Phox2b expression in the taste centers of fish

R E S E A RC H AR T I C L E Phox2b Expression in the Taste Centers of Fish Eva Coppola,1,2,3 Fabien D’autr
eaux,1,2,3 Marc Nomaksteinsky,1,2,3 and Jean-François Brunet1,2,3* 1 École normale sup
erieure, Institut de Biologie de l’École normale sup
erieure (IBENS), Paris F-75005, France Centre National de la Recherche Scientifique (CNRS) UMR8197, Paris F-75005, France 3 Institut National de la Sant
e et de la Recherche M
edicale (INSERM) U1024, Paris F-75005, France 2 ABSTRACT The homeodomain transcription factor Phox2b controls the formation of the sensory-motor reflex circuits of the viscera in vertebrates. Among Phox2b-dependent structures characterized in rodents is the nucleus of the solitary tract, the first relay for visceral sensory input, including taste. Here we show that Phox2b is expressed throughout the primary taste centers of two cyprinid fish, Danio rerio and Carassius auratus, i.e., in their vagal, glossopharyngeal, and facial lobes, providing the first molecular evidence for their homology with the nucleus of the solitary tract of mammals and suggesting that a single ancestral Phox2b-positive neuronal type evolved to give rise to both fish and mammalian structures. In zebrafish larvae, the distribution of Phox2bþ neurons, combined with the expression pattern of Olig4 (a homologue of Olig3, determinant of the nucleus of the solitary tract in mice), reveals that the superficial position and sheet-like architecture of the viscerosensory column in cyprinid fish, ideally suited for the somatotopic representation of oropharyngeal and bodily surfaces, arise by radial migration from a dorsal progenitor domain, in contrast to the tangential migration observed in amniotes. J. Comp. Neurol. 520:3633– 3649, 2012. C 2012 Wiley Periodicals, Inc. V INDEXING TERMS: Nucleus of the solitary tract, Vagal lobe, Zebrafish, Goldfish, Gene expression pattern Taste is a prominent sensory modality in several clades of fish. In addition to numerous taste buds in their oropharyngeal cavity and gills, cypriniformes (which include zebrafish and goldfish) and to an even greater extent siluriformes (catfish) have taste buds on their lips, barbells, and body surface, whose stimulation triggers orientation or prehensile movements of the head and body (Herrick, 1905; Atema, 1971). In addition, carps have many taste buds on their palatal organ, a specialized structure of the roof of the oral cavity, capable of sorting edible from nonedible particles by spatially discrete muscular responses to gustatory input (Finger, 2008). All taste buds are innervated by sensory neurons in three cranial ganglia appended, respectively, to the facial, glossopharyngeal, and vagal nerves. The central axon of these sensory neurons projects to the visceral sensory column arranged in three gustatory ‘‘lobes’’: facial, glossopharyngeal, and vagal. Owing to their location (in the dorsal hindbrain) and connectivity (postsynaptic to primary gustatory afferents) the gustatory lobes of teleosts are considered homologous, collectively, to the gustatory portion of the nucleus of the solitary tract (nTS) of mammals (Herrick, 1905). Their architecture though, diverges strikingly from that of the mammalian nTS. The vagal lobes of goldfish C 2012 Wiley Periodicals, Inc. V (Carassius auratus) form two dorsal bulges posterior to the cerebellum, which account for about 20% of the total brain mass (Finger, 2009). Histologically, they are formed by 16 alternating layers of cells and fibers, according to the current classification (Meek and Nieuwenhuys, 1998), first established in Carassius carassius (Morita et al., 1983) and then extended to C. auratus (Morita and Finger, 1985). The facial lobe (or ‘‘tuberculum impar’’ of Herrick, 1905) is a midline structure, presumably resulting from the fusion of the left and right rostral ends of the visceral sensory column. Histologically, it is made up of a superficial layer of neurons overlying randomly distributed ones. The relatively inconspicuous glossopharyngeal lobe is tucked in between the facial and vagal lobes. A caudal extension of the viscerosensory column in the obex region, the commissural nucleus of Cajal (nCC)—subdivided into a medial and two Grant sponsors: Agence Nationale pour la Recherche; Grant number: 08-BLAN-0043 (to J.F.B.); Centre National de la Recherche Scientifique (CNRS); Institut National de la Sant
e et de la Recherche M
edicale (INSERM); École normale sup
erieure. *CORRESPONDENCE TO: Jean-François Brunet, Institut de Biologie de l’ENS, IBENS, 46 rue d’Ulm, 75005 Paris, France. E-mail: jfbrunet@biologie.ens.fr Received December 21, 2011; Revised March 22, 2012; Accepted March 23, 2012 DOI 10.1002/cne.23117 Published online April 2, 2012 in Wiley Online Library (wileyonlinelibrary. com) The Journal of Comparative Neurology | Research in Systems Neuroscience 520:3633–3649 (2012) 3633 Coppola et al. lateral subnuclei—receives input from the thoracic and abdominal viscera (Morita and Finger, 1987a). On that basis, and that of its ascending projections (Yoshimoto and Yamamoto, 2010), it is considered homologous with the general visceral sensory portion of the mammalian nucleus of the solitary tract. The gustatory lobes of zebrafish have been much less studied than those of the goldfish. They are similarly formed, except for a much smaller size and no lamination (Wullimann et al., 1996). Little is known about the genetic makeup of the fish taste lobes and nCC apart from markers of neurotransmitter phenotype. Motoneurons all express choline acetyltransferase (ChAT) (Mueller et al., 2004), and many coexpress nNOS (Giraldez-Perez et al., 2009) and the FMRF-related peptide C-RFamide (Wang et al., 2000). The vagal sensory zone contains interneurons that respond to glutamate and use glutamate as a neurotransmitter (Ikenaga et al., 2009), as well as glutamic acid decarboxylase-immunoreactive (and thus presumably c-aminobutyric acid [GABA]ergic) interneurons (Castro et al., 2006). In mammals, the gustatory centers occupy the rostral half of the nucleus of the solitary tract, a large group of interneurons clustered in the dorsal hindbrain, close to the fasciculated central axons of primary visceral neurons forming the geniculate, petrosal, and nodose ganglia (Blessing, 1997). Over the past ten years, much has been learned about the ontogeny of the nTS in mice, at least its glutamatergic contingent. Glutamatergic interneurons of the nTS are born in the dorsal alar plate of the hindbrain, from rhombomeres (r) 4 to 7, in the socalled dA3 domain of the neuroepithelium (Storm et al., 2009). As they leave the neuroepithelium and exit the mitotic cycle, nTS precursors switch on the homeodomain transcription factor Phox2b (Pattyn et al., 1997; Dauger et al., 2003; Kang et al., 2007) on which they depend for their differentiation (Dauger et al., 2003). In Phox2b knockouts, nTS precursors are born but switch their molecular code and adopt a fate akin to that of somatic sensory neurons in the spinal trigeminal nucleus (SpV) (D’Autr
eaux et al., 2011). Along the taste pathways, Phox2b is also expressed in the geniculate, petrose, and nodose ganglia (Pattyn et al., 1997), i.e., in primary taste neurons, and in neurons of the efferent pathways that execute local reflex motor responses to taste input, such as swallowing, salivation, and local vasodilation: the nucleus ambiguus (Pattyn et al., 2000), the salivatory motoneurons (Pattyn et al., 2000), and their postsynaptic targets, the parasympathetic ganglia (Pattyn et al., 1997). In Phox2b knockouts, primary taste afferents switch their identity to that of touch receptors (D’Autr
eaux et al., 2011), whereas the motoneurons and parasympathetic ganglionic cells are not born (Pattyn et al., 1999, 2000). 3634 Previously, Phox2b expression has been studied in the peripheral nervous system (PNS) of zebrafish (Elworthy et al., 2005; Lucas et al., 2006) but hardly at all in the central nervous system (CNS) or taste centers. Here, we describe for the first time the expression of the Phox2b orthologue in the primary taste centers of zebrafish and goldfish, and compare the development of the viscerosensory columns in zebrafish and mouse, providing molecular evidence for their homology and an explanation for their divergent morphology. MATERIALS AND METHODS Animals Fertilized zebrafish eggs were obtained from natural spawning of wild-type lines according to the The Zebrafish Book (Westerfield, 1995). Adult fish were maintained at 28
C with a lighting schedule of 14-hours light and 10hours dark. Embryos were collected within 3 hours of spawning, rinsed, placed into 100-mm Petri dishes containing embryo medium, and allowed to develop at 28.5
C to the required stage. To reduce pigmentation, embryos were transferred to embryo medium containing 0.003% phenylthiourea (PTU) starting from 24 hours post fertilization (hpf). Adult goldfish were obtained from local commercial sources. Transgenic mouse lines used in this work were the Crereporter line ROSAstoploxYFP (Srinivas et al., 2001) and the Phox2b::Cre(BAC), generated with the bacterial artificial chromosome (BAC) CHORI # RP24–95M11 (D’Autr
eaux et al., 2011). This BAC construct recapitulates endogenous expression of Phox2b, as ascertained by comparing the pattern of immunofluorescence with the anti-Phox2b antibodies and anti-GFP antibodies in Phox2b::Cre;ROSAloxstopYFP embryos at developmental stages embryonic (E)8.5, E9.5, E10.5, E11.5, E12.5, E13.5, and E18.5 (D’Autr
eaux et al., 2011). All experimental procedures were approved by the ethical committee in charge of the École normale sup
erieure. Tissue preparation for immunostaining and in situ hybridization Zebrafish embryos were collected at the required stage and killed with 0.04% MS222 (ethyl-m-aminobenzoate methanosulfonate; Sigma, St. Louis, MO) in embryo medium. Adult zebrafish and goldfish were anesthetized by using 0.04% MS222 and decapitated when spontaneous respiration ceased; the brains were dissected out in phosphate-buffered saline (PBS; pH 7.4). Embryos and brains were fixed in 4% paraformaldehyde (PFA) in PBS overnight at 4
C, cryoprotected overnight in 20% sucrose in PBS, The Journal of Comparative Neurology | Research in Systems Neuroscience Phox2b expression in the taste centers of fish and then embedded in OCT. Sections (12 lm) were cut in the transverse plane on a cryostat. Staged mouse embryos (day of vaginal plug was E0.5) were deeply anesthetized on ice, decapitated, and subsequently treated in the same way. A total of 17 mouse embryos, 5 adult goldfish, 8 adult zebrafish, and 30 larval zebrafish were used for this study. Western blots E14.5 mouse embryo and adult goldfish and zebrafish tissues were lysed in cold RIPA buffer (3 ml/g of tissue) containing protease and phosphatase inhibitors (Complete Protease and PhosSTOP Phosphatase Inhibitor Cocktail Tablets; Roche, Penzberg, Germany), by using the Tissuelyser (Qiagen, Chatsworth, CA). The samples were then centrifuged at 12,000 rpm for 20 minutes at 4
C to get rid of the cellular debris. The supernatants were stored at 80
C. Lysates were denatured in LDS sample loading buffer (Thermo Scientific Pierce, Waltham, MA) containing 1.3 mM b-mercaptoethanol, at 90
C for 5 minutes. Samples were electrophoresed on precast 12% polyacrylamide gels (NuPAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE] gels; Invitrogen, Carlsbad, CA) and transferred on Hybond-C Extra nitrocellulose membrane (Amersham, Arlington Heights, IL). Membranes were washed in 20 mM Tris-HCl, pH 7.5, 130 mM NaCl, 0.1% Tween (TBS-T), blocked with 5% skim milk in TBS-T, and incubated overnight at 4
C with rabbit antimouse Phox2b antibody (1:2,500) diluted in 1% skim milk in TBS-T. After being washed three times with TBS-T, the membranes were incubated for 2 hours at room temperature with horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (1:5,000; Jackson ImmunoResearch, West Grove, PA) in 1% skim milk in TBS-T. After three washes with TBS-T, the membranes were developed with enhanced chemiluminescence (ECL) substrate (SuperSignal West Femto Chemiluminescent Substrate; Thermo Scientific Pierce ) for 2 minutes. The ECL signal was recorded by using the ImageQuant imager LAS 4000 mini (GE Healthcare Life Sciences, Buckinghamshire, UK). Immunostaining The sections were incubated with the primary antibody solution overnight at 4
C in PBS/0.1% Triton X-100/20% fetal calf serum (FCS) and revealed for fluorescent observation by using DyLight 488- or Alexa Fluor 488-, Cy3-, and Cy5-labeled secondary antibodies of the appropriate specificity (Jackson ImmunoResearch), for 2 hours at room temperature. In some experiments, following the immunofluorescence protocol, sections were incubated with NeuroTrace 640/660 (cat. no. N-21483; Molecular Probes, Eugene, OR) diluted 1:100 in PBS/0.1% Triton X100 for 20 minutes at room temperature. Sections were washed twice in PBS/0.1% Triton X-100 for 10 minutes and then in PBS for 2 hours. Primary antibodies See Tables 1 and 2 for details on primary and secondary antibodies. An anti-Phox2b rabbit polyclonal antiserum (1:500) (Pattyn et al., 1997) was raised against the 14-amino acid C-terminal sequence of the Phox2b protein (coupled to bovine serum albumin [BSA]), and its specificity was confirmed in mouse by the perfect match between in situ hybridization and immunohistochemistry (Pattyn et al., 1997), and the absence of reactivity on Phox2b knockout animals (J.-F.B., unpublished data), and in rats by suppression of reactivity with the original peptide (Kang et al., 2007). The guinea pig polyclonal antiserum against Phox2b (1:500) (Dubreuil et al., 2009) was obtained against the same peptide, and its specificity was demonstrated in the same way. Because the last 14 amino acids of zebrafish Phox2b differ from the mouse sequence by three residues (Fig. 1A), we tested whether the antibody still recognized specifically zebrafish Phox2b by comparing its pattern of immunoreactivity with that revealed by the zebrafish cRNA probe, which matched perfectly (Figs. 1B–D, 2). Finally, the antibody recognizes a band of the expected molecular weight in mouse hindbrain and zebrafish and goldfish medulla but not in mouse cortex (Fig. 1E). The mouse anti-Nestin monoclonal (rat-401) antibody (1:300; CA556309; BD Pharmingen, San Diego, CA) was produced in mouse, by using amino acid residues 544– 776 as immunogen (Hockfield and McKay, 1985). It recognizes a single band of 198-kDa molecular weight in homogenates of developing neural tube cells by western blot and labels neuroepithelial progenitor cells by immunohistochemical analysis (Hockfield and McKay, 1985). Immunohistochemistry and western blot analysis on neural tube of E15 rats showed that the Nestin antibody only recognizes radial glial cells and dividing neural stem cells in the embryonic but not adult rat CNS (Lendahl et al., 1990). Finally, in mouse, it recognizes a single band in cell lysates of neuroprogenitors from wild-type but not Nestin knockout animals, and immunoreactivity is seen in neuroprogenitor cultures only in cells from wild type but not Nestin knockout animals (Park et al., 2009). The Islet-1 antibodies (1:100; a mixture in equal parts of 40.2D6 and 39.4D5; Developmental Studies Hybridoma Bank, Iowa City, IA) were raised against the C-terminal portion of rat Islet-1. Their staining pattern in zebrafish neural tube coincides with the sum of the patterns revealed by in situ hybridization with probes for Islet-1 The Journal of Comparative Neurology | Research in Systems Neuroscience 3635 Coppola et al. TABLE 1. Primary and Secondary Antibodies Used in This Study Primary antibodies Antibody Phox2b Antigen Host type Titer Company or reference Cat. no. Rabbit polyclonal 1:500 Pattyn et al., 1997 Guinea pig polyclonal 1:500 1:50 Dubreuil et al., 2009 Chemicon AB114P ChAT Islet-1 Carboxy-terminal residues 301–314 (PNGAKAALVKSSMF) of mouse Phox2b Carboxy-terminal residues 301–314 (PNGAKAALVKSSMF) of mouse Phox2b Human ChAT (placental choline acetyltransferase) Carboxy-terminal residues 178–349 of rat Islet-1 Goat polyclonal Mouse monoclonal 1:100 Islet-1 Carboxy-terminal residues 178–349 of rat Islet-1 Mouse monoclonal 1:100 NeuN Zrf-1 Mouse brain nuclei GFAP Mouse monoclonal Mouse monoclonal 1:200 1:2,500 Nestin Olig3 GFP Residues 544–776 of rat Nestin Full-length mouse Olig3 Recombinant GFP Mouse monoclonal Rabbit polyclonal Chicken polyclonal 1:300 1:20,000 1:500 Developmental Studies Hybridoma Bank Developmental Studies Hybridoma Bank Millipore Zebrafish International Resource Center (ZIRC) DB Pharmigen Mueller et al., 2004 Aves Phox2b1 Antigen Rabbit IgG Rabbit IgG Guinea pig IgG Goat IgG Mouse IgG Chicken IgG Rabbit IgG Fluorophore/enzyme Alexa Fluor 488 Cy3 DyLight 488 Cy3 Cy3 Cy5 Horseradish peroxidase Secondary antibodies Host Titer Donkey 1:500 Donkey 1:500 Donkey 1:500 Donkey 1:500 Goat 1:500 Donkey 1:500 Goat 1:5,000 Company Jackson ImmunoTesearch Jackson ImmunoTesearch Jackson ImmunoTesearch Jackson ImmunoTesearch Jackson ImmunoTesearch Jackson ImmunoTesearch Jackson ImmunoTesearch 39.4D5 40.2D6 MAB377 556309 GFP-1020 Cat. no. 711-545-152 711-165-152 706-485-148 705-165-147 115-165-003 703-175-155 111-035-003 1 The guinea pig anti-Phox2b antibody was used only for immunostaining on mouse in Figure 5A. TABLE 2. Probes Used for In Situ Hybridization and Enzymes for Antisense Probe Preparation Gene DR DR DR DR CA Phox2b Olig4 VGlut2.1 VGlut2.2 VGlut2 Reference GenBank accession no. Fragment Vector Restriction enzyme RNA polymerase Elworthy et al., 2005 Tiso et al., 2009 Higashijima et al., 2004 Higashijima et al., 2004 This study AY846871 AJ488293 AB183386 AB183387 JQ435859 1–2165 300–1096 906–1855 1469 –2069 105–1022 105–1022 pGEMT pCRTOPO pBluescript-SK pBluescript-SK pGEMT-easy pGEMT-easy NotI, T7 KpnI, T7 EcoRI, T3 HindIII, T3 NcoI, Sp6 NdeI, T7 sense probe and Islet-2 (Lewis and Eisen, 2003), and is eliminated by injection of one-cell embryos with a mixture of spliceblocking morpholinos directed against Islet-1 and Islet-2 (Hutchinson and Eisen, 2006). They label all hindbrain motoneurons, independently indentified by retrograde labeling (Chandrasekhar, 1997). The rabbit anti-Olig3 antibody (1:20,000) (Muller et al., 2005) was raised against the affinity-purified fusion of the full-length protein with a HIS-Tag. The staining pattern detected in the spinal cord was indistinguishable from that obtained by in situ hybridization with an Olig3 probe (Zechner et al., 2007) and was abrogated in Olig3 knockout embryos (L.R. Hernandez-Miranda and C. Birchmeier, personnal communication). 3636 Chicken anti-GFP (1:500; GFP-1020; Aves, Tigard, OR) was raised by using purified recombinant enhanced green fluorescent protein (EGFP) as immunogen. This anti-EGFP recognizes the YFP molecules expressed from the Rosa locus in ROSAstoploxYFP mice after recombination with a Cre, according to the expected pattern of the Cre allele, and gives no signal in mice that do not express Cre (D’Autr
eaux et al., 2011). The goat-anti ChAT antibody (1:50; AB114P; Chemicon, Temecula,CA) raised against human placental ChAT, has already been used to describe cholinergic neurons in zebrafish (Clemente et al., 2004; Mueller et al., 2004) and goldfish (Giraldez-Perez et al., 2009) brain. This antibody recognized a single band of 70 kDa in lysate of 293 The Journal of Comparative Neurology | Research in Systems Neuroscience Figure 1. Specificity of Phox2b antibodies in Danio and cloning of Carassius VGluT2. A: Alignment of the amino acid sequence of mouse (Mm) and zebrafish (Dr) Phox2b. Asterisks indicate identical residues, and colons indicate conservative changes. The homeodomain is underlined in black and the region used as immunogen in gray. B–D: DIC (B,C) and epifluorescence (D) images of adjacent transverse sections of a 1-month zebrafish head at the level of the nodose ganglion (Xg), hybridized with a Dr-Phox2b probe (B,) or stained with an antiPhox2b antibody, raised in rabbit (D). The cytoplasmic signal outside the ganglion is due to autofluorescence of red blood cells, also visible in Nomarsky optic (asterisks). The perfect match between the in situ hybridization and the immunostaining confirms the specificity of the anti-Phox2b antibody. C corresponds to the boxed area in B. E: Western blot of mouse E14.5 cortex and medulla of adult zebrafish and goldfish medulla labeled with the rabbit anti-mouse Phox2b antiserum. All medullary extracts, but not the cortical extract, show a band compatible with the theoretical MW of Phox2b (31 kDa in mouse and 30 kDa in zebrafish). F,G: Sequence analysis of Carassius auratus Vglut2. F: Alignment of the amino acid sequence of Dr-VGlut2.1, Dr-VGlut2.2, and Mm-VGluT2 with the conceptual translation of the PCR-cloned fragment of Carassius VGluT2. G: Unrooted tree showing the clustering of Ca-VGluT2 with Dr-VGluT2.2 (see Materials and Methods for details). Numbers indicate support of posterior probabilities. Species abbreviations: Ca, Carassius auratus; Dr, Danio rerio; Mm, Mus musculus. Scale bar ¼ 100 lm in B and D (also applies to C). Coppola et al. cells transfected with an expression vector containing the sequence of zebrafish ChAT (Volkmann et al., 2010). Western blot analysis of brain extracts of rat, dogfish, sturgeon, trout (Anad
on et al., 2000), and goldfish (Giraldez-Perez et al., 2009) showed that this antibody recognized similar bands between 68 and 72 kDa. The mouse monoclonal zrf-1 antibody (1:2,500; Zebrafish International Resource Center, Eugene, OR) was raised against a basal lamina and cytoskeleton preparation from adult zebrafish hindbrains and spinal cords (Trevarrow et al., 1990). It was later found to recognize glial fibrillary acidic protein (GFAP) by western blot analysis (Marcus and Easter, 1995). The staining pattern obtained in zebrafish larvae matches that obtained with EGFP expressed under the control of Gfap regulatory sequences in the Tg-gfap:GFP transgenic line (Kim et al., 2008). The mouse NeuN antibody (1:200) (clone A60; NEUronal Nuclei) specifically recognizes the DNA-binding, neuron-specific protein NeuN, present in most CNS and PNS neuronal cell types (Mullen et al., 1992). In the goldfish optic tectum it was shown to recognize neurons but not non-neuronal cells (astrocytes, oligodendrocytes, endothelial cells, and microglia) (King et al., 2004). For the experiments on zebrafish we used the antiPhox2b raised in rabbit, the anti-ChAT, anti-Islet-1, zrf-1, and NeuN antibodies. For the experiments on mouse we used the anti-Phox2b raised in guinea pig, the anti-Olig3, anti-GFP, and anti-Nestin antibodies. For experiments on goldfish we used the anti-Phox2b raised in rabbit, the anti-ChAT and NeuN antibodies. In situ hybridization For in situ hybridization, sections were briefly washed with PBS, followed by treatment with RIPA buffer (150 mM NaCl, 0.1% SDS, 1 mM EDTA, pH 8.5, 50 mM TrisHCl, pH 8, 0.5% sodium deoxycholate, 1% NP-40) for 20 minutes. The sections were postfixed with 4% paraformaldehyde in PBS and washed three times in PBS. Then they were treated with triethanolamine buffer (100 mM triethanolamine, 0.2% acetic acid) containing 0.25% acetic anhydride. The samples were washed three times in PBS and then prehybridized with prewarmed (70
C) hybridization buffer (50% formamide, 5X SSC, 5X Denhardt’s solution, 500 lg/ml salmon sperm, 250 lg/ml yeast RNA) for at least 1 hour. The slides were incubated overnight 70
C with 250 ll hybridization buffer containing 200– 400 ng/ml of digoxigenin (DIG; Roche) RNA probe, in a humidified chamber (5X SSC, 50% formamide). The slides were washed twice for 1 hour in prewarmed post-hybridization solution (50% formamide, 2X SSC, 0.1% Tween) at 70
C, then washed three times with buffer B1 (100 mM maleic acid, 150 mM NaCl, 0.1% Tween), followed by a 1-hour incubation with B2 solution (10% FCS in B1). 3638 Alkaline phosphatase-conjugated anti-DIG antibodies were diluted 1:2,000 in solution B2 and incubated at 4
C overnight. The following day the samples were washed twice in B1 and incubated in solution B3 (100 mM NaCl, 50 mM MgCl2, 100 mM Tris-HCl, pH 9.5, 0.1% Tween) for 30 minutes before the alkaline phosphatase substrate BCIP-NBT (Liquid Substrate Solution; Sigma) was added. Tween (0.1%) was added to the substrate solution to obtain a bluish color of the precipitate. The color reaction was allowed to develop in the dark and stopped with several washes in PBS 0.1% Tween (PBT); then the slides were rinsed briefly in H2O, dried, and mounted in Aquatex (Merck, Darmstadt, Germany). The probes used for zebrafish were the following: Phox2b, Olig4, and a mixture of VGlut2.1 and VGlut2.2 to detect glutamatergic neurons (Higashijima et al., 2004). Cloning of goldfish VGluT2 Two degenerate oligonucleotide primers were designed, based on two stretches of nine amino acids in VGlut2 shared among mouse and zebrafish VGluT2: GGI-GTI-CA(ACGT)-TA(TC)-CC(ACGT)-GC(ACGT)-TG(TC)CA(TC)-GG (I standing for inosine) (corresponding to the amino acid sequence GVTYPACHG) and GG(GA)-TCIGCC-CA(ACGT)-GG(TC)-TG(TC)-TT(TC)-TC(ACGT)-CC (corresponding to the amino acid sequence GEKQPWADP). An 891-bp DNA fragment was amplified by polymerase chain reaction (PCR) from whole brain-derived cDNA and subcloned in pGEMT-easy (Promega, Madison, WI). Orthology with Dr-VGlut2.2 was established by aligning nucleotide sequences using ClustalW2 (Larkin et al., 2007) from the EMBL-European Bioinformatics Institute (http://www. ebi.ac.uk/Tools/msa/clustalw2) and Bayesian inference analysis. The latter was performed on a conserved block of 896 nucleotides with MrBayes (Ronquist et al., 2003). Four chains were run for 2,000,000 generations with the following parameters: General Time Reversible model, four gamma-shaped rate variation categories with a proportion of invariant sites, sampling frequency 250, burn in 2000. The Sialin genes were used as an outgroup. The tree is unrooted and designed as radial logarithmic with TreeIllustrator version 0.52 (Trookens et al., 2005). The plasmid was digested with NcoI, transcribed with the Sp6 polymerase (Roche) for synthesis of the antisense probe, digested with NdeI, and transcribed with T7 polymerase for synthesis of the sense probe. The sequence of Ca-VGluT2 has been deposited in Genebank under the accession number JQ435859. Analysis and imaging Images were taken with a Leica (Nussloch, Germany) microscope CTR5000, equipped with a Leica DFC420C camera for brightfield, and a Hamamatsu (Hamamatsu The Journal of Comparative Neurology | Research in Systems Neuroscience Phox2b expression in the taste centers of fish City, Japan) Orca ER camera for fluorescent observation. Digital images were processed with Adobe (San Jose, CA) Photoshop for color merging, and, when necessary, for adjustment of brightness and contrast of the entire image. In the case of goldfish, because of the large size of the brain, the photomerge function was used to assemble several fields into one picture. RESULTS Expression of Phox2b in the visceromotor and viscerosensory columns of adult zebrafish We first analyzed Phox2b expression in the medulla of adult zebrafish. Phox2bþ neurons could be divided into two large groups. The first group included all visceral motoneurons (general [or preganglionic parasympathetic neurons] and special [or branchiomotor neurons] hereafter collectively designated as BM/VM neurons), as identified by their position, compared with previously published maps (Wullimann et al., 1996). These were, from rostral to caudal: the trigeminal motor nucleus (with its two divisions, ventral and dorsal [Fig. 2A]), the facial motor nucleus (Fig. 2B), and the juxtaventricular column formed by the visceromotor neurons (Fig. 2C,D). BM/VM neurons coexpressed ChAT (Fig. 2E and inset), in agreement with their cholinergic phenotype. In the isthmus and midbrain, the trochlear and oculomotor nuclei expressed Phox2b (Fig. 3A,B and insets), as they do in mouse (Pattyn et al., 1997; and see Discussion). The second large group of Phox2bþ neurons was situated in the viscerosensory column, i.e., the facial (Fig. 2C) and vagal lobes (Fig. 2D,E) and the general viscerosensory zone or nCC (Fig. 4D). Coexpression of NeuN indicated that the vast majority of Phox2bþ cells in the taste lobes were neurons (Fig. 2F–H). Expression in these lobes could be further subdivided based on spatial arrangement: cells packed in a dense superficial layer (black arrowheads) overlaid cells situated more deeply, scattered in no overt pattern (magenta arrowheads, Fig. 2C,D). Most Phox2bþ neurons in the facial and vagal lobes coexpressed Vglut2 (Fig. 4 and data not shown), an established marker for glutamatergic neurons in fish and mammals (Higashijima et al., 2004 and references therein), showing that they correspond to glutamatergic neurons, as is the case in the rodent nTS (Kang et al., 2007). Thus, in fish as in mammals, Phox2b is expressed in BM/VM neurons and in the glutamatergic neurons of the viscerosensory column. However, the latter has a strikingly different structure from the mammalian nTS. To understand the source of this difference, we next com- pared the formation of the viscerosensory column in zebrafish and mouse. Formation of the visceromotor and viscerosensory columns compared between mouse and zebrafish In mice, glutamatergic nTS precursors are born between E9.5 and E12.5 (Pattyn et al., 2006) from a neuroepithelial domain termed dA3, located in the dorsal alar plate, which expresses the bHLH transcription factor Olig3 (Storm et al., 2009) (Fig. 5A). Neuronal precursors emerge from dA3 and migrate tangentially (Fig. 5C,D), i.e., perpendicularly to radial glia, to settle in more ventral position (Fig. 5E–H). The dorsal motor nucleus of the vagus nerve concomitantly migrates dorsally, from the ventralmost pMNv (for ‘‘progenitor of visceral motoneurons’’) progenitor domain (Briscoe et al., 1999) to a level just ventral to the nTS (Dauger et al., 2003) (Fig. 5E,F). There, vagal motoneuronal precursors reestablish contacts with the ventricle, through processes (Fig. 5G,H and insets) that have been described earlier, although they were thought at the time to be ‘‘remnants’’ of an original ventricular attachment (Rinaman and Levitt, 1993). In zebrafish, at 24 hpf, Phox2b was only detected in the paramedian domain of the neuroepithelium (Fig. 6A and insets), likely corresponding to the pMNv domain of the mouse hindbrain, where visceral motoneurons are born. Accordingly, early postmitotic cells coexpressed Islet-1, an early marker of motoneurons (Ericson et al., 1992) (Fig. 6A). The dorsal alar plate was devoid of Phox2b expression at caudal levels but expressed high levels of Olig4, the orthologue of mouse Olig3 (Tiso et al., 2009) (Fig. 6A,B). At 33 hours the cell arrangement was basically the same, except that postmitotic precursors of vagal motoneurons had accumulated (Fig. 6C,D). At 48 hpf, a chain of Phox2b-positive neurons, roughly parallel to the lateral edge of the hindbrain, emerged from the Olig4-positive dorsal neuroepithelial domain (Fig. 6E,F). At the same stage, VGluT2 was switched on in a column overlapping the Phox2bþcells, as judged from adjacent sections (Fig. 6E,G) and as previously described (Higashijima et al., 2004). Thus, this dorsolateral column of Phox2bþcells, by its neurotransmitter identity [VGluT2þ] and region of origin [Olig4þ domain of the neuroepithelium] corresponds to the anlage of the viscerosensory column. Double labeling for Phox2b and Zrf-1, a marker of zebrafish radial glia, showed that the migration of the viscerosensory neuron precursors was aligned with the radial glia (Fig. 6H–J). At 3 days post fertilization (dpf) the arrangement of the dorsal Phox2bþ cells was basically unchanged, except that a superficial layer of Phox2b cells was no longer The Journal of Comparative Neurology | Research in Systems Neuroscience 3639 Coppola et al. Figure 2. Expression pattern of Phox2b in the adult zebrafish brain. A–E: Transverse sections of zebrafish brain at the level of the hindbrain. The sections are shown in a rostral (top) to caudal (bottom) order. A–D: DIC images of in situ hybridization with a Phox2b probe showing expression in the ventral (Vmv) and dorsal (Vmd) division of the trigeminal motor nucleus (A), in the facial motor neurons (VIIm) (B), in the visceromotor neurons associated with glossopharyngeal (IXm) and vagal (Xm) nerves (C,D), and in the facial (LVII) and vagal (LX) lobes (C,D). E: Epifluorescence image of a section at the same level as D, double-immunostained with anti-Phox2b (green) and anti-ChAT (magenta) antibodies, showing that the Phox2b-positive Xm neurons are cholinergic. Inset: Higher magnification of the boxed area in E. F–H: Epifluorescence images of LX neurons labeled with anti-Phox2b (F) and NeuN (G) antibodies, showing coexpression of the two proteins (H) in the viscerosensory neurons. Magenta arrowheads, scattered Phox2bþ cells in the LVII (C) and LX (D). CC, crista cerebellaris. Scale bar ¼ 100 lm in E (applies to A–E); 50 lm in H (applies to F–H and inset in E). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] present, bringing the column of Phox2bþ cells just beneath the pia (Fig. 6K), whereas motoneurons tended to occupy a wider area (Fig. 6K) and Olig4 expression had considerably decreased, most likely reflecting the narrowing of the progenitor zone (Fig. 6N). The pattern was basically unchanged at 7 dpf (Fig. 6L,O). At 1 month post fertilization, the number of Phox2bþ cells had vastly 3640 increased (Fig. 6M) and, consistent with a continuous production, Olig4 expression persisted in the neuroepithelium (Fig. 6P). The motoneurons, expressing ChAT (Fig. 6M), were lined up dorsoventrally along the ventricular surface. At none of the developmental stages examined (i.e., up to 7 dpf) could we distinguish the vagal from the glossopharyngeal and facial lobes. The Journal of Comparative Neurology | Research in Systems Neuroscience Phox2b expression in the taste centers of fish Figure 3. Expression pattern of Phox2b in the oculomotor and trochlear nuclei of zebrafish. A,B: Brightfield images of transverse sections at the level of the midbrain showing the oculomotor (IIIm; A and inset) and trochlear (IVm; B and inset) motoneurons stained by in situ hybridization by using a Phox2b probe. OT, optic tectum. Scale bar ¼ 200 lm in A (applies to A,B); 100 lm in insets. Expression of Phox2b in the facial and vagal lobes of Carassius auratus We next examined expression of Phox2b by immunohistochemistry in the viscerosensory column of adult goldfish. Phox2b immunoreactivity was detected in many cells throughout the vagal, glossopharyngeal, and facial lobes (Fig. 7A). As in zebrafish, the vast majority of Phox2bþ cells coexpressed NeuN and were therefore neuronal (Fig. 7B–D). On closer inspection, four of the five neuronal layers identified in the ‘‘vagal sensory zone’’ of Carassius (Morita and Finger, 1985) contained Phox2bþ neurons: layers III (where they were by far the most abundant) and V (which project to the secondary gustatory zone) as well as VII and IX (which project [together with V] to the ‘‘vagal motor zone’’ (Fig. 8A,B). Layer XI contained only occasional Phox2bþ neurons (green arrowhead in Fig. 8B). Layer XIV, where motoneurons of the vagal lobe reside (projecting their axons into layer XV), also contained Phox2bþ cells (Fig. 8B) whose moto- Figure 4. Glutamatergic phenotype of the viscerosensory neurons of zebrafish. A–H: Epifluorescent (A–D) and DIC (E–H) images of transverse sections through a zebrafish hindbrain stained with the anti-Phox2b antibody (label in A applies to A–D) or a VGluT2 probe (label in E applies to E-H). The Phox2b-positive neurons of the facial lobe (LVII; A,B), of the vagal lobe (LX; B,C), and of the general viscerosensory zone (D) express VGlut2. CC, crista cerebellaris; nCC, nucleus commisuralis of Cajal. Scale bar ¼ 100 lm in D (applies to A–H). neuronal identity was confirmed by coexpression of ChAT (Fig. 9A and inset). The smaller intrinsic neurons described in the superficial region of layer XIV (Morita et al., 1983) were Phox2b-negative (white arrows in Fig. 8B). We cloned goldfish Vglut2.2 (Fig. 1F,G) and verified that it was massively expressed in layer III (Fig. 9B), most likely in Phox2bþ neurons, showing that they correspond to the principal relay neurons, which are glutamatergic (Ikenaga et al., 2009). Sandwiched between the sensory and motor zones, the fiber layers XII and XIII (which contain, respectively, primary afferent fibers and efferent fibers to the secondary gustatory center) were largely devoid of Phox2bþ cells (Fig. 8A,B). The Journal of Comparative Neurology | Research in Systems Neuroscience 3641 Coppola et al. Figure 5. Development of the viscerosensory and visceromotor neurons in mouse. A–D: Epifluorescent images of transverse sections through a wild-type mouse embryo at the level of the caudal hindbrain at E11.5. A: Phox2b (green) and Olig3 (magenta) double staining showing that Phox2b-positive precursors of the nTS emerge from the Olig3-expressing domain dA3. B–D: Double labeling using anti-nestin (B,D) and antiPhox2b (C,D) antibodies, showing the orientation of the radial glial fibers (B,D) and the tangential migration (i.e., perpendicular to radial glia) of Phox2b-positive nTS neurons (C,D). E–H: Transverse sections of transgenic embryos expressing YFP following the action of a Phox2b::Cre, double-stained with anti-GFP (green) and anti-Phox2b (magenta) antibodies (label in E applies to E–H) at the indicated ages, showing the migration of nTS and dorsal vagal motoneuronal precursors toward each other. E–G: The double labeling allows one to distinguish the weakly YFP-positive nTS precursors (magenta arrowheads) from the strongly YFP-positive motoneurons (green arrowheads), the lower levels of the reporter in the former being likely due to the later expression of Phox2b. H: The nTS and dmnX have settled in their final position. Insets in G and H: De novo cytoplasmic projections of dmnX neurons toward the ventricle. White arrowhead, solitary tract (TS). Scale bar ¼ 100 lm in D (applies to A–G); 100 lm in H; 50 lm in insets. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] The facial lobe also contained many Phox2bþ neurons (Fig. 9C), organized in one dense superficial layer with a seemingly random pattern underneath, matching their appearance by the Golgi impregnation method in C. carassius (Morita et al., 1983). The superficial neurons and at least some of the deep ones expressed Vglut2 (Fig. 9D). Ventrally to the facial lobe, the facial motoneurons coexpressed ChAT and Phox2b (Fig. 9C, inset). Caudally to the vagal lobes, Phox2bþ neurons were found in medial and paramedian groups of cells corresponding to the lateral and medial subnuclei of the commissural nucleus of 3642 Cajal, i.e., the general viscerosensory zone and the area postrema (Morita and Finger, 1987b) (Fig. 10). DISCUSSION Homology of the viscerosensory nuclei in aquatic and terrestrial vertebrates The taste lobes of fish have long been considered homologous to the gustatory portion of the nTS of mammals on hodological grounds, i.e., based on their status as projection sites for gustatory afferents of the facial, The Journal of Comparative Neurology | Research in Systems Neuroscience Figure 6. Development of the viscerosensory and visceromotor neurons in zebrafish. A–P: Transverse sections of zebrafish larvae (A– L,N,O) and adult zebrafish brain (M,P) labeled with the indicated antibodies (imaged by epifluorescence) or probes (imaged by DIC). A–D: Transverse sections at 24 hpf (A,B) and 33 hpf (C,D), double labeled with anti-Phox2b (green) and anti-Islet-1 (magenta) antibodies (A,C)—showing early postmitotic precursors (magenta arrowhead and closeups)—or labeled with an Olig4 probe (B–D). E–G: Transverse sections through the caudal hindbrain at 48 hpf immunostained with anti-Phox2b and anti-Islet1 antibodies, or hybridized with Olig4 or Vglut2 probe. Green arrowhead, vagal lobe precursors; magenta arrow, vagal motoneuronal precursors. At this stage Olig4 labels three discrete neuroepithelial region as previously described (Tiso et al., 2009). H–J: Transverse sections at 48 hpf double-stained with anti-Phox2b and an antibody against radial glia (Zrf-1), as indicated. Note that the migration of the vagal lobe precursors is aligned with the radial glia. K–M: Transverse sections at 3 dpf, 7 dpf, and 1 month, immunostained for Phox2b (green) and Islet-1 or ChAT (magenta). Insets: Closeups of the motoneurons. N–P: Transverse sections adjacent to K–M respectively, hybridized with Olig4. Scale bar ¼ 50 lm in A (applies to A–D); 100 lm in O (applies to E–O) and P (applies to M–P); 25 lm in closeup from A (also applies to C; 20 lm in insets of K (applies to K–M). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Coppola et al. Contrasted topology of the viscerosensory nuclei in cyprinid fish and mammals Figure 7. Phox2b distribution in the viscerosensory column of goldfish. A: Epifluorescent image of a transverse section of goldfish brain through the medulla, stained with Phox2b antibody, showing expression of Phox2b in the facial (LVII), glossopharyngeal (LIX), and vagal (LX) lobes. B–D: Epifluorescent images of LX neurons double-labeled with anti-Phox2b (B) and anti-NeuN (C) antibodies, showing coexpression of the two proteins (D) in the viscerosensory neurons. Scale bar ¼ 200 lm in A; 50 lm in D (applies to B–D). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] glossopharyngeal, and vagal nerves (e.g., Herrick, 1899). In fact, homology was first proposed before the gustatory nucleus of mammals was properly identified (as is clear from Herrick, 1905, p. 381), a curious case of an a priori statement of homology. The widespread expression of Phox2b in both the nTS and fish taste lobes that we document here provides a strong additional argument, and the first molecular one, for this homology, as well as the homology of the nCC with the general viscerosensory zone of the nTS. Herrick (1899, p. 216) cautioned that ‘‘if we should attempt to draw up a detailed comparison, the various elements would doubtless not be exactly equivalent in the two groups of animals.’’ It is indeed doubtful that each class of interneurons identified in the goldfish vagal lobe by its position in different layers, its morphology, and its connections (Morita et al., 1983) has a homologous counterpart in the nonlaminated mammalian nTS. However, many excitatory interneurons of the goldfish taste lobes, including putative synapomorphic types, have recruited the same transcription factor, Phox2b, which makes them likely ‘‘sister cell types’’ (Arendt, 2008), produced from an ancestral relay taste neuron. 3644 In his landmark monograph on Menidia, Herrick (1899, p. 213) tried to reconcile the homology of the teleost and mammalian viscerosensory columns with their blatantly different form and position. He argued that the vagal lobes were not ‘‘dorsal’’ as they appeared, because they were ventral to the roof plate, and that their inordinate development had crowded out (laterally and ventrally) a topologically—Herrick says ‘‘morphologically’’—more dorsal structure, the somatosensory column, or SpV. This theoretical distortion allowed Herrick to fit the viscerosensory column of fish into the intermediodorsal position assigned by the columnar model of the hindbrain inherited from Gaskell (reviewed in Nieuwenhuys et al., 1998). Implicitly, this interpretation displaced ventrolaterally the junction of the tela chorioidea (‘‘beneath’’ which the lobes were supposed to develop [Herrick, 1905, p. 384]) with the pia mater (which covers the SpV, and made the entire surface of the vagal lobe ventricular), an intriguing notion, with no further history to our knowledge. Our data in zebrafish show that the topology of the vagal lobe is in fact irreconcilable with that of the mammalian nTS, the reason lying in a different migration pattern from a similar origin. In mammals, the intermediodorsal position of the nTS arises by a tangential migration from the dA3 neuropithelial domain of the alar plate, in a ventral direction (Fig. 5A–D). On the second axis of the tube, radial (or ventriculo-pial), this tangential migration maintains the nTS close to the ventricle, with the tractus solitarius at its pial border (Fig. 5H). In fish, the Phox2bþ, Vglut2þ interneurons of the taste lobes are born in the same dA3 domain, marked by Olig4, but their migration is radial, so that they remain dorsal (Fig. 6J); they become even more so once their dorsal neighbors apparently migrate out in a ventral direction, in ways that we have not analyzed. The latter could correspond to the barlh2þ cells described at a similar position (Kinkhabwala et al., 2011) and could be homologous to dA1-derived precerebellar neurons in mouse (Bermingham et al., 2001), also known to express Barhl1/2 and to settle ventrally. In addition, Phox2bþ neurons migrate to various extents— some remaining ventricular, some ending up pial, and others everywhere in between—so that, viewed in transverse sections, the cells form a column rather than a nucleus, lined up along the radial axis. This cellular arrangement in the developing hindbrain of zebrafish has been described for other neuron types (Higashijima et al., 2004; Kinkhabwala et al., 2011). Note that, relative to body axes, these radial columns of neurons assume a dorsoventral appearance at the hindbrain level due to a ventral deflection of the radial axis in the alar plate The Journal of Comparative Neurology | Research in Systems Neuroscience Phox2b expression in the taste centers of fish Figure 8. Phox2b distribution in the sensory and motor layers of the goldfish vagal lobe. A,B: Epifluorescent image of a transverse section of the goldfish brain stained with NeuroTrace alone (A) or combined with anti-Phox2b antibody (B) showing the laminar organization of the vagal lobe. Phox2b-positive neurons are present in neuron containing layers III, V, VII, IX, and XIV. Only an occasional Phox2bþ neuron can be found in layer XI (green arrowhead). Inset in B: Higher magnification of the boxed area showing Phox2b-positive and Phox2b-negative (arrowhead) neurons in layer V. The white arrows point to Phox2b-negative intrinsic neurons of layer XIV. Scale bar ¼ 100 lm in A (applies to A,B); 50 lm in closeups. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] (Fig. 6H )—and, at more rostral levels, of the ventricular surface itself, where it becomes T-shaped (e.g., Lyons et al., 2003). This distortion is transient, and later on, the fish viscerosensory column shifts from a lateral to a more dorsal position (in a Cartesian space). Our data on the formation of the zebrafish vagal lobe are compatible with previous descriptions made in the goldfish (Lamb and Kiyohara, 2005) where proliferation was said to occur ‘‘mainly at the tip of the vagal lobe’’ forming ‘‘cohorts that are older at the base of the lobe and progressively younger dorsally.’’ This mode of growth was likened by the authors to the ‘‘tangential morphogenetic pattern’’ of the fish optic tectum (Nguyen et al., 1999). Topologically, however, i.e., relative to the axes of the developing tube rather than the shape of the adult structure, this growth, at least in zebrafish, is not tangential but radial. From this perspective, the main differences between zebrafish and goldfish would be the much higher number of cells produced in the latter, feeding the bulge of the lobe, as well as lamination, whose mechanism remains to be explored (Finger, 2009). The Journal of Comparative Neurology | Research in Systems Neuroscience 3645 Coppola et al. Figure 9. Neurotransmitter phenotype of Phox2b-positive neurons in the vagal and facial lobes of goldfish. A–E: Transverse sections through the vagal (A–C) and facial (C–E) lobes immunostained for Phox2b (green) and ChAT (magenta; A,C; imaged by epifluorescence) or hybridized with an antisense (B,D) or sense (E) VGlut2 probe (imaged by DIC). Insets: Closeups of the boxed area. Asterisk, fiber layers XII and XIII; NX, vagal nerve; Xm, visceromotor layer of vagal lobe. Scale bar ¼ 200 lm in C (applies to A,C); 100 lm in B (applies to B,D,E); 100 lm in inset to C (also applies to inset in A). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] One can speculate that this mode of migration, resulting in a two-dimensional sheet of cells, was key in achieving the fine viscerotopic representation of the oropharynx in the vagal lobe (Morita and Finger, 1985). This representation, together with an equivalent one in the motor nucleus (see below) allows for pointby-point projections between the two (Morita and Finger, 1985), the anatomic basis for the reflex system, whereby local taste sensation triggers local muscle contraction in the palatal organ, enabling food sorting (Finger, 2008). Whether this topology and the mode of migration that brings it about is primitive, or a derived feature of teleosts, will require the study of additional fish clades. The facial lobe also contains a topological representation of the periphery—a somatotopic representation of the body surface or ‘‘pisciculus’’ (Puzdrowski, 1987)—but it differs markedly from the vagal lobe in that it is a medial, 3646 unpaired structure. Our data do not elucidate the developmental events leading to this configuration. Migratory behavior of visceromotor neurons In contrast to sensory neurons, the migratory behavior of BM/VM neurons is globally conserved in mammals and fish: they are born ventrally from the pMNv domain and migrate tangentially in a dorsal direction (Dauger et al., 2003; Guthrie, 2007; Ohata et al., 2009; this study) to settle dorsal to somatic motoneurons, in the intermedioventral position described by the columnar model of the hindbrain. This migration keeps them close to the ventricle, with which they reestablish direct contacts, at least in rodents. However, the fine settling pattern of motoneurons also diverges between fish and mammals. In the former, and particularly in goldfish, the special visceromotoneurons form an arrayed layer along the dorsoventral The Journal of Comparative Neurology | Research in Systems Neuroscience Phox2b expression in the taste centers of fish is their determinant Phox2a, a paralogue of the BM/VM determinant Phox2b (Pattyn et al., 1997, 2000; Guo et al., 1999a,b). Again, like BM/VM neurons and unlike somatic neurons, they postmitotically express Phox2b (Pattyn et al., 1997) (and Tbx20 for the trochlear neurons; Grillet et al., 2003)—but neither Hb9 (Thaler et al., 1999) nor Lhx3/4 (Sharma et al., 1998). Finally, their targets, the extraocular muscles, arise from the nonsegmented preotic paraxial mesoderm and are independent of Pax3, like branchial muscles, with which they might share an evolutionary origin (Sambasivan et al., 2011). All in all, oculomotor and trochlear motoneurons appear to have arisen by tinkering with the identity of BM/VM neurons and should be classified either as BM/VM-like or in a class of their own. ACKNOWLEDGMENTS We thank members of the F. Rosa laboratory and Y. Bouchoucha, C. Labalette-Peaucelle, Rapha€el Corre, and L. Bailly-Cuif for advice and kindly providing us with zebrafish embryos, N. Tiso and S. Higashijima for probes, Thomas Müller for antibodies, and Carmen Le Moal for handling mouse strains. LITERATURE CITED Figure 10. Phox2b expression in the caudal medulla of goldfish. A,B: Epifluorescent images of transverse sections through the caudal medulla (B caudal to A) showing expression of Phox2b in the general visceral columns and area postrema. LX, vagal lobe; NCCm, nucleus commissuralis of Cajal, pars medialis; NCCl, nucleus commissuralis of Cajal, pars lateralis; AP, area postrema. 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