Energetics of Acidianus ambivalens growth in response to oxygen availability

All life requires energy to drive metabolic reactions such as growth and cell maintenance; therefore, fluctuations in energy availability can alter microbial activity. There is a gap in our knowledge concerning how energy availability affects the growth of extreme chemolithoautotrophs. Toward this end, we investigated the growth of thermoacidophile Acidianus ambivalens during sulfur oxidation under aerobic to microaerophilic conditions. Calorimetry was used to measure enthalpy (ΔH_inc) of microbial activity, and chemical changes in growth media were measured to calculate Gibbs energy change (ΔG_inc) during incubation. In all experiments, Gibbs energy was primarily dissipated through the release of heat, which suggests enthalpy‐driven growth. In microaerophilic conditions, growth was significantly more efficient in terms of biomass yield (defined as C‐mol biomass per mole sulfur consumed) and resulted in lower ΔG_inc and ΔH_inc. ΔG_inc in oxygen‐limited (OL) and oxygen‐ and CO₂‐limited (OCL) microaerophilic growth conditions resulted in averages of ?1.44 × 10³ kJ/C‐mol and ?7.56 × 10² kJ/C‐mol, respectively, and average ΔH_inc values of ?1.11 × 10⁵ kJ/C‐mol and ?4.43 × 10⁴ kJ/C‐mol, respectively. High‐oxygen experiments resulted in lower biomass yield values, an increase in ΔG_inc to ?1.71 × 10⁴ kJ/C‐mol, and more exothermic ΔH_inc values of ?4.71 × 10⁵ kJ/C‐mol. The observed inefficiency in high‐oxygen conditions may suggest larger maintenance energy demands due to oxidative stresses and a preference for growth in microaerophilic environments.     

类胰岛素生长因子信号途径及其对脊椎动物生长和发育的调控作用:以斑马鱼为模式动物的新进展

类胰岛素生长因子(包括IGF-I和IGF-Ⅱ)是进化上保守性很强的多肽。IGFs对脊椎动物的生长和早期发育有极其重要的调控作用。IGF的生理作用是由IGF受体中介并受几个分泌性的IGF结合蛋白调节。本文主要介绍了以斑马鱼为模式动物，用基因敲除、转基因动物和培养细胞系等现代实验方法对IGF信号途径的最新研究进展，综述了IGF配体、受体和结合蛋白的结构特点、基因表达和调节和生物学功能。此外，也对斑马鱼作为模式动物的许多优点进行了探讨。     

Phenothiourea sensitizes zebrafish cranial neural crest and extraocular muscle development to changes in retinoic acid and IGF signaling

1-Phenyl 2-thiourea (PTU) is a tyrosinase inhibitor commonly used to block pigmentation and aid visualization of zebrafish development. At the standard concentration of 0.003% (200 μM), PTU inhibits melanogenesis and reportedly has minimal other effects on zebrafish embryogenesis. We found that 0.003% PTU altered retinoic acid and insulin-like growth factor (IGF) regulation of neural crest and mesodermal components of craniofacial development. Reduction of retinoic acid synthesis by the pan-aldehyde dehydrogenase inhibitor diethylbenzaldehyde, only when combined with 0.003% PTU, resulted in extraocular muscle disorganization. PTU also decreased retinoic acid-induced teratogenic effects on pharyngeal arch and jaw cartilage despite morphologically normal appearing PTU-treated controls. Furthermore, 0.003% PTU in combination with inhibition of IGF signaling through either morpholino knockdown or pharmacologic inhibition of tyrosine kinase receptor phosphorylation, disrupted jaw development and extraocular muscle organization. PTU in and of itself inhibited neural crest development at higher concentrations (0.03%) and had the greatest inhibitory effect when added prior to 22 hours post fertilization (hpf). Addition of 0.003% PTU between 4 and 20 hpf decreased thyroxine (T4) in thyroid follicles in the nasopharynx of 96 hpf embryos. Treatment with exogenous triiodothyronine (T3) and T4 improved, but did not completely rescue, PTU-induced neural crest defects. Thus, PTU should be used with caution when studying zebrafish embryogenesis as it alters the threshold of different signaling pathways important during craniofacial development. The effects of PTU on neural crest development are partially caused by thyroid hormone signaling.     

Reactive oxygen species signaling in primordial germ cell development in Drosophila embryos

REDOX mechanisms that induce biosynthesis of the reactive oxygen species (ROS) have attracted considerable attention due to both the deleterious and beneficial responses elicited by the reactive radical. In several organisms including Drosophila melanogaster, modulation of ROS activity is thought to be crucial for the maintenance of cell fates in developmental contexts. Interestingly, REDOX mechanisms have been shown to be involved in maintaining progenitor fate of stem cells as well as their proliferation and differentiation. Here, we have explored the possible functions of ROS during proper specification and developmental progression of embryonic primordial germ cells (PGCs). Indicating its potential involvement in these processes, ROS can be detected in the embryonic PGCs and the surrounding somatic cells from very early stages of embryogenesis. Using both “loss” and “gain” of function mutations in two different components of the REDOX pathway, we show that ROS levels are likely to be critical in maintaining germ cell behavior, including their directed migration. Altering the activity of a putative regulator of ROS also adversely influences the ability of PGCs to adhere to one another in cellular blastoderm embryos, suggesting potential involvement of this pathway in orchestrating different phases of germ cell migration.     

APC mutant zebrafish uncover a changing temporal requirement for wnt signaling in liver development

Developmental signaling pathways hold the keys to unlocking the promise of adult tissue regeneration, and to inhibiting carcinogenesis. Patients with mutations in the Adenomatous Polyposis Coli (APC) gene are at increased risk of developing hepatoblastoma, an embryonal form of liver cancer, suggesting that Wnt affects hepatic progenitor cells. To elucidate the role of APC loss and enhanced Wnt activity in liver development, we examined APC mutant and wnt inducible transgenic zebrafish. APC^+/− embryos developed enlarged livers through biased induction of hepatic gene programs and increased proliferation. Conversely, APC^−/− embryos formed no livers. Blastula transplantations determined that the effects of APC loss were cell autonomous. Induction of wnt modulators confirmed biphasic consequences of wnt activation: endodermal pattern formation and gene expression required suppression of wnt signaling in early somitogenesis; later, increased wnt activity altered endodermal fate by enhancing liver growth at the expense of pancreas formation; these effects persisted into the larval stage. In adult APC^+/− zebrafish, increased wnt activity significantly accelerated liver regeneration after partial hepatectomy. Similarly, liver regeneration was significantly enhanced in APC^Min/+ mice, indicating the conserved effect of Wnt pathway activation in liver regeneration across vertebrate species. These studies reveal an important and time-dependent role for wnt signaling during liver development and regeneration.     

Genetic analysis of melanophore development in zebrafish embryos

Vertebrate pigment cells are derived from neural crest, a tissue that also forms most of the peripheral nervous system and a variety of ectomesenchymal cell types. Formation of pigment cells from multipotential neural crest cells involves a number of common developmental processes. Pigment cells must be specified; their migration, proliferation, and survival must be controlled and they must differentiate to the final pigment cell type. We previously reported a large set of embryonic mutations that affect pigment cell development from neural crest (R. N. Kelsh et al., 1996, Development 123, 369-389). Based on distinctions in pigment cell appearance between mutants, we proposed hypotheses as to the process of pigment cell development affected by each mutation. Here we describe the cloning and expression of an early zebrafish melanoblast marker, dopachrome tautomerase. We used this marker to test predictions about melanoblast number and pattern in mutant embryos, including embryos homozygous for mutations in the colourless, sparse, touchdown, sunbleached, punkt, blurred, fade out, weiss, sandy, and albino genes. We showed that in homozygous mutants for all loci except colourless and sparse, melanoblast number and pattern are normal. colourless mutants have a pronounced decrease in melanoblast cell number from the earliest stages and also show poor melanoblast differentiation and migration. Although sparse mutants show normal numbers of melanoblasts initially, their number is reduced later. Furthermore, their distribution indicates a defect in melanoblast dispersal. These observations permit us to refine our model of the genetic control of melanophore development in zebrafish embryos.     

Spatial and temporal expression patterns of chitinase genes in developing zebrafish embryos

Chitinases and chitinase like proteins play an important role in mammalian immunity and functions in early zebrafish development have been suggested. Here we report identification of six zebrafish chitinases and chitinase like proteins (called CHIA.1–6) belonging to the glycoside hydrolase family 18, and determine their spatial and temporal expression at 10 stages of zebrafish development.CHIA.4 is highly maternally expressed and it is expressed 100 fold above any other CHIA gene at zygote through to blastula stage. Later, after the maternal to zygotic transition, CHIA.4 expression decreases to the same level as CHIA.5 and CHIA.6. Subsequently, CHIA.1, CHIA.2, CHIA.3 and CHIA.4, CHIA.5, CHIA.6 each follow distinct paths in terms of expression levels.Until 4 days post fertilization the spatial expression patterns of all six CHIA genes overlap extensively, with expression detected predominantly in vascular, ocular and intestinal tissues. At 5 days post fertilization CHIA.1, CHIA.2 and CHIA.3 are expressed almost exclusively in the stomach, whereas CHIA.4, CHIA.5 and CHIA.6 are also prominently expressed in the liver. These different expression patterns may contribute to the establishment of a basis on which functional analysis in older larvae may be founded.     

Role of fibroblast growth factor receptor signaling in kidney development

Fibroblast growth factor receptors (Fgfrs) consist of four signaling family members and one nonsignaling "decoy" receptor, Fgfr-like 1 (Fgfrl1), all of which are expressed in the developing kidney. Several studies have shown that exogenous fibroblast growth factors (Fgfs) affect growth and maturation of the metanephric mesenchyme (MM) and ureteric bud (UB) in cultured tissues. Transgenic and conditional knockout approaches in whole animals have shown that Fgfr1 and Fgfr2 (predominantly the IIIc isoform) in kidney mesenchyme are critical for early MM and UB formation. Conditional deletion of the ligand, Fgf8, in nephron precursors or global deletion of Fgfrl1 interrupts nephron formation. Fgfr2 (likely the IIIb isoform signaling downstream of Fgf7 and Fgf10) is critical for ureteric morphogenesis. Moreover, Fgfr2 appears to act independently of Frs2α (the major signaling adapter for Fgfrs) in regulating UB branching. Loss of Fgfr2 in the MM leads to many kidney and urinary tract anomalies, including vesicoureteral reflux. Thus Fgfr signaling is critical for patterning of virtually all renal lineages at early and later stages of development.     

miR-8 microRNAs regulate the response to osmotic stress in zebrafish embryos

MicroRNAs (miRNAs) are highly conserved small RNAs that act as translational regulators of gene expression, exerting their influence by selectively targeting mRNAs bearing complementary sequence elements. These RNAs function in diverse aspects of animal development and physiology. Because of an ability to act as rapid responders at the level of translation, miRNAs may also influence stress response. In this study, we show that the miR-8 family of miRNAs regulates osmoregulation in zebrafish embryos. Ionocytes, which are a specialized cell type scattered throughout the epidermis, are responsible for pH and ion homeostasis during early development before gill formation. The highly conserved miR-8 family is expressed in ionocytes and enables precise control of ion transport by modulating the expression of Nherf1, which is a regulator of apical trafficking of transmembrane ion transporters. Ultimately, disruption of miR-8 family member function leads to an inability to respond to osmotic stress and blocks the ability to properly traffic and/or cluster transmembrane glycoproteins at the apical surface of ionocytes.     

Dichloroacetate-induced developmental toxicity and production of reactive oxygen species in zebrafish embryos

Dichloroacetate (DCA) is one of the toxic by products that are formed during the chlorine disinfection process of drinking water. In this study, the developmental toxicity of DCA has been determined in zebrafish (Danio rerio) embryos. Embryos were exposed to different concentrations (4, 8, 16, and 32 mM) of the compound at the 4 h postfertilization (hpf) stage of development, and were observed for different developmental toxic effects at 8, 24, 32, 55, 80, and 144 hpf. Exposure of embryos to 8-32 mM of DCA resulted in significant increases in the heart rate and blood flow of the 55 and 80 hpf embryos that turned into significant decreases at the 144 hpf time point. At 144 hpf, malformations of mouth structure, notochord bending, yolk sac edema and behavioral effects including perturbed swimming and feeding behaviors were also observed. DCA was also found to produce time- and concentration-dependent increases in embryonic levels of superoxide anion (O2*-) and nitric oxide (NO), at various stages of development. The results of the study suggest that DCA-induced developmental toxic effects in zebrafish embryos are associated with production of reactive oxygen species in those embryos.     

Targeted knockdown of insulin-like growth factor binding protein-2 disrupts cardiovascular development in zebrafish embryos

IGF binding protein-2 (IGFBP-2) is an evolutionarily conserved protein that binds IGFs and modulates their biological activities. Although the actions of IGFBP-2 have been well studied in vitro, we have a poor understanding of its in vivo functions, particularly during early development. Using the transparent zebrafish embryo as a model, we show that IGFBP-2 mRNA is expressed in lens epithelium and cranial boundary regions during early embryonic development and becomes localized to the liver by the completion of embryogenesis. Targeted knock-down of IGFBP-2 by antisense morpholino-modified oligonucleotides resulted in delayed development, reduced body growth, reduced IGF-I mRNA levels, and disruptions to cardiovascular development, including reduced blood cell number, reduced blood circulation, cardiac dysfunction, and brain ventricle edema. Detailed examination of vascular tissues, using a stable transgenic line of zebrafish expressing green fluorescent protein in vascular endothelial cells, revealed specific angiogenic (vessel sprouting) defects in IGFBP-2 knockdown embryos, with effects being localized in regions associated with IGFBP-2 mRNA expression. These findings suggest that IGFBP-2 is required for general embryonic development and growth and plays a local role in regulating vascular development in a model vertebrate organism.     

Ontogeny of intestinal motility in correlation to neuronal development in zebrafish embryos and larvae

For the first time, spontaneous intestinal activity was demonstrated and quantified before the onset of exogenous feeding in zebrafish Danio rerio embryos and larvae in vivo , using digital motion analysis. At 3 days post fertilization (dpf), erratic and spontaneous contraction waves were observed in the gut. Later (4–7 dpf), more distinct contraction patterns were distinguished, and anterograde and retrograde contraction waves projecting anally and orally along the intestine, respectively, as well as local rectal contraction waves could be identified and quantified. The frequency of both anterograde intestinal and local rectal contractions increased significantly during the first days of development. There was a tendency towards shorter anterograde contraction waves in the first dpf stage investigated, but the velocity of the waves did not differ significantly between the different dpf stages. The presence of developing neurones in the gut of zebrafish was established using immunohistochemistry, staining for a suite of marker proteins (Hu C/D, HNK-1 and acetylated tubulin). Structural neurones were present in the developing gut from the first dpf stage investigated (2 dpf). In conclusion, during the period (3–7 dpf) when erratic contraction waves turn into a more organized pattern of motility there is also a pronounced development of the innervation, suggesting a correlation in time of the development of gut motility and its neuronal control.     

Individual variation in the rate of oxygen consumption by zebrafish embryos

A sensitive microsensor‐based method was used to measure oxygen consumption of individual zebrafish Danio rerio embryos at 6 h intervals from 24 to 75 h post‐fertilization. An increase in oxygen consumption rates from 4·54 to 8·29 nmol O₂ h⁻¹ was found during this period. At the individual level the differences in oxygen consumption rates caused the total oxygen consumption from 24 to 75 h post‐fertilization to vary between 0·261 and 0·462 μmol O₂ per individual with a mean of 0·379 μmol O₂ per individual. A separate carbon mass balance study corroborated the mean total oxygen consumption obtained by yielding a respiratory quotient of 0·80 for this period. These results suggest that there is significant intraspecific variation in the metabolic rate of developing zebrafish embryos, which may influence other early life‐history traits such as growth and starvation resistance.     

Reptin and pontin antagonistically regulate heart growth in zebrafish embryos

Organ size is precisely regulated during development, but the control mechanisms remain obscure. We have isolated a mutation in zebrafish, liebeskummer (lik), which causes development of hyperplastic embryonic hearts. lik encodes Reptin, a component of a DNA-stimulated ATPase complex. The mutation activates ATPase activity of Reptin complexes and causes a cell-autonomous proliferation of cardiomyocytes to begin well after progenitors have fashioned the primitive heart tube. With regard to heart growth, beta-catenin and Pontin, a DNA-stimulated ATPase that is often part of complexes with Reptin, are in the same genetic pathways. Pontin reduction phenocopies the cardiac hyperplasia of the lik mutation. Thus, the Reptin/Pontin ratio serves to regulate heart growth during development, at least in part via the beta-catenin pathway.