miRNAs) are short RNAs that are players in the regulation of this process. The miRNA miR-7 shows highly conserved expression in insulin-producing cells across the animal kingdom. However, its conserved functions in regulation of insulin-like peptides (ILPs) remain unknown. Using Drosophila as a model, we demonstrate that miR-7 limits ILP availability by inhibiting its production and secretion. Increasing miR-7 alters body growth and metabolism in an ILP-dependent manner, elevating circulating sugars and total body triglycerides, while decreasing animal growth. These effects are not due to direct targeting of ILP mRNA, but instead arise through alternate targets that affect the function of ILP-producing cells. The Drosophila F-actin capping protein alpha (CPA) is a direct target of miR-7, and knockdown of CPA in insulin-producing cells phenocopies the effects of miR-7 on ILP secretion. This regulation of CPA is conserved in mammals, with the mouse ortholog Capza1 also targeted by miR-7 in ß-islet cells. Taken together, these results support a role for miR-7 regulation of an actin capping protein in insulin regulation, and highlight a conserved mechanism of action for an evolutionarily ancient microRNA.

Differential Masking of Natural Genetic Variation by miR-9a in Drosophila.

Genetic variation is prevalent among individuals of the same species and yet the potential effects of genetic variation on developmental outcomes are frequently suppressed. Understanding the mechanisms that are responsible for this suppression is an important goal. Previously, we found that the microRNA miR-9a mitigates the impact of natural genetic variants that promote the development of scutellar bristles in adult Drosophila. Here we find that miR-9a does not affect the impact of genetic variants that inhibit the development of scutellar bristles. We show this using both directional and stabilizing selection in the laboratory. This specificity of action suggests that miR-9a does not interact with all functional classes of developmental genetic variants affecting sensory organ development. We also investigate the impact of miR-9a on a fitness trait, which is adult viability. At elevated physiological temperatures, miR-9a contributes to viability through masking genetic variants that hinder adult viability. We conclude that miR-9a activity in different developmental networks contributes to suppression of natural variants from perturbing development.

Silencing by small RNAs is linked to endosomal trafficking.

Small RNAs direct RNA-induced silencing complexes (RISCs) to regulate stability and translation of mRNAs. RISCs associated with target mRNAs often accumulate in discrete cytoplasmic foci known as GW-bodies. However, RISC proteins can associate with membrane compartments such as the Golgi and endoplasmic reticulum. Here, we show that GW-bodies are associated with late endosomes (multivesicular bodies, MVBs). Blocking the maturation of MVBs into lysosomes by loss of the tethering factor HPS4 (ref. 5) enhances short interfering RNA (siRNA)- and micro RNA (miRNA)-mediated silencing in Drosophila melanogaster and humans. It also triggers over-accumulation of GW-bodies. Blocking MVB formation by ESCRT (endosomal sorting complex required for transport) depletion results in impaired miRNA silencing and loss of GW-bodies. These results indicate that active RISCs are physically and functionally coupled to MVBs. We further show that MVBs promote the competence of RISCs in loading small RNAs. We suggest that the recycling of RISCs is promoted by MVBs, resulting in RISCs more effectively engaging with small RNA effectors and possibly target RNAs. It may provide a means to enhance the dynamics of RNA silencing in the cytoplasm.

Swimming of larval zebrafish: fin-axis coordination and implications for function and neural control.

Adult actinopterygian fishes typically perform steady forward swimming using either their pectoral fins or their body axis as the primary propulsor. In most species, when axial undulation is employed for swimming, the pectoral fins are tucked (i.e. adducted) against the body; conversely, when pectoral fins are beating, the body axis is held straight. In contrast to adults, larval fishes can combine their pectoral fin and body-axis movements during locomotion; however, little is known about how these locomotor modes are coordinated. With this study we provide a detailed analysis of the coordinated fin and axial movements during slow and fast swimming by examining forward locomotion in larval zebrafish (Danio rerio L.). In addition, we describe the musculature that powers pectoral fin movement in larval zebrafish and discuss its functional implications. As larvae, zebrafish alternate their pectoral fins during slow swimming (0.011+/-0.001 mm ms(-1)) in conjunction with axial undulations of the same frequency (18-28 Hz). During fast swimming (0.109+/-0.030 mm ms(-1); 36-67 Hz), the fins are tucked against the body and propulsion occurs by axial undulation alone. We show that during swimming, larval fishes can use a similar limb-axis coordination pattern to that of walking and running salamanders. We suggest that the fin-axis coordination observed in larval zebrafish may be attributed to a primitive neural circuit and that early terrestrial vertebrates may have gained the ability to coordinate limbs and lateral bending by retaining a larval central pattern generator for limb-axis coordination in the adult life history stage.