Articles in PresS. Am J Physiol Regul Integr Comp Physiol (April 10, 2013). doi:10.1152/ajpregu.00411.2012
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Ontogeny of ornithine-urea cycle gene expression in zebrafish (Danio rerio)
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Christophe M.R. LeMoine* and Patrick .J. Walsh
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Department of Biology, University of Ottawa,
30 Marie Curie private, Ottawa, ON K1N 6N5
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*Author for Correspondence :
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Christophe LeMoine, PhD
Department of Biology
University of Ottawa
30 Marie Curie private
Ottawa, ON K1N 6N5
[email protected]
Tel: 1-613-562-5800 ext 2794
Fax: 1-613-562-5486
Running title: O-UC gene expression in zebrafish
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Copyright © 2013 by the American Physiological Society.
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Abstract
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Although the majority of adult teleosts excrete most of their nitrogenous wastes as ammonia,
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several fish species are capable of producing urea early in development. In zebrafish, it is unclear
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if this results from a functional ornithine-urea cycle (O-UC) and, if so, how it might be regulated.
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This study examined the spatiotemporal patterns of gene expression of four major O-UC
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enzymes, carbamoyl phosphate synthase III (CPSIII), ornithine transcarboxylase,
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arginosuccinate synthetase and arginosuccinate lyase, using real-time PCR and whole mount in
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situ hybridization. In addition, we hypothesized that CPSIII gene expression was epigenetically
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regulated through methylation of its promoter, a widespread mode of differential gene regulation
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between tissues and lifestages in vertebrates. Further, to assess CPSIII functionality , we used
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morpholinos to silence CPSIII in zebrafish embryos and assessed their nitrogenous waste
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handling during development, and in response to ammonia injections. Our results suggest that O-
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UC enzymes mRNAs are expressed early in zebrafish development and colocalize to the
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embryonic endoderm. In addition, the methylation status of CPSIII promoter is not consistent
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with the patterns of expression observed in developing larvae or adult tissues, suggesting other
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means of transcriptional regulation of this enzyme. Finally, CPSIII morphants exhibited a
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transient reduction in CPSIII enzyme activity 24 hours post fertilization, which was paralleled by
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reduced urea production during development and in response to an ammonia challenge. Overall,
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we conclude that the O-UC is functional in zebrafish embryos, providing further evidence that
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the capacity to produce urea via the O-UC is widespread in developing teleosts.
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Keywords: Ureogenesis, nitrogen metabolism, development, ammonia challenge
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Introduction
In animals, ammonia is the primary metabolic waste formed from the breakdown of
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proteins. Ammonia is highly toxic and therefore must be excreted or detoxified to prevent
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harmful accumulation within the body. Most tetrapods have adopted the strategy to convert the
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bulk of their ammonia into less noxious compounds, urea and uric acid. In contrast, the majority
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of adult teleost fish directly excrete most of their nitrogenous waste in the form of ammonia to
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the surrounding water. This strategy is thought to be advantageous, as in permissive
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environments ammonia can diffuse down its gradient from the blood to the water via the gills at
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little or no cost, whereas the production of urea is energetically expensive. Indeed in fish, the
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ornithine-urea cycle (O-UC) yields 1 mol of urea for 5 mol of ATP consumed, and it requires
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several enzyme-catalyzed steps via: carbamoyl phosphate synthase III (CPSIII), ornithine
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transcarboxylase (OTC), arginosuccinate synthetase (ASS), arginosuccinate lyase (ASL),
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arginase, and the accessory enzyme glutamine synthetase that catalyzes the formation of
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glutamine from ammonia for the entry into the O-UC through CPSIII (57). Although additional
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pathways (e.g., uricolysis, arginine catabolism) may contribute to urea production in
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ammonotelic teleosts, these pathways are not thought to be functionally related to ammonia
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detoxification in these species (see 1).
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Although the majority of adult teleosts are primarily ammonotelic, a few exceptional
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species can excrete substantial amounts of urea in challenging or stressful environmental
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conditions (30, 38, 46, 56, 60). In addition, a variety of fish that are primarily ammonotelic as
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adults are also capable of producing significant amounts of urea early on in their development (4,
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7, 13, 51, 62). This ureogenic capacity may be of crucial importance to prevent ammonia toxicity
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considering the nutritional, developmental and biophysical constraints imposed upon developing
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teleost fish. Indeed, developing embryos and larvae rely heavily on amino acid catabolism for
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energy production resulting in the formation of ammonia (12, 15,16,23, 42, 43,44, 48). This
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ammonia, however, cannot be efficiently excreted to the external medium as, prior to hatching,
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fish typically lack functional gills and are enclosed in a semi-permeable chorion (41, 48).
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Therefore the biophysical characteristics that allow adult fish to readily excrete ammonia in their
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environment are absent or minimized early in development, as suggested by the accumulation of
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nitrogenous wastes in embryos and yolk of several teleost species (4, 7, 18,62). Thus, the
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detoxification of ammonia via urea production may be of crucial importance to prevent ammonia
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toxicity during embryogenesis. In addition, in the few fish species examined, ureogenesis during
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early life stages capability has been attributed to the presence of the gene transcripts and
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functional activities of several enzymes of the O-UC (7, 13, 25, 27, 50, 62). Further, recent work
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in trout showed that most O-UC enzymes activities were primarily located in the embryonic
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body, not the yolk sac or liver but due to technical limitation of the assays the exact localization
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of the enzymes couldn’t be further refined (50). Similarly in the African catfish the enzymes
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localized to the muscle tissues not the liver (51).
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In regulatory terms, the ontogenic switch from larval fish able to produce significant
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levels of urea to adults with much lower capacity is intriguing. Indeed, it suggests that all the
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genetic material necessary for making up the various components of the O-UC are present in
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fish, primarily functional during early life stages and silenced thereafter (7, 62). Typically, the
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regulation of physiological processes can be achieved at several levels. Traditionally,
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transcriptional regulation via changes in the abundance or activity of specific transcription
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factors has been found to play a major role in physiological adaptations. More recently, an
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additional layer of regulation termed epigenetics has emerged to be an important mechanism in
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both ontogenic and tissue specific mRNA expression patterns in a variety of models (8, 31, 40,
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49). In particular, the methylation status of cytosine/guanine dinucleotide rich domains (CpG
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islands) of regulatory regions of specific genes has a profound impact on the activation or
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silencing of these genes under certain conditions. Interestingly, in the case of CPSI in mammals
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(homologous to CPSIII in fish), the promoter has been well characterized (9, 10, 11, 19, 28), and
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the methylation status of cytosine residues in the rat promoter correlates well with its tissue and
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developmental mRNA expression patterns (45).
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With the above background in mind, we undertook the current study to further explore
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the ontogeny of ureogenesis in a teleost species. Since the zebrafish has been found to be
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ureogenic during its early lifestages (4), we wished to further exploit the numerous advantages of
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the zebrafish as an animal model in genetics, developmental biology and physiology. The first
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aim of the present study was to assess the presence and developmental patterns of expression of
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the O-UC enzymes transcripts in developing zebrafish embryos. In addition, using in situ
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hybridization techniques we wished to further refine the spatial mRNA expression patterns of
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selected O-UC enzymes in the developing embryo. Secondly, we hypothesized that the
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differences in expression patterns of CPSIII, the enzyme limiting nitrogen entry in the OUC, that
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were observed during development in zebrafish larvae and in specific tissues of adult fish are
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epigenetically regulated by the methylation status of its proximate promoter. Finally, although
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the transcript and enzyme activity of several O-UC enzymes has previously been detected in
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several species (e.g., 7, 62), the functionality of the O-UC in developing fish has rarely been
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directly tested (13). Therefore, we used morpholino-based gene silencing to knockdown CPSIII
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expression in developing zebrafish to assess if urea production during early development is
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primarily via the O-UC and thus determine if this pathway is physiologically relevant in
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developing fish embryos.
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Methods
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Animals
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Adult wild type zebrafish (Danio rerio) were kept in 10 l plastic tanks in aerated and
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dechlorinated Ottawa tap water at 28°C. The fish were maintained in a 10:14 h light dark cycle
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and fed once daily on Adult zebrafish complete diet (Zeigler Gardners, PA, USA). Embryos
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were obtained following standard breeding procedures (59) and raised in E3 embryo medium (in
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mM: 5 NaCl, 0.17 KCl, 0.33 CaCl, 0.33 MgSO4 and 0.00001% methylene blue) in Petri dishes
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at 28°C until needed. All procedures were approved under Protocol BL-255 by the Animal Care
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Committee of the University of Ottawa and are in accordance with guidelines of the Canadian
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Council on Animal Care.
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Real-time PCR analysis
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Embryos were harvested at 5, 24, 32, 48 and 72 h post-fertilization (hpf), immediately flash
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frozen in liquid nitrogen and stored at -80°C until further analysis. For each time point 30 to 50
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embryos were homogenized in Trizol, total RNA was extracted, treated with DNAseI and
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reverse transcribed with Superscript II reverse transcriptase following manufacturer’s
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instructions (Invitrogen, Burlington, ON). Gene expression analysis was carried with real-time
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PCR technology on a Stratagene MX3000P real-time cycler (La Jolla, CA, USA). The cycling
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conditions were 3 min of 95°C denaturation and 40 cycles of 15 sec denaturation (95°C), 30 sec
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annealing (58°C) and 30 sec extension (72°C). Each sample was assayed in duplicate in a 12.5 μl
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mix containing 6.5 μl Brilliant SYBR mix, 2 μl cDNA, and 0.2 μM of each primer (Table 1). No
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template controls were run in parallel to ensure the absence of contamination. Relative levels of
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gene expression were obtained following ΔΔCt using elongation factor 1α (EF1α) as a reference
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gene. The specificity of the primers used was ensured through post-real-time PCR dissociation
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curve analysis.
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Whole mount in situ hybridization
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Digoxygenin-labelled RNA probes were generated as previously described (54). Briefly, gene
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specific antisense sequences were amplified using gene-specific primers (Table 1) and cloned in
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pDrive vectors (Qiagen, Mississauga, ON). Plasmids were then linearized with either BamHI or
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SacI, and transcribed using the DIG RNA labelling kit (Sp6/T7) following manufacturer’s
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instructions (Roche, Laval, QC). DIG-labelled RNA was ethanol purified, quantified through
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native gel electrophoresis, and stored at -80°C until needed. Wild type embryos were raised until
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30-32 hpf in embryo medium containing 0.003% 1-phenyl-2-thiourea (Sigma-Aldrich, Oakville,
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ON) to minimize pigment formation. After collection, embryos were fixed, manually
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dechorionated, dehydrated and stored at -20°C in 100% methanol until further manipulation. The
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whole mount in situ hybridization was performed as described in Thisse and Thisse (54), after
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which the embryos were postfixed in a 4% paraformaldehyde solution overnight and transferred
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to glycerol prior to image acquisition. Embryos were manually deyolked and images were
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captured on a Nikon SMZ1500 stereomicroscope (Melville, NY, USA).
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Morpholino gene knockdown
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A splice blocking morpholino was designed to target the 2nd intron-exon boundary of the
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zebrafish CPSIII gene (ZDB-GENE-081105-17) (Gene tools, Philomath, OR). This morpholino
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prevents the excision of the second intron of CPSIII from the pre-mRNA transcript and
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introduces 9 stop codons in frame. Using a microinjector (IM 300, Narishige, Long Island, NY)
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and a pulled 1.0 mm borosilicate glass micropipette (Stutton Instrument, Novato, CA), one-cell
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embryos were injected with 1 nl of either the splice blocking morpholino for CPSIII
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[TTCAGAGGATGCCTTCTCACCCAAC] or a control morpholino
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[CCTCTTACCTCAGTTACAATTTATA] at a concentration of 0.5mM in Danieau buffer (in
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mM: 58 NaCl, 0.7 KCl, 0.4 MgSO4, 0.6 Ca(NO3)2, 5.0 Hepes pH 7.6) with 0.05% Phenol Red.
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This concentration was found to be the optimal amount of morpholino required to silence CPSIII
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in CPSIII morphants at 24hpf as checked by standard PCR with primers flanking the exon-intron
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boundary (Table 1), while also reducing the risks of non-specific effects on the developmental
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rates, morphology and nitrogenous waste excretion patterns of both control and CPSIII
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morphants. Both control and CPSIII morpholinos were labelled with a fluorescent dye,
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carboxyfluorescein, allowing to screen in vivo the successful uptake of the morpholino by the
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injected embryos. Only the embryos exhibiting widespread fluorescence by 6-24 hpf were kept
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for further analysis.
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Urea and ammonia determination
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At different stages of development (n = 4-7), approximately 50 embryos (corresponding to n = 1)
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were incubated in a well of a 6 well cell culture plates (BD Biosciences, Mississauga, ON) for
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evaluation of their nitrogenous waste excretion flux rates. The fluxes were conducted for 6 and
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18 h respectively in 3 or 6 ml of filtered dechlorinated tap water at 28°C. These flux periods
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were selected for practical reasons (light vs. dark) and to allow for reliable measurements of
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nitrogenous wastes in the water. For each series a well containing water only was included to
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serve as a blank.The water samples were immediately frozen at -20°C for later determination of
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ammonia and urea concentration following standard procedures (37, 56). The excretion rates
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were calculated accounting for the duration of the flux, initial and final ammonia or urea
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concentration in the water, the volume of water in the well as well as the number of embryos
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fluxed. For whole body urea and ammonia concentration in 24 hpf and 48 hpf morphants, the
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embryos were collected, flash frozen in liquid nitrogen and stored at -80°C until further
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manipulation. Each sample (~50 embryos) was homogenized in 100 μl 8% perchloric acid, and
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centrifuged at 13,000 rpm for 15 min. The supernatant was then neutralized with saturated
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K2CO3, and assayed for urea as described above and for ammonia using a commercially
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available assay (AA0100, Sigma). Urea concentrations (flux and body content) were multiplied
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by two to account for the two nitrogen atoms present in urea, and the detection limit of the assay
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is estimated to be 0.5 μmol.L-1 (35).
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In addition, we subjected 24 hpf morphants with an ammonia challenge as described before (6).
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We chose this route of administration to test the capacity of the embryo to detoxify ammonia due
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to a rapid surge within the embryo/yolk sac. In addition, this technique allowed us to precisely
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control the level of exposure of each embryo, and circumvent any potential problems due to the
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chorion and its permeability to ammonia seen before in young zebrafish (5). Briefly, 24 hpf
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embryos were enzymatically dechorionated in pronase (Roche; 1 mg l-1), and lightly
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anaesthesized in tricaine methanesulfonate (0.01 mg l-1; Sigma Aldrich). The embryos were then
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microinjected in their yolk sac with 3 nl of a solution of 1 mM NH4Cl in Danieau buffer. The
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embryos (50 per well, n = 4-6 replicates) were then fluxed in 6 well plates for 2 h, and water and
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body samples were taken for determination of their nitrogen waste excretion and whole body
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content as described above.
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Enzyme assays
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The activities of CPSII (a cytoplasmic isoform of CPS involved in pyrimidine synthesis and not
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the O-UC) and CPSIII of morphants were assayed radiometrically as described before (27).
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Embryos were homogenized on ice in 3 volumes of homogenization buffer (0.05 M Hepes, pH
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7.5, 0.05 M KCl, 1mM DTT and 0.5 mM EDTA), briefly sonicated, and centrifugated at 14,500
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x g for 10 min at 4°C. and immediately 100 μl of this supernatant was added to 200 μl of
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reaction buffer such that final concentrations were: 20 mM ATP, 25 mM MgCl2, 25 mM
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phosphoenolpyruvate, 2 U pyruvate kinase, 5 mM [14C] sodium bicarbonate (3 x 106 cpm), 0.04
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M Hepes pH7.6, 0.04 M KCl, 0.5mM DTT, 0.05 mM EDTA, 20 mM glutamine and when
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applicable 1.7 mM AGA, 1.7 mM UTP. After 1 h of incubation at 26°C, the reaction was
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stopped with 40 μl of freshly prepared 1 M NH4OH in 0.5 M NaOH containing 0.5 mM
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carbamoyl phosphate and incubated for 5 min at room temperature. The reaction was then boiled
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for 15 min in a fume hood immediately after addition of 100 μl of 2 M NH4Cl pH 8.7. The
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solution was allowed to cool, after which it was passed through a DOWEX anion exchange
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column (OH-, 50-100 mesh, Sigma), washed into the resin with 500 μl of 0.5 mM urea. The 14C
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urea formed was eluted from the column with 5 ml of 0.5 mM urea into a scintillation vial. The
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eluate was throroughly mixed with 10 ml of Ultima Gold XR scintillation fluid (Perkin Elmer,
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Waltham, MA), and the radioactivity in each sample was measured on a Tricarb LSC 2910 TR
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liquid scintillation spectrometer (Perkin Elmer). All reactions were carried out in duplicate and
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each assay series included t = 0 controls in which the reaction was immediately stopped to
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evaluate background values. Protein contents of the homogenates were evaluated using the
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bicinchoninic acid assay following manufacturer’s instructions (Sigma). As the homogenates
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were not subjected to gel filtration described before (7), the enzymatic rates obtained are not
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maximal, but could be more reflective of their in vivo activity.
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Bisulfite sequencing
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In order to examine methylation status, bisulfite sequencing was employed. This method consists
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in treating genomic DNA with sodium bisulfite under acidic condition (17). This treatment
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converts only cytosine residues to uracil if they are unmethylated, leaving all methylated
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cytosines unchanged (17). Thus, following PCR amplification and sequencing, one can identify
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the methylation state of each cytosine residues in a given genomic DNA region (for review, see
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22). Genomic DNA was extracted using the standard phenol/chloroform technique and subjected
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to bisulfite conversion using the Imprint DNA modification kit following manufacturer’s
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instructions (Sigma). The bisulfite treated DNA was then PCR amplified with specific primers
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(Table 1, Fig. 1) flanking a major CpG island situated -920 to -630bp upstream of the translation
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start site of CPSIII (Fig. 1). The PCR products were then purified and cloned using commercially
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available kits (QIAGEN). The clones for each amplified product (n=6-10) were then sequenced
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in both directions at the Genome Quebec Sequencing center (Montreal, QC, Canada). Results
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were analyzed with BiQ Analyzer v2.0 (2).
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Statistical analyses
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All data were analyzed in SigmaStat v 3.5 (Systat Software, San Jose, CA, USA). Gene
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expression for larval zebrafish was analyzed with one-way ANOVA on ranks (Fig. 2A-D) and
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with t-tests (Fig. 2E) for adult tissues. Ammonia and urea excretion fluxes (Fig. 5A,B) were
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rank-transformed and analyzed using a two-way ANOVA. Ammonia and urea content of
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morphants (Fig. 5C, D) and CPS enzyme activities (Fig. 4) were analyzed using a two way
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ANOVA. Data pertaining to the ammonia injected morphants (Fig. 6) were subjected to t-test
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analyses. All multiple comparisons were followed by post-hoc Student Newman Keuls analysis.
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Results
The expression of CPSIII, OTC, ASL and ASS mRNAs was detected as early as 5 hpf
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(data not shown). Over the course of the first 96 hpf, the transcripts for all genes had a tendency
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to increase (Fig. 2A-D). CPSIII mRNA increased 2-fold between 24 and 32 hpf to return to basal
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levels afterward. The other enzyme transcripts slowly increased over the course of development
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to only become significantly elevated by 72 hpf (ASL) and 96 hpf (OTC and ASL). As a
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qualitative comparison, we also evaluated the gene expression of these enzymes in the liver and
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muscle of adult fish (Fig. 2E). In adult liver, most enzymes were expressed at very low levels
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except for ASL. In comparison, the urea enzymes transcripts were expressed at levels
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comparable to 24 hpf larvae in the adult muscle, with a markedly high expression of ASL in
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adult muscle.
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The methylation status of cytosine residues in the proximal promoter of CPSIII appeared
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to be variable both across developmental stages and adult tissues (Table 2.). Through the course
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of development, there was a tendency for a reduction in the proportion of methylated residues as
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the larvae grew older, but there did not appear to be any consistent patterns at individual
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residues. Similarly, when comparing adult tissues, the CPSIII promoter in liver was overall
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hypomethylated when compared to muscle. However, when looking at each residue individually,
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the differences in methylation status were more variable. For example site 1 is hypomethylated
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while site 2 is hypermethylated (Table 2) in muscle compared to adult liver tissue.
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When we examined the localization of transcripts of O-UC enzymes through whole
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mount in situ hybridization in zebrafish larvae at 32 hpf, transcripts of all 4 enzymes examined
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were detectable (Fig. 3). The O-UC enzyme transcripts appeared to primarily localize in the
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embryonic endoderm adjacent to the anterior part of the yolk sac (i.e. the yolk ball), where in
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contrast no marking was observable on the posterior trunk region surrounding the yolk extension
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(Fig. 3). Transcripts for the enzymes can also be detected, albeit more diffusely, in the anterior
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part of brain for ASL and ASS1, and in the region ventral to the hindbrain for CPSIII and OTC
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(Fig. 3).
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Next, knowing that the transcripts levels for these four enzymes of the O-UC were
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present in developing zebrafish, we decided to test the functionality of the cycle in these fish
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with a CPSIII morpholino knockdown. To test the effectiveness of this knockdown, we assayed
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the activity of CPSIII, and its cytosolic homologue CPSII, in control and CPSIII morphants (Fig.
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4). There was a significant decrease in CPSII activity at 48 hpf independent of the type of
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morpholino injected. Overall, CPSII enzyme activity was unaffected by the morpholino
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treatment (Fig. 4A). In contrast, CPSIII morphants showed a 50% reduction in CPSIII enzyme
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activity compared to control morphants at 24 hpf. However, by 48 hpf CPSIII morphants mostly
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recovered to exhibit CPSIII enzyme activities comparable to their control morphants
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counterparts. In addition, regardless of the morpholino injected, morphants’ CPSIII activity
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approximately doubled from 24 to 48 hpf.
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We monitored the nitrogenous waste excretion in the morphants over the course of 72 h
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(Fig. 5). Regardless of the morpholino treatment, ammonia excretion significantly increased over
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the course of development (Fig. 5A). Ammonia excretion rates were overall comparable between
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the CPSIII and control morphants (Fig. 5A). However, between 30 and 48 hpf ammonia
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excretion was elevated in control morphants, and significantly lower after 58 hpf (Fig. 5A). In
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contrast, overall urea excretion was more variable during development (Fig. 5B), significantly
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elevated during the nocturnal fluxes (30-48 hpf and 55-72 hpf; Fig. 5B). Compared to the control
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morphants, the CPSIII morphants did not excrete any significant levels of urea between 24 and
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30 hpf (Fig. 5B). It should be noted that although low levels of urea were excreted in some
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treated fish, we could not detect any urea in most water samples. After 30 hpf, the CPSIII
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morphants excreted urea at levels comparable to control morphants (Fig. 5B). As a result,
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between 23 and 30 hpf urea production as a percentage of total nitrogenous waste was 4-fold
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lower in the CPSIII morphants than in the control morphants, but relatively similar at later
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developmental stages. We also measured the urea and ammonia body content at 24 and 48 hpf
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(Fig. 5C-D). Ammonia body content showed no significant differences between CPSIII and
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control morphants at both time points examined (Fig. 5C). In contrast, by 24 hpf the urea body
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content was approximately 10 times lower in CPSIII morphants compared to their control
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counterparts, but this difference disappeared by 48 hpf (Fig. 5D).
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At 24 hpf, morphants were injected in the yolk sac with exogenous ammonia and their
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nitrogenous waste excretion monitored. Both control and CPSIII morphants excreted high levels
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of ammonia for 2 h post-injection (Fig. 6A). In contrast, while the control morphants also
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excreted high levels of urea in response to ammonia loading, CPSIII morphants only excreted
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approximately half the amount of the urea excreted by their control morphants (Fig. 6A). When
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we looked at their body contents, CPSIII morphants had higher concentrations of ammonia while
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their urea content was marginally lower than the control morphants (Fig. 6B). Overall, the
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nitrogenous budget (excreted nitrogenous waste + nitrogenous body content) of both morphant
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types were comparable over the flux period (0.76 ± 0.07 nmol N/embryo for controls vs. 0.75 ±
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0.07 nmol N/embryo in CPSIII morphants) with the CPSIII morphants excreting and
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accumulating less urea and more ammonia than control morphants.
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Discussion
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In the current study, we demonstrate that mRNA for four of the six urea cycle enzymes is
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expressed early on in the development of zebrafish. We also established that differences
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associated in the gene expression of CPSIII across developmental stages and between adult
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tissues could not be attributed to drastic changes in the methylation status of its promoter,
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suggesting other means of transcriptional regulation of this gene. Further, the use of a
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morpholino based silencing of CPSIII allowed us for the first time to directly assess the
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functionality of the O-UC in zebrafish larvae, suggesting that at least very early on in
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development the O-UC is functional, and has an impressive capacity to detoxify ammonia as
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tested via injections of supraphysiological levels of ammonia.
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Gene expression of O-UC enzymes
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In a variety of teleosts, several enzymes of the O-UC are detectable at relatively early life
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stages (7, 25, 27, 50, 62). To our knowledge, the only other study to examine mRNA expression
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patterns of the O-UC in a developing teleost focused on CPSIII mRNA in rainbow trout (27). In
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rainbow trout, CPSIII transcripts are present shortly after fertilization, and mRNA expression
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maximizes within two weeks of development (27). Our results suggest that not only CPSIII but
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three other enzymes of the O-UC cycles are transcriptionally active as early as 24 hpf (Fig. 2). In
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addition, we detected transcripts for these enzymes as early as 5 hpf, which is barely after the
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start of zygotic transcription, suggesting that the transcripts detected at this point may be of
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maternal origin (24). Another interesting point is that there is a surge in CPSIII mRNA at
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approximately 32 hpf (Fig. 2A). Given that zebrafish larvae typically hatch around 48-72 hpf, it
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is reasonable to think that this CPSIII mRNA increase “primes” the fish for the upcoming
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hatching event. Indeed during hatching, the weakened chorion is first opened and then shed by
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the movements of the larvae. Metabolically speaking, this pre-hatch surge in activity of the
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embryo must be demanding, and thus must be exacerbating production of metabolic wastes (i.e.,
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ammonia) that need to be detoxified (15, 16, 33). Interestingly, zebrafish larvae accumulate
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significant amounts of urea (a four-fold increase!) during hatching, while their excretion of both
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ammonia and urea remain stable (4). These observations would tend to suggest that the increase
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in CPSIII mRNA prior to hatching is of physiological importance as it will help the embryo
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detoxify the ammonia produced during hatching. Although the other enzymes transcripts didn’t
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increase until later (Fig. 2A-D), this situation is not atypical. For example, in fetal mice CPSI,
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the CPSIII mammalian homologue, is one of the first O-UC genes to be expressed during
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development, while other enzymes such as OTC follow a few days later (32). It is possible that
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the other enzymes are in sufficient quantities before hatching, and considering the importance of
355
CPSIII as a regulatory enzyme (controlling then entry into the O-UC) it might be most important
356
to tightly regulate that enzyme to control overall urea production rate through the cycle.
357
Previous work has examined the localization of some of the O-UC enzymes in newly
358
hatched rainbow trout (50), and determined that CPSIII enzyme activity was primarily detected
359
in the embryonic body of developing trout, while OTC was found to be also expressed in hepatic
360
tissues. However due to the technical limitation of the assays, these spatial patterns could not be
361
further refined or include other O-UC enzymes (i.e. ASL, ASS). Thus we took advantage of
362
established techniques in zebrafish to localize the transcripts of major O-UC enzymes (Fig. 3).
363
Overall, most of the expression of the transcripts was restricted to the anterior endoderm adjacent
364
to the yolk sac (Fig. 3). This finding is not surprising when we consider the
365
compartmentalization of nitrogen waste in teleost embryos. Indeed in zebrafish, as in other fish
366
species, urea, and to a lesser extent ammonia, accumulate at much higher levels in the yolk sac
367
than in the embryonic body (6, 15, 16, 17, 36, 42, 50). This compartmentalization would suggest
17
368
that there is a transport mechanism that allows these nitrogenous wastes to be concentrated in the
369
yolk sac, and in the case of urea, that the site of formation is either in the yolk sac of the embryo
370
or in adjacent areas. Our results as well as previous work suggest that not only are ammonia and
371
urea transporters present in the region surrounding the yolk sac in zebrafish (4), but that O-UC
372
enzymes predominantly colocalize to embryonic tissues surrounding the yolk sac (Fig. 3). Thus,
373
both synthesis and transport of urea could potentially occur very effectively in that vascularised
374
region allowing rapid detoxification of ammonia and segregation of nitrogenous wastes within
375
the yolk. In terms of gene expression in adults, the higher expression of O-UC enzymes mRNA
376
in muscular than in hepatic tissues is in agreement with previous work in several other
377
ammonotelic teleosts at both the mRNA and enzyme levels (26, 27).
378
Considering these very specific patterns of expression both in developing fish and in
379
adult tissues, we hypothesized that they could be dictated by epigenetic mechanisms of
380
regulation as opposed to transcriptional regulation. Indeed, epigenetic regulation has recently
381
emerged as a powerful mean to establish both temporal and tissue specific gene expression
382
patterns (8, 31, 40, 49, 63). Specifically, we focused on the methylation status of cytosine
383
residues located in a region rich in cytosine-guanosine dinucleotides within the proximal
384
promoter of CPSIII. Overall, the patterns we observed both across developmental stages and
385
between adult tissues are not consistent with our initial hypothesis (Table 2). The methylation
386
state in embryos seemed to decrease over time, while the gene expression peaked at 32 hpf (Fig.
387
2A). Similarily, in adult fish while CPSIII mRNA is expressed at levels that are orders of
388
magnitude higher in muscle than in liver (Fig. 2E), the methylation status at both individual sites
389
and of the region as a whole show little difference between the two tissues, if anything the
390
promoter is slightly hypomethylated in liver compared to muscle (Table 2.). This situation is
18
391
very different from mammalian systems, where there is clearly an epigenetic impact on gene
392
expression of CPSI (45). Further, the characterization of the mammalian CPSI promoter
393
uncovered a complex mode of transcriptional regulation, with multiple regulatory units
394
controlling its gene expression (9, 10, 11, 19, 20, 28, 47, 55). We performed a preliminary in
395
silico analysis of the zebrafish CPSIII promoter, and failed to identify most of the regulatory
396
markers present in mammals CPSI promoter (e.g. glucocorticoid and hepatocyte nuclear factor
397
response elements). However, our results suggest the possibility that transcriptional, and not
398
epigenetic, regulation may be coordinating the spatiotemporal expression of CPSIII in zebrafish,
399
though this speculation warrants further investigation beyond the scope of the present study. We
400
should keep in mind though that in mammals the functionality of the urea cycle is of crucial
401
importance in all life stages, as defects in this cycle are highly detrimental and can be lethal
402
based on their severity (see 3). In contrast, in ammonotelic teleost the urea cycle is apparently
403
mostly active only at early life stages, and defects in the cycle (i.e., transient knockdown of
404
CPSIII) are not lethal in zebrafish embryos (personal observation).Thus it is reasonable to think
405
that the two evolutionary and physiologically distant taxa present very different means of
406
regulating components of the urea cycle. In that respect, a comparative study of the mechanism
407
of regulation of the O-UC in closely related ammonotelic and ureotelic teleosts (e.g., Opsanus
408
beta and Porhychis notatus) in comparison to the mammalian model could provide invaluable
409
information on the evolution and the regulation of ureotelism in vertebrates.
410
CPSIII silencing
411
We employed reverse genetic techniques to assess the functionality of CPSIII and by
412
extension the O-UC in developing zebrafish (Fig. 4-6). Overall our results suggest that in
413
zebrafish CPSIII and the O-UC are active at early life stages. CPSIII was efficiently silenced for
19
414
the first 24 hpf in the larvae as indicated by enzyme assays (Fig. 4B). Indeed, the activity of the
415
enzyme was reduced by approximately 50% in CPSIII morphants, but quickly recovered to
416
control levels thereafter (Fig. 4B). The patterns observed at the enzyme level were mirrored by
417
the urea fluxes and body contents of the morphants, as by 24 hpf there was nearly no urea
418
excreted or accumulated by the CPSIII morphants, but a full recovery is evident by 48 hpf (Fig.
419
5B, D). As development progresses, ammonia excretion tends to increase regardless of the
420
morpholino treatment as described elsewhere (Fig. 5A; 3, 5), however there does not seem to be
421
a compensatory increase in ammonia fluxes from the CPSIII morphants compared to their
422
control counterparts. The rapid recovery observed in CPSIII morphants may be due to several
423
factors. First, this compensation may reflect the normal increase in gene expression that occurs in
424
wild type embryos (Fig. 2A), and a large increase in CPSIII mRNA could buffer in part or
425
completely the morpholino effect. Another possible cause for this rescue of CPSIII could be a
426
specific attempt of the embryo to compensate for the morpholino silencing, again resulting in the
427
reduction of the morpholino effect (13, 34). These two possibilities could superimpose on the
428
fact that as the embryo grows, the morpholinos get diluted over time, and in the case of a site
429
specific expression of the target sequence (as evidenced by Fig. 3), the initial effective
430
morpholino concentration becomes diluted and the silencing is overwhelmed by the relative
431
increase in the mRNA target’s expression.
432
To extend our findings, we ammonia challenged 24 hpf morphants with direct injection
433
and assessed their nitrogenous wastes fluxes and body contents (Fig. 6). Both control and CPSIII
434
morphants showed an elevated excretion of ammonia within 2 h of injection compared to 24-30
435
hpf uninjected embryos (Fig. 5A, Fig. 6A). In addition, control morphants also showed a rate of
436
urea excretion similar in magnitude to their ammonia flux (Fig. 6A). In contrast, CPSIII
20
437
morphants only excreted half the amount of urea the control morphants did (Fig. 6A), further
438
establishing that the O-UC is impaired in CPIII morphants. Moreover, the accumulation of
439
nitrogenous wastes in the embryos corroborates these results, as CPSIII accumulated more
440
ammonia and less urea than control morphants (Fig. 6 A). The effects of the exogenous ammonia
441
injection on the control morphants are similar to previous reports of embryos exposed to high
442
environmental ammonia (50, 61), as embryos increased their urea excretion to prevent harmful
443
ammonia accumulation. These results also suggest that this rapid detoxification of ammonia
444
occurs primarily through the O-UC as a 50% decrease in CPSIII activity results in a similar
445
reduction in urea production in the ammonia injected CPSIII morphants (Fig. 4B).
446
However when considering these rapid changes in urea production, the activities for CPS
447
enzyme activities obtained in zebrafish larvae seem relatively modest to accomplish this feat. But
448
when approximated per g of tissue, we estimate them to be about 0.05-0.2 nmole/min/g for
449
CPSII and 0.01-0.04 nmole/min/g for CPSIII, which is lower, but in the range of previous reports
450
for teleost embryos (e.g., 7). However, even at these relatively low levels of activities, the flux
451
through CPSIII in optimal conditions (though unlikely to happen in vivo), would be more than
452
sufficient to produce the large amounts of urea observed when presented with an excessive
453
ammonia load (Fig. 5). Thus, zebrafish embryos, as other teleost species, have the capacity to
454
produce significant levels of urea even early in their development, but do not completely activate
455
that metabolic pathway until presented with an ammonia challenge. Although seemingly
456
excessive in comparison to their normal nitrogenous budget, this capacity could be crucial at
457
specific point in development (i.e. hatching) where conditions may require rapid detoxification
458
of ammonia.
459
Conclusion and perspectives
21
460
Overall, we demonstrated that several enzymes of the O-UC are expressed at early
461
developmental stages of the zebrafish larvae. In addition, the localization of the enzyme
462
transcripts suggest that they are optimally located to compartmentalize urea within the yolk of
463
the embryo. Further, using reverse genetics and manipulation of ammonia load in embryo, we
464
firmly establish the functionality of the O-UC and its potential importance over other ureogenic
465
pathways for ammonia detoxification at these stages of development. Considering the
466
localization of the O-UC enzymes and their capacity to rapidly detoxify relatively large amounts
467
of urea, it appears that the retention of ureogenic capacity in several developing teleost embryos
468
could act as a protective mechanism against endogenous or exogenous surges in ammonia. This
469
report in zebrafish, a warm acclimated freshwater species, corroborate previous results obtained
470
in phylogenetically distant species from diverse freshwater and seawater environments (e.g., cod,
471
rainbow trout, halibut, african catfish; 7, 51, 52, 62). Thus our results provide additional support
472
to the emerging view that O-UC detoxifying capacity is active and maintained in a variety of
473
teleost species during early development, but this capacity is repressed at later life stages (21).
474
22
475
Acknowledgements
The authors wish to thank Marc Ekker and Steve Perry for helpful discussions, Vishal
476
477
Saxhena and Melanie Debiais for technical assistance with fish husbandry, in situ hybridization
478
and microinjection work.
479
Grants
480
This work was funded by the Canada Foundation for Innovation and a Natural Science and
481
Engineering Research Council Discovery grant to P. J. Walsh who is also supported by the
482
Canada Research Chair Program.
483
23
484
485
486
487
488
489
490
491
492
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Figure 1. Location of the CpG nucleotides in the zebrafish CPSIII promoter. This sequence
encompasses the region -920 bp to -630 bp upstream of the CPSIII translation start site. The 12
putative methylation sites are boxed, while the underlined sequences indicate the locations of the
primer used for bisulfite sequencing (see Table 1.).
Figure 2. Ornithine-urea cycle mRNA expression in developing larvae and adult tissues of
zebrafish. Real time PCR analysis was used to evaluate the mRNA levels of A) CPSIII, B) OTC,
C)ASS and D) ASL in developing zebrafish using EF1α as a reference gene. E) The relative
levels of these transcripts were also measured in liver (white bars) and muscle (black bars)
tissues of adult zebrafish. In both developing embryos and adults, for each gene the levels of
expression are normalized to the expression in 24 hours post fertilization embryos. Different
letters denote statistical significance between time points (n=5).
Figure 3. Spatial expression of O-UC enzymes in 32 hours post fertilization zebrafish embryos.
Whole mounts in situ hybridization was performed to localize the mRNA expression of A)
CPSIII, B) OTC, C) ASS1 and D) ASL. The arrows indicate the pronounced marking for each
gene on the embryonic endoderm. The scale represents 100μm.
Figure 4. CPSII and CPSIII enzyme activities in developing control (white bars) and CPSIII
morphants (black bars). The relative enzyme activity of A) CPSII and B) CPSIII was evaluated
in 24 and 48 hours post fertilization zebrafish embryos. Different letters indicate statistically
significant difference between developmental times or between treatments (n=4-6).
Figure 5. Nitrogen fluxes and body contents in developing control and CPSIII morphants.
Control morphants (white bars) and CPSIII morphants (black bars) were evaluated for (A)
ammonia and (B) urea excretion, as well as whole body content for C) ammonia and D) urea at
different stages of their development. Different letters indicate statistically significant difference
between developmental times or between treatments (n=4-7).
Figure 6. Nitrogen fluxes and body contents in ammonia injected control and CPSIII morphants.
Control morphants (white bars) and CPSIII morphants (black bars) were yolk injected with
ammonia and we evaluated A) their ammonia and urea excretion rates for 2hrs, and B) their
ammonia and urea body contents. Different letters indicate statistically significant difference
between treatments (n=4-6).
30
723
Table 1. List of primers used in this study (see footnotes for accession numbers).
Gene target
Real time
PCR
In situ
hybridization
Bisulfite PCR
724
725
726
727
728
729
Forward primer
(5′-3′)
Reverse primer
(5′-3′)
EF1α1
CPSIII2
OTC3
ASS14
ASL5
gtgctgtgctgattgttgct
agcagcgtctgggtcagtat
gtgggaaaagcaactttgga
aaccctgagcattctcctga
ctggctttgcaactcatcaa
tgtatgcgctgacttccttg
cgatctgtttgacccaaggt
tgggagatgctctcctctgt
tgctttcctccgatctcatt
catccctgcttattgccact
CPSIII2
OTC3
ASS14
ASL5
gcgtgtcaggagctctttct
gcgtggacatcctctgactt
ggcgcactgaataaaatgaa
caggtgctccagagacgagt
acgcgtctgatccaattcat
cgctttagttttccgtttgc
caaagcgaacctggtcattt
CPSIII6
ttggttttgaagtttttatttatttatt
aatttacatattctccctacattcac
gcaataccatcccgtaacca
aaatcactgaggctgggatg
tgaatccgatcagactccac
Morpholino CPSIII2
PCR
1
AY422992 (29); 2ENSDART00000004742; 3ENSDART00000089526;
4
ENSDART00000039903; 5ENSDART00000040190 ; 6ENSDARG00000020028.
31
730
731
732
733
734
Table 2. Methylation status of the CPSIII proximal promoter in developing embryos and adult
tissues. The proportion of methylated cytosines residues is calculated as a percentage at each
individual site (n=2 for each embryonic stage, n=3 for adult tissues). See Fig. 1 for location of
the CpG sites in the DNA sequence.
CpG site
735
736
24hpf embryo 36hpf embryo 48hpf embryo
Adult liver
Adult muscle
1
100
100
83
83
75
2
100
100
83
83
100
3
75
100
17
100
100
4
50
100
83
100
89
5
100
100
83
70
89
6
75
100
83
70
89
7
100
100
83
70
89
8
100
33
83
80
96
9
100
33
67
47
96
10
75
50
17
47
96
11
100
67
0
43
21
12
0
0
17
8
0
Total
81
74
58
67
78
TTGGTCTTGAAGCCTTCATTCATTTATTCGTTT
TCTTGTCGGCTTAGTCCCTTTATTAATCTGGGG
TCACCACAGCGGAATGAACCGCCAACTTATCC
AGCACATTTTTACGCAGCGAATGCCCATCTAA
C CG CAACCCATCTCTGGGAAACATCCAC
AAACACACATTCACTCACACACTACGGACAAT
TTAGCTTACCCAATTCACCTGTACCGCATGTCT
TTGGACTGTGGAGGAAACCGGAGCACCCGG
AGGAAACCCA CG TGAATGCAGGGAGAAC
ATGCAAACT
Relative mRNA
expression
A) CPSIII
3
B) OTC
4
b
b
3
2
a
a
a
a
1
a
2
1
0
0
C) ASS
D) ASL
a
a
a
Relative mRNA
expression
b
2
1
0
a
2
a
a
a
24
1
32
48
72
96
Hours post fertilization
E) Adult tissues
Relative mRNA
expression
b
10
1
0.1
0
CPSIII
OTC
ASS1
ASL
0
b
ab
a
24
a
32
48
72
96
Hours post fertilization
A) CPS III
B) OTC
C) ASS
D) ASL
16
B)
a
12
b
8
4
0
24
48
Hours post fertilization
CPSIII activity (pmole/min/mg protein)
CPSII activity (pmole/min/mg protein)
A)
b
e
3
e
a
2
c
d
1
0
24
48
Hours post fertilization
A) Ammonia
d
Rate of nitrogen excretion
(nmol N/h/embryo)
0.4
f
B) Urea
b
0.08
f
b
0.3
c
e
e
0.06
e
0.2
e
0.04
f
e
a
a
e
e
a
e
0.1
0.02
e e
e
f
0
0
24-30
30-48
48-55
55-72
24-30
30-48
48-55
55-72
Hours post fertilization
Hours post fertilization
C) Ammonia
Nitrogen concentration
(nmol N/h/embryo)
b
e
D) Urea
a
a
0.8
a
0.16
a
0.6
a
0.4
a
a
0.2
0.12
0.08
0.04
b
0
0
24
48
Hours post fertilization
24
48
Hours post fertilization
Nitrogen body content
(nmol N/embryo)
Rate of nitrogen excretion
(nmol N/h/embryo)
A)
0.08
a
0.5
0.4
a
0.2
Ammonia
a
0.06
b
0.04
0.02
0
Ammonia
Urea
B)
b
a
0.3
a
b
0.1
0
Urea