The Endoderm Plays an Important Role in Patterning the Segmented Pharyngeal Region in Zebrafish (Danio rerio)

AbstractThe development of the vertebrate head is a highly complex process involving tissues derived from all three germ layers. The endoderm forms pharyngeal pouches, the paraxial mesoderm gives rise to endothelia and muscles, and the neural crest cells, which originate from the embryonic midbrain and hindbrain, migrate ventrally to form cartilage, connective tissue, sensory neurons, and pigment cells. All three tissues form segmental structures: the hindbrain compartmentalizes into rhombomeres, the mesoderm into somitomeres, and the endoderm into serial gill slits. It is not known whether the different segmented tissues in the head develop by the same molecular mechanism or whether different pathways are employed. It is also possible that one tissue imposes segmentation on the others. Most recent studies have emphasized the importance of neural crest cells in patterning the head. Neural crest cells colonize the segmentally arranged arches according to their original position in the brain and convey positional information from the hindbrain into the periphery. During the screen for mutations that affect embryonic development of zebrafish, one mutant, called van gogh (vgo), in which segmentation of the pharyngeal region is absent, was isolated. In vgo, even though hindbrain segmentation is unaffected, the pharyngeal endoderm does not form reiterated pouches and surrounding mesoderm is not patterned correctly. Accordingly, migrating neural crest cells initially form distinct streams but fuse when they reach the arches. This failure to populate distinct pharyngeal arches is likely due to the lack of pharyngeal pouches. The results of our analysis suggest that the segmentation of the endoderm occurs without signaling from neural crest cells but that tissue interactions between the mesendoderm and the neural crest cells are required for the segmental appearance of the neural crest-derived cartilages in the pharyngeal arches. The lack of distinct patches of neural crest cells in the pharyngeal region is also seen in mutants of one-eyed pinhead and casanova, which are characterized by a lack of endoderm, as well as defects in mesodermal structures, providing evidence for the important role of the endoderm and mesoderm in governing head segmentation

Rapid mapping of visual receptive fields by filtered back-projection: application to multi-neuronal electrophysiology and imaging

Neurons in the visual system vary widely in the spatiotemporal properties of their receptive fields (RFs), and understanding these variations is key to elucidating how visual information is processed. We present a new approach for mapping RFs based on the filtered back projection (FBP), an algorithm used for tomographic reconstructions. To estimate RFs, a series of bars were flashed across the retina at pseudo‐random positions and at a minimum of five orientations. We apply this method to retinal neurons and show that it can accurately recover the spatial RF and impulse response of ganglion cells recorded on a multi‐electrode array. We also demonstrate its utility for in vivo imaging by mapping the RFs of an array of bipolar cell synapses expressing a genetically encoded Ca2+ indicator. We find that FBP offers several advantages over the commonly used spike‐triggered average (STA): (i) ON and OFF components of a RF can be separated; (ii) the impulse response can be reconstructed at sample rates of 125 Hz, rather than the refresh rate of a monitor; (iii) FBP reveals the response properties of neurons that are not evident using STA, including those that display orientation selectivity, or fire at low mean spike rates; and (iv) the FBP method is fast, allowing the RFs of all the bipolar cell synaptic terminals in a field of view to be reconstructed in under 4 min. Use of the FBP will benefit investigations of the visual system that employ electrophysiology or optical reporters to measure activity across populations of neurons

Effect of lighting conditions on zebrafish growth and development

In the underwater environment, the properties of light (intensity and spectrum) change rapidly with depth and water quality. In this article, we have described how and to what extent lighting conditions can influence the development, growth, and survival of zebrafish. Fertilized eggs and the corresponding larvae were exposed to different visible light wavelengths (violet, blue, green, yellow, red, and white) in a 12-h light-12-h dark (LD) cycle until 30 days posthatching (dph), when the expression of morphometric parameters and growth (igf1a, igf2a)- and stress-related (crh and pomca) genes were examined. Another group of larvae was raised under constant darkness (DD) until 5 or 10 dph, after which they were transferred to a LD of white light. A third group remained under DD to investigate the effects of light deprivation upon zebrafish development. The results revealed that the hatching rate was highest under blue and violet light, while total length at 30 dph was greatest under blue, white, and violet light. Red light led to reduced feeding activity and poor survival (100% mortality). Larvae raised under constant white light (LL) showed a higher proportion of malformations, as did larvae raised under LD violet light. The expression of growth and stress factors was upregulated in the violet (igf1a, igf2a, pomca, and chr) and blue (igf2a) groups, which is consistent with the higher growth recorded and the higher proportion of malformations detected under the violet light. All larvae kept under DD died before 18 dph, but the survival rates improved in larvae transferred to LD at 5 dph and at 10 dph. In summary, these findings revealed that lighting conditions are crucial factors influencing zebrafish larval development and growth

Optimizing information flow in small genetic networks. II: Feed forward interactions

Central to the functioning of a living cell is its ability to control the readout or expression of information encoded in the genome. In many cases, a single transcription factor protein activates or represses the expression of many genes. As the concentration of the transcription factor varies, the target genes thus undergo correlated changes, and this redundancy limits the ability of the cell to transmit information about input signals. We explore how interactions among the target genes can reduce this redundancy and optimize information transmission. Our discussion builds on recent work [Tkacik et al, Phys Rev E 80, 031920 (2009)], and there are connections to much earlier work on the role of lateral inhibition in enhancing the efficiency of information transmission in neural circuits; for simplicity we consider here the case where the interactions have a feed forward structure, with no loops. Even with this limitation, the networks that optimize information transmission have a structure reminiscent of the networks found in real biological systems

Effects of Cyclin Dependent Kinase 9 inhibition on zebrafish larvae

CDK9 is a known regulator of cellular transcription, growth and proliferation. Small molecule inhibitors are currently being developed and assessed in clinical trials as anti-cancer drugs. The zebrafish embryo provides an ideal model to explore the effects of CDK9 inhibition in-vivo. This has not been adequately explored previously at the level of a whole organism. We have compared and contrasted the effects of pharmacological and molecular inhibition of CDK9 on somatic growth, apoptosis and cellular proliferation in zebrafish larvae between 0 to 120 hours post fertilisation (hpf) using flavopiridol, a selective CDK9 antagonist, and CDK9-targeting morpholino. We demonstrate that the inhibition of CDK9 diminishes cellular proliferation and increases apoptosis. Subsequently, it affects somatic growth and development of a number of key embryonic structures including the brain, heart, eye and blood vessels. For the first time, we have localized CDK9 at a subcellular level in whole-mounted larvae. This works shows, at a high-throughput level, that CDK9 clearly plays a fundamental role in early cellular growth and proliferation

The Drosophila Mitochondrial Translation Elongation Factor G1 Contains a Nuclear Localization Signal and Inhibits Growth and DPP Signaling

Mutations in the human mitochondrial elongation factor G1 (EF-G1) are recessive lethal and cause death shortly after birth. We have isolated mutations in iconoclast (ico), which encodes the highly conserved Drosophila orthologue of EF-G1. We find that EF-G1 is essential during fly development, but its function is not required in every tissue. In contrast to null mutations, missense mutations exhibit stronger, possibly neomorphic phenotypes that lead to premature death during embryogenesis. Our experiments show that EF-G1 contains a secondary C-terminal nuclear localization signal. Expression of missense mutant forms of EF-G1 can accumulate in the nucleus and cause growth and patterning defects and animal lethality. We find that transgenes that encode mutant human EF-G1 proteins can rescue ico mutants, indicating that the underlying problem of the human disease is not just the loss of enzymatic activity. Our results are consistent with a model where EF-G1 acts as a retrograde signal from mitochondria to the nucleus to slow down cell proliferation if mitochondrial energy output is low

The role of the segmentation gene hairy in Tribolium

Hairy stripes in Tribolium are generated during blastoderm and germ band extension, but a direct role for Tc-h in trunk segmentation was not found. We have studied here several aspects of hairy function and expression in Tribolium, to further elucidate its role. First, we show that there is no functional redundancy with other hairy paralogues in Tribolium. Second, we cloned the hairy orthologue from Tribolium confusum and show that its expression mimics that of Tribolium castaneum, implying that stripe expression should be functional in some way. Third, we show that the dynamics of stripe formation in the growth zone is not compatible with an oscillatory mechanism comparable to the one driving the expression of hairy homologues in vertebrates. Fourth, we use parental RNAi experiments to study Tc-h function and we find that mandible and labium are particularly sensitive to loss of Tc-h, reminiscent of a pair-rule function in the head region. In addition, lack of Tc-h leads to cell death in the gnathal region at later embryonic stages, resulting in a detachment of the head. Cell death patterns are also altered in the midline. Finally, we have analysed the effect of Tc-h knockdown on two of the target genes of hairy in Drosophila, namely fushi tarazu and paired. We find that the trunk expression of Tc-h is required to regulate Tc-ftz, although Tc-ftz is itself also not required for trunk segmentation in Tribolium. Our results imply that there is considerable divergence in hairy function between Tribolium and Drosophila

Information transmission in genetic regulatory networks: a review

Genetic regulatory networks enable cells to respond to the changes in internal and external conditions by dynamically coordinating their gene expression profiles. Our ability to make quantitative measurements in these biochemical circuits has deepened our understanding of what kinds of computations genetic regulatory networks can perform and with what reliability. These advances have motivated researchers to look for connections between the architecture and function of genetic regulatory networks. Transmitting information between network's inputs and its outputs has been proposed as one such possible measure of function, relevant in certain biological contexts. Here we summarize recent developments in the application of information theory to gene regulatory networks. We first review basic concepts in information theory necessary to understand recent work. We then discuss the functional complexity of gene regulation which arrises from the molecular nature of the regulatory interactions. We end by reviewing some experiments supporting the view that genetic networks responsible for early development of multicellular organisms might be maximizing transmitted 'positional' information.Comment: Submitted to J Phys: Condens Matter, 31 page

Network Evolution of Body Plans

Segmentation in arthropod embryogenesis represents a well-known example of body plan diversity. Striped patterns of gene expression that lead to the future body segments appear simultaneously or sequentially in long and short germ-band development, respectively. Regulatory genes relevant for stripe formation are evolutionarily conserved among arthropods, therefore the differences in the observed traits are thought to have originated from how the genes are wired. To reveal the basic differences in the network structure, we have numerically evolved hundreds of gene regulatory networks that produce striped patterns of gene expression. By analyzing the topologies of the generated networks, we show that the characteristics of stripe formation in long and short germ-band development are determined by Feed-Forward Loops (FFLs) and negative Feed-Back Loops (FBLs) respectively. Network architectures, gene expression patterns and knockout responses exhibited by the artificially evolved networks agree with those reported in the fly Drosophila melanogaster and the beetle Tribolium castaneum. For other arthropod species, principal network architectures that remain largely unknown are predicted.Comment: 35 pages, 4 figures and 1 tabl

A Software Tool to Model Genetic Regulatory Networks. Applications to the Modeling of Threshold Phenomena and of Spatial Patterning in Drosophila

We present a general methodology in order to build mathematical models of genetic regulatory networks. This approach is based on the mass action law and on the Jacob and Monod operon model. The mathematical models are built symbolically by the Mathematica software package GeneticNetworks. This package accepts as input the interaction graphs of the transcriptional activators and repressors of a biological process and, as output, gives the mathematical model in the form of a system of ordinary differential equations. All the relevant biological parameters are chosen automatically by the software. Within this framework, we show that concentration dependent threshold effects in biology emerge from the catalytic properties of genes and its associated conservation laws. We apply this methodology to the segment patterning in Drosophila early development and we calibrate the genetic transcriptional network responsible for the patterning of the gap gene proteins Hunchback and Knirps, along the antero-posterior axis of the Drosophila embryo. In this approach, the zygotically produced proteins Hunchback and Knirps do not diffuse along the antero-posterior axis of the embryo of Drosophila, developing a spatial pattern due to concentration dependent thresholds. This shows that patterning at the gap genes stage can be explained by the concentration gradients along the embryo of the transcriptional regulators