Changes in the amino acid profiles during embryonic development of the blacklip abalone (Haliotis Rubra)

Changes in the total amino acid (TAA) and the free amino acid (FAA) contents during embryonic development, through newly spawned eggs, to pre-settled larvae of blacklip abalone (Haliotis rubra) are described. The TAA (protein bound + free) and the FAA contents increased prior to hatching but decreased towards settlement, but the changes were not always significant between different stages of development. Threonine, arginine, lysine and leucine accounted for nearly 50 % of the total essential amino acids (TEAA) in all developmental stages. The mean FAA content of newly spawned eggs was 262.8 ± 28.2 pmol·ind^–1 and accounted for 11.5 ± 8.3 % of the TAA. Free essential amino acid (FEAA) content increased significantly as development progressed (P < 0.05), in which threonine, arginine and lysine accounted for over 63 % of this pool. In all developmental stages, the FAA pool was dominated by the non-essential amino acids taurine + proline which accounted for 79.5 % of the total. Generally, the FAA accounted for between 10 to 15 % of the TAA in the different developmental stages of blacklip abalone. All evidence appears to indicate that in blacklip abalone the energy requirements during early ontogeny are mostly met with from the lipid reserves, and that there is a tendency to conserve amino acids until pre-settlement.

Adamts5, the gene encoding a proteoglycan-degrading metalloprotease, is expressed by specific cell lineages during mouse embryonic development and in adult tissues.

The secreted metalloprotease ADAMTS5 is implicated in destruction of the cartilage proteoglycan aggrecan in arthritis, but its physiological functions are unknown. Its expression profile during embryogenesis and in adult tissues is therefore of considerable interest. β-Galactosidase (β-gal) histochemistry, enabled by a LacZ cassette inserted in the Adamts5 locus, and validated by in situ hybridization with an Adamts5 cRNA probe and ADAMTS5 immunohistochemistry, was used to profile Adamts5 expression during mouse embryogenesis and in adult mouse tissues. Embryonic expression was scarce prior to 11.5 days of gestation (E11.5) and noted only in the floor plate of the developing brain at E9.5. After E11.5 there was continued expression in brain, especially in the choroid plexus, peripheral nerves, dorsal root ganglia, cranial nerve ganglia, spinal and cranial nerves, and neural plexuses of the gut. In addition to nerves, developing limbs have Adamts5 expression in skeletal muscle (from E13.5), tendons (from E16.5), and inter-digital mesenchyme of the developing autopod (E13.5–15.5). In adult tissues, there is constitutive Adamts5 expression in arterial smooth muscle cells, mesothelium lining the peritoneal, pericardial and pleural cavities, smooth muscle cells in bronchi and pancreatic ducts, glomerular mesangial cells in the kidney, dorsal root ganglia, and in Schwann cells of the peripheral and autonomic nervous system. Expression of Adamts5 during neuromuscular development and in smooth muscle cells coincides with the broadly distributed proteoglycan versican, an ADAMTS5 substrate. These observations suggest the major contexts in which developmental and physiological roles could be sought for this protease.

High atmospheric temperatures and 'ambient incubation' drive embryonic development and lead to earlier hatching in a passerine bird

Tropical and subtropical species typically experience relatively high atmospheric temperatures during reproduction, and are subject to climate-related challenges that are largely unexplored, relative to more extensive work conducted in temperate regions. We studied the effects of high atmospheric and nest temperatures during reproduction in the zebra finch. We characterized the temperature within nests in a subtropical population of this species in relation to atmospheric temperature. Temperatures within nests frequently exceeded the level at which embryo's develop optimally, even in the absence of parental incubation. We experimentally manipulated internal nest temperature to demonstrate that an average difference of 6°C in the nest temperature during the laying period reduced hatching time by an average of 3% of the total incubation time, owing to 'ambient incubation'. Given the avian constraint of laying a single egg per day, the first eggs of a clutch are subject to prolonged effects of nest temperature relative to later laid eggs, potentially increasing hatching asynchrony. While birds may ameliorate the negative effects of ambient incubation on embryonic development by varying the location and design of their nests, high atmospheric temperatures are likely to constitute an important selective force on avian reproductive behaviour and physiology in subtropical and tropical regions, particularly in the light of predicted climate change that in many areas is leading to a higher frequency of hot days during the periods when birds breed.

Citalopram and sertraline exposure compromises embryonic bone development

Selective serotonin reuptake inhibitors (SSRIs) are the most commonly prescribed treatments for depression and, as a class of drugs, are among the most used medications in the world. Concern regarding possible effects of SSRI treatment on fetal development has arisen recently as studies have suggested a link between maternal SSRI use and an increase in birth defects such as persistent pulmonary hypertension, seizures and craniosynostosis. Furthermore, SSRI exposure in adults is associated with decreased bone mineral density and increased fracture risk, and serotonin receptors are expressed in human osteoblasts and osteoclasts. To determine possible effects of SSRI exposure on developing bone, we treated both zebrafish, during embryonic development, and human mesenchymal stem cells (MSCs), during differentiation into osteoblasts, with the two most prescribed SSRIs, citalopram and sertraline. SSRI treatment in zebrafish decreased bone mineralization, visualized by alizarin red staining and decreased the expression of mature osteoblast-specific markers during embryogenesis. Furthermore, we showed that this inhibition was not associated with increased apoptosis. In differentiating human MSCs, we observed a decrease in osteoblast activity that was associated with a decrease in expression of the osteoblast-specific genes Runx2, Sparc and Spp1, measured with quantitative real-time PCR (qRT-PCR). Similar to the developing zebrafish, no increase in expression of the apoptotic marker Caspase 3 was observed. Therefore, we propose that SSRIs inhibit bone development by affecting osteoblast maturation during embryonic development and MSC differentiation. These results highlight the need to further investigate the risks of SSRI use during pregnancy in exposing unborn babies to potential skeletal abnormalities.Molecular Psychiatry advance online publication, 8 September 2015; doi:10.1038/mp.2015.135.

Induced spawning, larval development and rearing of two indigenous Malaysian mahseer, Tor tambroides and T. douronensis

The Mahseers (Tor spp.) are highly valued freshwater fishes across the Himalayan and South-east Asian regions. Over exploitation of natural stocks because of high demand and the deteriorating environmental conditions have resulted in marked decline of mahseers in the wild. Malaysian mahseers, T. tambroides (Bleeker) and T. douronensis (Valenciennes), locally known as empurau, kelah or belian and semah, respectively, have significant cultural and economic importance but both species are now threatened in the wild because of environmental degradation and over fishing. A captive breeding programme was instigated to attempt to propagate these two species artificially for conservation and aquaculture purposes. Both pond-reared and tank-held T. tambroides and T. douronensis reached sexual maturity in captivity and were successfully induced to spawn using hormone treatments. Ovaprim (0.5 mL kg⁻¹) was the most successful hormone treatment for both species. Pre-treatment of fish with Ovaplant (28–68 μg kg⁻¹, 2–7 weeks before spawning induction) greatly improved the success rate of spawning induction. Repeat spawning (within 4 months of initial spawning) was induced in some captive fish. Use of formalin baths improved hatching by preventing fungal infections. Embryonic development and hatching are described. Juveniles were reared in static greenwater ponds. Tor tambroides reached 142–179 g (max 270 g) in 60 weeks. These results represent the first successful captive spawning and rearing of both species. Options for future research to improve production are discussed.

Suppressor of cytokine signaling 4 and 5 in zebrafish development

This research provides insight into the evolution and function of zebrafish cell signaling proteins, Suppressor of Cytokine Signaling 4a, 4b and 5a. These signaling proteins were found to play essential roles during early and late stages of embryonic development, with specific roles in sensory processing and immune cell development.

Sex-specific developmental plasticity in response to yolk corticosterone in an oviparous lizard

Corticosterone exposure during prenatal development as a result of maternal upregulation of circulating hormone levels has been shown to have effects on offspring development in mammals. Corticosterone has also been documented in egg yolk in oviparous vertebrates, but the extent to which this influences phenotypic development is less studied. We show that maternal corticosterone is transferred to egg yolk in an oviparous lizard (the mallee dragon, Ctenophorus fordi Storr), with significant variation among clutches in hormone levels. Experimental elevation of yolk corticosterone did not affect hatching success, incubation period or offspring sex ratio. However, corticosterone did have a sex-specific effect on skeletal growth during embryonic development. Male embryos exposed to relatively high levels of corticosterone were smaller on average than control males at hatching whereas females from hormone-treated eggs were larger on average than control females. The data thus suggest that males are not just more sensitive to the detrimental effects of corticosterone but rather that the sexes may have opposite responses to corticosterone during development. Positive selection on body size at hatching for both sexes in this species further suggests that increased corticosterone in egg yolk may have sex-specific fitness consequences, with potential implications for sex allocation and the evolution of hormone-mediated maternal effects.

Ontogeny of putative GABAergic neurons and their receptors in the nervous system of the crayfish Cherax destructor

Inhibitory neurons exert control the expression of many aspects of behaviour by regulating the effectiveness of excitatory neural function. By comparison with excitatory neural systems, relatively little is known about the development of inhibitory neurons and the influence which these neurons exert on the development of other neural systems. Two issues which relate to the development of inhibitory neurons are of particular interest. First, a paradox arises when inhibitory neurons are considered in terms of modern models of synaptic development which involve activity-dependent mechanisms of synaptic plasticity. Second, there is some evidence that inhibitory neurotransmitters may act in a special trophic manner during the early development of nervous systems. Investigations of these issues would be greatly facilitated in a neural system in which it was possible to experimentally control aspects of the development of individual pre- and postsynaptic cells. The aim of the results presented in this thesis was to characterise the normal development of one such system: the GABAergic inhibitory system of the Australian freshwater crayfish, Cherax destructor. The ontogeny of the inhibitory neurotransmitter GABA across the embryonic period of 30% to 100% development was investigated using immunohistochemical techniques. GABA-like immunoreactive cells and fibres were first detected in the embryonic brain region. The expression of GABA-like immunoreactivity progressed along a rostro-caudal gradient, with GABA-like immunoreactive cells being detected in the most anterior thoracic ganglia at 45% development and in all ganglia by 65% development. GABA-like immunoreactive fibres were evident in peripheral nerves as early as 55% development and ramified extensively throughout the neuropil of the nervous system by 65% development. By contrast, immunoreactivity to the primary excitatory neurotransmitter, glutamate, was not detected until 60-65% development. Glutamate-like immunoreactivity at 60-65% development was evident only in the form of punctate staining in the midline of the ventral nerve cord. Cell body staining was observed only at 90% development and was restricted to only a few cells on the periphery of the ventral nerve cord. Radio-labelled ligand binding methods and autoradiography were used to study the expression of putative GABA receptors in the Cherax embryos from 30% to 100% development. Specific binding was evident in the earliest embryos studies at 30% development. There was an initial increase in binding from 30% to 40% development, followed by a dramatic drop to almost zero binding at 50-55% development. This was followed by a gradual increase in binding levels with age, reaching a plateau at 85% development. Preliminary pharmacological evaluation of binding indicated that at least three GABA receptor types were expressed during embryonic development. Methods for culturing, dissociated neural tissues explanted form Cherax embryos at 85% development were established. The success of cultures was demonstrated by neurite extension, and neuronal networks in which neurons appeared to form connections with other neurons and with explanted muscle cells after two days in culture. Immunohistochemical studies demonstrated that some explanted neurons expressed GABA-like immunoreactivity within two days of explanting. These studies have provided a comprehensive description of the development of GABAergic neurons and their receptors in Cherax destructor embryos. The very early expression of GABA-like immunoreactivity, coupled with the early onset of specific GABA binding, strongly indicates that the GABAergic neurons are functional and able to exert an effect on other cells during much of the period of nervous system development in crayfish embryos. These results support the hypothesis that inhibitory neurons may play an important role as regulators of the overall process of assembly and maturation of the nervous system and provide a substantial basis for future experimental studies in which the specific action of inhibitory neurons on the development of discrete components of the crayfish nervous system may be investigated.

Consistent paternity skew through ontogeny in Peron's tree frog (Litoria peronii)

Background
A large number of studies in postcopulatory sexual selection use paternity success as a proxy for fertilization success. However, selective mortality during embryonic development can lead to skews in paternity in situations of polyandry and sperm competition. Thus, when assessment of paternity fails to incorporate mortality skews during early ontogeny, this may interfere with correct interpretation of results and subsequent evolutionary inference. In a previous series of in vitro sperm competition experiments with amphibians (Litoria peronii), we showed skewed paternity patterns towards males more genetically similar to the female.

Methodology/Principal Findings
Here we use in vitro fertilizations and sperm competition trials to test if this pattern of paternity of fully developed tadpoles reflects patterns of paternity at fertilization and if paternity skews changes during embryonic development. We show that there is no selective mortality through ontogeny and that patterns of paternity of hatched tadpoles reflects success of competing males in sperm competition at fertilization.

Conclusions/Significance
While this study shows that previous inferences of fertilization success from paternity data are valid for this species, rigorous testing of these assumptions is required to ensure that differential embryonic mortality does not confound estimations of true fertilization success.

Analysis of the Stat5 genes in zebrafish

This dissertation identified and characterised a key genetic regulator called Stat5 using zebrafish. Up-regulation of Stat5 led to an increase in blood cells, indicative of pre-leukaemia, whilst down-regulation decreased these cells and caused other defects. This work shows that Stat5 is critical in blood cell maturation and early embryonic development.

Mutation of the LUNATIC FRINGE gene in humans Causes spondylocostal dysostosis with a severe vertebral phenotype

The spondylocostal dysostoses (SCDs) are a heterogeneous group of vertebral malsegmentation disorders that arise during embryonic development by a disruption of somitogenesis. Previously, we had identified two genes that cause a subset of autosomal recessive forms of this disease: DLL3 (SCD1) and MESP2 (SCD2). These genes are important components of the Notch signaling pathway, which has multiple roles in development and disease. Here, we have used a candidate-gene approach to identify a mutation in a third Notch pathway gene, LUNATIC FRINGE (LFNG), in a family with autosomal recessive SCD. LFNG encodes a glycosyltransferase that modifies the Notch family of cell-surface receptors, a key step in the regulation of this signaling pathway. A missense mutation was identified in a highly conserved phenylalanine close to the active site of the enzyme. Functional analysis revealed that the mutant LFNG was not localized to the correct compartment of the cell, was unable to modulate Notch signaling in a cell-based assay, and was enzymatically inactive. This represents the first known mutation in the human LFNG gene and reinforces the hypothesis that proper regulation of the Notch signaling pathway is an absolute requirement for the correct patterning of the axial skeleton.

Two novel missense mutations in hairy-and-enhancer-of-split-7 in a family with spondylocostal dysostosis

Spondylocostal dysostosis (SCD) is an inherited disorder with abnormal vertebral segmentation that results in extensive hemivertebrae, truncal shortening and abnormally aligned ribs. It arises during embryonic development by a disruption of formation of somites (the precursor tissue of the vertebrae, ribs and associated tendons and muscles). Four genes causing a subset of autosomal recessive forms of this disease have been identified: DLL3 (SCDO1: MIM 277300), MESP2 (SCDO2: MIM 608681), LFNG (SCDO3: MIM609813) and HES7 (SCDO4). These genes are all essential components of the Notch signalling pathway, which has multiple roles in development and disease. Previously, only a single SCD-causative missense mutation was described in HES7. In this study, we have identified two new missense mutations in the HES7 gene in a single family, with only individuals carrying both mutant alleles being affected by SCD. In vitro functional analysis revealed that one of the mutant HES7 proteins was unable to repress gene expression by DNA binding or protein heterodimerization.

Estimating nephron number in the developing kidney using the physical disector/fractionator combination

Over the last decade the development of new molecular biology tools, advanced microscopy, live imaging and systems biology approaches have revolutionized our conception of how embryonic development proceeds. One fundamental aspect of development biology is the concept of morphogenesis: understanding how a group of multipotent cells organize and differentiate into a complex organ. In Kidney Development: Methods and Protocols, expert researchers in the field detail different approaches to tackle kidney development. These approaches include culture and live imaging aspects of kidney development, analyzing the 3-dimensional aspects of branching morphogenesis as well as nephrogenesis, manipulation of the gene/protein expression during kidney development as well as in the adult kidney, and how to assess kidney malformation and disease. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Kidney Development: Methods and Protocols seeks to aid scientists in the further study of the process of morphogenesis which is fundamental important not only for studying developmental biology but also for regenerative medicine.

Metabolic profile analysis of zebrafish embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.