Long-term imaging of living adult zebrafish.

The zebrafish has become a widely used animal model due, in large part, to its accessibility to and usefulness for high-resolution optical imaging. Although zebrafish research has historically focused mostly on early development, in recent years the fish has increasingly been used to study regeneration, cancer metastasis, behavior and other processes taking place in juvenile and adult animals. However, imaging of live adult zebrafish is extremely challenging, with survival of adult fish limited to a few tens of minutes using standard imaging methods developed for zebrafish embryos and larvae. Here, we describe a new method for imaging intubated adult zebrafish using a specially designed 3D printed chamber for long-term imaging of adult zebrafish on inverted microscope systems. We demonstrate the utility of this new system by nearly day-long observation of neutrophil recruitment to a wound area in living double-transgenic adult casper zebrafish with fluorescently labeled neutrophils and lymphatic vessels, as well as intubating and imaging the same fish repeatedly. We also show that Mexican cavefish can be intubated and imaged in the same way, demonstrating this method can be used for long-term imaging of adult animals from diverse aquatic species.

Two-Photon Absorbing AIEgens: Influence of Stereoconfiguration on Their Crystallinity and Spectroscopic Properties and Applications in Bioimaging.

This paper aims at designing chromophores with efficient aggregation-induced emission (AIE) properties for two-photon fluorescence microscopy (2PFM), which is one of the best-suited types of microscopy for the imaging of living organisms or thick biological tissues. Tetraphenylethylene (TPE) derivatives are common building blocks in the design of chromophores with efficient AIE properties. Therefore, in this study, extended TPE AIEgens specifically optimized for two-photon absorption (2PA) are synthesized and the resulting (E/Z) isomers are separated using chromatography on chiral supports. Comparative characterization of the AIE properties is performed on the pure (Z) and (E) isomers and the mixture, allowing us, in combination with powder X-ray diffraction and solid-state NMR, to document a profound impact of crystallinity on solid-state fluorescence properties. In particular, we show that stereopure AIEgens form aggregates of superior crystallinity, which in turn exhibit a higher fluorescence quantum yield compared to diastereoisomers mixtures. Preparation of stereopure organic nanoparticles affords very bright fluorescent contrast agents, which are then used for cellular and intravital two-photon microscopy on human breast cancer cells and on zebrafish embryos.

The denticle surface of thresher shark tails: Three-dimensional structure and comparison to other pelagic species.

Shark skin denticles (scales) are diverse in morphology both among species and across the body of single individuals, although the function of this diversity is poorly understood. The extremely elongate and highly flexible tail of thresher sharks provides an opportunity to characterize gradients in denticle surface characteristics along the length of the tail and assess correlations between denticle morphology and tail kinematics. We measured denticle morphology on the caudal fin of three mature and two embryo common thresher sharks (Alopias vulpinus), and we compared thresher tail denticles to those of eleven other shark species. Using surface profilometry, we quantified 3D-denticle patterning and texture along the tail of threshers (27 regions in adults, and 16 regions in embryos). We report that tails of thresher embryos have a membrane that covers the denticles and reduces surface roughness. In mature thresher tails, surfaces have an average roughness of 5.6 µm which is smoother than some other pelagic shark species, but similar in roughness to blacktip, porbeagle, and bonnethead shark tails. There is no gradient down the tail in roughness for the middle or trailing edge regions and hence no correlation with kinematic amplitude or inferred magnitude of flow separation along the tail during locomotion. Along the length of the tail there is a leading-to-trailing-edge gradient with larger leading edge denticles that lack ridges (average roughness = 9.6 µm), and smaller trailing edge denticles with 5 ridges (average roughness = 5.7 µm). Thresher shark tails have many missing denticles visible as gaps in the surface, and we present evidence that these denticles are being replaced by new denticles that emerge from the skin below.

In vivo analysis of renal epithelial cells in zebrafish.

The zebrafish kidney has been used effectively for studying kidney development, repair and disease. New gene editing capability makes it a more versatile in vivo vertebrate model system to investigate renal epithelial cells in their native environment. In this chapter we focus on dissecting gene function in basic cellular biology of renal epithelial cells, including lumen formation and cell polarity, in intact zebrafish embryos.

Visualizing gene expression during zebrafish pronephros development and regeneration.

The vertebrate kidney is comprised of functional units known as nephrons. Defects in nephron development or activity are a common feature of kidney disease. Current medical treatments are unable to ameliorate the dire consequences of nephron deficit or injury. Although there have been tremendous advancements in our understanding of nephron ontogeny and the response to damage, many significant knowledge gaps still remain. The zebrafish embryo kidney, or pronephros, is an ideal model for many renal development and regeneration studies because it is comprised of nephrons that share conserved features with the nephron units that comprise the mammalian metanephric kidney. In this chapter, we provide an overview about the benefits of using the zebrafish pronephros to study the mechanisms underlying nephrogenesis as well as epithelial repair and regeneration. We subsequently detail methods for the spatiotemporal assessment of gene and protein expression in zebrafish embryos that can be used to extend the understanding of nephron development and disease, and thereby create new opportunities to identify therapeutic strategies for regenerative medicine.

Morphometric signatures of exposure to endocrine disrupting chemicals in zebrafish eleutheroembryos.

Understanding the mode of action of the different pollutants in human and wildlife health is a key step in environmental risk assessment. The aim of this study was to determine signatures that could link morphological phenotypes to the toxicity mechanisms of four Endocrine Disrupting Chemicals (EDCs): bisphenol A (BPA), perfluorooctanesulfonate potassium salt (PFOS), tributyltin chloride (TBT), and 17-ß-estradiol (E2). Zebrafish (Danio rerio) eleutheroembryos were exposed from 2 to 5 dpf to a wide range of BPA, PFOS, TBT and E2 concentrations. At the end of the exposures several morphometric features were assessed. Common and non-specific effects on larvae pigmentation or swim bladder area were observed after exposures to all compounds. BPA specifically induced yolk sac malabsorption syndrome and altered craniofacial parameters, whereas PFOS had specific effects on the notochord formation presenting higher rates of scoliosis and kyphosis. The main effect of E2 was an increase in the body length of the exposed eleutheroembryos. In the case of TBT, main alterations on the morphological traits were related to developmental delays. When integrating all morphometrical parameters, BPA showed the highest rates of malformations in terms of equilethality, followed by PFOS and, distantly, by TBT and E2. In the case of BPA and PFOS, we were able to relate our results with effects on the transcriptome and metabolome, previously reported. We propose that methodized morphometric analyses in zebrafish embryo model can be used as an inexpensive and easy screening tool to predict modes of action of a wide-range number of contaminants.

Toxicological Evaluation of SiO2 Nanoparticles by Zebrafish Embryo Toxicity Test.

As the use of nanoparticles (NPs) is increasing, the potential toxicity and behavior of NPs in living systems need to be better understood. Our goal was to evaluate the developmental toxicity and bio-distribution of two different sizes of fluorescently-labeled SiO2 NPs, 25 and 115 nm, with neutral surface charge or with different surface functionalization, rendering them positively or negatively charged, in order to predict the effect of NPs in humans. We performed a zebrafish embryo toxicity test (ZFET) by exposing the embryos to SiO2 NPs starting from six hours post fertilization (hpf). Survival rate, hatching time, and gross morphological changes were assessed at 12, 24, 36, 48, 60, and 72 hpf. We evaluated the effect of NPs on angiogenesis by counting the number of sub-intestinal vessels between the second and seventh intersegmental vessels and gene expression analysis of vascular endothelial growth factor (VEGF) and VEGF receptors at 72 hpf. SiO2 NPs did not show any adverse effects on survival rate, hatching time, gross morphology, or physiological angiogenesis. We found that SiO2 NPs were trapped by the chorion up until to the hatching stage. After chemical removal of the chorion (dechorionation), positively surface-charged SiO2 NPs (25 nm) significantly reduced the survival rate of the fish compared to the control group. These results indicate that zebrafish chorion acts as a physical barrier against SiO2 NPs, and removing the chorions in ZFET might be necessary for evaluation of toxicity of NPs.

Developmental toxicity and cardiac effects of butyl benzyl phthalate in zebrafish embryos.

Phthalic acid esters (PAEs), commonly called phthalates, have become ubiquitous environment pollutants. Studies have focused on reproductive toxicity, neurotoxicity, teratogenicity, tumourigenesis, and mutagenesis of phthalates. However, relatively little is known about the phthalates effects on the heart. Butyl benzyl phthalate (BBP), a member of PAEs, is classified by the US Environmental Protection Agency as a priority environmental pollutant. We studied the developmental toxicity of BBP, especially its effects on the heart development, in zebrafish (Danio rerio) embryos. Embryos at 4hr post-fertilization (hpf) were exposed to 0, 0.1, 0.6 and 1.2mg/L BBP until 72hpf. BBP caused abnormalities in embryo morphology, including yolk-sac edema, spinal curvature, tail deformity, uninflated swim bladder and cardiac defects. Exposure to 0.6mg/L BBP significantly increased the malformation rate, caused growth inhibition, increased the cardiac malformation rate as well as the distance between the sinus venosus (SV) and bulbus arteriosus (BA), and reduced the heart rate of embryos. Exposure to 1.2mg/L BBP significantly affected all endpoints, except survival rate at 24hpf. To preliminarily elucidate the potential mechanism of heart developmental toxicity caused by BBP, we examined the expression of two genes related to heart development, Nkx2.5 and T-box transcription factor 5, by real-time quantitative PCR. The expression of the two genes was dose-dependently downregulated with BBP. BBP could induce developmental toxicity, with adverse effects on the heart development in zebrafish embryos, and alter the expression of genes related to heart development.

Descriptions and revised key to the eggcases of the skates (Rajiformes: Rajidae) and catsharks (Carcharhiniformes: Scyliorhinidae) of the British Isles.

Updated descriptions and measurements for the eggcases of 10 rajiform and three scyliorhinid species occurring in the shelf seas around the British Isles are given, based on museum material, specimens collected during the 'Great Eggcase Hunt' (a Shark Trust recording project), and specimens obtained from fishery surveys. Quantitative data are given for Amblyraja radiata (n = 94), Dipturus batis (n = 24), D. cf. intermedia (n = 33), Leucoraja naevus (n = 94), Raja brachyura (n = 53), R. clavata (n = 52), R. microocellata (n = 57), R. montagui (n = 52), R. undulata (n = 52), Rostroraja alba (n = 5), Galeus melastomus (n = 7), Scyliorhinus canicula (n = 52) and S. stellaris (n = 58). An updated key for the identification of elasmobranch eggcases is provided, incorporating recent changes in skate taxonomy.

External morphological features of the gills of Ichthyophis bannanicus embryo (Amphibia, Gymnophiona) / Características morfológicas externas de las branquias del embrión de Ichthyophis bannanicus (Amphibia, Gymnophiona)

The gills of Ichthyophis bannanicus have yet to be investigated. This paper describes the external morphological features of the gills of mature I. bannanicus embryos exhibiting three pairs of gills on their neck region. Each gill is composed of an axis and filaments. In newly released embryos, the filaments and the axis form at approximately 90° relative to each other; eventually, this angle decreases and the color of the gill fades. The filaments on the axis are arranged alternately, and their spacing varies. The mid-pair of gills is significantly longer by nearly twofold than the front and rear pairs. Likewise, the lengths of the front and rear pairs of gills are not significantly different (P >0.05); for the same pair of gills, the lengths of the left and right parts are not significantly different (P >0.05). The number of filaments is greater in the mid-pair of gills than in the front and rear pairs (P <0.05); the number of filaments in the front pair is not significantly different from that of the rear pair (P >0.05); the number of filaments in the left part does not significantly differ from that of the right parts (P >0.05). Results showed that the gills of I. bannanicus embryo are more similar to those of other species in Ichthyophiidae than to those of species in other families.

Las branquias del Ichthyophis bannanicus aún no se han investigado. En este trabajo se describen las características morfológicas externas de las branquias de embriones maduros de I. bannanicus, que exhiben tres pares de branquias en la región del cuello. Cada branquia está compuesta de un eje y filamentos. En embriones recién liberados los filamentos y la forma del eje es de aproximadamente 90° respecto a la otra; finalmente, este ángulo disminuye y el color de las branquias se desvanece. Los filamentos en el eje están dispuestos en forma alternada y su separación varía. La media de par de branquias es significativamente más larga, por casi el doble que los pares anterior y posterior. Del mismo modo, las longitudes de la parte delantera y posterior de pares de branquias no son significativamente diferentes (p >0,05); para el mismo par de branquias, las longitudes de las partes izquierda y derecha no son significativamente diferentes (p >0,05). El número de filamentos es mayor en el par medio de branquias, que en la parte delantera y posterior de pares (p <0,05); el número de filamentos en el par frontal no es significativamente diferente de la del par trasero (p >0,05); el número de filamentos en la parte izquierda no varía significativamente del de las partes derechas (p >0,05). Los resultados mostraron que las branquias del embrión de I. bannanicus son más similares a los de otras especies en Ichthyophiidae que de otras especies.

Patterning mechanisms of the sub-intestinal venous plexus in zebrafish.

Despite considerable interest in angiogenesis, organ-specific angiogenesis remains less well characterized. The vessels that absorb nutrients from the yolk and later provide blood supply to the developing digestive system are primarily venous in origin. In zebrafish, these are the vessels of the Sub-intestinal venous plexus (SIVP) and they represent a new candidate model to gain an insight into the mechanisms of venous angiogenesis. Unlike other vessel beds in zebrafish, the SIVP is not stereotypically patterned and lacks obvious sources of patterning information. However, by examining the area of vessel coverage, number of compartments, proliferation and migration speed we have identified common developmental steps in SIVP formation. We applied our analysis of SIVP development to obd mutants that have a mutation in the guidance receptor PlexinD1. obd mutants show dysregulation of nearly all parameters of SIVP formation. We show that the SIVP responds to a unique combination of pathways that control both arterial and venous growth in other systems. Blocking Shh, Notch and Pdgf signaling has no effect on SIVP growth. However Vegf promotes sprouting of the predominantly venous plexus and Bmp promotes outgrowth of the structure. We propose that the SIVP is a unique model to understand novel mechanisms utilized in organ-specific angiogenesis.

Identification of the optic recess region as a morphogenetic entity in the zebrafish forebrain.

Regionalization is a critical, highly conserved step in the development of the vertebrate brain. Discrepancies exist in how regionalization of the anterior vertebrate forebrain is conceived since the "preoptic area" is proposed to be a part of the telencephalon in tetrapods but not in teleost fish. To gain insight into this complex morphogenesis, formation of the anterior forebrain was analyzed in 3D over time in zebrafish embryos, combining visualization of proliferation and differentiation markers, with that of developmental genes. We found that the region containing the preoptic area behaves as a coherent morphogenetic entity, organized around the optic recess and located between telencephalon and hypothalamus. This optic recess region (ORR) makes clear borders with its neighbor areas and expresses a specific set of genes (dlx2a, sim1a and otpb). We thus propose that the anterior forebrain (secondary prosencephalon) in teleosts contains three morphogenetic entities (telencephalon, ORR and hypothalamus), instead of two (telencephalon and hypothalamus). The ORR in teleosts could correspond to "telencephalic stalk area" and "alar hypothalamus" in tetrapods, resolving current inconsistencies in the comparison of basal forebrain among vertebrates.

Gill dimensions in near-term embryos of Amazonian freshwater stingrays (Elasmobranchii: Potamotrygonidae) and their relationship to the lifestyle and habitat of neonatal pups

This comparative study of gill morphometrics in near-term embryos of freshwater stingray potamotrygonids examines gill dimensions in relation to neonatal lifestyle and habitat. In embryos of the potamotrygonids Paratrygon aiereba, Plesiotrygon iwamae, Potamotrygon motoro, Potamotrygon orbignyi, and cururu ray Potamotrygon sp. the number and length of filaments, total gill surface area, mass-specific surface area, water-blood diffusion distance, and anatomical diffusion factor were analysed. In all potamotrygonids, the 3rd branchial arch possessed a larger respiratory surface than the other gill arches. Larger embryos had more gill surface area and large spiracles, which are necessary to maintain the high oxygen uptake needed due to their larger body size. However, the higher mass-specific gill surface area observed in near-term embryos may be advantageous because neonates can use hypoxic environments as refuges against predators, as well as catch small prey that inhabit the same environment. As expected from their benthic mode of life, freshwater stingrays are sluggish animals compared to pelagic fishes. However, based on gill respiratory morphometry (such as gill area, mass-specific gill area, the water-blood diffusion barrier, anatomical diffusion factor, and relative opening of the spiracle), subtypes of lifestyles can be observed corresponding to: active, intermediate, and sluggish species according to Gray's scale.

Este estudo realizado com embriões a termo de arraias de água doce (Potamotrygonidae) compara e analisa as dimensões branquiais em relação ao estilo de vida e habitat dos neonatos. Nos embriões de Paratrygon aiereba, Plesiotrygon iwamae, Potamotrygon motoro, Potamotrygon orbignyi e Potamotrygon sp. (arraia cururu) foram analisados número e comprimento dos filamentos, área branquial, área superficial branquial massa-específica, barreira de difusão água-sangue e fator de difusão anatômico. Em todos os potamotrigonídeos estudados, o 3º arco branquial possui uma superfície respiratória maior que os demais arcos. Embriões de espécies de maior porte possuem grandes espiráculos e maior área de superfície branquial. Isso ajuda a manter a taxa de absorção de oxigênio proporcional ao requerimento do animal. No entanto, a grande área de superfície branquial massa-específica observadas nos embriões a termo pode ser vantajosa, pois os neonatos podem usar ambientes hipóxicos como refúgios contra predadores, bem como capturar pequenas presas que habitam o mesmo ambiente. Devido ao modo de vida bentônico, as arraias de água doce são nadadoras lentas comparadas aos peixes pelágicos. No entanto, com base na morfometria branquial (área de superfície branquial, área branquial massa-específica, barreira de difusão água-sangue, fator de difusão anatômico e abertura relativa do espiráculo), subtipos de estilos de vida podem ser observados: ativas, intermediárias e lentas, conforme escala definida por Gray.

Imaging without fluorescence: nonlinear optical microscopy for quantitative cellular imaging.

Quantitative single-cell analysis enables the characterization of cellular systems with a level of detail that cannot be achieved with ensemble measurement. In this Feature we explore quantitative cellular imaging applications with nonlinear microscopy techniques. We first offer an introductory tutorial on nonlinear optical processes and then survey a range of techniques that have proven to be useful for quantitative live cell imaging without fluorescent labels.

Symmetry breakage in the vertebrate embryo: when does it happen and how does it work?

Asymmetric development of the vertebrate embryo has fascinated embryologists for over a century. Much has been learned since the asymmetric Nodal signaling cascade in the left lateral plate mesoderm was detected, and began to be unraveled over the past decade or two. When and how symmetry is initially broken, however, has remained a matter of debate. Two essentially mutually exclusive models prevail. Cilia-driven leftward flow of extracellular fluids occurs in mammalian, fish and amphibian embryos. A great deal of experimental evidence indicates that this flow is indeed required for symmetry breaking. An alternative model has argued, however, that flow simply acts as an amplification step for early asymmetric cues generated by ion flux during the first cleavage divisions. In this review we critically evaluate the experimental basis of both models. Although a number of open questions persist, the available evidence is best compatible with flow-based symmetry breakage as the archetypical mode of symmetry breakage.

Craniofacial development of hagfishes and the evolution of vertebrates.

Cyclostomes, the living jawless vertebrates including hagfishes and lampreys, represent the most basal lineage of vertebrates. Although the monophyly of cyclostomes has been supported by recent molecular analyses, the phenotypic traits of hagfishes, especially the lack of some vertebrate-defining features and the reported endodermal origin of the adenohypophysis, have been interpreted as hagfishes exhibiting a more ancestral state than those of all other vertebrates. Furthermore, the adult anatomy of hagfishes cannot be compared easily with that of lampreys. Here we describe the craniofacial development of a series of staged hagfish embryos, which shows that their adenohypophysis arises ectodermally, consistent with the molecular phylogenetic data. This finding also allowed us to identify a pan-cyclostome pattern, one not shared by jawed vertebrates. Comparative analyses indicated that many of the hagfish-specific traits can be explained by changes secondarily introduced into the hagfish lineage. We also propose a possibility that the pan-cyclostome pattern may reflect the ancestral programme for the craniofacial development of all living vertebrates.

Molecular and cellular changes associated with the evolution of novel jaw muscles in parrots.

Vertebrates have achieved great evolutionary success due in large part to the anatomical diversification of their jaw complex, which allows them to inhabit almost every ecological niche. While many studies have focused on mechanisms that pattern the jaw skeleton, much remains to be understood about the origins of novelty and diversity in the closely associated musculature. To address this issue, we focused on parrots, which have acquired two anatomically unique jaw muscles: the ethmomandibular and the pseudomasseter. In parrot embryos, we observe distinct and highly derived expression patterns for Scx, Bmp4, Tgfß2 and Six2 in neural crest-derived mesenchyme destined to form jaw muscle connective tissues. Furthermore, immunohistochemical analysis reveals that cell proliferation is more active in the cells within the jaw muscle than in surrounding connective tissue cells. This biased and differentially regulated mode of cell proliferation in cranial musculoskeletal tissues may allow these unusual jaw muscles to extend towards their new attachment sites. We conclude that the alteration of neural crest-derived connective tissue distribution during development may underlie the spatial changes in jaw musculoskeletal architecture found only in parrots. Thus, parrots provide valuable insights into molecular and cellular mechanisms that may generate evolutionary novelties with functionally adaptive significance.

The anti-cancer agent SU4312 unexpectedly protects against MPP(+) -induced neurotoxicity via selective and direct inhibition of neuronal NOS.

BACKGROUND AND PURPOSE: SU4312, a potent and selective inhibitor of VEGF receptor-2 (VEGFR-2), has been designed to treat cancer. Recent studies have suggested that SU4312 can also be useful in treating neurodegenerative disorders. In this study, we assessed neuroprotection by SU4312 against 1-methyl-4-phenylpyridinium ion (MPP(+) )-induced neurotoxicity and further explored the underlying mechanisms. EXPERIMENTAL APPROACH: MPP(+) -treated neurons and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated zebrafish were used to study neuroprotection by SU4312. NOS activity was assayed in vitro to examine direct interactions between SU4312 and NOS isoforms. KEY RESULTS: SU4312 unexpectedly prevented MPP(+) -induced neuronal apoptosis in vitro and decreased MPTP-induced loss of dopaminergic neurons, reduced expression of mRNA for tyrosine hydroxylase and impaired swimming behaviour in zebrafish. In contrast, PTK787/ZK222584, a well-studied VEGFR-2 inhibitor, failed to prevent neurotoxicity, suggesting that the neuroprotective actions of SU4312 were independent of its anti-angiogenic action. Furthermore, SU4312 exhibited non-competitive inhibition of purified neuronal NOS (nNOS) with an IC(50) value of 19.0 µM but showed little or no effects on inducible and endothelial NOS. Molecular docking simulations suggested an interaction between SU4312 and the haem group within the active centre of nNOS. CONCLUSIONS AND IMPLICATION: SU4312 exhibited neuroprotection against MPP(+) at least partly via selective and direct inhibition of nNOS. Because SU4312 could reach the brain in rats, our study also offered a support for further development of SU4312 to treat neurodegenerative disorders, particularly those associated with NO-mediated neurotoxicity.

Development of head and trunk mesoderm in the dogfish, Scyliorhinus torazame: I. Embryology and morphology of the head cavities and related structures.

Vertebrate head segmentation has attracted the attention of comparative and evolutionary morphologists for centuries, given its importance for understanding the developmental body plan of vertebrates and its evolutionary origin. In particular, the segmentation of the mesoderm is central to the problem. The shark embryo has provided a canonical morphological scheme of the head, with its epithelialized coelomic cavities (head cavities), which have often been regarded as head somites. To understand the evolutionary significance of the head cavities, the embryonic development of the mesoderm was investigated at the morphological and histological levels in the shark, Scyliorhinus torazame. Unlike somites and some enterocoelic mesodermal components in other vertebrates, the head cavities in S. torazame appeared as irregular cyst(s) in the originally unsegmented mesenchymal head mesoderm, and not via segmentation of an undivided coelom. The mandibular cavity appeared first in the paraxial part of the mandibular mesoderm, followed by the hyoid cavity, and the premandibular cavity was the last to form. The prechordal plate was recognized as a rhomboid roof of the preoral gut, continuous with the rostral notochord, and was divided anteroposteriorly into two parts by the growth of the hypothalamic primordium. Of those, the posterior part was likely to differentiate into the premandibular cavity, and the anterior part disappeared later. The head cavities and somites in the trunk exhibited significant differences, in terms of histological appearance and timing of differentiation. The mandibular cavity developed a rostral process secondarily; its homology to the anterior cavity reported in some elasmobranch embryos is discussed.

Early development of New Zealand hapuku Polyprion oxygeneios eggs and larvae.

This study describes for the first time the normal development of New Zealand hapuku Polyprion oxygeneios embryos and larvae reared from fertilization to 11 days post-hatch (dph) at a constant temperature. Fertilized eggs were obtained from natural spawnings from communally reared captive wild broodstock. Eggs averaged 2 mm in diameter and had single or multiple oil globules. Embryos developed following the main fish embryological stages and required an average of 1859·50 degree hours post-fertilization (dhpf) to hatch. The newly hatched larvae (4·86 mm mean total length, L(T) ) were undifferentiated, with unpigmented eyes, a single and simple alimentary tube and a finfold that covered the entire body. Larvae relied on the energy from the yolk-sac reserves until 11 dph (7·33 mm mean L(T) ), when yolk-sac reabsorption was almost completed. Some of the major developmental stages from hatching to yolk-sac reabsorption were eye pigmentation (5 dph), upper jaw formation (7 dph), lower jaw formation (8 dph) and mouth opening (8-9 dph). By 9 dph, the digestive system consisted of pancreas, liver, primordial stomach, anterior and posterior gut; therefore, P. oxygeneios larvae would be capable of feeding on live prey. The developmental, morphological and histological data described constitutes essential baseline information on P. oxygeneios biology and normal development.