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During development, tissues and organs must coordinate growth and patterning so they reach the right size and shape. During larval stages, a dramatic increase in size and cell number of Drosophila wing imaginal discs is controlled by the action of several signaling pathways. Complex cross-talk between these pathways also pattern these discs to specify different regions with different fates and growth potentials. We show that the Notch signaling pathway is both required and sufficient to inhibit the activity of Yorkie (Yki), the Salvador/Warts/Hippo (SWH) pathway terminal transcription activator, but only in the central regions of the wing disc, where the TEAD factor and Yki partner Scalloped (Sd) is expressed. We show that this cross-talk between the Notch and SWH pathways is mediated, at least in part, by the Notch target and Sd partner Vestigial (Vg). We propose that, by altering the ratios between Yki, Sd and Vg, Notch pathway activation restricts the effects of Yki mediated transcription, therefore contributing to define a zone of low proliferation in the central wing discs. [ABSTRACT FROM AUTHOR]

The postnatal proliferation and maturation of insulin-secreting pancreatic β-cells are critical for glucose metabolism and disease development in adults. Elucidation of the molecular mechanisms underlying these events will be beneficial to direct the differentiation of stem cells into functional β-cells. Maturation of β-cells is accompanied by increased expression of MafA, an insulin gene transcription factor. Transcriptome analysis of MafA knockout islets revealed MafA is required for the expression of several molecules critical for β-cell function, including Glut2, ZnT8, Granuphilin, Vdr, Pcsk1 and Urocortin 3, as well as Prolactin receptor (Prlr) and its downstream target Cyclin D2 (Ccnd2). Inhibition of MafA expression in mouse islets or β-cell lines resulted in reduced expression of Prlr and Ccnd2, and MafA transactivated the Prlr promoter. Stimulation of β-cells by prolactin resulted in the phosphorylation and translocation of Stat5B and an increased nuclear pool of Ccnd2 via Prlr and Jak2. Consistent with these results, the loss of MafA resulted in impaired proliferation of β-cells at 4 weeks of age. These results suggest that MafA regulates the postnatal proliferation of β-cells via prolactin signaling. [ABSTRACT FROM AUTHOR]

The RIKEN Center for Developmental Biology in Kobe, Japan, hosted a meeting entitled 'Regeneration of Organs: Programming and Self- Organization' in March, 2014. Scientists from across the globe met to discuss current research on regeneration, organ morphogenesis and self-organization - and the links between these fields. A diverse range of experimental models and organ systems was presented, and the speakers aptly illustrated the unique power of each. This Meeting Review describes the major advances reported and themes emerging from this exciting meeting. [ABSTRACT FROM AUTHOR]

As animals grow, many early born structures grow by cell expansion rather than cell addition; thus growth of distinct structures must be coordinated to maintain proportionality. This phenomenon is particularly widespread in the nervous system, with dendrite arbors of many neurons expanding in concert with their substrate to sustain connectivity and maintain receptive field coverage as animals grow. After rapidly growing to establish body wall coverage, dendrites of Drosophila class IV dendrite arborization (C4da) neurons grow synchronously with their substrate, the body wall epithelium, providing a system to study how proportionality is maintained during animal growth. Here, we show that the microRNA bantam (ban) ensures coordinated growth of C4da dendrites and the epithelium through regulation of epithelial endoreplication, a modified cell cycle that entails genome amplification without cell division. In Drosophila larvae, epithelial endoreplication leads to progressive changes in dendrite-extracellular matrix (ECM) and dendrite-epithelium contacts, coupling dendrite/substrate expansion and restricting dendrite growth beyond established boundaries. Moreover, changes in epithelial expression of cell adhesion molecules, including the beta-integrin myospheroid (mys), accompany this developmental transition. Finally, endoreplication and the accompanying changes in epithelial mys expression are required to constrain late-stage dendrite growth and structural plasticity. Hence, modulating epithelium-ECM attachment probably influences substrate permissivity for dendrite growth and contributes to the dendrite-substrate coupling that ensures proportional expansion of the two cell types. [ABSTRACT FROM AUTHOR]

Regeneration of lost tissues depends on the precise interpretation of molecular signals that control and coordinate the onset of proliferation, cellular differentiation and cell death. However, the nature of those molecular signals and the mechanisms that integrate the cellular responses remain largely unknown. The planarian flatworm is a unique model in which regeneration and tissue renewal can be comprehensively studied in vivo. The presence of a population of adult pluripotent stem cells combined with the ability to decode signaling after wounding enable planarians to regenerate a complete, correctly proportioned animal within a few days after any kind of amputation, and to adapt their size to nutritional changes without compromising functionality. Here, we demonstrate that the stress-activated c-jun–NH2–kinase (JNK) links wound-induced apoptosis to the stem cell response during planarian regeneration. We show that JNK modulates the expression of wound-related genes, triggers apoptosis and attenuates the onset of mitosis in stem cells specifically after tissue loss. Furthermore, in pre-existing body regions, JNK activity is required to establish a positive balance between cell death and stem cell proliferation to enable tissue renewal, remodeling and the maintenance of proportionality. During homeostatic degrowth, JNK RNAi blocks apoptosis, resulting in impaired organ remodeling and rescaling. Our findings indicate that JNK-dependent apoptotic cell death is crucial to coordinate tissue renewal and remodeling required to regenerate and to maintain a correctly proportioned animal. Hence, JNK might act as a hub, translating wound signals into apoptotic cell death, controlled stem cell proliferation and differentiation, all of which are required to coordinate regeneration and tissue renewal. [ABSTRACT FROM AUTHOR]

Organisms in the wild develop with varying food availability. During periods of nutritional scarcity, development may slow or arrest until conditions improve. The ability to modulate developmental programs in response to poor nutritional conditions requires a means of sensing the changing nutritional environment and limiting tissue growth. The mechanisms by which organisms accomplish this adaptation are not well understood. We sought to study this question by examining the effects of nutrient deprivation on Caenorhabditis elegans development during the late larval stages, L3 and L4, a period of extensive tissue growth and morphogenesis. By removing animals from food at different times, we show here that specific checkpoints exist in the early L3 and early L4 stages that systemically arrest the development of diverse tissues and cellular processes. These checkpoints occur once in each larval stage after molting and prior to initiation of the subsequent molting cycle. DAF-2, the insulin/insulin-like growth factor receptor, regulates passage through the L3 and L4 checkpoints in response to nutrition. The FOXO transcription factor DAF-16, a major target of insulin-like signaling, functions cell-nonautonomously in the hypodermis (skin) to arrest developmental upon nutrient removal. The effects of DAF-16 on progression through the L3 and L4 stages are mediated by DAF-9, a cytochrome P450 ortholog involved in the production of C. elegans steroid hormones. Our results identify a novel mode of C. elegans growth in which development progresses from one checkpoint to the next. At each checkpoint, nutritional conditions determine whether animals remain arrested or continue development to the next checkpoint. [ABSTRACT FROM AUTHOR]

Understanding biological phenomena requires a systemic approach that incorporates different mechanisms acting on different spatial and temporal scales, since in organisms the workings of all components, such as organelles, cells, and organs interrelate. This inherent interdependency between diverse biological mechanisms, both on the same and on different scales, provides the functioning of an organism capable of maintaining homeostasis and physiological stability through numerous feedback loops. Thus, developing models of organisms and their constituents should be done within the overall systemic context of the studied phenomena. We introduce such a method for modeling growth and regeneration of livers at the organ scale, considering it a part of the overall multi-scale biochemical and biophysical processes of an organism. Our method is based on the earlier discovered general growth law, postulating that any biological growth process comprises a uniquely defined distribution of nutritional resources between maintenance needs and biomass production. Based on this law, we introduce a liver growth model that allows to accurately predicting the growth of liver transplants in dogs and liver grafts in humans. Using this model, we find quantitative growth characteristics, such as the time point when the transition period after surgery is over and the liver resumes normal growth, rates at which hepatocytes are involved in proliferation, etc. We then use the model to determine and quantify otherwise unobservable metabolic properties of livers. [ABSTRACT FROM AUTHOR]

Throughout Metazoa, developmental processes are controlled by a surprisingly limited number of conserved signaling pathways. Precisely how these signaling cassettes were assembled in early animal evolution remains poorly understood, as do the molecular transitions that potentiated the acquisition of their myriad developmental functions. Here we analyze the molecular evolution of the proto-oncogene yes-associated protein (Yap)/Yorkie, a key effector of the Hippo signaling pathway that controls organ size in both Drosophila and mammals. Based on heterologous functional analysis of evolutionarily distant Yap/Yorkie orthologs, we demonstrate that a structurally distinct interaction interface between Yap/Yorkie and its partner TEAD/Scalloped became fixed in the eumetazoan common ancestor. We then combine transcriptional profiling of tissues expressing phylogenetically diverse forms of Yap/Yorkie with ChIP-seq validation to identify a common downstream gene expression program underlying the control of tissue growth in Drosophila. Intriguingly, a subset of the newly identified Yorkie target genes are also induced by Yap in mammalian tissues, thus revealing a conserved Yap-dependent gene expression signature likely to mediate organ size control throughout bilaterian animals. Combined, these experiments provide new mechanistic insights while revealing the ancient evolutionary history of Hippo signaling. [ABSTRACT FROM PUBLISHER]

Maintenance of cell survival is essential for proper embryonic development. In the mouse, Notchless homolog 1 (Drosophila) (Nle1) is instrumental for survival of cells of the inner cell mass upon implantation. Here, we analyze the function of Nle1 after implantation using the Meox2tm1(cre)Sor mouse that expresses the Cre recombinase specifically in the epiblast at E5.5. First, we find that NLE1 function is required in epiblast cells, as Nle1-deficient cells are rapidly eliminated. In this report, we also show that the Meox2Cre transgene is active in specific tissues during organogenesis. In particular, we detect high Cre expression in the vertebral column, ribs, limbs and tailbud. We took advantage of this dynamic expression profile to analyze the effects of inducing mosaic deletion of Nle1 in the embryo. We show that Nle1 deletion in this context, results in severe developmental anomalies leading to lethality at birth. Mutant embryos display multiple developmental defects in particular during axial skeletal formation. We also provide evidence that axial defects are due to an increase in apoptotic cell death in the somite at E9.5. These data demonstrate an essential role for Nle1 during organogenesis and in particular during axial development. [ABSTRACT FROM AUTHOR]

The actin-Capping Protein heterodimer, composed of the α and β subunits, is a master F-actin regulator. In addition to its role in many cellular processes, Capping Protein acts as a main tumor suppressor module in Drosophila and in humans, in part, by restricting the activity of Yorkie/YAP/TAZ oncogenes. We aimed in this report to understand how both subunits regulate each other in vivo. We show that the levels and capping activities of both subunits must be tightly regulated to control F-actin levels and consequently growth of the Drosophila wing. Overexpressing capping protein α and β decreases both F-actin levels and tissue growth, while expressing forms of Capping Protein that have dominant negative effects on F-actin promote tissue growth. Both subunits regulate each other's protein levels. In addition, overexpressing one of the subunit in tissues knocked-down for the other increases the mRNA and protein levels of the subunit knocked-down and compensates for its loss. We propose that the ability of the α and β subunits to control each other's levels assures that a pool of functional heterodimer is produced in sufficient quantities to restrict the development of tumor but not in excess to sustain normal tissue growth. [ABSTRACT FROM AUTHOR]

Temperature affects both the timing and outcome of animal development, but the detailed effects of temperature on the progress of early development have been poorly characterized. To determine the impact of temperature on the order and timing of events during Drosophila melanogaster embryogenesis, we used time-lapse imaging to track the progress of embryos from shortly after egg laying through hatching at seven precisely maintained temperatures between 17.5°C and 32.5°C. We employed a combination of automated and manual annotation to determine when 36 milestones occurred in each embryo. D. melanogaster embryogenesis takes 33 hours at 17.5°C, and accelerates with increasing temperature to a low of 16 hours at 27.5°C, above which embryogenesis slows slightly. Remarkably, while the total time of embryogenesis varies over two fold, the relative timing of events from cellularization through hatching is constant across temperatures. To further explore the relationship between temperature and embryogenesis, we expanded our analysis to cover ten additional Drosophila species of varying climatic origins. Six of these species, like D. melanogaster, are of tropical origin, and embryogenesis time at different temperatures was similar for them all. D. mojavensis, a sub-tropical fly, develops slower than the tropical species at lower temperatures, while D. virilis, a temperate fly, exhibits slower development at all temperatures. The alpine sister species D. persimilis and D. pseudoobscura develop as rapidly as tropical flies at cooler temperatures, but exhibit diminished acceleration above 22.5°C and have drastically slowed development by 30°C. Despite ranging from 13 hours for D. erecta at 30°C to 46 hours for D. virilis at 17.5°C, the relative timing of events from cellularization through hatching is constant across all species and temperatures examined here, suggesting the existence of a previously unrecognized timer controlling the progress of embryogenesis that has been tuned by natural selection as each species diverges. [ABSTRACT FROM AUTHOR]

Brassinosteroids, which control plant growth and development, are sensed by the membrane receptor kinase BRASSINOSTEROID INSENSITIVE 1 ( BRI1). Brassinosteroid binding to the BRI1 leucine-rich repeat ( LRR) domain induces heteromerisation with a SOMATIC EMBRYOGENESIS RECEPTOR KINASE ( SERK)-family co-receptor. This process allows the cytoplasmic kinase domains of BRI1 and SERK to interact, trans-phosphorylate and activate each other. Here we report crystal structures of the BRI1 kinase domain in its activated form and in complex with nucleotides. BRI1 has structural features reminiscent of both serine/threonine and tyrosine kinases, providing insight into the evolution of dual-specificity kinases in plants. Phosphorylation of Thr1039, Ser1042 and Ser1044 causes formation of a catalytically competent activation loop. Mapping previously identified serine/threonine and tyrosine phosphorylation sites onto the structure, we analyse their contribution to brassinosteroid signaling. The location of known genetic missense alleles provide detailed insight into the BRI1 kinase mechanism, while our analyses are inconsistent with a previously reported guanylate cyclase activity. We identify a protein interaction surface on the C-terminal lobe of the kinase and demonstrate that the isolated BRI1, SERK2 and SERK3 cytoplasmic segments form homodimers in solution and have a weak tendency to heteromerise. We propose a model in which heterodimerisation of the BRI1 and SERK ectodomains brings their cytoplasmic kinase domains in a catalytically competent arrangement, an interaction that can be modulated by the BRI1 inhibitor protein BKI1. [ABSTRACT FROM AUTHOR]

Successful completion of development requires coordination of patterning events with morphogenetic movements. Environmental variability challenges this coordination. For example, developing organisms encounter varying environmental temperatures that can strongly influence developmental rates. We hypothesized that the mechanics of morphogenesis would have to be finely adjusted to allow for normal morphogenesis across a wide range of developmental rates. We formulated our hypothesis as a simple model incorporating time-dependent application of force to a viscoelastic tissue. This model suggested that the capacity to maintain normal morphogenesis across a range of temperatures would depend on how both tissue viscoelasticity and the forces that drive deformation vary with temperature. To test this model we investigated how the mechanical behavior of embryonic tissue (Xenopus laevis) changed with temperature; we used a combination of micropipette aspiration to measure viscoelasticity, electrically induced contractions to measure cellular force generation, and confocal microscopy to measure endogenous contractility. Contrary to expectations, the viscoelasticity of the tissues and peak contractile tension proved invariant with temperature even as rates of force generation and gastrulation movements varied three-fold. Furthermore, the relative rates of different gastrulation movements varied with temperature: the speed of blastopore closure increased more slowly with temperature than the speed of the dorsal-to-ventral progression of involution. The changes in the relative rates of different tissue movements can be explained by the viscoelastic deformation model given observed viscoelastic properties, but only if morphogenetic forces increase slowly rather than all at once. [ABSTRACT FROM AUTHOR]

During the summer of 2013 seven Italian Tyrolean Grey calves were born with abnormally short limbs. Detailed clinical and pathological examination revealed similarities to chondrodysplastic dwarfism. Pedigree analysis showed a common founder, assuming autosomal monogenic recessive transmission of the defective allele. A positional cloning approach combining genome wide association and homozygosity mapping identified a single 1.6 Mb genomic region on BTA 6 that was associated with the disease. Whole genome re-sequencing of an affected calf revealed a single candidate causal mutation in the Ellis van Creveld syndrome 2 (EVC2) gene. This gene is known to be associated with chondrodysplastic dwarfism in Japanese Brown cattle, and dwarfism, abnormal nails and teeth, and dysostosis in humans with Ellis-van Creveld syndrome. Sanger sequencing confirmed the presence of a 2 bp deletion in exon 19 (c.2993_2994ACdel) that led to a premature stop codon in the coding sequence of bovine EVC2, and was concordant with the recessive pattern of inheritance in affected and carrier animals. This loss of function mutation confirms the important role of EVC2 in bone development. Genetic testing can now be used to eliminate this form of chondrodysplastic dwarfism from Tyrolean Grey cattle. [ABSTRACT FROM AUTHOR]

Multivesicular endosome (MVE) sorting depends on proteins of the Endosomal Sorting Complex Required for Transport (ESCRT) family. These are organized in four complexes (ESCRT-0, -I, -II, -III) that act in a sequential fashion to deliver ubiquitylated cargoes into the internal luminal vesicles (ILVs) of the MVE. Drosophila genes encoding ESCRT-I, -II, -III components function in sorting signaling receptors, including Notch and the JAK/STAT signaling receptor Domeless. Loss of ESCRT-I, -II, -III in Drosophila epithelia causes altered signaling and cell polarity, suggesting that ESCRTs genes are tumor suppressors. However, the nature of the tumor suppressive function of ESCRTs, and whether tumor suppression is linked to receptor sorting is unclear. Unexpectedly, a null mutant in Hrs, encoding one of the components of the ESCRT-0 complex, which acts upstream of ESCRT-I, -II, -III in MVE sorting is dispensable for tumor suppression. Here, we report that two Drosophila epithelia lacking activity of Stam, the other known components of the ESCRT-0 complex, or of both Hrs and Stam, accumulate the signaling receptors Notch and Dome in endosomes. However, mutant tissue surprisingly maintains normal apico-basal polarity and proliferation control and does not display ectopic Notch signaling activation, unlike cells that lack ESCRT-I, -II, -III activity. Overall, our in vivo data confirm previous evidence indicating that the ESCRT-0 complex plays no crucial role in regulation of tumor suppression, and suggest re-evaluation of the relationship of signaling modulation in endosomes and tumorigenesis. [ABSTRACT FROM AUTHOR]

Multivesicular endosome (MVE) sorting depends on proteins of the Endosomal Sorting Complex Required for Transport (ESCRT) family. These are organized in four complexes (ESCRT-0, -I, -II, -III) that act in a sequential fashion to deliver ubiquitylated cargoes into the internal luminal vesicles (ILVs) of the MVE. Drosophila genes encoding ESCRT-I, -II, -III components function in sorting signaling receptors, including Notch and the JAK/STAT signaling receptor Domeless. Loss of ESCRT-I, -II, -III in Drosophila epithelia causes altered signaling and cell polarity, suggesting that ESCRTs genes are tumor suppressors. However, the nature of the tumor suppressive function of ESCRTs, and whether tumor suppression is linked to receptor sorting is unclear. Unexpectedly, a null mutant in Hrs, encoding one of the components of the ESCRT-0 complex, which acts upstream of ESCRT-I, -II, -III in MVE sorting is dispensable for tumor suppression. Here, we report that two Drosophila epithelia lacking activity of Stam, the other known components of the ESCRT-0 complex, or of both Hrs and Stam, accumulate the signaling receptors Notch and Dome in endosomes. However, mutant tissue surprisingly maintains normal apico-basal polarity and proliferation control and does not display ectopic Notch signaling activation, unlike cells that lack ESCRT-I, -II, -III activity. Overall, our in vivo data confirm previous evidence indicating that the ESCRT-0 complex plays no crucial role in regulation of tumor suppression, and suggest re-evaluation of the relationship of signaling modulation in endosomes and tumorigenesis. [ABSTRACT FROM AUTHOR]

Since its discovery in 1923, the biology of photoperiodism remains a mystery in many ways. We sought the link connecting the circadian system to an endocrine switch, using Antheraea pernyi. PER-, CLK- and CYC-ir were co-expressed in two pairs of dorsolateral neurons of the protocerebrum, suggesting that these are the circadian neurons that also express melatonin-, NAT- and HIOMT-ir. The results suggest that a melatonin pathway is present in the circadian neurons. Melatonin receptor (MT2 or MEL-1B-R)-ir in PTTH-ir neurons juxtaposing clock neurons suggests that melatonin gates PTTH release. RIA showed a melatonin rhythm with a peak four hours after lights off in adult brain both under LD16∶8 (LD) and LD12∶12 (SD), and both the peak and the baseline levels were higher under LD than SD, suggesting a photoperiodic influence. When pupae in diapause were exposed to 10 cycles of LD, or stored at 4°C for 4 months under constant darkness, an increase of NAT activity was observed when PTTH released ecdysone. DNA sequence upstream of nat contained E-boxes to which CYC/CLK could bind, and nat transcription was turned off by clk or cyc dsRNA. dsRNANAT caused dysfunction of photoperiodism. dsRNAPER upregulated nat transcription as anticipated, based on findings in the Drosophila melanogaster circadian system. Transcription of nat, cyc and clk peaked at ZT12. RIA showed that dsRNANAT decreased melatonin while dsRNAPER increased melatonin. Thus nat, a clock controlled gene, is the critical link between the circadian clock and endocrine switch. MT-binding may release PTTH, resulting in termination of diapause. This study thus examined all of the basic functional units from the clock: a photoperiodic counter as an accumulator of mRNANAT, to endocrine switch for photoperiodism in A. pernyi showing this system is self-complete without additional device especially for photoperiodism. [ABSTRACT FROM AUTHOR]

Vertebrate limbs originate from the lateral plate mesoderm (LPM) and the overlying ectoderm. While normal limb formation in defined regions has been well studied, the question of whether other positions retain limb-forming potential has not been fully investigated in mice. By ectopically activating β-catenin in the ectoderm with Msx2-cre, we observed that local tissue outgrowths were induced, which either progressed into limb-like structure within the inter-limb flank or formed extra tissues in other parts of the mouse embryo. In the presumptive abdominal region of severely affected embryos, ectopic limb formation was coupled with impaired abdominal ventral body wall (AVBW) closure, which indicates the existence of a potential counterbalance of limb formation and AVBW closure. At the molecular level, constitutive β-catenin activation was sufficient to trigger, but insufficient to maintain the ectopic expression of a putative limb-inducing factor, Fgf8, in the ectoderm. These findings provide new insight into the mechanism of limb formation and AVBW closure, and the crosstalk between the Wnt/β-catenin pathway and Fgf signal. [ABSTRACT FROM AUTHOR]

Callipyge sheep exhibit postnatal muscle hypertrophy due to the up-regulation of DLK1 and/or RTL1. The up-regulation of PARK7 was identified in hypertrophied muscles by microarray analysis and further validated by quantitative PCR. The expression of PARK7 in hypertrophied muscle of callipyge lambs was confirmed to be up-regulated at the protein level. PARK7 was previously identified to positively regulate PI3K/AKT pathway by suppressing the phosphatase activity of PTEN in mouse fibroblasts. The purpose of this study was to investigate the effects of PARK7 in muscle growth and protein accretion in response to IGF1. Primary myoblasts isolated from Park7 (+/+) and Park7 (−/−) mice were used to examine the effect of differential expression of Park7. The Park7 (+/+) myotubes had significantly larger diameters and more total sarcomeric myosin expression than Park7 (−/−) myotubes. IGF1 treatment increased the mRNA abundance of Myh4, Myh7 and Myh8 between 20-40% in Park7 (+/+) myotubes relative to Park7 (−/−). The level of AKT phosphorylation was increased in Park7 (+/+) myotubes at all levels of IGF1 supplementation. After removal of IGF1, the Park7 (+/+) myotubes maintained higher AKT phosphorylation through 3 hours. PARK7 positively regulates the PI3K/AKT pathway by inhibition of PTEN phosphatase activity in skeletal muscle. The increased PARK7 expression can increase protein synthesis and result in myotube hypertrophy. These results support the hypothesis that elevated expression of PARK7 in callipyge muscle would increase levels of AKT activity to cause hypertrophy in response to the normal IGF1 signaling in rapidly growing lambs. Increasing expression of PARK7 could be a novel mechanism to increase protein accretion and muscle growth in livestock or help improve muscle mass with disease or aging. [ABSTRACT FROM AUTHOR]

The mantled floral phenotype of oil palm (Elaeis guineensis) affects somatic embryogenesis-derived individuals and is morphologically similar to mutants defective in the B-class MADS-box genes. This somaclonal variation has been previously demonstrated to be associated to a significant deficit in genome-wide DNA methylation. In order to elucidate the possible role of DNA methylation in the transcriptional regulation of EgDEF1, the APETALA3 ortholog of oil palm, we studied this epigenetic mark within the gene in parallel with transcript accumulation in both normal and mantled developing inflorescences. We also examined the methylation and expression of two neighboring retrotransposons that might interfere with EgDEF1 regulation. We show that the EgDEF1 gene is essentially unmethylated and that its methylation pattern does not change with the floral phenotype whereas expression is dramatically different, ruling out a direct implication of DNA methylation in the regulation of this gene. Also, we find that both the gypsy element inserted within an intron of the EgDEF1 gene and the copia element located upstream from the promoter are heavily methylated and show little or no expression. Interestingly, we identify a shorter, alternative transcript produced by EgDEF1 and characterize its accumulation with respect to its full-length counterpart. We demonstrate that, depending on the floral phenotype, the respective proportions of these two transcripts change differently during inflorescence development. We discuss the possible phenotypical consequences of this alternative splicing and the new questions it raises in the search for the molecular mechanisms underlying the mantled phenotype in the oil palm. [ABSTRACT FROM AUTHOR]

The proper control of tissue growth is essential during normal development and an important problem in human disease. Merlin, the product of the Neurofibromatosis 2 tumor suppressor gene, has been extensively studied to understand its functions in growth control. Here we describe experiments in which we used Drosophila as an in vivo system to test the functions of the normal human NF2 gene products and patient-derived mutant alleles. Although the predominant NF2 gene isoform, isoform 1, could functionally replace the Drosophila Merlin gene, a second isoform with a distinct C-terminal tail could not. Immunofluorescence studies show that the two isoforms have distinct subcellular localizations when expressed in the polarized imaginal epithelium, and function in genetic rescue assays correlates with apical localization of the NF2 protein. Interestingly, we found that a patient-derived missense allele, NF2L64P, appears to be temperature sensitive. These studies highlight the utility of Drosophila for in vivo functional analysis of highly conserved human disease genes. [ABSTRACT FROM AUTHOR]

Mechanisms that regulate the number of cell division rounds during embryogenesis have remained largely elusive. To investigate this issue, we used the ascidian, which develops into a tadpole larva with a small number of cells. The embryonic cells divide 11.45 times on average from fertilization to hatching. The number of cell division rounds varies depending on embryonic lineages. Notochord and muscle consist of large postmitotic cells and stop dividing early in developing embryos. Here we show that conversion of mesenchyme to muscle cell fates by inhibition of inductive FGF signaling or mis-expression of a muscle-specific key transcription factor for muscle differentiation, Tbx6, changed the number of cell divisions in accordance with the altered fate. Tbx6 likely activates a putative mechanism to halt cell division at a specific stage. However, precocious expression of Tbx6 has no effect on progression of the developmental clock itself. Zygotic expression of a cyclin-dependent kinase inhibitor, CKI-b, is initiated in muscle and then in notochord precursors. CKI-b is possibly downstream of tissue-specific key transcription factors of notochord and muscle. In the two distinct muscle lineages, postmitotic muscle cells are generated after 9 and 8 rounds of cell division depending on lineage, but the final cell divisions occur at a similar developmental stage. CKI-b gene expression starts simultaneously in both muscle lineages at the 110-cell stage, suggesting that CKI-b protein accumulation halts cell division at a similar stage. The difference in the number of cell divisions would be due to the cumulative difference in cell cycle length. These results suggest that muscle cells do not count the number of cell division rounds, and that accumulation of CKI-b protein triggered by tissue-specific key transcription factors after cell fate determination might act as a kind of timer that measures elapsed time before cell division termination. [ABSTRACT FROM AUTHOR]

Since its discovery in 1923, the biology of photoperiodism remains a mystery in many ways. We sought the link connecting the circadian system to an endocrine switch, using Antheraea pernyi. PER-, CLK- and CYC-ir were co-expressed in two pairs of dorsolateral neurons of the protocerebrum, suggesting that these are the circadian neurons that also express melatonin-, NAT- and HIOMT-ir. The results suggest that a melatonin pathway is present in the circadian neurons. Melatonin receptor (MT2 or MEL-1B-R)-ir in PTTH-ir neurons juxtaposing clock neurons suggests that melatonin gates PTTH release. RIA showed a melatonin rhythm with a peak four hours after lights off in adult brain both under LD16∶8 (LD) and LD12∶12 (SD), and both the peak and the baseline levels were higher under LD than SD, suggesting a photoperiodic influence. When pupae in diapause were exposed to 10 cycles of LD, or stored at 4°C for 4 months under constant darkness, an increase of NAT activity was observed when PTTH released ecdysone. DNA sequence upstream of nat contained E-boxes to which CYC/CLK could bind, and nat transcription was turned off by clk or cyc dsRNA. dsRNANAT caused dysfunction of photoperiodism. dsRNAPER upregulated nat transcription as anticipated, based on findings in the Drosophila melanogaster circadian system. Transcription of nat, cyc and clk peaked at ZT12. RIA showed that dsRNANAT decreased melatonin while dsRNAPER increased melatonin. Thus nat, a clock controlled gene, is the critical link between the circadian clock and endocrine switch. MT-binding may release PTTH, resulting in termination of diapause. This study thus examined all of the basic functional units from the clock: a photoperiodic counter as an accumulator of mRNANAT, to endocrine switch for photoperiodism in A. pernyi showing this system is self-complete without additional device especially for photoperiodism. [ABSTRACT FROM AUTHOR]

The whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) harbors several bacterial symbionts. Among the secondary (facultative) symbionts, Hamiltonella has high prevalence and high infection frequencies, suggesting that it may be important for the biology and ecology of its hosts. Previous reports indicated that Hamiltonella increases whitefly fitness and, based on the complete sequencing of its genome, may have the ability to synthesize cofactors and amino acids that are required by its host but that are not sufficiently synthesized by the host or by the primary endosymbiont, Portiera. Here, we assessed the effects of Hamiltonella infection on the growth of B. tabaci reared on low-, standard-, or high-nitrogen diets. When B. tabaci was reared on a standard-nitrogen diet, no cost or benefit was associated with Hamiltonella infection. But, if we reared whiteflies on low-nitrogen diets, Hamiltonella-infected whiteflies often grew better than uninfected whiteflies. Furthermore, nitrogen levels in field-collected whiteflies indicated that the nutritional conditions in the field were comparable to the low-nitrogen diet in our laboratory experiment. These data suggest that Hamiltonella may play a previously unrecognized role as a nutritional mutualist in B. tabaci. [ABSTRACT FROM AUTHOR]

Cell–cell communication, facilitating the exchange of small metabolites, ions and second messengers, takes place via aqueous proteinaceous channels called gap junctions. Connexins (cx) are the subunits of a gap junction channel. Mutations in zebrafish cx43 produces the short fin (sof b123) phenotype and is characterized by short fins due to reduced segment length of the bony fin rays and reduced cell proliferation. Previously established results from our lab demonstrate that Cx43 plays a dual role regulating both cell proliferation (growth) and joint formation (patterning) during the process of skeletal morphogenesis. In this study, we show that Hapln1a (Hyaluronan and Proteoglycan Link Protein 1a) functions downstream of cx43. Hapln1a belongs to the family of link proteins that play an important role in stabilizing the ECM by linking the aggregates of hyaluronan and proteoglycans. We validated that hapln1a is expressed downstream of cx43 by in situ hybridization and quantitative RT-PCR methods. Moreover, in situ hybridization at different time points revealed that hapln1a expression peaks at 3 days post amputation. Expression of hapln1a is located in the medial mesenchyme and the in the lateral skeletal precursor cells. Furthermore, morpholino mediated knock-down of hapln1a resulted in reduced fin regenerate length, reduced bony segment length and reduced cell proliferation, recapitulating all the phenotypes of cx43 knock-down. Moreover, Hyaluronic Acid (HA) levels are dramatically reduced in hapln1a knock-down fins, attesting the importance of Hapln1a in stabilizing the ECM. Attempts to place hapln1a in our previously defined cx43–sema3d pathway suggest that hapln1a functions in a parallel genetic pathway. Collectively, our data suggest that Cx43 mediates independent Sema3d and Hapln1a pathways in order to coordinate skeletal growth and patterning. [ABSTRACT FROM AUTHOR]

The Caenorhabditis elegans heterochronic gene pathway, which consists of a set of regulatory genes, plays an important regulatory role in the timing of stage-specific cell lineage development in nematodes. Research into the heterochronic gene pathway gave rise to landmark microRNA (miRNA) studies and showed that these genes are important in stem cell and cancer biology; however, their functions in vertebrate development are largely unknown. To elucidate the function of the heterochronic gene pathway during vertebrate development, we cloned the zebrafish homologs of the C. elegans let-7 miRNA-binding protein, Lin-28, and analyzed their function in zebrafish development. The zebrafish genome contains two Lin28-related genes, lin-28a and lin-28b. Similar to mammalian Lin28 proteins, both zebrafish Lin-28a and Lin-28b have a conserved cold-shock domain and a pair of CCHC zinc finger domains, and are ubiquitously expressed during early embryonic development. In a reciprocal fashion, the expression of downstream heterochronic genes, let-7 and lin-4/miR-125 miRNA, occurred subsequent to lin-28 expression. The knockdown of Lin-28a or Lin-28b function by morpholino microinjection into embryos resulted in severe cell proliferation defects during early morphogenesis. We found that the expression of let-7 miRNA was upregulated and its downstream target gene, lin-41, was downregulated in these embryos. Interestingly, the expression of miR-430, a key regulator of maternal mRNA decay, was downregulated in lin-28a and lin-28b morphant embryos, suggesting a role for Lin-28 in the maternal-to-zygotic transition in zebrafish. Taken together, our results suggest an evolutionarily conserved and pivotal role of the heterochronic gene pathway in early vertebrate embryogenesis. [ABSTRACT FROM AUTHOR]

Regulation of cell growth and cell division plays fundamental roles in tissue morphogenesis. However, the mechanisms of regulating tissue elongation through cell growth and cell division are still not well understood. The wing imaginal disc of Drosophila provides a model system that has been widely used to study tissue morphogenesis. Here we use a recently developed two-dimensional cellular model to study the mechanisms of regulating tissue elongation in Drosophila wing. We simulate the effects of directional cues on tissue elongation. We also computationally analyze the role of reduced cell size. Our simulation results indicate that oriented cell divisions, oriented mechanical forces, and reduced cell size can all mediate tissue elongation, but they function differently. We show that oriented cell divisions and oriented mechanical forces act as directional cues during tissue elongation. Between these two directional cues, oriented mechanical forces have a stronger influence than oriented cell divisions. In addition, we raise the novel hypothesis that reduced cell size may significantly promote tissue elongation. We find that reduced cell size alone cannot drive tissue elongation. However, when combined with directional cues, such as oriented cell divisions or oriented mechanical forces, reduced cell size can significantly enhance tissue elongation in Drosophila wing. Furthermore, our simulation results suggest that reduced cell size has a short-term effect on cell topology by decreasing the frequency of hexagonal cells, which is consistent with experimental observations. Our simulation results suggest that cell divisions without cell growth play essential roles in tissue elongation. [ABSTRACT FROM AUTHOR]

There is a long and active tradition of land use planning in the United States, and planners have tried to subordinate regulation, primarily zoning, to prior planning. However, this project has met with only limited success. Americans prefer to make small-scale, ad hoc land use decisions. The reasons include the tradition of plans as blueprints for unlimited growth, the legal ability of local governments to ignore the regional impacts of their decisions, America’s preference for sprawling suburbia, the marginal status of plans in zoning law, and the chilling impact of the Supreme Court’s takings jurisprudence. [ABSTRACT FROM PUBLISHER]

The morphogenesis of organs necessarily involves mechanical interactions and changes in mechanical properties of a tissue. A long standing question is how such changes are directed on a cellular scale while being coordinated at a tissular scale. Growing evidence suggests that mechanical cues are participating in the control of growth and morphogenesis during development. We introduce a mechanical model that represents the deposition of cellulose fibers in primary plant walls. In the model both the degree of material anisotropy and the anisotropy direction are regulated by stress anisotropy. We show that the finite element shell model and the simpler triangular biquadratic springs approach provide equally adequate descriptions of cell mechanics in tissue pressure simulations of the epidermis. In a growing organ, where circumferentially organized fibers act as a main controller of longitudinal growth, we show that the fiber direction can be correlated with both the maximal stress direction and the direction orthogonal to the maximal strain direction. However, when dynamic updates of the fiber direction are introduced, the mechanical stress provides a robust directional cue for the circumferential organization of the fibers, whereas the orthogonal to maximal strain model leads to an unstable situation where the fibers reorient longitudinally. Our investigation of the more complex shape and growth patterns in the shoot apical meristem where new organs are initiated shows that a stress based feedback on fiber directions is capable of reproducing the main features of in vivo cellulose fiber directions, deformations and material properties in different regions of the shoot. In particular, we show that this purely mechanical model can create radially distinct regions such that cells expand slowly and isotropically in the central zone while cells at the periphery expand more quickly and in the radial direction, which is a well established growth pattern in the meristem. [ABSTRACT FROM AUTHOR]

The Notch signaling pathway is thought to regulate multiple stages of inner ear development. Mutations in the Notch signaling pathway cause disruptions in the number and arrangement of hair cells and supporting cells in sensory regions of the ear. In this study we identify an insertional mutation in the mouse Sfswap gene, a putative splicing factor, that results in mice with vestibular and cochlear defects that are consistent with disrupted Notch signaling. Homozygous Sfswap mutants display hyperactivity and circling behavior consistent with vestibular defects, and significantly impaired hearing. The cochlea of newborn Sfswap mutant mice shows a significant reduction in outer hair cells and supporting cells and ectopic inner hair cells. This phenotype most closely resembles that seen in hypomorphic alleles of the Notch ligand Jagged1 (Jag1). We show that Jag1; Sfswap compound mutants have inner ear defects that are more severe than expected from simple additive effects of the single mutants, indicating a genetic interaction between Sfswap and Jag1. In addition, expression of genes involved in Notch signaling in the inner ear are reduced in Sfswap mutants. There is increased interest in how splicing affects inner ear development and function. Our work is one of the first studies to suggest that a putative splicing factor has specific effects on Notch signaling pathway members and inner ear development. [ABSTRACT FROM AUTHOR]

The collagen type II alpha 1 (COL2A1) mutation causes severe skeletal malformations, but the pathogenic mechanisms of how this occurs are unclear. To understand how this may happen, a col2a1 p.Gly1170Ser mutated mouse model was constructed and in homozygotes, the chondrodysplasia phenotype was observed. Misfolded procollagen was largely synthesized and retained in dilated endoplasmic reticulum and the endoplasmic reticulum stress (ERS)-unfolded protein response (UPR)-apoptosis cascade was activated. Apoptosis occurred prior to hypertrophy, prevented the formation of a hypertrophic zone, disrupted normal chondrogenic signaling pathways, and eventually caused chondrodysplasia. Heterozygotes had normal phenotypes and endoplasmic reticulum stress intensity was limited with no abnormal apoptosis detected. Our results suggest that earlier chondrocyte death was related to the ERS-UPR-apoptosis cascade and that this was the chief cause of chondrodysplaia. The col2a1 p.Gly1170Ser mutated mouse model offered a novel connection between misfolded collagen and skeletal malformation. Further investigation of this mouse mutant model can help us understand mechanisms of type II collagenopathies. [ABSTRACT FROM AUTHOR]

Abelson tyrosine kinase (Abl) is known to regulate axon guidance, muscle development, and cell-cell interaction in vivo. The Drosophila primary culture system offers advantages in exploring the cellular mechanisms mediated by Abl with utilizing various experimental manipulations. Here we demonstrate that single-embryo cultures exhibit stage-dependent characteristics of cellular differentiation and developmental progression in neurons and myocytes, as well as nerve-muscle contacts. In particular, muscle development critically depends on the stage of dissociated embryos. In wild-type (WT) cultures derived from embryos before stage 12, muscle cells remained within cell clusters and were rarely detected. Interestingly, abundant myocytes were spotted in Abl mutant cultures, exhibiting enhanced myocyte movement and fusion, as well as neuron-muscle contacts even in cultures dissociated from younger, stage 10 embryos. Notably, Abl myocytes frequently displayed well-expanded lamellipodia. Conversely, Abl neurons were characterized with fewer large veil-like lamellipodia, but instead had increased numbers of filopodia and darker nodes along neurites. These distinct phenotypes were equally evident in both homo- and hetero-zygous cultures (Abl/Abl vs. Abl/+) of different alleles (Abl1 and Abl4) indicating dominant mutational effects. Strikingly, in WT cultures derived from stage 10 embryos, high temperature (HT) incubation promoted muscle migration and fusion, partially mimicking the advanced muscle development typical of Abl cultures. However, HT enhanced neuronal growth with increased numbers of enlarged lamellipodia, distinct from the characteristic Abl neuronal morphology. Intriguingly, HT incubation also promoted Abl lamellipodia expansion, with a much greater effect on nerve cells than muscle. Our results suggest that Abl is an essential regulator for myocyte and neuron development and that high-temperature incubation partially mimics the faster muscle development typical of Abl cultures. Despite the extensive alterations by Abl mutations, we observed myocyte fusion events and nerve-muscle contact formation between WT and Abl cells in mixed WT and Abl cultures derived from labeled embryos. [ABSTRACT FROM AUTHOR]

Angiogenesis is regulated by the local microenvironment, including the mechanical interactions between neovessel sprouts and the extracellular matrix (ECM). However, the mechanisms controlling the relationship of mechanical and biophysical properties of the ECM to neovessel growth during sprouting angiogenesis are just beginning to be understood. In this research, we characterized the relationship between matrix density and microvascular topology in an in vitro 3D organ culture model of sprouting angiogenesis. We used these results to design and calibrate a computational growth model to demonstrate how changes in individual neovessel behavior produce the changes in vascular topology that were observed experimentally. Vascularized gels with higher collagen densities produced neovasculatures with shorter vessel lengths, less branch points, and reduced network interconnectivity. The computational model was able to predict these experimental results by scaling the rates of neovessel growth and branching according to local matrix density. As a final demonstration of utility of the modeling framework, we used our growth model to predict several scenarios of practical interest that could not be investigated experimentally using the organ culture model. Increasing the density of the ECM significantly reduced angiogenesis and network formation within a 3D organ culture model of angiogenesis. Increasing the density of the matrix increases the stiffness of the ECM, changing how neovessels are able to deform and remodel their surroundings. The computational framework outlined in this study was capable of predicting this observed experimental behavior by adjusting neovessel growth rate and branching probability according to local ECM density, demonstrating that altering the stiffness of the ECM via increasing matrix density affects neovessel behavior, thereby regulated vascular topology during angiogenesis. [ABSTRACT FROM AUTHOR]