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Na,K-ATPase α and β subunit genes exhibit unique expression patterns during zebrafish embryogenesis
Mechanisms of Development 116 (2002) 51–59 www.elsevier.com/locate/modo
Na,K-ATPase a and b subunit genes exhibit unique expression patterns during zebrafish embryogenesis Victor A. Canfield a,1, Benjamin Loppin b,1, Bernard Thisse b, Christine Thisse b, John H. Postlethwait c, Manzoor-Ali P.K. Mohideen d, S. Johannes R. Rajarao a, Robert Levenson a,* b
a Department of Pharmacology, Penn State College of Medicine, Hershey, PA 17033, USA Insitut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM/ULP, CU de Strasbourg, Strasbourg, France c Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA d Department of Pathology, Penn State College of Medicine, Hershey, PA 17033, USA
Received 20 February 2002; received in revised form 17 April 2002; accepted 17 April 2002
Abstract We have used in situ hybridization to analyze Na,K-ATPase a and b subunit gene expression during zebrafish embryogenesis. The most striking finding is that each of the 14 Na,K-ATPase genes exhibits a distinct expression profile. All a and b subunit genes are expressed in the nervous system, although the pattern of expression in different regions varies dramatically. In peripheral tissues, three of the five a1-like genes are expressed in pronephros and mucous cells, one is expressed in heart, and one is predominant in skeletal muscle. The a2 gene is expressed in brain and heart but is most prominent in skeletal muscle, while the two a3 genes are restricted in their expression to the nervous system. Of the six b subunit genes, b1a is expressed at highest abundance in lens, pronephros, and heart, while b1b transcripts are abundant in mucous cells. The two b2-like genes are differentially expressed in the nervous system. One b3 gene is expressed exclusively in brain while the other is abundantly expressed in skeletal muscle. Based on these expression patterns, we predict that at least 14 a/b subunit pairs are likely to be formed in different tissues. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Zebrafish embryogenesis; Na,K-ATPase a subunit gene; Na,K-ATPase b subunit gene; In situ hybridization
1. Results and discussion Na,K-ATPase generates and maintains the Na 1 and K 1 gradients that underlie electrical excitability in nerve and muscle (Thomas, 1972), as well as the transport of numerous solutes and water across epithelia (Lingrel et al., 1990). This active enzyme consists of a catalytic a subunit and a b subunit of unknown function. We have previously identified eight a and six b subunit genes (Rajarao et al., 2001, 2002). Among the a subunit genes, five are members of the a1-like subfamily, two are orthologous to the mammalian a3 subunit, and one appears to represent an a2 subunit gene. Each of the three mammalian b subunit genes is represented by a pair of zebrafish genes. Here we have used whole mount in situ hybridization to study the expression pattern of the 14 zebrafish Na,K-ATPase a and b subunit genes. Comparison of the expression profiles of each a and b subunit gene
* Corresponding author. Tel.: 11-717-531-4545; fax: 11-717-531-5013. E-mail address: [email protected] (R. Levenson). 1 Contributed equally to this work.
allows predictions about which combinations of a and b subunits are likely to be formed in vivo. 1.1. Overview Na,K-ATPase mRNA expression was examined during zebrafish embryogenesis from 4 to 72 h post-fertilization (hpf). In zebrafish, there are five a1-like genes, two a3like genes, and one ortholog of the mammalian a2 subunit. There are also six b subunit genes, among which are two each of the b1, b2 and b3 isoforms. Each of the a and b subunit genes becomes transcriptionally active by midsomitogenesis. An overview of the expression patterns at 36 hpf is shown in Fig. 1. The most striking aspect of Na,K-ATPase transcription is that within the a and b gene families, each gene exhibits a unique expression profile. All 14 genes are expressed in the nervous system. Some genes, such as a1a.1, a2, and b2b are expressed throughout the nervous system, while other genes, such as a1a.4 and b1b, show a much more restricted expression profile. Outside the nervous system, the a1a.1 (A), a1a.2 (B), and a1a.4 (D) genes are expressed at high levels in pronephros and at
0925-4773/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(02)00135-1
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Fig. 1. Expression of Na,K-ATPase a and b subunit genes during zebrafish embryogenesis analyzed by in situ hybridization. Lateral views of 36 hpf embryos. B, blood islands; L, lens; MC, mucous cells; S, somites; PD, pronephric duct; H, heart; SC, spinal cord; E, epiphysis; OV, otic vesicle.
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Fig. 2. Expression of Na,K-ATPase a and b subunit genes during zebrafish embryogenesis. Dorsal views of 36 hpf embryos. Embryos in panels A, E, and I were partially dissected from yolk. L, lens; TL, telencephalon; D, diencephalon; TG, tegmentum; HB, hindbrain; OL, olfactory placode; OV, otic vesicle; E, epiphysis. Asterisks indicate labeled neurons in anterior spinal cord. Arrowheads in panel F indicate paired columns of cells within hindbrain.
variable levels in mucous cells. The b1a (H) and b2b genes (K) appear to be the only b subunit genes expressed in pronephros, while the b1b gene (I) is the only b subunit gene expressed in mucous cells. It is also apparent that the a1a.3 (C), a2 (F), and b3b (M) genes are coexpressed in skeletal muscle. The a1a.1 (A) and b1a (H) genes appear to be coexpressed in lens, while a1a.1 (A), a2 (F) and b1a (H) are the three genes expressed in heart. Comparing expression of a versus b subunit genes, it is apparent that the a1a.3 (C) and b3b (M) genes show nearly identical expression profiles. The a3a (G) and b2a (J) genes also show strong similarities in expression. These results suggest the likelihood that enzymes composed of a1a.3 and b3b subunits are formed in skeletal muscle and brain, and that a3a/b2a combinations are also present in brain. 1.2. Expression of Na,K-ATPase genes in the zebrafish central nervous system A more detailed picture of the expression pattern of Na,K-ATPase genes in central nervous system (CNS) can
be obtained from an examination of a and b subunit transcripts in dorsal views of zebrafish embryos. The results for selected a and b subunit genes at 36 hpf are presented in Fig. 2. From an overall perspective, it appears that the a1a.1 (A), a1a.3 (B), a3a (E), b2a (I), and b3b (K) genes are expressed within a common set of CNS structures. These genes show an overlapping pattern of expression in nuclei located within telencephalon, diencephalon, ventral tegmentum, and ventro-lateral regions of the hindbrain rhombomeres. However, it is also apparent that the relative abundance of transcripts within these structures differs for each of the genes, and that the expression profiles are not completely concordant. Several Na,KATPase genes including a1a.4 (C), a3a (E), b1a (G), and b1b (H) show expression in olfactory placode, while a1a.3 (B), b1b (H), b2a (I), and b3b (K) are expressed in a distinct subset of cells in anterior spinal cord. Three genes, a1a.4 (C), b1a (G), and b2b (J), are expressed in otic vesicle. Within this structure, expression of the a1a.4 and b1a genes appears to precisely overlap, whereas b2b transcripts are localized to two distinct regions within the otic
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vesicle. Two genes, a1a.1 (A) and b1a (G), show strong expression in lens, while a3b (F) is strongly expressed in retina. The expression patterns of the a1b (D) and a3b (F) genes appear distinct from those of any of the other a subunit genes. The high expression levels of of these isoforms in midline structures are particularly apparent. Although in dorsal views only the b2b gene appears to be expressed in epiphysis (J), other views demonstrate that the a1a.1, a1a.3, a3a, b2a, and b3b genes are also expressed in this structure (data not shown). 1.3. Expression of Na,K-ATPase a subunit genes during embryogenesis 1.3.1. a 1-like genes a1a.1 mRNA expression is detectable by early somitogenesis, and is uniformly distributed throughout the embryo (data not shown). Expression throughout the embryo is also observed at the 15 somite stage (Fig. 3A), with highest levels of expression present in pronephric duct, otic vesicle, and developing brain. Staining of pronephric duct remains strong through 72 hpf, the last stage examined. Expression of a1a.1 transcripts in lens, developing heart, and mucous cells is visible at 24 hpf (Fig. 3B), while staining of otic vesicle is no longer prominent at this stage. At 36 hpf (Fig. 1A), expression is visible in blood islands in the tail. This overall pattern of a1a.1 expression remains essentially unchanged throughout 72 hpf. Within CNS, highest levels of expression are found in posterior midbrain, within nuclei of the hindbrain, and spinal cord (Fig. 2A). A similar expression pattern has been published (a1B1) by Serluca et al. (2001). The expression of the a1a.2 gene at the 15 somite stage (Fig. 3C) is particularly prominent in pronephric duct, with lower levels of expression in brain and otic vesicle. At 36 hpf, transcripts of the a1a.2 gene continue to be abundant in pronephric duct and mucous cells, while lower expression levels are visible in lens, posterior midbrain, and otic vesicle (Fig. 1B). By 72 hpf, the levels of a1a.2 transcripts are most abundant in eye and brain, with moderate mRNA levels detectable in pronephric duct, mucous cells, and ear (Fig. 3D). It is striking that while expression in brain is very robust, no a1a.2 expression is detectable in spinal cord. Diffuse expression of the a1a.3 gene is visible at the15 somite stage, with stronger expression detectable in the head region and somitic mesoderm (Fig. 3E). Levels of a1a.3 mRNA are most abundant at 24 hpf in somites, with lower levels detectable in spinal cord and brain (Fig. 3F). At 36 hpf, a1a.3 transcripts are detectable in many brain regions including telencephalon, diencephalon, epiphysis, tegmentum, and hindbrain rhombomeres (Figs. 1C and 2B). At 48 hpf, a1a.3 expression is readily apparent in somites. However, the strongest expression of a1a.3 mRNA is in CNS, with highest expression levels detectable in hindbrain (Fig. 3G,H). Expression of the a1a.4 and a1b genes is best visualized
in 36 hpf embryos. mRNA transcripts of the a1a.4 gene are most apparent in pronephric duct and ear (Fig. 1D). Lower levels of a1a.4 gene expression are also detectable in olfactory placode and mucous cells (Fig. 2C). This expression pattern remains virtually unchanged during the first 72 h of zebrafish development. Similar results for a1a.4 expression (designated a1A1) have been reported previously (Serluca et al., 2001). At 36 hpf, the a1b gene is expressed most prominently in brain and spinal cord. In lateral views (Fig. 1E), a1b transcripts display a segmental expression pattern in hindbrain and are clearly present within distinct cell populations in dorsal and ventral spinal cord. In dorsal views (Fig. 2D), a1b mRNA is visible in hindbrain nuclei, but is most prominently expressed in midline CNS structures. 1.3.2. a 2 gene To date, only one a2-like gene has been identified in zebrafish (Rajarao et al., 2001). The a2 gene is expressed throughout the embryo from gastrulation to early somitogenesis (data not shown). By the 15 somite stage, a2 transcripts are clearly detectable in a segmental pattern characteristic of somitic mesoderm (Fig. 4A). Expression at 24 hpf is strong in somites and the head region (Fig. 4B), and is also visible in pronephric duct (Fig. 4B). A similar pattern for a2 expression is seen at 36 hpf (Fig. 1F). By 48 hpf, a2 mRNA is clearly visible in heart (Fig. 4C). In contrast to our results, Serluca et al. (2001) did not observe expression in brain before 48 hpf. 1.3.3. a 3-like genes At the 15 somite stage, expression of the a3a gene is detectable within four discrete structures; telencephalon, epiphysis, trigeminal ganglia, and spinal cord (Fig. 4D). By 24 hpf, a3a transcripts are also present in blood islands, spinal cord, and anterior and posterior lateral line ganglia (Fig. 4E). a3a transcripts are clearly visible within discrete structures in telencephalon, epiphysis, olfactory placode, ventral diencephalon, and hindbrain at 24 hpf (Fig. 4E). In the midbrain, a3a expression is detectable specifically within tegmentum. A similar expression profile is seen at 36 hpf (Fig. 1G). Expression of the paralogous a3b gene becomes visible at the anterior neural plate by 10 hpf (Fig. 4F), while diffuse staining throughout brain can be seen at the 15 somite stage (Fig. 4G). By 24 hpf, a3b mRNA expression is prominent in brain and retina and becomes visible in spinal cord (Fig. 4H). A similar expression profile for a3b is seen at 36 hpf (Fig. 1N). 1.4. Expression of Na,K-ATPase b subunit genes 1.4.1. b 1-like genes Expression of the b1a gene becomes detectable during gastrulation, and is clearly visible in the anterior neural plate by the 10 somite stage (Fig. 5A). At the 15 somite stage (Fig. 5B), expression is strong in pronephric duct, heart, and
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otic vesicle, while at 24 hpf, b1a transcripts are also apparent in lens, olfactory placode, and blood islands (Fig. 5C). A similar expression profile is visible from 36 to 48 hpf, with expression detectable in parapineal gland at 36 hpf (Fig. 1H) and retina at 48 hpf (Fig. 5D). At 72 hpf, expression is most prominent in retina, foregut, heart, and ear (Fig. 5E). At this stage, staining of b1a transcripts is visible within brain but not spinal cord (Fig. 5E). The paralogous b1b gene exhibits a markedly different expression pattern. At the 15 somite
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stage, b1b transcripts are present at high abundance in mucous cells and at lower levels in the nervous system (Fig. 5F). At 24 hpf, b1b expression also becomes visible within cells located in hindbrain and anterior spinal cord (Fig. 5G), while at 36 hpf, b1b transcripts are restricted to mucous cells, epiphysis, olfactory tubercle, tegmentum, and hindbrain (Fig. 1I). By 72 hpf, strong expression is detected in midbrain, lens, as well as anterior and posterior lateral line ganglia (Fig. 5H).
Fig. 3. Expression of zebrafish Na,K-ATPase a1-like genes. (A, B) a1a.1; (C, D) a1a.2; (E–H) a1a.3. (A) a1a.1 at mid-somitogenesis (15 somite stage, lateral view); (B) a1a.1 at 24 hpf (lateral view); (C) a1a.2 at mid-somitogenesis (15 somite stage, lateral view); (D) a1a.2 at 72 hpf (lateral view); (E) a1a.3 at midsomitogenesis (15 somite stage, lateral view); (F) a1a.3 at 24 hpf (lateral view); (G) a1a.3 at 48 hpf (lateral view); (H) a1a.3 at 48 hpf (dorsal view). OV, otic vesicle; PD, pronephric duct; H, heart; MC, mucous cells; SC, spinal cord; S, somites; HB, hindbrain.
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1.4.2. b 2-like genes The b2a and b2b genes are predominantly expressed in nervous system. Within the nervous system, however, these two genes exhibit dramatically different expression patterns. In 36 hpf embryos, b2a transcripts are present at high abundance throughout brain and in spinal cord neurons (Fig. 1J). A dorsal view of a 36 hpf embryo shows high levels of b2a
transcripts in telencephalon, diencephalon, ventral tegmentum, and ventro-lateral regions of the hindbrain rhombomeres (Fig. 2I). In contrast to b1a, b1b mRNA expression is diffuse in brain and is also visible at low levels in pronephric duct (Fig. 1K). In dorsal views of 36 hpf embryos, b2b transcripts were diffusely distributed throughout brain with higher expression levels visible in otic vesicles and epiphysis
Fig. 4. Expression of zebrafish a2 and a3-like genes. (A–C) a2; (D–E) a3a; (F–H) a3b. (A) a2 at mid-somitogenesis (15 somite stage, lateral view); (B) a2 at 24 hpf lateral view); (C) a2 at 48 hpf (lateral view); (D) a3a at mid-somitogenesis (15 somite stage, lateral view); (E) a3a, at 24 hpf (lateral view); (F) a3b, early somitogenesis (10 somite stage, lateral view); (G) a3b, mid-somitogenesis (15 somite stage, lateral view); (H) a3b at 24 hpf (lateral view). S, somites; PD, pronephric duct; H, heart; TGG, trigeminal ganglion; SC, spinal cord; E, epiphysis; TL, telencephalon; D, diencephalon.
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Fig. 5. In situ hybridization analysis of zebrafish b subunit gene expression. (A–E) b1a; (F–H) b1b; (I–J) b3a; (K–L) b3b. (A) b1a at early somitogenesis (10 somite stage, lateral view); (B) b1a at mid-somitogenesis (15 somite stage, lateral view); (C) b1a at 24 hpf (lateral view); (D) b1a at 48 hpf (lateral view); (E) b1a at 72 hpf (lateral view); (F) b1b at mid-somitogenesis (15 somite stage, dorsal view); (G) b1b at 24 hpf (dorsal view); (H) b1b at 72 hpf (lateral view); (I) b3a at 48 hpf (dorsal view); (J) b3a at 72 hpf (lateral view); (K) b3b at 24 hpf (lateral view); (L) b3b at 48 hpf (lateral view). OV, otic vesicle; H, heart; PD, pronephric duct; E, epiphysis; L, lens; B, blood island; MC, mucous cells; SC, spinal cord; LL, lateral line ganglia; R, retina; S, somites.
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(Fig. 2J). As development proceeds, abundant expression of the b2b gene occurs within sensory organs (Rajarao et al., 2002). A more complete description of b2a and b2b gene expression can be found in Rajarao et al. (2002). 1.4.3. b 3-like genes The expression profile of the two zebrafish b3-like genes is shown in Fig. 5. From gastrulation through 22 hpf, the b3a gene exhibits a changing segmental pattern of expression in the CNS (Appel et al., 1996). At 24 hpf, we find very low levels of b3a expression in the embryo (data not shown). This contrasts to some degree with previous results reported by Appel et al. (1996), who found that the b3a gene is expressed predominantly in brain and eye throughout embryogenesis. Commencing at 36 hpf (Fig. 1L), and continuing through 48 hpf (Fig. 5I), strongest expression of b3a mRNA is seen in retina and brain. At 72 hpf, b3a mRNA is most prominent in the photoreceptor layer of the retina (Fig. 5J), as also observed by Appel et al. (1996). The b3b gene shows a markedly distinct expression pattern from that of the b3a gene. At 24 hpf, b3b mRNA expression is apparent in somitic mesoderm and CNS, with very low expression detectable in pronephric duct (Fig. 5K). The pattern is similar at 36 hpf (Fig. 1M), although pronephric staining is no longer visible at this or subsequent stages. By 48 hpf, strong expression is visible throughout the CNS and is especially prominent in retina as well as lateral line ganglia (Fig. 5L). 1.5. Comparison of a and b subunit expression Comparison of expression profiles of Na,K-ATPase a and b genes makes it possible to predict which a/b subunit pairs may combine to form functional enzyme. A summary of these predictions is shown in Table 1. The most obvious similarity in expression is shared by the a1a.3 and the b3b genes. These isoforms exhibit nearly identical expression patterns, suggesting that a1a.3/b3b subunit pairs are produced in a variety of structures including skeletal muscle, telencephalon, diencephalon, epiphysis, and hindbrain. a3a and b2a also show strong similarities in expression. Although we predict the existence of isoenzymes composed of a3a/b2a subunits in brain, it should be noted that the overlap in expression between a3a and b2a subunits is largely within CNS structures that also express the a1a.1, a1a.3, and b3b subunits. Stronger evidence for the pairing of specific a and b subunit isoforms can be obtained by examining tissues which express only a single a or a single b subunit. For example, three Na,K-ATPase genes are expressed in zebrafish heart: a1a.1; a2; and b1a. Assuming that both of the a subunits present in heart must pair with the b1a subunit, it seems likely that enzymes composed of a1a.1/b1a and a2/ b1a subunit combinations are active in myocardium. A distinctive set of a and b subunit genes are expressed in mucous cells including a1a.1, a1a.2, a1a.4, and b1b. Mucous cells are therefore likely to express three different
Na,K-ATPase isoenzymes which are composed of each of the three a1-like subunits in combination with the single b1b subunit. The same three a subunits (a1a.1, a1a.2, and a1a.4) are the predominant a subunits expressed in pronephros, while the b1a gene is the only detectable b subunit produced in this tissue. It therefore seems likely that embryonic kidney contains three distinct Na,K-ATPase isoenzymes composed of a1a.1/b1a, a1a.2/b1a, and a1a.4/ b1a subunit combinations. Another isoenzyme, formed from a3a and b1a subunits, is predicted based on expression of these genes in olfactory placode. The a1a.3 and a2 genes are the predominant a subunit genes expressed in skeletal muscle, while b3b is the only b subunit gene expressed in this tissue. This expression pattern allows us to predict the formation of an additional isoenzyme, consisting of a2/b3b subunits, in skeletal muscle (The a1a.3/b3b enzyme is also present in brain). We also detect expression of a discrete set of Na,K-ATPase genes within a subset of neurons in anterior spinal cord. Three b subunit genes (b1b, b2a, and b3b) and only one a subunit gene (a1a.3) are expressed in these neurons. This profile predicts two additional isoenzymes composed of a1.3/b1b and a1a.3/b2a in spinal cord (a1a.3/b3b is also present in skeletal muscle and brain). From their expression profiles, several of the a and b subunits do not have obvious binding partners. For example, the a3b, b2b and b3a subunit genes exhibit a diffuse expression pattern throughout brain and retina. It is possible that a3b/b2b and a3b/b3a subunit combinations may be formed in brain. However, a3b, b2b, and b3a subunits might also combine with other a and b subunit isoforms that have overlapping expression within CNS. The inability to identify specific cell types or compartments expressing the a3b, b2b and b3a genes makes it difficult to predict which subunit pairs containing these isoforms actually combine to form Table 1 Expression sites of predicted zebrafish Na,K-ATPase a/b subunit combinations a/b Combination a Subunit
b Subunit
a1a.1 a1a.1 a1a.2 a1a.2 a1a.3 a1a.3 a1a.3 a1a.4 a1a.4 a1b a2 a2 a3a a3a a3b Unknown Unknown
b1a b1b b1a b1b b1b b2a b3b b1a b1b Unknown b1a b3b b1a b2a Unknown b2b b3a
Site of expression
Heart, pronephric duct, lens Mucous cells Pronephric duct Mucous cells Spinal cord Spinal cord Skeletal muscle, spinal cord, brain Pronephric duct, ear Mucous cells Brain and spinal cord Heart Skeletal muscle Olfactory placode Brain (probable combination) Brain Brain and spinal cord Brain
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functional Na,K-ATPase. The a1b subunit represents another example of an a subunit without an obvious b subunit binding partner. The a1b gene is most highly expressed in CNS along the midline. No b subunit gene shows a similar expression pattern, and three of the b subunits (b1b, b2a, and b3b) are expressed at lowest levels along the midline. It is not clear which of the other b subunit isoforms might combine with a1b. At least two additional isoenzymes containing a1b and a3b, or b2b and b3a subunits must be produced in the CNS. This analysis points to a minimum of 14 distinct Na,K-ATPase isoenzymes in zebrafish (Table 1), although additional isoenzymes could potentially be formed from additional a/b subunit pairings. 2. Experimental procedures Whole mount in situ hybridization was performed as previously described (Thisse et al., 1994). The following probes were used to analyze a and b subunit gene expression. 2.1. a Subunit genes a1a.1 (atp1a1a.1; Accession number AF286372), nucleotides 1–3474. a1a.2 (atp1a1a.2; Accession number AF286374), nucleotides 296–3255. a1a.3 (atp1a1a.3; Accession number AF308598), nucleotides 623–3275. a1a.4 (atp1a1a.4; Accession number AY008376), nucleotides 112–3262. a1b (atp1a1b; Accession number AY008375), nucleotides 234–2764. a2 (atp1a2; Accession number AF286373), nucleotides 1461–3889. a3a (atp1a3a; Accession number AF308599), nucleotides 185–3256. a3b (atp1a3b; Accession number AY008374), nucleotides 282–2251. 2.2. b Subunit genes 1-b1a (atp1b1a; Accession number AF286375), nucleotides 1–1477.
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b1b (atp1b1b; Accession number AF3085975), nucleotides 1–2384. b2a (atp1b2a; Accession number AF286376), nucleotides 1–1076. b2b (atp1b2b; Accession number AF373976), nucleotides 1–1227. b3a (atp1b3a; Accession number AF469651), nucleotides 1–834. 2 b3b (atp1b3b; Accession number AF293369), nucleotides 1–1755.
Acknowledgements This work was supported by NIH grants to R.L., J.H.P., and B.T. B.T. and C.T acknowledge support from the Institut National de la Sante´ et de la Recherche Me´ dicale, the Centre National de la Recherche Scientifique, the Hoˆ pital Universitaire de Strasbourg, the Association pour la Recherche sur le Cancer and the Ligue Nationale Contre le Cancer. References Appel, C., Gloor, S., Schmalzing, G., Schachner, M., Bernhardt, R.R., 1996. Expression of a Na,K-ATPase b3 subunit during development of the zebrafish central nervous system. J. Neurosci. Res. 46,, 551–564. Lingrel, J.B., Orlowski, J., Shull, M.M., Price, E.M., 1990. Molecular genetics of Na,K-ATPase. Prog. Nucleic Acid Res. Mol. Biol. 38, 37–89. Rajarao, S.J.R., Canfield, V.A., Mohideen, M.A., Yan, Y.L., Postlethwait, J.H., Cheng, K.C., Levenson, R., 2001. The repertoire of Na,K-ATPase a and b subunit genes expressed in the zebrafish, Danio rerio. Genome Res. 11, 1211–1220. Rajarao, S.J.R., Canfield, V.A., Loppin, B., Thisse, B., Thisse, C., Yan, Y.L., Postlethwait, J.H., Levenson, R., 2002. Two Na,K-ATPase b2 subunit isoforms are differentially expressed within the central nervous system and sensory organs during zebrafish embryogenesis. Dev. Dyn. 223, 254–261. Serluca, F.C., Sidow, A., Mably, J.D., Fishman, M.C., 2001. Partitioning of tissue expression accompanies multiple duplications of the Na 1/K 1 ATPase a subunit gene. Genome Res. 11, 1625–1631. Thisse, C., Thisse, B., Halpern, M.E., Postlethwait, J.H., 1994. Goosecoid expression in neurectoderm and mesendoderm is disrupted in zebrafish cyclops gastrulas. Dev. Biol. 164, 420–429. Thomas, R.C., 1972. Electrogenic sodium pump in nerve and muscle cells. Physiol. Rev. 52, 563–594.
2 The sequence of the b3 cDNA used here was found to vary from the b3a sequence (Accession number X89722) published previously (Appel et al., 1996).
Na,K-ATPase a and b subunit genes exhibit unique expression patterns during zebrafish embryogenesis Victor A. Canfield a,1, Benjamin Loppin b,1, Bernard Thisse b, Christine Thisse b, John H. Postlethwait c, Manzoor-Ali P.K. Mohideen d, S. Johannes R. Rajarao a, Robert Levenson a,* b
a Department of Pharmacology, Penn State College of Medicine, Hershey, PA 17033, USA Insitut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM/ULP, CU de Strasbourg, Strasbourg, France c Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA d Department of Pathology, Penn State College of Medicine, Hershey, PA 17033, USA
Received 20 February 2002; received in revised form 17 April 2002; accepted 17 April 2002
Abstract We have used in situ hybridization to analyze Na,K-ATPase a and b subunit gene expression during zebrafish embryogenesis. The most striking finding is that each of the 14 Na,K-ATPase genes exhibits a distinct expression profile. All a and b subunit genes are expressed in the nervous system, although the pattern of expression in different regions varies dramatically. In peripheral tissues, three of the five a1-like genes are expressed in pronephros and mucous cells, one is expressed in heart, and one is predominant in skeletal muscle. The a2 gene is expressed in brain and heart but is most prominent in skeletal muscle, while the two a3 genes are restricted in their expression to the nervous system. Of the six b subunit genes, b1a is expressed at highest abundance in lens, pronephros, and heart, while b1b transcripts are abundant in mucous cells. The two b2-like genes are differentially expressed in the nervous system. One b3 gene is expressed exclusively in brain while the other is abundantly expressed in skeletal muscle. Based on these expression patterns, we predict that at least 14 a/b subunit pairs are likely to be formed in different tissues. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Zebrafish embryogenesis; Na,K-ATPase a subunit gene; Na,K-ATPase b subunit gene; In situ hybridization
1. Results and discussion Na,K-ATPase generates and maintains the Na 1 and K 1 gradients that underlie electrical excitability in nerve and muscle (Thomas, 1972), as well as the transport of numerous solutes and water across epithelia (Lingrel et al., 1990). This active enzyme consists of a catalytic a subunit and a b subunit of unknown function. We have previously identified eight a and six b subunit genes (Rajarao et al., 2001, 2002). Among the a subunit genes, five are members of the a1-like subfamily, two are orthologous to the mammalian a3 subunit, and one appears to represent an a2 subunit gene. Each of the three mammalian b subunit genes is represented by a pair of zebrafish genes. Here we have used whole mount in situ hybridization to study the expression pattern of the 14 zebrafish Na,K-ATPase a and b subunit genes. Comparison of the expression profiles of each a and b subunit gene
* Corresponding author. Tel.: 11-717-531-4545; fax: 11-717-531-5013. E-mail address: [email protected] (R. Levenson). 1 Contributed equally to this work.
allows predictions about which combinations of a and b subunits are likely to be formed in vivo. 1.1. Overview Na,K-ATPase mRNA expression was examined during zebrafish embryogenesis from 4 to 72 h post-fertilization (hpf). In zebrafish, there are five a1-like genes, two a3like genes, and one ortholog of the mammalian a2 subunit. There are also six b subunit genes, among which are two each of the b1, b2 and b3 isoforms. Each of the a and b subunit genes becomes transcriptionally active by midsomitogenesis. An overview of the expression patterns at 36 hpf is shown in Fig. 1. The most striking aspect of Na,K-ATPase transcription is that within the a and b gene families, each gene exhibits a unique expression profile. All 14 genes are expressed in the nervous system. Some genes, such as a1a.1, a2, and b2b are expressed throughout the nervous system, while other genes, such as a1a.4 and b1b, show a much more restricted expression profile. Outside the nervous system, the a1a.1 (A), a1a.2 (B), and a1a.4 (D) genes are expressed at high levels in pronephros and at
0925-4773/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(02)00135-1
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Fig. 1. Expression of Na,K-ATPase a and b subunit genes during zebrafish embryogenesis analyzed by in situ hybridization. Lateral views of 36 hpf embryos. B, blood islands; L, lens; MC, mucous cells; S, somites; PD, pronephric duct; H, heart; SC, spinal cord; E, epiphysis; OV, otic vesicle.
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Fig. 2. Expression of Na,K-ATPase a and b subunit genes during zebrafish embryogenesis. Dorsal views of 36 hpf embryos. Embryos in panels A, E, and I were partially dissected from yolk. L, lens; TL, telencephalon; D, diencephalon; TG, tegmentum; HB, hindbrain; OL, olfactory placode; OV, otic vesicle; E, epiphysis. Asterisks indicate labeled neurons in anterior spinal cord. Arrowheads in panel F indicate paired columns of cells within hindbrain.
variable levels in mucous cells. The b1a (H) and b2b genes (K) appear to be the only b subunit genes expressed in pronephros, while the b1b gene (I) is the only b subunit gene expressed in mucous cells. It is also apparent that the a1a.3 (C), a2 (F), and b3b (M) genes are coexpressed in skeletal muscle. The a1a.1 (A) and b1a (H) genes appear to be coexpressed in lens, while a1a.1 (A), a2 (F) and b1a (H) are the three genes expressed in heart. Comparing expression of a versus b subunit genes, it is apparent that the a1a.3 (C) and b3b (M) genes show nearly identical expression profiles. The a3a (G) and b2a (J) genes also show strong similarities in expression. These results suggest the likelihood that enzymes composed of a1a.3 and b3b subunits are formed in skeletal muscle and brain, and that a3a/b2a combinations are also present in brain. 1.2. Expression of Na,K-ATPase genes in the zebrafish central nervous system A more detailed picture of the expression pattern of Na,K-ATPase genes in central nervous system (CNS) can
be obtained from an examination of a and b subunit transcripts in dorsal views of zebrafish embryos. The results for selected a and b subunit genes at 36 hpf are presented in Fig. 2. From an overall perspective, it appears that the a1a.1 (A), a1a.3 (B), a3a (E), b2a (I), and b3b (K) genes are expressed within a common set of CNS structures. These genes show an overlapping pattern of expression in nuclei located within telencephalon, diencephalon, ventral tegmentum, and ventro-lateral regions of the hindbrain rhombomeres. However, it is also apparent that the relative abundance of transcripts within these structures differs for each of the genes, and that the expression profiles are not completely concordant. Several Na,KATPase genes including a1a.4 (C), a3a (E), b1a (G), and b1b (H) show expression in olfactory placode, while a1a.3 (B), b1b (H), b2a (I), and b3b (K) are expressed in a distinct subset of cells in anterior spinal cord. Three genes, a1a.4 (C), b1a (G), and b2b (J), are expressed in otic vesicle. Within this structure, expression of the a1a.4 and b1a genes appears to precisely overlap, whereas b2b transcripts are localized to two distinct regions within the otic
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vesicle. Two genes, a1a.1 (A) and b1a (G), show strong expression in lens, while a3b (F) is strongly expressed in retina. The expression patterns of the a1b (D) and a3b (F) genes appear distinct from those of any of the other a subunit genes. The high expression levels of of these isoforms in midline structures are particularly apparent. Although in dorsal views only the b2b gene appears to be expressed in epiphysis (J), other views demonstrate that the a1a.1, a1a.3, a3a, b2a, and b3b genes are also expressed in this structure (data not shown). 1.3. Expression of Na,K-ATPase a subunit genes during embryogenesis 1.3.1. a 1-like genes a1a.1 mRNA expression is detectable by early somitogenesis, and is uniformly distributed throughout the embryo (data not shown). Expression throughout the embryo is also observed at the 15 somite stage (Fig. 3A), with highest levels of expression present in pronephric duct, otic vesicle, and developing brain. Staining of pronephric duct remains strong through 72 hpf, the last stage examined. Expression of a1a.1 transcripts in lens, developing heart, and mucous cells is visible at 24 hpf (Fig. 3B), while staining of otic vesicle is no longer prominent at this stage. At 36 hpf (Fig. 1A), expression is visible in blood islands in the tail. This overall pattern of a1a.1 expression remains essentially unchanged throughout 72 hpf. Within CNS, highest levels of expression are found in posterior midbrain, within nuclei of the hindbrain, and spinal cord (Fig. 2A). A similar expression pattern has been published (a1B1) by Serluca et al. (2001). The expression of the a1a.2 gene at the 15 somite stage (Fig. 3C) is particularly prominent in pronephric duct, with lower levels of expression in brain and otic vesicle. At 36 hpf, transcripts of the a1a.2 gene continue to be abundant in pronephric duct and mucous cells, while lower expression levels are visible in lens, posterior midbrain, and otic vesicle (Fig. 1B). By 72 hpf, the levels of a1a.2 transcripts are most abundant in eye and brain, with moderate mRNA levels detectable in pronephric duct, mucous cells, and ear (Fig. 3D). It is striking that while expression in brain is very robust, no a1a.2 expression is detectable in spinal cord. Diffuse expression of the a1a.3 gene is visible at the15 somite stage, with stronger expression detectable in the head region and somitic mesoderm (Fig. 3E). Levels of a1a.3 mRNA are most abundant at 24 hpf in somites, with lower levels detectable in spinal cord and brain (Fig. 3F). At 36 hpf, a1a.3 transcripts are detectable in many brain regions including telencephalon, diencephalon, epiphysis, tegmentum, and hindbrain rhombomeres (Figs. 1C and 2B). At 48 hpf, a1a.3 expression is readily apparent in somites. However, the strongest expression of a1a.3 mRNA is in CNS, with highest expression levels detectable in hindbrain (Fig. 3G,H). Expression of the a1a.4 and a1b genes is best visualized
in 36 hpf embryos. mRNA transcripts of the a1a.4 gene are most apparent in pronephric duct and ear (Fig. 1D). Lower levels of a1a.4 gene expression are also detectable in olfactory placode and mucous cells (Fig. 2C). This expression pattern remains virtually unchanged during the first 72 h of zebrafish development. Similar results for a1a.4 expression (designated a1A1) have been reported previously (Serluca et al., 2001). At 36 hpf, the a1b gene is expressed most prominently in brain and spinal cord. In lateral views (Fig. 1E), a1b transcripts display a segmental expression pattern in hindbrain and are clearly present within distinct cell populations in dorsal and ventral spinal cord. In dorsal views (Fig. 2D), a1b mRNA is visible in hindbrain nuclei, but is most prominently expressed in midline CNS structures. 1.3.2. a 2 gene To date, only one a2-like gene has been identified in zebrafish (Rajarao et al., 2001). The a2 gene is expressed throughout the embryo from gastrulation to early somitogenesis (data not shown). By the 15 somite stage, a2 transcripts are clearly detectable in a segmental pattern characteristic of somitic mesoderm (Fig. 4A). Expression at 24 hpf is strong in somites and the head region (Fig. 4B), and is also visible in pronephric duct (Fig. 4B). A similar pattern for a2 expression is seen at 36 hpf (Fig. 1F). By 48 hpf, a2 mRNA is clearly visible in heart (Fig. 4C). In contrast to our results, Serluca et al. (2001) did not observe expression in brain before 48 hpf. 1.3.3. a 3-like genes At the 15 somite stage, expression of the a3a gene is detectable within four discrete structures; telencephalon, epiphysis, trigeminal ganglia, and spinal cord (Fig. 4D). By 24 hpf, a3a transcripts are also present in blood islands, spinal cord, and anterior and posterior lateral line ganglia (Fig. 4E). a3a transcripts are clearly visible within discrete structures in telencephalon, epiphysis, olfactory placode, ventral diencephalon, and hindbrain at 24 hpf (Fig. 4E). In the midbrain, a3a expression is detectable specifically within tegmentum. A similar expression profile is seen at 36 hpf (Fig. 1G). Expression of the paralogous a3b gene becomes visible at the anterior neural plate by 10 hpf (Fig. 4F), while diffuse staining throughout brain can be seen at the 15 somite stage (Fig. 4G). By 24 hpf, a3b mRNA expression is prominent in brain and retina and becomes visible in spinal cord (Fig. 4H). A similar expression profile for a3b is seen at 36 hpf (Fig. 1N). 1.4. Expression of Na,K-ATPase b subunit genes 1.4.1. b 1-like genes Expression of the b1a gene becomes detectable during gastrulation, and is clearly visible in the anterior neural plate by the 10 somite stage (Fig. 5A). At the 15 somite stage (Fig. 5B), expression is strong in pronephric duct, heart, and
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otic vesicle, while at 24 hpf, b1a transcripts are also apparent in lens, olfactory placode, and blood islands (Fig. 5C). A similar expression profile is visible from 36 to 48 hpf, with expression detectable in parapineal gland at 36 hpf (Fig. 1H) and retina at 48 hpf (Fig. 5D). At 72 hpf, expression is most prominent in retina, foregut, heart, and ear (Fig. 5E). At this stage, staining of b1a transcripts is visible within brain but not spinal cord (Fig. 5E). The paralogous b1b gene exhibits a markedly different expression pattern. At the 15 somite
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stage, b1b transcripts are present at high abundance in mucous cells and at lower levels in the nervous system (Fig. 5F). At 24 hpf, b1b expression also becomes visible within cells located in hindbrain and anterior spinal cord (Fig. 5G), while at 36 hpf, b1b transcripts are restricted to mucous cells, epiphysis, olfactory tubercle, tegmentum, and hindbrain (Fig. 1I). By 72 hpf, strong expression is detected in midbrain, lens, as well as anterior and posterior lateral line ganglia (Fig. 5H).
Fig. 3. Expression of zebrafish Na,K-ATPase a1-like genes. (A, B) a1a.1; (C, D) a1a.2; (E–H) a1a.3. (A) a1a.1 at mid-somitogenesis (15 somite stage, lateral view); (B) a1a.1 at 24 hpf (lateral view); (C) a1a.2 at mid-somitogenesis (15 somite stage, lateral view); (D) a1a.2 at 72 hpf (lateral view); (E) a1a.3 at midsomitogenesis (15 somite stage, lateral view); (F) a1a.3 at 24 hpf (lateral view); (G) a1a.3 at 48 hpf (lateral view); (H) a1a.3 at 48 hpf (dorsal view). OV, otic vesicle; PD, pronephric duct; H, heart; MC, mucous cells; SC, spinal cord; S, somites; HB, hindbrain.
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1.4.2. b 2-like genes The b2a and b2b genes are predominantly expressed in nervous system. Within the nervous system, however, these two genes exhibit dramatically different expression patterns. In 36 hpf embryos, b2a transcripts are present at high abundance throughout brain and in spinal cord neurons (Fig. 1J). A dorsal view of a 36 hpf embryo shows high levels of b2a
transcripts in telencephalon, diencephalon, ventral tegmentum, and ventro-lateral regions of the hindbrain rhombomeres (Fig. 2I). In contrast to b1a, b1b mRNA expression is diffuse in brain and is also visible at low levels in pronephric duct (Fig. 1K). In dorsal views of 36 hpf embryos, b2b transcripts were diffusely distributed throughout brain with higher expression levels visible in otic vesicles and epiphysis
Fig. 4. Expression of zebrafish a2 and a3-like genes. (A–C) a2; (D–E) a3a; (F–H) a3b. (A) a2 at mid-somitogenesis (15 somite stage, lateral view); (B) a2 at 24 hpf lateral view); (C) a2 at 48 hpf (lateral view); (D) a3a at mid-somitogenesis (15 somite stage, lateral view); (E) a3a, at 24 hpf (lateral view); (F) a3b, early somitogenesis (10 somite stage, lateral view); (G) a3b, mid-somitogenesis (15 somite stage, lateral view); (H) a3b at 24 hpf (lateral view). S, somites; PD, pronephric duct; H, heart; TGG, trigeminal ganglion; SC, spinal cord; E, epiphysis; TL, telencephalon; D, diencephalon.
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Fig. 5. In situ hybridization analysis of zebrafish b subunit gene expression. (A–E) b1a; (F–H) b1b; (I–J) b3a; (K–L) b3b. (A) b1a at early somitogenesis (10 somite stage, lateral view); (B) b1a at mid-somitogenesis (15 somite stage, lateral view); (C) b1a at 24 hpf (lateral view); (D) b1a at 48 hpf (lateral view); (E) b1a at 72 hpf (lateral view); (F) b1b at mid-somitogenesis (15 somite stage, dorsal view); (G) b1b at 24 hpf (dorsal view); (H) b1b at 72 hpf (lateral view); (I) b3a at 48 hpf (dorsal view); (J) b3a at 72 hpf (lateral view); (K) b3b at 24 hpf (lateral view); (L) b3b at 48 hpf (lateral view). OV, otic vesicle; H, heart; PD, pronephric duct; E, epiphysis; L, lens; B, blood island; MC, mucous cells; SC, spinal cord; LL, lateral line ganglia; R, retina; S, somites.
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(Fig. 2J). As development proceeds, abundant expression of the b2b gene occurs within sensory organs (Rajarao et al., 2002). A more complete description of b2a and b2b gene expression can be found in Rajarao et al. (2002). 1.4.3. b 3-like genes The expression profile of the two zebrafish b3-like genes is shown in Fig. 5. From gastrulation through 22 hpf, the b3a gene exhibits a changing segmental pattern of expression in the CNS (Appel et al., 1996). At 24 hpf, we find very low levels of b3a expression in the embryo (data not shown). This contrasts to some degree with previous results reported by Appel et al. (1996), who found that the b3a gene is expressed predominantly in brain and eye throughout embryogenesis. Commencing at 36 hpf (Fig. 1L), and continuing through 48 hpf (Fig. 5I), strongest expression of b3a mRNA is seen in retina and brain. At 72 hpf, b3a mRNA is most prominent in the photoreceptor layer of the retina (Fig. 5J), as also observed by Appel et al. (1996). The b3b gene shows a markedly distinct expression pattern from that of the b3a gene. At 24 hpf, b3b mRNA expression is apparent in somitic mesoderm and CNS, with very low expression detectable in pronephric duct (Fig. 5K). The pattern is similar at 36 hpf (Fig. 1M), although pronephric staining is no longer visible at this or subsequent stages. By 48 hpf, strong expression is visible throughout the CNS and is especially prominent in retina as well as lateral line ganglia (Fig. 5L). 1.5. Comparison of a and b subunit expression Comparison of expression profiles of Na,K-ATPase a and b genes makes it possible to predict which a/b subunit pairs may combine to form functional enzyme. A summary of these predictions is shown in Table 1. The most obvious similarity in expression is shared by the a1a.3 and the b3b genes. These isoforms exhibit nearly identical expression patterns, suggesting that a1a.3/b3b subunit pairs are produced in a variety of structures including skeletal muscle, telencephalon, diencephalon, epiphysis, and hindbrain. a3a and b2a also show strong similarities in expression. Although we predict the existence of isoenzymes composed of a3a/b2a subunits in brain, it should be noted that the overlap in expression between a3a and b2a subunits is largely within CNS structures that also express the a1a.1, a1a.3, and b3b subunits. Stronger evidence for the pairing of specific a and b subunit isoforms can be obtained by examining tissues which express only a single a or a single b subunit. For example, three Na,K-ATPase genes are expressed in zebrafish heart: a1a.1; a2; and b1a. Assuming that both of the a subunits present in heart must pair with the b1a subunit, it seems likely that enzymes composed of a1a.1/b1a and a2/ b1a subunit combinations are active in myocardium. A distinctive set of a and b subunit genes are expressed in mucous cells including a1a.1, a1a.2, a1a.4, and b1b. Mucous cells are therefore likely to express three different
Na,K-ATPase isoenzymes which are composed of each of the three a1-like subunits in combination with the single b1b subunit. The same three a subunits (a1a.1, a1a.2, and a1a.4) are the predominant a subunits expressed in pronephros, while the b1a gene is the only detectable b subunit produced in this tissue. It therefore seems likely that embryonic kidney contains three distinct Na,K-ATPase isoenzymes composed of a1a.1/b1a, a1a.2/b1a, and a1a.4/ b1a subunit combinations. Another isoenzyme, formed from a3a and b1a subunits, is predicted based on expression of these genes in olfactory placode. The a1a.3 and a2 genes are the predominant a subunit genes expressed in skeletal muscle, while b3b is the only b subunit gene expressed in this tissue. This expression pattern allows us to predict the formation of an additional isoenzyme, consisting of a2/b3b subunits, in skeletal muscle (The a1a.3/b3b enzyme is also present in brain). We also detect expression of a discrete set of Na,K-ATPase genes within a subset of neurons in anterior spinal cord. Three b subunit genes (b1b, b2a, and b3b) and only one a subunit gene (a1a.3) are expressed in these neurons. This profile predicts two additional isoenzymes composed of a1.3/b1b and a1a.3/b2a in spinal cord (a1a.3/b3b is also present in skeletal muscle and brain). From their expression profiles, several of the a and b subunits do not have obvious binding partners. For example, the a3b, b2b and b3a subunit genes exhibit a diffuse expression pattern throughout brain and retina. It is possible that a3b/b2b and a3b/b3a subunit combinations may be formed in brain. However, a3b, b2b, and b3a subunits might also combine with other a and b subunit isoforms that have overlapping expression within CNS. The inability to identify specific cell types or compartments expressing the a3b, b2b and b3a genes makes it difficult to predict which subunit pairs containing these isoforms actually combine to form Table 1 Expression sites of predicted zebrafish Na,K-ATPase a/b subunit combinations a/b Combination a Subunit
b Subunit
a1a.1 a1a.1 a1a.2 a1a.2 a1a.3 a1a.3 a1a.3 a1a.4 a1a.4 a1b a2 a2 a3a a3a a3b Unknown Unknown
b1a b1b b1a b1b b1b b2a b3b b1a b1b Unknown b1a b3b b1a b2a Unknown b2b b3a
Site of expression
Heart, pronephric duct, lens Mucous cells Pronephric duct Mucous cells Spinal cord Spinal cord Skeletal muscle, spinal cord, brain Pronephric duct, ear Mucous cells Brain and spinal cord Heart Skeletal muscle Olfactory placode Brain (probable combination) Brain Brain and spinal cord Brain
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functional Na,K-ATPase. The a1b subunit represents another example of an a subunit without an obvious b subunit binding partner. The a1b gene is most highly expressed in CNS along the midline. No b subunit gene shows a similar expression pattern, and three of the b subunits (b1b, b2a, and b3b) are expressed at lowest levels along the midline. It is not clear which of the other b subunit isoforms might combine with a1b. At least two additional isoenzymes containing a1b and a3b, or b2b and b3a subunits must be produced in the CNS. This analysis points to a minimum of 14 distinct Na,K-ATPase isoenzymes in zebrafish (Table 1), although additional isoenzymes could potentially be formed from additional a/b subunit pairings. 2. Experimental procedures Whole mount in situ hybridization was performed as previously described (Thisse et al., 1994). The following probes were used to analyze a and b subunit gene expression. 2.1. a Subunit genes a1a.1 (atp1a1a.1; Accession number AF286372), nucleotides 1–3474. a1a.2 (atp1a1a.2; Accession number AF286374), nucleotides 296–3255. a1a.3 (atp1a1a.3; Accession number AF308598), nucleotides 623–3275. a1a.4 (atp1a1a.4; Accession number AY008376), nucleotides 112–3262. a1b (atp1a1b; Accession number AY008375), nucleotides 234–2764. a2 (atp1a2; Accession number AF286373), nucleotides 1461–3889. a3a (atp1a3a; Accession number AF308599), nucleotides 185–3256. a3b (atp1a3b; Accession number AY008374), nucleotides 282–2251. 2.2. b Subunit genes 1-b1a (atp1b1a; Accession number AF286375), nucleotides 1–1477.
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b1b (atp1b1b; Accession number AF3085975), nucleotides 1–2384. b2a (atp1b2a; Accession number AF286376), nucleotides 1–1076. b2b (atp1b2b; Accession number AF373976), nucleotides 1–1227. b3a (atp1b3a; Accession number AF469651), nucleotides 1–834. 2 b3b (atp1b3b; Accession number AF293369), nucleotides 1–1755.
Acknowledgements This work was supported by NIH grants to R.L., J.H.P., and B.T. B.T. and C.T acknowledge support from the Institut National de la Sante´ et de la Recherche Me´ dicale, the Centre National de la Recherche Scientifique, the Hoˆ pital Universitaire de Strasbourg, the Association pour la Recherche sur le Cancer and the Ligue Nationale Contre le Cancer. References Appel, C., Gloor, S., Schmalzing, G., Schachner, M., Bernhardt, R.R., 1996. Expression of a Na,K-ATPase b3 subunit during development of the zebrafish central nervous system. J. Neurosci. Res. 46,, 551–564. Lingrel, J.B., Orlowski, J., Shull, M.M., Price, E.M., 1990. Molecular genetics of Na,K-ATPase. Prog. Nucleic Acid Res. Mol. Biol. 38, 37–89. Rajarao, S.J.R., Canfield, V.A., Mohideen, M.A., Yan, Y.L., Postlethwait, J.H., Cheng, K.C., Levenson, R., 2001. The repertoire of Na,K-ATPase a and b subunit genes expressed in the zebrafish, Danio rerio. Genome Res. 11, 1211–1220. Rajarao, S.J.R., Canfield, V.A., Loppin, B., Thisse, B., Thisse, C., Yan, Y.L., Postlethwait, J.H., Levenson, R., 2002. Two Na,K-ATPase b2 subunit isoforms are differentially expressed within the central nervous system and sensory organs during zebrafish embryogenesis. Dev. Dyn. 223, 254–261. Serluca, F.C., Sidow, A., Mably, J.D., Fishman, M.C., 2001. Partitioning of tissue expression accompanies multiple duplications of the Na 1/K 1 ATPase a subunit gene. Genome Res. 11, 1625–1631. Thisse, C., Thisse, B., Halpern, M.E., Postlethwait, J.H., 1994. Goosecoid expression in neurectoderm and mesendoderm is disrupted in zebrafish cyclops gastrulas. Dev. Biol. 164, 420–429. Thomas, R.C., 1972. Electrogenic sodium pump in nerve and muscle cells. Physiol. Rev. 52, 563–594.
2 The sequence of the b3 cDNA used here was found to vary from the b3a sequence (Accession number X89722) published previously (Appel et al., 1996).