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Neuronal expression of fibroblast growth factor receptors in zebrafish
Gene Expression Patterns 13 (2013) 354–361
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Neuronal expression of fibroblast growth factor receptors in zebrafish Patricia Rohs, Alicia M. Ebert, Ania Zuba, Sarah McFarlane ⇑ Hotchkiss Brain Institute, Department of Cell Biology and Anatomy, University of Calgary, Canada
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Article history: Received 23 December 2012 Received in revised form 27 May 2013 Accepted 22 June 2013 Available online 9 July 2013 Keywords: mRNA expression Development Embryonic Danio rerio
a b s t r a c t Fibroblast growth factor (FGF) signaling is important for a host of developmental processes such as proliferation, differentiation, tissue patterning, and morphogenesis. In vertebrates, FGFs signal through a family of four fibroblast growth factor receptors (FGFR 1-4), one of which is duplicated in zebrafish (FGFR1). Here we report the mRNA expression of the five known zebrafish fibroblast growth factor receptors at five developmental time points (24, 36, 48, 60, and 72 h postfertilization), focusing on expression within the central nervous system. We show that the receptors have distinct and dynamic expression in the developing zebrafish brain, eye, inner ear, lateral line, and pharynx. In many cases, the expression patterns are similar to those of homologous FGFRs in mouse, chicken, amphibians, and other teleosts. Ó 2013 Elsevier B.V. All rights reserved.
Fibroblast growth factors (FGFs) are found in all metazoans and control many aspects of embryonic development. In particular, FGF signaling is required for proper proliferation, differentiation, and coordinated cell movement (Dorey and Amaya, 2010), and has known roles in processes such as mesoderm formation, axis specification, and limb development (Thisse and Thisse, 2005). Furthermore, FGFs function in many stages of neural development, including tissue patterning, neuronal migration, axon guidance, and synaptogenesis (Guillemot and Zimmer, 2011). There are 22 identified FGFs in vertebrates (Itoh and Ornitz, 2011; Pownall and Isaacs, 2010). These diffusible ligands signal through a family of four tyrosine kinase receptors, the FGF receptors (FGFR1-4). Upon formation of an FGF-hepar an sulfate-FGFR complex, FGFRs dimerize and initiate a variety of intracellular signal transduction events (Bottcher and Niehrs, 2005; Dorey and Amaya, 2010; Pownall and Isaacs, 2010). Multiple factors determine which FGFs and FGFRs can interact in vivo. The four FGF receptors have distinct expression patterns, which change over time (Ota et al., 2010). Furthermore, each FGFR contains three extracellular immunoglobulin-like domains, which control FGF binding specificity and affinity. In vitro studies have characterized the activity of different FGF-FGFR combinations, demonstrating significant selectivity (Zhang et al., 2006). Complexity is added by multiple splice forms of each FGFR (Zhang et al., 2006), and in zebrafish, the existence of a second copy of FGFR1 resulting from genome duplication (Rohner et al., 2009). Abbreviations: Hpf, hours postfertilization; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor. ⇑ Corresponding author. Address: Hotchkiss Brain Institute, University of Calgary, 3330 Hospital Dr., NW Calgary, AB T2N 4N1, Canada. Tel.: +1 403 220 2539. E-mail address: [email protected] (S. McFarlane). 1567-133X/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gep.2013.06.006
Given that FGF signaling is important for a host of developmental events, expression patterns can aid us in identifying novel roles for these molecules. Zebrafish is an amenable and versatile model organism for the study of FGF signaling during development. Although expression of the five FGFRs has been well characterized through the period of somitogenesis up until 24 h post fertilization (hpf; Ota et al., 2010), expression data for later embryogenesis, when neurons are migrating and sending out axons, is only available for select genes, developmental times, and in some cases, select organs (Nakayama et al., 2008; Nechiporuk et al., 2005; Regan et al., 2009; Rohner et al., 2009; Scholpp et al., 2004; SleptsovaFriedrich et al., 2001; Tamimi et al., 2006; Thisse and Thisse, 2005; Thisse et al., 2008; Tonou-Fujimori et al., 2002). This paper provides expression data for the five known zebrafish FGFRs at five developmental time points between 24 and 72 h postfertilization hpf, focusing mainly on neuronal expression.
1. Results 1.1. fgfr1a expression At 24 hpf (Fig. 1A–D), fgfr1a is expressed weakly throughout the central nervous system, with stronger expression in the midbrain– hindbrain boundary (Fig. 1B), lens (Fig. 1C), ventral midbrain (Fig. 1D), and tailbud (not shown). At 36 hpf (Fig. 1E–H), fgfr1a continues to be expressed in the lens (Fig. 1G), tailbud (not shown), midbrain (Fig. 1H) and midbrain–hindbrain boundary (Fig. 1E and F), with additional expression in the thalamus (Fig. 1G). Tissue surrounding the midbrain tectal ventricle expresses fgfr1a at 48 hpf (Fig. 1L). From 48 to 72 hpf, expression is observed in the
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Fig. 1. In situ hybridization of fgfr1a gene expression. In situ hybridization of fgfr1a gene expression at 24 hpf (A–D), 36 hpf (E–H), 48 hpf (I–L), 60 hpf (M–P), and 72 hpf (Q–T). Lateral views (A, E, I, M, Q) with anterior to the left, dorsal at the top, dorsal views (B, F, J, N, R) with anterior to the left, rostral transverse brain sections (C, G, K, O, S), and caudal transverse brain sections (D, H, L, P, T). Dotted lines indicate approximate orientation of imaged sections. Solid arrowheads indicate the location of the midbrain– hindbrain boundary. Arrowheads in C point to weak brain expression. Labels point to expression in the ciliary marginal zone (CMZ), dorsal thalamus (DT), fin bud (FB), jaw (J), lens (L), midbrain (MB), midbrain–hindbrain boundary (MHB), midbrain tegmentum (T), neuromasts (NM), optic tectum (Te), pharyngeal arches (PA), periocular mesenchyme (POM), splanchnocranium (SC), tectal ventricle (TeVe), telencephalon (Tel), and ventral midbrain (VMB).
pharyngeal arches (Fig. 1I, M, Q), fin bud (Fig. 1J and N), neuromasts (Fig. 1Q and R) and lateral line (data not shown), periocular mesenchyme (Fig. 1K, O, S), and the proliferative ciliary marginal zone of the peripheral retina (Fig. 1K0 , O0 , S0 ). At 72 hpf, fgfr1a mRNA is also present in the jaw (Fig. 1S).
the eye (Fig. 2K, O, S). During this time fgfr1b is also expressed near the midbrain–hindbrain boundary, and in lens and tissue, potentially periocular mesenchyme or blood vessels, surrounding the lens (Fig. 2K, O, S). In addition, fgfr1b is expressed in the ciliary marginal zone at 48 and 60 hpf (Fig. 2K and O) and in neuromasts at 72 hpf (Fig. 2R).
1.2. fgfr1b expression 1.3. fgfr2 expression At 24 hpf (Fig. 2A–D) and 36 hpf (Fig. 2E–H), fgfr1b expression in the central nervous system is diffuse, with higher intensity staining in the ventral diencephalon and ventral retina (Fig. 2C and G). From 48 to 72 hpf (Fig. 2I–T), fgfr1b is expressed in the ventricular zones of the brain (Fig. 2J, K, L, N, O, R, S), pharyngeal arches (Fig. 2I, M, Q), fin bud (Fig. 2J, N, R), periocular mesenchyme (Fig. 2K, O, S), and the optic nerve head, where retinal ganglion cell axons exit
At 24 hpf, fgfr2 is expressed in the diencephalon (Fig. 3A and B), midbrain (Fig. 3A), hindbrain (Fig. 3D), otic placodes (Fig. 3B), lens (Fig. 3C), and ventricles (Fig. 3B and C). At 36 hpf (Fig. 3E–H), expression is present in the midbrain (Fig. 3E, H), diencephalon (Fig. 3E and G), and hindbrain (Fig. 3E and F), while expression in the otic placodes (Fig. 3F), and lens (Fig. 3G) is sustained. There
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Fig. 2. In situ hybridization of fgfr1b gene expression. In situ hybridization of fgfr1b gene expression at 24 hpf (A–D), 36 hpf (E–H), 48 hpf (I–L), 60 hpf (M–P), and 72 hpf (Q–T). Lateral views (A, E, I, M, Q) with anterior to the left, dorsal at the top, dorsal views (B, F, J, N, R) with anterior to the left, rostral transverse brain sections (C, G, K, O, S), and caudal transverse brain sections (D, H, L, P, T). Dotted lines indicate approximate orientation of imaged sections. Solid arrowheads indicate the location of the midbrain– hindbrain boundary. Labels point to expression in the fin bud (FB), lens (L), neuromasts (NM), optic nerve head (ONH), pharyngeal arches (PA), periocular mesenchyme (POM), tectal ventricle (TeVe), ventral diencephalon (VDi), ventral retina (VR), and ventricle (Ve). White arrowheads point to expression surrounding the lens (K, O, S).
is also transient hypochord expression at 36 hpf (data not shown). By 48 hpf (Fig. 3I–L), fgfr2 expression becomes more restricted with in situ signal found in the rhombomere boundaries of the hindbrain (Fig. 3J), midbrain ventricular zones, dorsal forebrain (Fig. 3I and K), fin bud (Fig. 3J), jaw (Fig. 3K), pharyngeal arches (Fig. 3I), and tissue in or near the lens (Fig. 3K). At 60 hpf (Fig. 3M–P), fgfr2 continues to be expressed in the rhombomere boundaries (Fig. 3N), dorsal midbrain (Fig. 3O), pharyngeal arches (Fig. 3M) and fin bud (Fig. 3N), with additional expression in the forebrain ventricular zone (Fig. 3M and N) and periocular mesenchyme (Fig. 3O). At 72 hpf (Fig. 3Q–T), fgfr2 is expressed in the fin bud (Fig. 3Q and R), periocular mesenchyme (Fig. 3S), pharynx (Fig. 3Q), regions of the midbrain and hindbrain (Fig. 3Q and R), and the lens (Fig. 3S). Although fgfr2 is expressed in the posterior midbrain and first rhombomere, expression is notably absent from the midbrain–hindbrain boundary at all time points examined (arrowheads).
1.4. fgfr3 expression At 24 hpf (Fig. 4A–D) and 36 hpf (Fig. 4E–H), fgfr3 is expressed strongly throughout the brain and spinal cord, with the exception of the telencephalon, ventral diencephalon, posterior tectum, and midbrain–hindbrain boundary (Fig. 4A, B, E, F, data for the spinal cord not shown). Expression is also present transiently in the lens at 36 hpf (Fig. 4F and G) and retinal pigment epithelium at 24 and 36 hpf (Fig. 4C and G). By 48 hpf (Fig. 4I–L), expression (not shown) in the brain restricts to select regions, including the rhombomere boundaries (Fig. 4J), dorsal diencephalon (Fig. 4K), forebrain ventricular zone (Fig. 4I) and hypothalamus (Fig. 4K). This brain expression is sustained at 60 hpf (Fig. 4M–P). At 72 hpf (Fig. 4Q–T), regions of strongest expression include the first rhombomere (Fig. 4Q and R), forebrain ventricular zone (Fig. 4Q) and vagal ganglion (Fig. 4T). Outside of the nervous system, in situ signal is evident in the somites from 24 to 36 hpf
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Fig. 3. In situ hybridization of fgfr2 gene expression. In situ hybridization of fgfr2 gene expression at 24 hpf (A–D), 36 hpf (E–H), 48 hpf (I–L), 60 hpf (M–P), and 72 hpf (Q–T). Lateral views (A, E, I, M, Q) with anterior to the left, dorsal at the top, dorsal views (B, F, J, N, R) with anterior to the left, rostral transverse brain sections (C, G, K, O, S), and caudal transverse brain sections (D, H, L, P, T). Dotted lines indicate approximate orientation of imaged sections. Solid arrowheads indicate the location of the midbrain– hindbrain boundary. Labels point to expression in the cerebellar plate (CeP), diencephalon (Di), dorsal forebrain (DFrb), dorsal midbrain (DMB), dorsal thalamus (DT), fin bud (FB), forebrain (FrB), forebrain ventricular zone (FrVe), hindbrain (HB), inner ear (E), jaw (J), lens (L), medulla oblongata (MO), midbrain (MB), otic placode (OP), periocular mesenchyme (POM), pharyngeal arches (PA), pharynx (P), rhombomere boundaries (RhB), and ventricle (Ve). In panels K and S, the asterisk draws attention to expression surrounding the lens.
(data not shown), fin bud from 48 to 72 hpf (Fig. 4J, M, Q), and in the splanchnocranium and mandibular cartilage at 72 hpf (Fig. 4Q and S). 1.5. fgfr4 expression At 24 hpf, fgfr4 is expressed broadly throughout the brain with stronger expression in the thalamus (Fig. 5A), choroid fissure (Fig. 5A), first rhombomere (Fig. 5B), dorsal hindbrain (Fig. 5A, D), and lens (Fig. 5C). At 36 hpf (Fig. 5E–H), fgfr4 also has strong expression in the forebrain (Fig. 5G) and based on its medial and bilateral placement ventral to the hindbrain, we suggest the octaval ganglion (Fig. 5H). In confirmation of previously reported zebrafish fgfr4 expression in the pineal nucleus, parapineal nucleus, and what
appears to be the habenula (Regan et al., 2009), we suggest that fgfr4 continues to be expressed in these diencephalic regions at 48 hpf (Fig. 5E, I, K). Of note, fgfr4 is expressed in the adult rat habenula (Itoh et al., 1994). From 24 to 36 hpf, and weakly at 48 hpf, fgfr4 is expressed in the tail bud (not shown). At 48 hpf (Fig. 5I–L), fgfr4 is expressed in the thalamus and transiently in tissue surrounding the lens (Fig. 5K). From 48 to 72 hpf, fgfr4 is expressed in the medial tectal proliferation zone of the dorsal midbrain (Fig. 5I, K, M, O, Q, S), cerebellar plate (Fig. 5I, M, Q), forebrain ventricular zone (Fig. 5I, M, Q), and fin bud (Fig. 5J and R), as well as select regions in the forebrain and hindbrain (Fig. 5 I, J, M, N, Q, R). fgfr4 is expressed in the periocular mesenchyme (Fig. 5O and S) and tail fins (not shown) at 60 and 72 hpf, and at 72 hpf expression is present in the cephalic musculature (Fig. 5Q).
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Fig. 4. In situ hybridization of fgfr3 gene expression. In situ hybridization of fgfr3 gene expression at 24 hpf (A–D), 36 hpf (E–H), 48 hpf (I–L), 60 hpf (M–P), and 72 hpf (Q–T). Lateral views (A, E, I, M, Q) with anterior to the left, dorsal at the top, dorsal views (B, F, J, N, R) with anterior to the left, rostral transverse brain sections (C, G, K, O, S), and caudal transverse brain sections (D, H, L, P, T). Dotted lines indicate approximate orientation of imaged sections. Solid arrowheads indicate the location of the midbrain– hindbrain boundary. Labels point to expression in the diencephalon (Di), hypothalamus (H), hindbrain (HB), fin bud (FB), forebrain ventricular zone (FrVe), lens (L), mandibular cartilage (MC), midbrain (MB), periocular mesenchyme (POM), rhombomere boundaries (RhB), rhombomere 1 (r1), retinal pigment epithelium (RPE), spinal cord (SC), splanchnocranium (SCr), and vagal ganglion (VG).
2. Discussion In this paper we characterize the expression patterns of the different fgfr genes in the developing nervous system of zebrafish. All five receptors show initial broad expression patterns, in particular at 36 hpf, which rapidly resolve within the next 12–14 h to more restricted patterns of expression. In various tissues, including the midbrain–hindbrain boundary, rhombomere 1, and the lining of the brain ventricles, two or more receptors are co-expressed, suggesting that certain receptors have redundant roles. In other neuronal and non-neuronal tissues, however, the five receptors have distinct and highly dynamic expression patterns, both spatially and temporally. 2.1. fgfr1a and 1b have partially overlapping expression patterns The two fgfr1 paralogs, fgfr1a and 1b, exhibit overlapping expression in several brain areas including the diencephalon,
ventricular zones, and regions near the MHB, as well as in the neuromasts, anterior lateral line, pharyngeal arches, and fin buds. Yet, fgfr1a and 1b expression patterns do differ, most noticeably in the posterior lateral line, splanchnocranium and in the eye. For instance, in the latter, fgfr1a is expressed in the lens and ciliary marginal zone, while fgfr1b is also expressed in the optic nerve head and ventral retina. Each fgfr1 paralog is thought to partially compensate for loss of the other, as zebrafish fgfr1a mutants have no obvious embryonic phenotype (Rohner et al., 2009). fgfr1 has broad central nervous system expression in several developing organisms. For instance, fgfr1 is expressed throughout the developing mouse and chick neural tube (Blak et al., 2005; Lunn et al., 2007; Trokovic et al., 2003; Walshe and Mason, 2000; Yamaguchi et al., 1992). In zebrafish, although fgfr1a and 1b are initially expressed broadly, they are localized to specific brain regions, most noticeably in proliferative zones lining the ventricles, by 72 hpf.
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Fig. 5. In situ hybridization of fgfr4 gene expression. In situ hybridization of fgfr4 gene expression at 24 hpf (A–D), 36 hpf (E–H), 48 hpf (I–L), 60 hpf (M–P), and 72 hpf (Q–T). Lateral views (A, E, I, M, Q) with anterior to the left, dorsal at the top, dorsal views (B, F, J, N, R) with anterior to the left, rostral transverse brain sections (C, G, K, O, S), and caudal transverse brain sections (D, H, L, P, T). Dotted lines indicate approximate orientation of imaged sections. Solid arrowheads indicate the location of the midbrain– hindbrain boundary. Labels point to expression in the cephalic musculature (CM), cerebellar plate (CeP), choroid fissure (CF), diencephalon (Di), dorsal midbrain (DMB), fin bud (FB), forebrain ventricular zone (FrVe), ganglion (G), habenula (HA), lens (L), mandibular cartilage (MC), periocular mesenchyme (POM), rhombomere 1 (r1), and thalamus (TH). In panel K, expression is present in tissue surrounding the lens (asterisk).
Given the importance of fgfr signaling in MHB development (Scholpp et al., 2003), it is not surprising that zebrafish fgfr1a and 1b are expressed early in the MHB (Ota et al., 2010; Rohner et al., 2009; Tonou-Fujimori et al., 2002). Indeed, fgfr1 is found in the MHB and/or rhombomere 1 of many vertebrates (Blak et al., 2005; Golub et al., 2000; Walshe and Mason, 2000). Interestingly, we find the early expression of fgfr1a and 1b in the MHB continues through to 72 hpf. Further, in the hindbrain fgfr1a and 1b localize primarily to the ventricular zone, in contrast to the broader expression domains of other zebrafish fgfrs. These data argue that FGFR1 signaling has ongoing roles through midbrain/hindbrain development. fgfr1a is present in the zebrafish telencephalon at 24 hpf (Tonou-Fujimori et al. 2002), with expression continuing through 72 hpf, and eventually becomes restricted to the ventricular zone and possibly the olfactory bulb. Telencephalic expression is generally conserved amongst various species (Yamaguchi et al., 1992; Walshe and Mason, 2000; Atkinson-Leadbeater et al.,
2010), though fgfr1 is absent from the telencephalon of medaka and Pleurodeles waltl (Launay et al., 1994; Carl and Wittbrodt, 1999). Medaka is also relatively unique in missing fgfr1 from the MHB (Carl and Wittbrodt, 1999). 2.2. fgfr2 expression in the brain In zebrafish, fgfr2 is initially expressed in the diencephalon and ventral telencephalon (Tonou-Fujimori et al., 2002; Fig. 3), and by 48–72 hpf is confined to forebrain ventricular zones. Similarly, fgfr2 is expressed in the medaka posterior forebrain (Carl and Wittbrodt, 1999), Pleurodeles diencephalon (Shi et al., 1994), and in chick becomes refined over time to first the dorsal diencephalon and then dorsal telencephalon (Walshe and Mason, 2000; Lunn et al., 2007). Zebrafish fgfr2 is expressed throughout the midbrain but excluded from the MHB from the 8-somite stage through to 36 hpf (Tonou-fujimori et al., 2002; Fig. 3), similar to what is seen
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in medaka, chick and mouse (Blak et al., 2005; Carl and Wittbrodt, 1999; Walshe and Mason, 2000). Midbrain expression in zebrafish then restricts to the ventricular zones and a band just anterior to the MHB from 48 to 72 hpf, whereas Xenopus fgfr2 is expressed in two stripes just anterior to, and potentially overlapping with, the MHB (Friesel and Brown, 1992; Golub et al., 2000). In the zebrafish hindbrain, fgfr2 initially localizes to specific rhombomeric stripes, expanding to encompass most rhombomeres by 48 hpf (Tonou-Fujimori et al., 2002), a pattern we find continues through to 72 hpf. fgfr2 restricts to chick rhombomere boundaries (Lunn et al., 2007; Walshe and Mason, 2000), and is expressed in the ventral hindbrain of medaka (Carl and Wittbrodt, 1999), and hindbrain abutting the MHB in mouse (Blak et al., 2005), suggesting roles in hindbrain development in various species. 2.3. fgfr3 nervous system expression pattern is conserved between species fgfr3 nervous system expression appears to be highly conserved between organisms. In embryonic zebrafish, mouse, chick, and medaka, this receptor is expressed in the diencephalon, the anterior part of the midbrain, throughout the hindbrain with particularly strong expression in rhombomere 1, and the spinal cord (Blak et al., 2005; Carl and Wittbrodt, 1999; Ota et al., 2010; Sleptsova-Friedrich et al., 2001; Walshe and Mason, 2000). In zebrafish, expression restricts by 48 hpf to ventricular zones and rhombomere boundaries, with no spinal cord expression (Fig. 4). Chick fgfr3 also becomes restricted to rhombomere boundaries (Walshe and Mason, 2000). Nevertheless, differences between species do exist. For instance, we find zebrafish fgfr3 expression is strongest in the dorsal hindbrain, but fgfr3 localizes mainly to the ventral mouse hindbrain. fgfr3 is expressed in a number of additional brain and non-neuronal tissues, including the hypothalamus from early stages through 72 hpf and into the adult (Sleptsova-Friedrich et al., 2001; Topp et al., 2008), the splanchnocranium at 72 hpf, the mandibular cartilage, vagal ganglion, and pharyngeal endoderm (Nechiporuk et al., 2005). 2.4. fgfr4 is expressed in the ventricular zones, midbrain, and first rhombomere Zebrafish fgfr4 is expressed most broadly at early stages (Ota et al., 2010; Sleptsova-Friedrich et al., 2001; Thisse et al., 1995; Tonou-Fujimori et al., 2002), from 24 to 48 hpf, in the diencephalon and ventral telencephalon. We find that through 72 hpf, fgfr4 is also present in the dorsal medial midbrain, midbrain ventricular zones, and the first rhombomere. Unlike other fgfrs, expression in the maturing brain is not restricted to proliferative ventricular zones, suggesting a role in differentiation of brain cells. Other authors describe a similar expression pattern for other lower vertebrates (Launay et al., 1994; Carl and Wittbrodt, 1999; Golub et al., 2000), though fgfr4 is reported to be absent from the murine hindbrain, except potentially in ventricular zones (Korhonen et al., 1992; Yaylaoglu et al., 2005). 2.5. Expression of fgfrs in the developing eye is regulated in a spatially and temporally dynamic fashion fgfr mRNA is spatially restricted in the developing eye, and different fgfrs show considerable overlap in their expression, arguing for functional redundancy. Expression of fgfrs starts early in eye development, with reported fgfr1a expression in zebrafish eye vesicles (Nakayama et al., 2008), though our data indicates that there is minimal fgfr1a expression at 24 hpf. Indeed at 24 hpf, fgfr expression appears mainly restricted to the lens, with the exceptions being fgfr3, which is absent (Nakayama et al., 2008; Fig. 3), and
fgfr1b, being present in the ventral eye domain. At some developmental time point, mRNA for each receptor appears in the lens. fgfr1a, fgfr1b, fgfr2, and fgfr4 are present shortly after lens formation (24 hpf) (Tonou-Fujimori et al., 2002; Nakayama et al., 2007), though only fgfr2 and fgfr4 persist past 36 hpf. fgfr3 turns on transiently at this point, and is gone by 48 hpf. fgfr3 and fgfr2 are also expressed in the lens in other species, including medaka and Pleurodeles (Launay et al., 1994; Carl and Wittbrodt, 1999). The other main area of fgfr expression is the ciliary marginal zone, a region that remains proliferative in the peripheral retina through adulthood. Here fgfr1a and fgfr1b turn on at 48 hpf, and fgfr4 at 60 hpf. Given the known roles for FGFs in regulating proliferation (Guillemot and Zimmer, 2011; Powers et al., 2000; Pownall and Isaacs, 2010), potentially fgfrs carry out a similar function in the ciliary marginal zone and proliferating lens epithelium. We noted several other domains of specific fgfr expression. fgfr3 is the lone FGFR in the developing retinal pigment epithelium, fgfr1b localizes to the ventral eye cup and subsequently the optic nerve head, and fgfr2 is present in the choroid fissure (Nakayma et al., 2007; Fig. 3). All fgfrs, except fgfr3, are expressed late in embryonic eye development (48–60 hpf) in the periocular mesenchyme that surrounds the eye and helps control eye patterning and development (Cvekl and Wang, 2009). Various species express fgfrs in the developing eye. For instance, fgfr1 and fgfr2 are expressed in the embryonic eye of medaka, chick, Xenopus and Pleurodeles (Carl and Wittbrodt, 1999; Golub et al., 2000; Walshe and Mason, 2000; Launay et al., 1994). While some expression is conserved, for instance chick fgfr1 is expressed in the lens (Walshe and Mason, 2000), in other cases distinct fgfrs are expressed. For example, in zebrafish the fgfr in the retinal pigment epithelium is fgfr3, whereas both fgfr3 and fgfr1 are expressed in Pleurodeles (Launay et al., 1994), and fgfr4 in medaka (Carl and Wittbrodt, 1999). 3. Experimental procedures 3.1. Zebrafish husbandry Tupfel Longfin (ZIRC, Eugene OR) embryos were raised at 28.5 °C in E3 medium supplemented with 0.25 mg/L methylene blue as previously described (Fishman et al., 1997). Embryos were staged by hours postfertilization (Kimmel et al., 1995). Pigmentation was inhibited by adding 0.003% (w/v) 1-pheny1–2-thiourea at 24 h pf. 3.2. Histology Digoxigenin-labeled RNA probes were synthesized as previously described (Thisse and Thisse, 2008). Probes were synthesized from image clones where available (fgfr1a-IMG 9037670, fgfr2-IMG 6524173, fgfr3-IMG 9038195, fgfr4-IMG 9039420; Open Biosystems, Lafayette CO). For fgfr1b, DNA was amplified from 72 hpf zebrafish cDNA using gene-specific primers (forward-AAGCCAT CATCGAGTAGCCAAGA, reverse-GCCAATGCAGTAGATCAGCA), then cloned into PCRII-topo (Invitrogen, Burlington ON). Vectors containing the probe template were linearized with appropriate restriction enzymes and mRNA was transcribed using SP6 RNA polymerase. In situ hybridization was performed using whole embryos as previously described (Thisse and Thisse, 2008). Whole embryos were mounted in 3% methyl cellulose (Sigma). Flat mounts were prepared by incubating embryos in glycerol for 24 h, dissecting away the yolk, and mounting in glycerol between a slide and coverslip. Embryos were also embedded using JB-4 embedding solutions (Polysciences, Warrington, PA) and sectioned at 7 lm on a Leica microtome to obtain transverse sections through the
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brain. All imaging was performed using a Stemi SV 11 microscope and an AxioCam HRc camera with AxioVision software. Images were edited and compiled using Adobe Photoshop. Acknowledgements AME was supported by fellowships from the Canadian Institutes of Health Research (CIHR), Foundation for Fighting Blindness and Alberta Innovates-Health Solutions (AI-HS). AZ was supported by an AI-HS Summer Studentship. SM is an AI-HS Scientist and was supported by a Canada Research Chair in Developmental Neurobiology. Research funding was provided by a CIHR operating grant to SM. References Atkinson-Leadbeater, K., Bertolesi, G.E., Hehr, C.L., Webber, C.A., Cechmanek, P.B., McFarlane, S., 2010. Dynamic expression of axon guidance cues required for optic tract development is controlled by fibroblast growth factor signaling. J. Neurosci. 30, 685–693. Blak, A.A., Naserke, T., Vogt Weisenhorn, D.M., Prakash, N., Partanen, J., Wurst, W., 2005. Expression of Fgf receptors 1, 2, and 3 in the developing mid- and hindbrain of the mouse. Dev. Dyn. 233, 1023–1030. Bottcher, R.T., Niehrs, C., 2005. Fibroblast growth factor signaling during early vertebrate development. Endocr. Rev. 26, 63–77. Carl, M., Wittbrodt, J., 1999. Graded inteference with FGF signalling reveals its dorsoventral asymmetry at the mid–hindbrain boundary. Development 126, 5659–5667. Cvekl, A., Wang, W., 2009. Retinoic acid signaling in mammalian eye development. Exp. Eye Res. 89, 280–291. Dorey, K., Amaya, E., 2010. FGF signalling: diverse roles during early vertebrate embryogenesis. Development 137, 3731–3742. Fishman, M.C., Stainier, D.Y., Breitbart, R.E., Westerfield, M., 1997. Zebrafish: genetic and embryological methods in a transparent vertebrate embryo. Methods Cell Biol. 52, 67–82. Friesel, R., Brown, S.A.N., 1992. Spatially restricted expression of fibroblast growth factor receptor-2 during Xenopus development. Development 116, 1051–1058. Golub, R., Adelman, Z., Clementi, J., Weiss, R., Bonasera, J., Servetnick, M., 2000. Evolutionarily conserved and divergent expression of members of the FGF receptor family among vertebrate embryos, as revealed by FGFR expression patterns in Xenopus. Dev. Genes. Evol. 210, 345–357. Guillemot, F., Zimmer, C., 2011. From cradle to grave: the multiple roles of fibroblast growth factors in neural development. Neuron 71, 574–588. Itoh, N., Yazaki, N., Tagashira, S., Miyake, A., Ozaki, K., Minami, M., Satoh, M., Ohta, M., Kawasaki, T., 1994. Rat FGF receptor-4 mRNA in the brain is expressed preferentially in the medial habenular nucleus. Brain Res. Mol. Brain Res. 21, 344–348. Itoh, N., Ornitz, D.M., 2011. Fibroblast growth factors: from molecular evolution to roles in development, metabolism and disease. J. Biochem. 149, 121–130. Kimmel, C.B., Ballard, W.W., Kimmel, S.R., Ullmann, B., Schilling, T.F., 1995. Stages of embryonic development of the zebrafish. Dev. Dyn. 203, 253–310. Korhonen, J., Partanen, J., Alitalo, K., 1992. Expression of FGFR-4 mRNA in developing mouse tissues. Int. J. Dev. Biol. 36, 323–329. Launay, C., Fromentoux, V., Thery, C., Shi, D., Boucaut, J., 1994. Comparative analysis of the tissue distribution of three fibroblast growth factor receptor mRNAs during amphibian morphogenesis. Differentiation 58, 101–111. Lunn, J.S., Fishwick, K.J., Halley, P.A., Storey, K.G., 2007. A spatial and temporal map of FGF/Erk1/2 activity and response repertoires in the early chick embryo. Dev. Biol. 302, 536–553. Nakayama, Y., Miyake, A., Nakagawa, Y., Mido, T., Yoshikawa, M., Konishi, M., Itoh, N., 2008. Fgf19 is required for zebrafish lens and retina development. Dev. Biol. 313, 752–766.
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Gene Expression Patterns journal homepage: www.elsevier.com/locate/gep
Neuronal expression of fibroblast growth factor receptors in zebrafish Patricia Rohs, Alicia M. Ebert, Ania Zuba, Sarah McFarlane ⇑ Hotchkiss Brain Institute, Department of Cell Biology and Anatomy, University of Calgary, Canada
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Article history: Received 23 December 2012 Received in revised form 27 May 2013 Accepted 22 June 2013 Available online 9 July 2013 Keywords: mRNA expression Development Embryonic Danio rerio
a b s t r a c t Fibroblast growth factor (FGF) signaling is important for a host of developmental processes such as proliferation, differentiation, tissue patterning, and morphogenesis. In vertebrates, FGFs signal through a family of four fibroblast growth factor receptors (FGFR 1-4), one of which is duplicated in zebrafish (FGFR1). Here we report the mRNA expression of the five known zebrafish fibroblast growth factor receptors at five developmental time points (24, 36, 48, 60, and 72 h postfertilization), focusing on expression within the central nervous system. We show that the receptors have distinct and dynamic expression in the developing zebrafish brain, eye, inner ear, lateral line, and pharynx. In many cases, the expression patterns are similar to those of homologous FGFRs in mouse, chicken, amphibians, and other teleosts. Ó 2013 Elsevier B.V. All rights reserved.
Fibroblast growth factors (FGFs) are found in all metazoans and control many aspects of embryonic development. In particular, FGF signaling is required for proper proliferation, differentiation, and coordinated cell movement (Dorey and Amaya, 2010), and has known roles in processes such as mesoderm formation, axis specification, and limb development (Thisse and Thisse, 2005). Furthermore, FGFs function in many stages of neural development, including tissue patterning, neuronal migration, axon guidance, and synaptogenesis (Guillemot and Zimmer, 2011). There are 22 identified FGFs in vertebrates (Itoh and Ornitz, 2011; Pownall and Isaacs, 2010). These diffusible ligands signal through a family of four tyrosine kinase receptors, the FGF receptors (FGFR1-4). Upon formation of an FGF-hepar an sulfate-FGFR complex, FGFRs dimerize and initiate a variety of intracellular signal transduction events (Bottcher and Niehrs, 2005; Dorey and Amaya, 2010; Pownall and Isaacs, 2010). Multiple factors determine which FGFs and FGFRs can interact in vivo. The four FGF receptors have distinct expression patterns, which change over time (Ota et al., 2010). Furthermore, each FGFR contains three extracellular immunoglobulin-like domains, which control FGF binding specificity and affinity. In vitro studies have characterized the activity of different FGF-FGFR combinations, demonstrating significant selectivity (Zhang et al., 2006). Complexity is added by multiple splice forms of each FGFR (Zhang et al., 2006), and in zebrafish, the existence of a second copy of FGFR1 resulting from genome duplication (Rohner et al., 2009). Abbreviations: Hpf, hours postfertilization; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor. ⇑ Corresponding author. Address: Hotchkiss Brain Institute, University of Calgary, 3330 Hospital Dr., NW Calgary, AB T2N 4N1, Canada. Tel.: +1 403 220 2539. E-mail address: [email protected] (S. McFarlane). 1567-133X/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gep.2013.06.006
Given that FGF signaling is important for a host of developmental events, expression patterns can aid us in identifying novel roles for these molecules. Zebrafish is an amenable and versatile model organism for the study of FGF signaling during development. Although expression of the five FGFRs has been well characterized through the period of somitogenesis up until 24 h post fertilization (hpf; Ota et al., 2010), expression data for later embryogenesis, when neurons are migrating and sending out axons, is only available for select genes, developmental times, and in some cases, select organs (Nakayama et al., 2008; Nechiporuk et al., 2005; Regan et al., 2009; Rohner et al., 2009; Scholpp et al., 2004; SleptsovaFriedrich et al., 2001; Tamimi et al., 2006; Thisse and Thisse, 2005; Thisse et al., 2008; Tonou-Fujimori et al., 2002). This paper provides expression data for the five known zebrafish FGFRs at five developmental time points between 24 and 72 h postfertilization hpf, focusing mainly on neuronal expression.
1. Results 1.1. fgfr1a expression At 24 hpf (Fig. 1A–D), fgfr1a is expressed weakly throughout the central nervous system, with stronger expression in the midbrain– hindbrain boundary (Fig. 1B), lens (Fig. 1C), ventral midbrain (Fig. 1D), and tailbud (not shown). At 36 hpf (Fig. 1E–H), fgfr1a continues to be expressed in the lens (Fig. 1G), tailbud (not shown), midbrain (Fig. 1H) and midbrain–hindbrain boundary (Fig. 1E and F), with additional expression in the thalamus (Fig. 1G). Tissue surrounding the midbrain tectal ventricle expresses fgfr1a at 48 hpf (Fig. 1L). From 48 to 72 hpf, expression is observed in the
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Fig. 1. In situ hybridization of fgfr1a gene expression. In situ hybridization of fgfr1a gene expression at 24 hpf (A–D), 36 hpf (E–H), 48 hpf (I–L), 60 hpf (M–P), and 72 hpf (Q–T). Lateral views (A, E, I, M, Q) with anterior to the left, dorsal at the top, dorsal views (B, F, J, N, R) with anterior to the left, rostral transverse brain sections (C, G, K, O, S), and caudal transverse brain sections (D, H, L, P, T). Dotted lines indicate approximate orientation of imaged sections. Solid arrowheads indicate the location of the midbrain– hindbrain boundary. Arrowheads in C point to weak brain expression. Labels point to expression in the ciliary marginal zone (CMZ), dorsal thalamus (DT), fin bud (FB), jaw (J), lens (L), midbrain (MB), midbrain–hindbrain boundary (MHB), midbrain tegmentum (T), neuromasts (NM), optic tectum (Te), pharyngeal arches (PA), periocular mesenchyme (POM), splanchnocranium (SC), tectal ventricle (TeVe), telencephalon (Tel), and ventral midbrain (VMB).
pharyngeal arches (Fig. 1I, M, Q), fin bud (Fig. 1J and N), neuromasts (Fig. 1Q and R) and lateral line (data not shown), periocular mesenchyme (Fig. 1K, O, S), and the proliferative ciliary marginal zone of the peripheral retina (Fig. 1K0 , O0 , S0 ). At 72 hpf, fgfr1a mRNA is also present in the jaw (Fig. 1S).
the eye (Fig. 2K, O, S). During this time fgfr1b is also expressed near the midbrain–hindbrain boundary, and in lens and tissue, potentially periocular mesenchyme or blood vessels, surrounding the lens (Fig. 2K, O, S). In addition, fgfr1b is expressed in the ciliary marginal zone at 48 and 60 hpf (Fig. 2K and O) and in neuromasts at 72 hpf (Fig. 2R).
1.2. fgfr1b expression 1.3. fgfr2 expression At 24 hpf (Fig. 2A–D) and 36 hpf (Fig. 2E–H), fgfr1b expression in the central nervous system is diffuse, with higher intensity staining in the ventral diencephalon and ventral retina (Fig. 2C and G). From 48 to 72 hpf (Fig. 2I–T), fgfr1b is expressed in the ventricular zones of the brain (Fig. 2J, K, L, N, O, R, S), pharyngeal arches (Fig. 2I, M, Q), fin bud (Fig. 2J, N, R), periocular mesenchyme (Fig. 2K, O, S), and the optic nerve head, where retinal ganglion cell axons exit
At 24 hpf, fgfr2 is expressed in the diencephalon (Fig. 3A and B), midbrain (Fig. 3A), hindbrain (Fig. 3D), otic placodes (Fig. 3B), lens (Fig. 3C), and ventricles (Fig. 3B and C). At 36 hpf (Fig. 3E–H), expression is present in the midbrain (Fig. 3E, H), diencephalon (Fig. 3E and G), and hindbrain (Fig. 3E and F), while expression in the otic placodes (Fig. 3F), and lens (Fig. 3G) is sustained. There
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Fig. 2. In situ hybridization of fgfr1b gene expression. In situ hybridization of fgfr1b gene expression at 24 hpf (A–D), 36 hpf (E–H), 48 hpf (I–L), 60 hpf (M–P), and 72 hpf (Q–T). Lateral views (A, E, I, M, Q) with anterior to the left, dorsal at the top, dorsal views (B, F, J, N, R) with anterior to the left, rostral transverse brain sections (C, G, K, O, S), and caudal transverse brain sections (D, H, L, P, T). Dotted lines indicate approximate orientation of imaged sections. Solid arrowheads indicate the location of the midbrain– hindbrain boundary. Labels point to expression in the fin bud (FB), lens (L), neuromasts (NM), optic nerve head (ONH), pharyngeal arches (PA), periocular mesenchyme (POM), tectal ventricle (TeVe), ventral diencephalon (VDi), ventral retina (VR), and ventricle (Ve). White arrowheads point to expression surrounding the lens (K, O, S).
is also transient hypochord expression at 36 hpf (data not shown). By 48 hpf (Fig. 3I–L), fgfr2 expression becomes more restricted with in situ signal found in the rhombomere boundaries of the hindbrain (Fig. 3J), midbrain ventricular zones, dorsal forebrain (Fig. 3I and K), fin bud (Fig. 3J), jaw (Fig. 3K), pharyngeal arches (Fig. 3I), and tissue in or near the lens (Fig. 3K). At 60 hpf (Fig. 3M–P), fgfr2 continues to be expressed in the rhombomere boundaries (Fig. 3N), dorsal midbrain (Fig. 3O), pharyngeal arches (Fig. 3M) and fin bud (Fig. 3N), with additional expression in the forebrain ventricular zone (Fig. 3M and N) and periocular mesenchyme (Fig. 3O). At 72 hpf (Fig. 3Q–T), fgfr2 is expressed in the fin bud (Fig. 3Q and R), periocular mesenchyme (Fig. 3S), pharynx (Fig. 3Q), regions of the midbrain and hindbrain (Fig. 3Q and R), and the lens (Fig. 3S). Although fgfr2 is expressed in the posterior midbrain and first rhombomere, expression is notably absent from the midbrain–hindbrain boundary at all time points examined (arrowheads).
1.4. fgfr3 expression At 24 hpf (Fig. 4A–D) and 36 hpf (Fig. 4E–H), fgfr3 is expressed strongly throughout the brain and spinal cord, with the exception of the telencephalon, ventral diencephalon, posterior tectum, and midbrain–hindbrain boundary (Fig. 4A, B, E, F, data for the spinal cord not shown). Expression is also present transiently in the lens at 36 hpf (Fig. 4F and G) and retinal pigment epithelium at 24 and 36 hpf (Fig. 4C and G). By 48 hpf (Fig. 4I–L), expression (not shown) in the brain restricts to select regions, including the rhombomere boundaries (Fig. 4J), dorsal diencephalon (Fig. 4K), forebrain ventricular zone (Fig. 4I) and hypothalamus (Fig. 4K). This brain expression is sustained at 60 hpf (Fig. 4M–P). At 72 hpf (Fig. 4Q–T), regions of strongest expression include the first rhombomere (Fig. 4Q and R), forebrain ventricular zone (Fig. 4Q) and vagal ganglion (Fig. 4T). Outside of the nervous system, in situ signal is evident in the somites from 24 to 36 hpf
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Fig. 3. In situ hybridization of fgfr2 gene expression. In situ hybridization of fgfr2 gene expression at 24 hpf (A–D), 36 hpf (E–H), 48 hpf (I–L), 60 hpf (M–P), and 72 hpf (Q–T). Lateral views (A, E, I, M, Q) with anterior to the left, dorsal at the top, dorsal views (B, F, J, N, R) with anterior to the left, rostral transverse brain sections (C, G, K, O, S), and caudal transverse brain sections (D, H, L, P, T). Dotted lines indicate approximate orientation of imaged sections. Solid arrowheads indicate the location of the midbrain– hindbrain boundary. Labels point to expression in the cerebellar plate (CeP), diencephalon (Di), dorsal forebrain (DFrb), dorsal midbrain (DMB), dorsal thalamus (DT), fin bud (FB), forebrain (FrB), forebrain ventricular zone (FrVe), hindbrain (HB), inner ear (E), jaw (J), lens (L), medulla oblongata (MO), midbrain (MB), otic placode (OP), periocular mesenchyme (POM), pharyngeal arches (PA), pharynx (P), rhombomere boundaries (RhB), and ventricle (Ve). In panels K and S, the asterisk draws attention to expression surrounding the lens.
(data not shown), fin bud from 48 to 72 hpf (Fig. 4J, M, Q), and in the splanchnocranium and mandibular cartilage at 72 hpf (Fig. 4Q and S). 1.5. fgfr4 expression At 24 hpf, fgfr4 is expressed broadly throughout the brain with stronger expression in the thalamus (Fig. 5A), choroid fissure (Fig. 5A), first rhombomere (Fig. 5B), dorsal hindbrain (Fig. 5A, D), and lens (Fig. 5C). At 36 hpf (Fig. 5E–H), fgfr4 also has strong expression in the forebrain (Fig. 5G) and based on its medial and bilateral placement ventral to the hindbrain, we suggest the octaval ganglion (Fig. 5H). In confirmation of previously reported zebrafish fgfr4 expression in the pineal nucleus, parapineal nucleus, and what
appears to be the habenula (Regan et al., 2009), we suggest that fgfr4 continues to be expressed in these diencephalic regions at 48 hpf (Fig. 5E, I, K). Of note, fgfr4 is expressed in the adult rat habenula (Itoh et al., 1994). From 24 to 36 hpf, and weakly at 48 hpf, fgfr4 is expressed in the tail bud (not shown). At 48 hpf (Fig. 5I–L), fgfr4 is expressed in the thalamus and transiently in tissue surrounding the lens (Fig. 5K). From 48 to 72 hpf, fgfr4 is expressed in the medial tectal proliferation zone of the dorsal midbrain (Fig. 5I, K, M, O, Q, S), cerebellar plate (Fig. 5I, M, Q), forebrain ventricular zone (Fig. 5I, M, Q), and fin bud (Fig. 5J and R), as well as select regions in the forebrain and hindbrain (Fig. 5 I, J, M, N, Q, R). fgfr4 is expressed in the periocular mesenchyme (Fig. 5O and S) and tail fins (not shown) at 60 and 72 hpf, and at 72 hpf expression is present in the cephalic musculature (Fig. 5Q).
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Fig. 4. In situ hybridization of fgfr3 gene expression. In situ hybridization of fgfr3 gene expression at 24 hpf (A–D), 36 hpf (E–H), 48 hpf (I–L), 60 hpf (M–P), and 72 hpf (Q–T). Lateral views (A, E, I, M, Q) with anterior to the left, dorsal at the top, dorsal views (B, F, J, N, R) with anterior to the left, rostral transverse brain sections (C, G, K, O, S), and caudal transverse brain sections (D, H, L, P, T). Dotted lines indicate approximate orientation of imaged sections. Solid arrowheads indicate the location of the midbrain– hindbrain boundary. Labels point to expression in the diencephalon (Di), hypothalamus (H), hindbrain (HB), fin bud (FB), forebrain ventricular zone (FrVe), lens (L), mandibular cartilage (MC), midbrain (MB), periocular mesenchyme (POM), rhombomere boundaries (RhB), rhombomere 1 (r1), retinal pigment epithelium (RPE), spinal cord (SC), splanchnocranium (SCr), and vagal ganglion (VG).
2. Discussion In this paper we characterize the expression patterns of the different fgfr genes in the developing nervous system of zebrafish. All five receptors show initial broad expression patterns, in particular at 36 hpf, which rapidly resolve within the next 12–14 h to more restricted patterns of expression. In various tissues, including the midbrain–hindbrain boundary, rhombomere 1, and the lining of the brain ventricles, two or more receptors are co-expressed, suggesting that certain receptors have redundant roles. In other neuronal and non-neuronal tissues, however, the five receptors have distinct and highly dynamic expression patterns, both spatially and temporally. 2.1. fgfr1a and 1b have partially overlapping expression patterns The two fgfr1 paralogs, fgfr1a and 1b, exhibit overlapping expression in several brain areas including the diencephalon,
ventricular zones, and regions near the MHB, as well as in the neuromasts, anterior lateral line, pharyngeal arches, and fin buds. Yet, fgfr1a and 1b expression patterns do differ, most noticeably in the posterior lateral line, splanchnocranium and in the eye. For instance, in the latter, fgfr1a is expressed in the lens and ciliary marginal zone, while fgfr1b is also expressed in the optic nerve head and ventral retina. Each fgfr1 paralog is thought to partially compensate for loss of the other, as zebrafish fgfr1a mutants have no obvious embryonic phenotype (Rohner et al., 2009). fgfr1 has broad central nervous system expression in several developing organisms. For instance, fgfr1 is expressed throughout the developing mouse and chick neural tube (Blak et al., 2005; Lunn et al., 2007; Trokovic et al., 2003; Walshe and Mason, 2000; Yamaguchi et al., 1992). In zebrafish, although fgfr1a and 1b are initially expressed broadly, they are localized to specific brain regions, most noticeably in proliferative zones lining the ventricles, by 72 hpf.
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Fig. 5. In situ hybridization of fgfr4 gene expression. In situ hybridization of fgfr4 gene expression at 24 hpf (A–D), 36 hpf (E–H), 48 hpf (I–L), 60 hpf (M–P), and 72 hpf (Q–T). Lateral views (A, E, I, M, Q) with anterior to the left, dorsal at the top, dorsal views (B, F, J, N, R) with anterior to the left, rostral transverse brain sections (C, G, K, O, S), and caudal transverse brain sections (D, H, L, P, T). Dotted lines indicate approximate orientation of imaged sections. Solid arrowheads indicate the location of the midbrain– hindbrain boundary. Labels point to expression in the cephalic musculature (CM), cerebellar plate (CeP), choroid fissure (CF), diencephalon (Di), dorsal midbrain (DMB), fin bud (FB), forebrain ventricular zone (FrVe), ganglion (G), habenula (HA), lens (L), mandibular cartilage (MC), periocular mesenchyme (POM), rhombomere 1 (r1), and thalamus (TH). In panel K, expression is present in tissue surrounding the lens (asterisk).
Given the importance of fgfr signaling in MHB development (Scholpp et al., 2003), it is not surprising that zebrafish fgfr1a and 1b are expressed early in the MHB (Ota et al., 2010; Rohner et al., 2009; Tonou-Fujimori et al., 2002). Indeed, fgfr1 is found in the MHB and/or rhombomere 1 of many vertebrates (Blak et al., 2005; Golub et al., 2000; Walshe and Mason, 2000). Interestingly, we find the early expression of fgfr1a and 1b in the MHB continues through to 72 hpf. Further, in the hindbrain fgfr1a and 1b localize primarily to the ventricular zone, in contrast to the broader expression domains of other zebrafish fgfrs. These data argue that FGFR1 signaling has ongoing roles through midbrain/hindbrain development. fgfr1a is present in the zebrafish telencephalon at 24 hpf (Tonou-Fujimori et al. 2002), with expression continuing through 72 hpf, and eventually becomes restricted to the ventricular zone and possibly the olfactory bulb. Telencephalic expression is generally conserved amongst various species (Yamaguchi et al., 1992; Walshe and Mason, 2000; Atkinson-Leadbeater et al.,
2010), though fgfr1 is absent from the telencephalon of medaka and Pleurodeles waltl (Launay et al., 1994; Carl and Wittbrodt, 1999). Medaka is also relatively unique in missing fgfr1 from the MHB (Carl and Wittbrodt, 1999). 2.2. fgfr2 expression in the brain In zebrafish, fgfr2 is initially expressed in the diencephalon and ventral telencephalon (Tonou-Fujimori et al., 2002; Fig. 3), and by 48–72 hpf is confined to forebrain ventricular zones. Similarly, fgfr2 is expressed in the medaka posterior forebrain (Carl and Wittbrodt, 1999), Pleurodeles diencephalon (Shi et al., 1994), and in chick becomes refined over time to first the dorsal diencephalon and then dorsal telencephalon (Walshe and Mason, 2000; Lunn et al., 2007). Zebrafish fgfr2 is expressed throughout the midbrain but excluded from the MHB from the 8-somite stage through to 36 hpf (Tonou-fujimori et al., 2002; Fig. 3), similar to what is seen
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in medaka, chick and mouse (Blak et al., 2005; Carl and Wittbrodt, 1999; Walshe and Mason, 2000). Midbrain expression in zebrafish then restricts to the ventricular zones and a band just anterior to the MHB from 48 to 72 hpf, whereas Xenopus fgfr2 is expressed in two stripes just anterior to, and potentially overlapping with, the MHB (Friesel and Brown, 1992; Golub et al., 2000). In the zebrafish hindbrain, fgfr2 initially localizes to specific rhombomeric stripes, expanding to encompass most rhombomeres by 48 hpf (Tonou-Fujimori et al., 2002), a pattern we find continues through to 72 hpf. fgfr2 restricts to chick rhombomere boundaries (Lunn et al., 2007; Walshe and Mason, 2000), and is expressed in the ventral hindbrain of medaka (Carl and Wittbrodt, 1999), and hindbrain abutting the MHB in mouse (Blak et al., 2005), suggesting roles in hindbrain development in various species. 2.3. fgfr3 nervous system expression pattern is conserved between species fgfr3 nervous system expression appears to be highly conserved between organisms. In embryonic zebrafish, mouse, chick, and medaka, this receptor is expressed in the diencephalon, the anterior part of the midbrain, throughout the hindbrain with particularly strong expression in rhombomere 1, and the spinal cord (Blak et al., 2005; Carl and Wittbrodt, 1999; Ota et al., 2010; Sleptsova-Friedrich et al., 2001; Walshe and Mason, 2000). In zebrafish, expression restricts by 48 hpf to ventricular zones and rhombomere boundaries, with no spinal cord expression (Fig. 4). Chick fgfr3 also becomes restricted to rhombomere boundaries (Walshe and Mason, 2000). Nevertheless, differences between species do exist. For instance, we find zebrafish fgfr3 expression is strongest in the dorsal hindbrain, but fgfr3 localizes mainly to the ventral mouse hindbrain. fgfr3 is expressed in a number of additional brain and non-neuronal tissues, including the hypothalamus from early stages through 72 hpf and into the adult (Sleptsova-Friedrich et al., 2001; Topp et al., 2008), the splanchnocranium at 72 hpf, the mandibular cartilage, vagal ganglion, and pharyngeal endoderm (Nechiporuk et al., 2005). 2.4. fgfr4 is expressed in the ventricular zones, midbrain, and first rhombomere Zebrafish fgfr4 is expressed most broadly at early stages (Ota et al., 2010; Sleptsova-Friedrich et al., 2001; Thisse et al., 1995; Tonou-Fujimori et al., 2002), from 24 to 48 hpf, in the diencephalon and ventral telencephalon. We find that through 72 hpf, fgfr4 is also present in the dorsal medial midbrain, midbrain ventricular zones, and the first rhombomere. Unlike other fgfrs, expression in the maturing brain is not restricted to proliferative ventricular zones, suggesting a role in differentiation of brain cells. Other authors describe a similar expression pattern for other lower vertebrates (Launay et al., 1994; Carl and Wittbrodt, 1999; Golub et al., 2000), though fgfr4 is reported to be absent from the murine hindbrain, except potentially in ventricular zones (Korhonen et al., 1992; Yaylaoglu et al., 2005). 2.5. Expression of fgfrs in the developing eye is regulated in a spatially and temporally dynamic fashion fgfr mRNA is spatially restricted in the developing eye, and different fgfrs show considerable overlap in their expression, arguing for functional redundancy. Expression of fgfrs starts early in eye development, with reported fgfr1a expression in zebrafish eye vesicles (Nakayama et al., 2008), though our data indicates that there is minimal fgfr1a expression at 24 hpf. Indeed at 24 hpf, fgfr expression appears mainly restricted to the lens, with the exceptions being fgfr3, which is absent (Nakayama et al., 2008; Fig. 3), and
fgfr1b, being present in the ventral eye domain. At some developmental time point, mRNA for each receptor appears in the lens. fgfr1a, fgfr1b, fgfr2, and fgfr4 are present shortly after lens formation (24 hpf) (Tonou-Fujimori et al., 2002; Nakayama et al., 2007), though only fgfr2 and fgfr4 persist past 36 hpf. fgfr3 turns on transiently at this point, and is gone by 48 hpf. fgfr3 and fgfr2 are also expressed in the lens in other species, including medaka and Pleurodeles (Launay et al., 1994; Carl and Wittbrodt, 1999). The other main area of fgfr expression is the ciliary marginal zone, a region that remains proliferative in the peripheral retina through adulthood. Here fgfr1a and fgfr1b turn on at 48 hpf, and fgfr4 at 60 hpf. Given the known roles for FGFs in regulating proliferation (Guillemot and Zimmer, 2011; Powers et al., 2000; Pownall and Isaacs, 2010), potentially fgfrs carry out a similar function in the ciliary marginal zone and proliferating lens epithelium. We noted several other domains of specific fgfr expression. fgfr3 is the lone FGFR in the developing retinal pigment epithelium, fgfr1b localizes to the ventral eye cup and subsequently the optic nerve head, and fgfr2 is present in the choroid fissure (Nakayma et al., 2007; Fig. 3). All fgfrs, except fgfr3, are expressed late in embryonic eye development (48–60 hpf) in the periocular mesenchyme that surrounds the eye and helps control eye patterning and development (Cvekl and Wang, 2009). Various species express fgfrs in the developing eye. For instance, fgfr1 and fgfr2 are expressed in the embryonic eye of medaka, chick, Xenopus and Pleurodeles (Carl and Wittbrodt, 1999; Golub et al., 2000; Walshe and Mason, 2000; Launay et al., 1994). While some expression is conserved, for instance chick fgfr1 is expressed in the lens (Walshe and Mason, 2000), in other cases distinct fgfrs are expressed. For example, in zebrafish the fgfr in the retinal pigment epithelium is fgfr3, whereas both fgfr3 and fgfr1 are expressed in Pleurodeles (Launay et al., 1994), and fgfr4 in medaka (Carl and Wittbrodt, 1999). 3. Experimental procedures 3.1. Zebrafish husbandry Tupfel Longfin (ZIRC, Eugene OR) embryos were raised at 28.5 °C in E3 medium supplemented with 0.25 mg/L methylene blue as previously described (Fishman et al., 1997). Embryos were staged by hours postfertilization (Kimmel et al., 1995). Pigmentation was inhibited by adding 0.003% (w/v) 1-pheny1–2-thiourea at 24 h pf. 3.2. Histology Digoxigenin-labeled RNA probes were synthesized as previously described (Thisse and Thisse, 2008). Probes were synthesized from image clones where available (fgfr1a-IMG 9037670, fgfr2-IMG 6524173, fgfr3-IMG 9038195, fgfr4-IMG 9039420; Open Biosystems, Lafayette CO). For fgfr1b, DNA was amplified from 72 hpf zebrafish cDNA using gene-specific primers (forward-AAGCCAT CATCGAGTAGCCAAGA, reverse-GCCAATGCAGTAGATCAGCA), then cloned into PCRII-topo (Invitrogen, Burlington ON). Vectors containing the probe template were linearized with appropriate restriction enzymes and mRNA was transcribed using SP6 RNA polymerase. In situ hybridization was performed using whole embryos as previously described (Thisse and Thisse, 2008). Whole embryos were mounted in 3% methyl cellulose (Sigma). Flat mounts were prepared by incubating embryos in glycerol for 24 h, dissecting away the yolk, and mounting in glycerol between a slide and coverslip. Embryos were also embedded using JB-4 embedding solutions (Polysciences, Warrington, PA) and sectioned at 7 lm on a Leica microtome to obtain transverse sections through the
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