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DEVELOPMENTAL BIOLOGY
The origin of the parathyroid gland
Masataka Okabe *,,, and
Anthony Graham *,
*Medical Research Council, Centre for Developmental Neurobiology, Guy's Campus, King's College London, London SE1 1UL, United Kingdom; and Department of Developmental Genetics, National Institute of Genetics, Shizuoka 411-8540, Japan
Edited by John C. Gerhart, University of California, Berkeley, CA, and approved November 3, 2004
(received for review August 20, 2004)
It has long been held that the parathyroid glands and parathyroidhormone evolved with the emergence of the tetrapods, reflectinga need for new controls on calcium homeostasis in terrestrial,rather than aquatic, environments. Developmentally, the parathyroidgland is derived from the pharyngeal pouch endoderm, and studiesin mice have shown that its formation is under the control ofa key regulatory gene, Gcm-2. We have used a phylogenetic analysisof Gcm-2 to probe the evolutionary origins of the parathyroidgland. We show that in chicks, as in mice, Gcm-2 is expressedin the pharyngeal pouches and the forming parathyroid gland.We find that Gcm-2 is present not only in tetrapods but alsoin teleosts and chondrichthyans, and that in these species,Gcm-2 is expressed within the pharyngeal pouches and internalgill buds that derive from them in zebrafish (Danio rerio),a teleost, and dogfish (Scyliorhinus canicula), a chondrichthyan.We further demonstrate that Gcm-2 is required for the formationof the internal gill buds in zebrafish. We also have identifiedparathyroid hormone 1/2-encoding genes in fish and show thatthese genes are expressed by the gills. We further show thatthe gills express the calcium-sensing receptor, which is usedin tetrapods to monitor serum calcium levels. These resultsindicate that the tetrapod parathyroid gland and the gills offish are evolutionarily related structures, and that the parathyroidlikely came into being as a result of the transformation ofthe gills during tetrapod evolution.
In tetrapods, the parathyroid glands play a pivotal role inregulating extracellular calcium homeostasis, which is importantto many physiological processes such as muscle contraction,blood coagulation, and synaptic activity. These glands detectchanges in the levels of calcium in blood by means of the calcium-sensingreceptor (CasR), which in turn modulates the secretion of parathyroidhormone (PTH), which acts to release calcium from internal storessuch as bone and modulates renal ion transport (1). Developmentally,the parathyroid glands arise from the endodermal pharyngealpouches; in humans and chickens, from the third and fourth pouches,and in mice, from the third pouch only. Importantly, studiesin mice have demonstrated that the transcription factor encodedby Gcm-2 is a key regulator of parathyroid gland development.The expression of this gene is restricted to the parathyroidglands, and if this gene is mutated, the parathyroid glandsfail to form (2–4).
Fish, however, have been believed to lack parathyroid glandsand PTH, and unlike tetrapods, the majority of calcium uptakein fish is from external sources. These differences are believedto reflect the fact that with the evolution of the tetrapodsand the shift from an aquatic to a terrestrial environment,new controls for regulating calcium homeostasis had to be putin place, and thus the evolution of the parathyroid glands andPTH was a key event in facilitating this transition. This eventfreed the tetrapods from relying on calcium uptake from thewater by giving them the ability to internally regulate theirserum calcium levels.
Although the evolution of the parathyroid gland was a key eventin the emergence of the tetrapods, there have been few studieson how this evolution was achieved. Here, we have exploitedthe rigid association between Gcm-2 and the parathyroid glandand conducted a phylogenetic analysis to gain insight into howthis structure evolved. Our results demonstrate that the parathyroidgland of tetrapods and the gills of fish most likely share acommon evolutionary origin; both express Gcm-2 and require thisgene for their formation, and both express PTH and CasR. Wethus suggest that the parathyroid gland came into being as theresult of the transformation of the gills into the parathyroidglands of tetrapods.
DNA Cloning. Partial sequences of CasR, PTH, and PTH-relatedprotein (PTHrP) in zebrafish and chicks were identified in expressionsequence tag databases in the National Center for BiotechnologyInformation, the European Bioinformatics Institute, and theUniversity of Delaware, Newark, and were isolated by RT-PCRfrom adult gills of zebrafish and thyroid and parathyroid inembryonic day (E) 11 chicken embryos, respectively. PTH genesin fugu were identified in the European Bioinformatics Institutedatabase. The partial sequences of zebrafish Gcm-2 and chickenGcm-1 were identified in the expressed sequence tag databasein the National Center for Biotechnology Information and theUniversity of Delaware. Partial sequences (278 bp) of Gcm-2of chicken, Xenopus laevis, rainbow trout, Australian lungfish,and dogfish were amplified from total RNA from embryonic pharyngealtissues by RT-PCR using degenerate primers. By using sequence-specificprimers, each full-length cDNA was isolated by 5' and 3' RACEusing the SMART RACE cDNA amplification kit (Clontech). Allsequences have been deposited in the DNA Data Bank of Japan.
In Situ Hybridization and Histology. Whole-mount in situ hybridizationfor chicken and zebrafish embryos was performed as describedin refs. 5 and 6. In situ hybridization for dogfish embryoswas performed by using the same protocol as for chicken embryos,except that soaking in DMSO/methanol (1:1) solution was substitutedfor the proteinase K treatment. The stained chicken embryoswere embedded in gelatin–albumin with 2.5% glutaraldehydeand sectioned at 50 µm with a Vibratome.
Morpholino-Modified Oligonucleotide (MO) Injection. Injectionof zebrafish Gcm-2 antisense and control MOs was performed asdescribed in ref. 7. MO antisense oligonucleotides (GeneTools,Philomath, OR) were designed against 25 bases around a splicingsite at the end of the third exon-encoding ORF. This MO wasdesigned as a splicing-blocking MO (8) to cause skipping ofan exon encoding the DNA-binding domain of Gcm-2. All injectionswere performed on one-cell-stage embryos at concentrations between2.5 and 5 µg/µl. The gene knockdown was confirmedby RT-PCR in eight individual injected embryos.
RT-PCR for Zebrafish PTH and CasR.PTH, CasR, and -actin geneswere amplified by the Onestep RT-PCR kit (Qiagen) using setsof sequence-specific primers from total RNA from whole gillsand whole brain of a wild-type adult male zebrafish.
Gcm-2 Is Expressed in the Pharyngeal Pouches and ParathyroidGland in a Nonmammalian Tetrapod. To date, Gcm-2 expressionhas been scrutinized in the mouse embryo only. We have thereforeisolated the chicken (Gallus gallus) Gcm-2 gene and analyzedits expression pattern to determine whether it is similarlyrestricted to the pharyngeal pouches and the parathyroid glandin nonmammalian tetrapods. We find that, as in mice, this geneis expressed exclusively in both the parathyroid gland and thepharyngeal pouches from which it derives. Fig. 1 A–D showsthe relative positions of the parathyroid and thyroid glandsin an E11 embryo. The parathyroid gland is unambiguously identifiedthrough its expression of PTH (Fig. 1B) and CasR (Fig. 1C),and the thyroid through expression of TTF-1 (9) (Fig. 1D). Wealso find that, at earlier developmental stages, this gene isexpressed in the pharyngeal pouches (Fig. 1 E–I) in thechick. Expression initiates in the third pouch, at stage 18(Fig. 1E), and then occurs in the fourth pouch, and additionallyin a small domain of the second pouch (Fig. 1F). It is clearwhen viewed in section that Gcm-2 expression is restricted tothe pharyngeal endoderm (Fig. 1H). As development proceeds,expression is lost from the second pharyngeal pouch but becomesstrongly up-regulated in the endodermal thickenings of the thirdand fourth pouches that are the primordia of the parathyroidglands in the chick (Fig. 1 G and I).
Fig. 1. Expression of Gcm-2 in the parathyroid gland and the pharyngeal pouches in the chick. (A–D) Whole-mount in situ hybridization of chick E11 thyroid (T) and parathyroid (P) glands for the following probes: Gcm-2 (A), PTH (B), CasR (C), and TTF-1 (D). Gcm-2 can be seen to be expressed in two round masses, the parathyroid glands, which are adjacent to the thyroid, which expresses TTF-1. The parathyroid glands additionally express PTH and CasR. (E–I) Expression of Gcm-2 in chicken embryos, staged as described in ref. 16. In these micrographs, anterior is to the left and ventral is to the bottom. OV, otic vesicle; pp, pharyngeal pouch; II–IV, pharyngeal arches. Expression of Gcm-2 starts in the third pharyngeal pouch at stage 18 (E), and then, as development proceeds, expression is also evident in the fourth pharyngeal pouch and additionally weakly in the second pouch (F). At stage 24, expression in the third and fourth pouches is concentrated in a region dorsal of the pharyngeal pouches and is lost from the second pouch (G). In Vibratome sections of stage-22 embryos (H), it is clear that Gcm-2 expression is localized to the pharyngeal endoderm and that, by stage 24, the region of the pharyngeal endoderm expressing Gcm-2 has thickened and given rise to round masses that are the parathyroid gland rudiments of the third and fourth pouches (I).
Gcm-2 Is Found Throughout the Gnathostomes. In mammals, twoGcm genes have been identified, Gcm-1 and Gcm-2 (2, 10). Importantly,it has been shown that, in the mice mutated for Gcm-2, PTH levelsin serum are normal and the result of Gcm-1, which is expressedin the thymus, causing the ectopic production of PTH from cellsin this organ (4). Given that Gcm-2 and, under certain circumstances,Gcm-1, can both elicit the production of PTH, we carried outan extensive phylogenetic analysis of the distribution of bothgenes to determine whether these genes evolved with the tetrapods.Surprisingly, we found that Gcm-2 is present not only in tetrapods(mammals Homo sapiens and Mus musculus, chicken, and Xenopus)but also in dipnoi fish (Australian lungfish, Neoceratodus forsteri),teleost fish (rainbow trout, Oncorhynchus mykiss; and zebrafish,Danio rerio), and from a chondrichthyan species (dogfish, Scyliorhinuscanicula). Contrastingly, we have been able to identify Gcm-1only in tetrapods. Searches of the fugu (Takifugu rubripes)and zebrafish genome fail to highlight the presence of a Gcm-1gene. A CLUSTAL analysis of the Gcm sequences shows clearlythat all of the Gcm-2 sequences that we have isolated grouptogether phylogenetically, as do all of the Gcm-1 sequences(Fig. 2A). We also find from an analysis of the genomes sequences(www.ensembl.org) that Gcm-2 is invariably physically linkedto another gene, ELOVL2, in humans (Chr6p24.2), chickens (Chr2ctg20.4),and zebrafish (Chr24ctg25479.1), further demonstrating thatthe fish Gcm-2 gene is the orthologue of the mammalian gene(Fig. 2B). Thus, Gcm-2 is widely distributed throughout thegnathostomes and is not restricted to species that possess aparathyroid gland.
Fig. 2. Phylogenetic analysis of the distribution of Gcm-2 and its expression in teleost (zebrafish) and chondrichthyan (dogfish) species. (A) A phylogenetic tree of vertebrate Gcm genes based on CLUSTAL analysis. Dr, zebrafish (Danio rerio); Gg, chicken (Gallus gallus); Hs, human (Homo sapiens); Mm, mouse (Mus musculus); Nf, Australian lungfish (Neoceratodus forsteri); Om, rainbow trout (Oncorhynchus mykiss); Sc, dogfish (Scyliorhinus canicula); Xl, Xenopus (Xenopus laevis). Branch lengths are in units of number of amino acid substitutions per site. (B) Schematic depicting the conserved linkage between Gcm2 and Elovl2 in humans on chromosome 6p24.2, in chickens on chromosome 2 ctg20.4, and in zebrafish on chromosome 24 ctg254791. (C–F) Gcm-2 expression in zebrafish embryos. In these micrographs, anterior is to the left and ventral is to the bottom. Gcm-2 initiates expression in the second pharyngeal pouch in early 3-day-old larval fish (indicated by arrowhead in C). Subsequently, Gcm-2 is expressed sequentially in the more posterior pouches (D), and, by day 4, Gcm-2 is expressed in all of the pouches (E). It is also apparent by day 4 that Gcm-2 is expressed in the developing internal gill buds emerging from the pharyngeal pouches (F). (G and H) Gcm-2 expression in dogfish embryos. This gene is expressed in the internal gill buds protruding from the pharyngeal pouches in stage-27 dogfish embryos (17). The pharyngeal arches are numbered II–VI.
Gcm-2 Is Expressed in the Pharyngeal Pouches and Their Derivatives,the Internal Gill Buds, in Teleost and Chondrichthyan Species.We have characterized the expression profiles of Gcm-2 in zebrafishand dogfish and compared these with the chick. We find that,in all species, this gene is expressed exclusively in the pharyngealpouch endoderm. In zebrafish larva, Gcm-2 expression initiatesin the second pharyngeal pouch at early E3 (Fig. 2C) and overthe next 24 h becomes established in all of the pharyngeal pouches(Fig. 2 D and E). On E4, it also is apparent that this geneis expressed in the buds of the internal gills that are derivedfrom the pharyngeal pouches (11) (Fig. 2F). Similarly, in dogfish,Gcm-2 expression is found in the pharyngeal pouches and theinternal gill buds that protrude from these structures (Fig.2 G and H). Thus, the presence of Gcm-2 and its expression inthe pharyngeal pouches and their elaborations was already inplace before the emergence of osteichthyans and the subsequentsplit into actinopterygian and sarcopterygian lineages. Theseresults indicate that the expression of Gcm-2 in the pharyngealpouches and their derivatives, the internal gill buds and parathyroidgland, are an ancient conserved feature of all jawed vertebrates.
Gcm-2 Is Required for the Elaboration of the Internal Gill Budsfrom the Pharyngeal Pouches. To further dissect the functionof Gcm-2 in fish, we have injected zebrafish with an antisenseMO that causes exon-skipping and thus interferes with Gcm-2function (8). In all embryos injected with the control MO (n= 49), gill buds formed as simply viewed with Nomarski optics(Fig. 3A) and also through Gcm-2 expression, which highlightsthese structures (Fig. 3B). Contrastingly, in 85% of the embryosthat received an injection of the Gcm-2 antisense MO (n = 34),gills buds did not form. This absence of gill buds is evidentboth from morphology (Fig. 3D) and from the lack of Gcm-2-expressingtissues associated with each pharyngeal pouch (Fig. 3E). Itis possible that the failure in the formation of the internalgill buds from the pharyngeal pouches could be due to earlydefects in the organization of the pouches themselves. To addressthis issue, we analyzed the expression profile of Pax-9a, whichmarks the pharyngeal pouches (12). In both control-injectedembryos and embryos injected with the Gcm-2 antisense MO, thegeneral patterning of the pharyngeal endoderm was unaffected,and Pax-9a expression clearly indicated the presence of thepharyngeal pouches (Fig. 3 C and F). Thus, in fish, Gcm-2 isspecifically required for the formation of the internal gillbuds from the pharyngeal pouches.
Fig. 3.Gcm-2 is required for the elaboration of the internal gill buds from the pharyngeal pouches in zebrafish. Zebrafish embryos were injected at the one-cell stage with either control or antisense Gcm-2 MOs. The embryos were then analyzed at day 5 for the presence of internal gill buds. (A–C) Five-day-old zebrafish larva injected with control MO. (A) Nomarski view of the pharyngeal region of a day-5 embryo injected with the control MO. The internal gill buds protruding from the pharyngeal pouches are clearly evident (arrowheads). (B) Embryo injected with control MO hybridized for Gcm-2. Gcm-2-expressing internal gill buds can be clearly seen protruding from the pharyngeal pouches. (C) Embryo injected with control MO, showing normal pharyngeal pouch formation as judged by Pax-9a expression. Each pharyngeal pouch is indicated by an arrowhead. (D–F) Five-day-old zebrafish larva injected with Gcm-2 antisense MO. (D) Nomarski view of the pharyngeal region of a E5 embryo injected with the antisense Gcm-2 MO. There are no internal gill buds protruding from the pharyngeal pouches. (E) Embryo injected with the antisense Gcm-2 MO hybridized for Gcm-2. There are no Gcm-2-expressing internal gill buds protruding from the pharyngeal pouches. (F) Embryo injected with the antisense Gcm-2 MO, showing normal pharyngeal pouch formation as judged by Pax-9a expression. Each pharyngeal pouch is indicated by an arrowhead. EY, eye; YK, yolk. Anterior is to left and ventral is to the bottom.
Teleosts Do Have PTH-Encoding Genes That Are Expressed in theGills, as Is CasR. The fact that the gill buds of fish and thetetrapod parathyroid both require Gcm-2 function for their formationsuggests that these structures are closely related. To furtherexamine this relationship, we have taken advantage of the availabilityof the zebrafish and fugu genome sequences to search for a PTH-encodinggene, and we have identified two such genes in both species(Fig. 4 A and B). CLUSTAL analysis of the protein sequencesencoded by these genes demonstrates that they group with thetetrapod PTH genes. These genes are distinct from the PTHrPgenes from these fish, which group with the tetrapod PTHrP genes(Fig. 4A). It is further clear from a comparison of the peptidesequences of the zebrafish and amniote PTH genes that key residueswithin the N-terminal 34 aa that are required for biologicalactivity are conserved (Fig. 4B). These results are in keepingwith another recent study that similarly identified two PTHgenes in zebrafish and fugu that are shown to be fully active(13, 14). We have further analyzed the tissues expression ofthe zebrafish PTH genes, and we find that these genes are expressedby the gills (Fig. 4C). Finally, we also have isolated CasRfrom zebrafish, and we demonstrate that this gene also is expressedby the gills (Fig. 4C).
Fig. 4.PTH genes in zebrafish. (A) A phylogenetic tree of PTH and PTHrP genes in vertebrates. Dr, zebrafish (Danio rerio); Gg, chicken (Gallus gallus); Hs, human (Homo sapiens); Mm, mouse (Mus musculus); Sa, seabream (Sparus aurata); Tr, fugu (Takifugu rubripes). (B) Comparison of the partial peptide sequences of zebrafish PTH and amniote PTH. The N-terminal 34 aa of mature human PTH peptide are sufficient for the biological activity of PTH. This alignment includes the N-terminal amino acids (1–34) with 2 aa before the final proteolytic cleavage site. (C) Gills in adult zebrafish express both the PTH genes and CasR. PTH and CasR were amplified from adult brain and gills by RT-PCR. Arrowhead indicates cDNA product.
In this study, we have analyzed the origin of the parathyroidgland. We find that Gcm-2, a gene that is expressed in the parathyroidgland and is absolutely required for its formation, is foundnot only in animals that possess a parathyroid gland, the tetrapods,but also in the jawed vertebrates. We also show that in amniotesand fish, Gcm-2 displays a conserved pattern of expression.In mouse, chicken, zebrafish, and dogfish, Gcm-2 is expressedin the pharyngeal pouches and, at later stages, in the structuresthat form from the pouches, the parathyroid gland in tetrapodsand the internal gill buds in fish. We further demonstrate thatGcm-2 is required for the formation of the internal gill budsin zebrafish. Finally, we demonstrate that fish possess PTH-encodinggenes that are expressed by the gills, which additionally expressCasR.
The results presented here demonstrate that the parathyroidgland and the internal gill buds of fish are related structuresand likely share a common evolutionary history. Both structuresoriginate from the pharyngeal pouch endoderm, express Gcm-2,and require this gene function for their formation. Thus, Gcm-2likely serves a conserved function across the gnathostomes withrespect to the elaboration of pharyngeal pouches into internalgill buds and parathyroid. It is known that calcium uptake isa particularly important function of the gills (15), and wealso show that fish do possess PTH genes, which have been shownby others to generate fully active peptides (13, 14), and thatthis gene is expressed in the gills. Thus, both the tetrapodparathyroid gland and the gills of fish contribute to the regulationof extracellular calcium levels. It is therefore reasonableto suggest that the parathyroid gland evolved as a result ofthe transformation of the gills into the parathyroid glandsof tetrapods and the transition from an aquatic to a terrestrialenvironment. This interpretation also would explain the positioningof the parathyroid gland within the pharynx in the tetrapodbody. Were the parathyroid gland to have emerged de novo withthe evolution of the tetrapods, it could, as an endocrine organ,have been placed anywhere in the body and still exert its effect.
Acknowledgements
We thank Jean Joss (Macquarie University, New South Wales, Australia),Mike Jones (Institute for Cancer Research, London), CorinneHouart and Gareth Fraser (both of King's College London), andthe London Aquarium for supplying material; Corinne Houart forhelping to establish the MO injections; Imelda McGonnell forhelp with protocols; and Christine Ferguson, Jo Begbie, andRobyn Quinlan for critical comments on the manuscript. Thiswork was supported by the Royal Society and grants from theMinistry of Education, Science, Sports, and Culture of Japanand the Japan Society for the Promotion of Science Research.
Footnotes
Author contributions: M.O. and A.G. performed research and wrotethe paper.
This paper was submitted directly (Track II) to the PNAS office.
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-actin genes were amplified by the Onestep RT-PCR kit (Qiagen) using sets of sequence-specific primers from total RNA from whole gills and whole brain of a wild-type adult male zebrafish. 
To whom correspondence may be addressed. E-mail: anthony.graham{at}kcl.ac.uk or maokabe{at}lab.nig.ac.jp. 
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