Lateral Gene Expression in Drosophila Early Embryos Is Supported by Grainyhead-Mediated Activation and Tiers of Dorsally-Localized Repression

The general consensus in the field is that limiting amounts of the transcription factor Dorsal establish dorsal boundaries of genes expressed along the dorsal-ventral (DV) axis of early Drosophila embryos, while repressors establish ventral boundaries. Yet recent studies have provided evidence that repressors act to specify the dorsal boundary of intermediate neuroblasts defective (ind), a gene expressed in a stripe along the DV axis in lateral regions of the embryo. Here we show that a short 12 base pair sequence (“the A-box”) present twice within the ind CRM is both necessary and sufficient to support transcriptional repression in dorsal regions of embryos. To identify binding factors, we conducted affinity chromatography using the A-box element and found a number of DNA-binding proteins and chromatin-associated factors using mass spectroscopy. Only Grainyhead (Grh), a CP2 transcription factor with a unique DNA-binding domain, was found to bind the A-box sequence. Our results suggest that Grh acts as an activator to support expression of ind, which was surprising as we identified this factor using an element that mediates dorsally-localized repression. Grh and Dorsal both contribute to ind transcriptional activation. However, another recent study found that the repressor Capicua (Cic) also binds to the A-box sequence. While Cic was not identified through our A-box affinity chromatography, utilization of the same site, the A-box, by both factors Grh (activator) and Cic (repressor) may also support a “switch-like” response that helps to sharpen the ind dorsal boundary. Furthermore, our results also demonstrate that TGF-β signaling acts to refine ind CRM expression in an A-box independent manner in dorsal-most regions, suggesting that tiers of repression act in dorsal regions of the embryo

The Evolution of Robust Development and Homeostasis in Artificial Organisms

During embryogenesis, multicellular animals are shaped via cell proliferation, cell rearrangement, and apoptosis. At the end of development, tissue architecture is then maintained through balanced rates of cell proliferation and loss. Here, we take an in silico approach to look for generic systems features of morphogenesis in multicellular animals that arise as a consequence of the evolution of development. Using artificial evolution, we evolved cellular automata-based digital organisms that have distinct embryonic and homeostatic phases of development. Although these evolved organisms use a variety of strategies to maintain their form over time, organisms of different types were all found to rapidly recover from environmental damage in the form of wounds. This regenerative response was most robust in an organism with a stratified tissue-like architecture. An evolutionary analysis revealed that evolution itself contributed to the ability of this organism to maintain its form in the face of genetic and environmental perturbation, confirming the results of previous studies. In addition, the exceptional robustness of this organism to surface injury was found to result from an upward flux of cells, driven in part by cell divisions with a stable niche at the tissue base. Given the general nature of the model, our results lead us to suggest that many of the robust systems properties observed in real organisms, including scar-free wound-healing in well-protected embryos and the layered tissue architecture of regenerating epithelial tissues, may be by-products of the evolution of morphogenesis, rather than the direct result of selection

BMP Signaling Modulates Hepcidin Expression in Zebrafish Embryos Independent of Hemojuvelin

Hemojuvelin (Hjv), a member of the repulsive-guidance molecule (RGM) family, upregulates transcription of the iron regulatory hormone hepcidin by activating the bone morphogenetic protein (BMP) signaling pathway in mammalian cells. Mammalian models have identified furin, neogenin, and matriptase-2 as modifiers of Hjv's function. Using the zebrafish model, we evaluated the effects of hjv and its interacting proteins on hepcidin expression during embryonic development. We found that hjv is strongly expressed in the notochord and somites of the zebrafish embryo and that morpholino knockdown of hjv impaired the development of these structures. Knockdown of hjv or other hjv-related genes, including zebrafish orthologs of furin or neogenin, however, failed to decrease hepcidin expression relative to liver size. In contrast, overexpression of bmp2b or knockdown of matriptase-2 enhanced the intensity and extent of hepcidin expression in zebrafish embryos, but this occurred in an hjv-independent manner. Furthermore, we demonstrated that zebrafish hjv can activate the human hepcidin promoter and enhance BMP responsive gene expression in vitro, but is expressed at low levels in the zebrafish embryonic liver. Taken together, these data support an alternative mechanism for hepcidin regulation during zebrafish embryonic development, which is independent of hjv

Identification of factors required for meristem function in Arabidopsis using a novel next generation sequencing fast forward genetics approach

<p>Abstract</p> <p>Background</p> <p>Phenotype-driven forward genetic experiments are powerful approaches for linking phenotypes to genomic elements but they still involve a laborious positional cloning process. Although sequencing of complete genomes now becomes available, discriminating causal mutations from the enormous amounts of background variation remains a major challenge.</p> <p>Method</p> <p>To improve this, we developed a universal two-step approach, named 'fast forward genetics', which combines traditional bulk segregant techniques with targeted genomic enrichment and next-generation sequencing technology</p> <p>Results</p> <p>As a proof of principle we successfully applied this approach to two Arabidopsis mutants and identified a novel factor required for stem cell activity.</p> <p>Conclusion</p> <p>We demonstrated that the 'fast forward genetics' procedure efficiently identifies a small number of testable candidate mutations. As the approach is independent of genome size, it can be applied to any model system of interest. Furthermore, we show that experiments can be multiplexed and easily scaled for the identification of multiple individual mutants in a single sequencing run.</p

Nlrp2, a Maternal Effect Gene Required for Early Embryonic Development in the Mouse

Maternal effect genes encode proteins that are produced during oogenesis and play an essential role during early embryogenesis. Genetic ablation of such genes in oocytes can result in female subfertility or infertility. Here we report a newly identified maternal effect gene, Nlrp2, which plays a role in early embryogenesis in the mouse. Nlrp2 mRNAs and their proteins (∼118 KDa) are expressed in oocytes and granulosa cells during folliculogenesis. The transcripts show a striking decline in early preimplantation embryos before zygotic genome activation, but the proteins remain present through to the blastocyst stage. Immunogold electron microscopy revealed that the NLRP2 protein is located in the cytoplasm, nucleus and close to nuclear pores in the oocytes, as well as in the surrounding granulosa cells. Using RNA interference, we knocked down Nlrp2 transcription specifically in mouse germinal vesicle oocytes. The knockdown oocytes could progress through the metaphase of meiosis I and emit the first polar body. However, the development of parthenogenetic embryos derived from Nlrp2 knockdown oocytes mainly blocked at the 2-cell stage. The maternal depletion of Nlrp2 in zygotes led to early embryonic arrest. In addition, overexpression of Nlrp2 in zygotes appears to lead to normal development, but increases blastomere apoptosis in blastocysts. These results provide the first evidence that Nlrp2 is a member of the mammalian maternal effect genes and required for early embryonic development in the mouse

Systematic Screening of Drosophila Deficiency Mutations for Embryonic Phenotypes and Orphan Receptor Ligands

This paper defines a collection of Drosophila deletion mutations (deficiencies) that can be systematically screened for embryonic phenotypes, orphan receptor ligands, and genes affecting protein localization. It reports the results of deficiency screens we have conducted that have revealed new axon guidance phenotypes in the central nervous system and neuromuscular system and permitted a quantitative assessment of the number of potential genes involved in regulating guidance of specific motor axon branches. Deficiency “kits” that cover the genome with a minimum number of lines have been established to facilitate gene mapping. These kits cannot be systematically analyzed for phenotypes, however, since embryos homozygous for many deficiencies in these kits fail to develop due to the loss of key gene products encoded within the deficiency. To create new kits that can be screened for phenotype, we have examined the development of the nervous system in embryos homozygous for more than 700 distinct deficiency mutations. A kit of ∼400 deficiency lines for which homozygotes have a recognizable nervous system and intact body walls encompasses >80% of the genome. Here we show examples of screens of this kit for orphan receptor ligands and neuronal antigen expression. It can also be used to find genes involved in expression, patterning, and subcellular localization of any protein that can be visualized by antibody staining. A subset kit of 233 deficiency lines, for which homozygotes develop relatively normally to late stage 16, covers ∼50% of the genome. We have screened it for axon guidance phenotypes, and we present examples of new phenotypes we have identified. The subset kit can be used to screen for phenotypes affecting all embryonic organs. In the future, these deficiency kits will allow Drosophila researchers to rapidly and efficiently execute genome-wide anatomical screens that require examination of individual embryos at high magnification

Zebrafish as a Model System to Study the Physiological Function of Telomeric Protein TPP1

Telomeres are specialized chromatin structures at the end of chromosomes. Telomere dysfunction can lead to chromosomal abnormalities, DNA damage responses, and even cancer. In mammalian cells, a six-protein complex (telosome/shelterin) is assembled on the telomeres through the interactions between various domain structures of the six telomere proteins (POT1, TPP1, TIN2, TRF1, TRF2 and RAP1), and functions in telomere maintenance and protection. Within the telosome, TPP1 interacts directly with POT1 and TIN2 and help to mediate telosome assembly. Mechanisms of telomere regulation have been extensively studied in a variety of model organisms. For example, the physiological roles of telomere-targeted proteins have been assessed in mice through homozygous inactivation. In these cases, early embryonic lethality has prevented further studies of these proteins in embryogenesis and development. As a model system, zebrafish offers unique advantages such as genetic similarities with human, rapid developmental cycles, and ease of manipulation of its embryos. In this report, we detailed the identification of zebrafish homologues of TPP1, POT1, and TIN2, and showed that the domain structures and interactions of these telosome components appeared intact in zebrafish. Importantly, knocking down TPP1 led to multiple abnormalities in zebrafish embryogenesis, including neural death, heart malformation, and caudal defect. And these embryos displayed extensive apoptosis. These results underline the importance of TPP1 in zebrafish embryogenesis, and highlight the feasibility and advantages of investigating the signaling pathways and physiological function of telomere proteins in zebrafish

Wnt, Hedgehog and Junctional Armadillo/β-Catenin Establish Planar Polarity in the Drosophila Embryo

To generate specialized structures, cells must obtain positional and directional information. In multi-cellular organisms, cells use the non-canonical Wnt or planar cell polarity (PCP) signaling pathway to establish directionality within a cell. In vertebrates, several Wnt molecules have been proposed as permissible polarity signals, but none has been shown to provide a directional cue. While PCP signaling components are conserved from human to fly, no PCP ligands have been reported in Drosophila. Here we report that in the epidermis of the Drosophila embryo two signaling molecules, Hedgehog (Hh) and Wingless (Wg or Wnt1), provide directional cues that induce the proper orientation of Actin-rich structures in the larval cuticle. We further find that proper polarity in the late embryo also involves the asymmetric distribution and phosphorylation of Armadillo (Arm or β-catenin) at the membrane and that interference with this Arm phosphorylation leads to polarity defects. Our results suggest new roles for Hh and Wg as instructive polarizing cues that help establish directionality within a cell sheet, and a new polarity-signaling role for the membrane fraction of the oncoprotein Arm