Zebrafish deadly seven Functions in Neurogenesis

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1 Developmental Biology 237, (2001) doi: /dbio , available online at on Zebrafish deadly seven Functions in Neurogenesis Michelle Gray,*, Cecilia B. Moens, Sharon L. Amacher, Judith S. Eisen, and Christine E. Beattie*,,,1 *Neurobiotechnology Center, Molecular, Cellular, and Developmental Biology Program, and Department of Neuroscience, Ohio State University, Columbus, Ohio 43210; Howard Hughes Medical Institute, The Fred Hutchinson Cancer Research Center, Seattle, Washington 98109; Department of Molecular and Cell Biology, University of California, Berkeley, California 94720; and Institute of Neuroscience, University of Oregon, Eugene, Oregon In a genetic screen, we isolated a mutation that perturbed motor axon outgrowth, neurogenesis, and somitogenesis. Complementation tests revealed that this mutation is an allele of deadly seven (des). By creating genetic mosaics, we demonstrate that the motor axon defect is non-cell autonomous. In addition, we show that the pattern of migration for some neural crest cell populations is aberrant and crest-derived dorsal root ganglion neurons are misplaced. Furthermore, our analysis reveals that des mutant embryos exhibit a neurogenic phenotype. We find an increase in the number of primary motoneurons and in the number of three hindbrain reticulospinal neurons: Mauthner cells, RoL2 cells, and MiD3cm cells. We also find that the number of Rohon Beard sensory neurons is decreased whereas neural crest-derived dorsal root ganglion neurons are increased in number supporting a previous hypothesis that Rohon Beard neurons and neural crest form an equivalence group during development. Mutations in genes involved in Notch Delta signaling result in defects in somitogenesis and neurogenesis. We found that overexpressing an activated form of Notch decreased the number of Mauthner cells in des mutants indicating that des functions via the Notch Delta signaling pathway to control the production of specific cell types within the central and peripheral nervous systems Academic Press Key Words: mutational analysis; primary motoneurons; neural crest; Rohon Beard neurons; somitogenesis; Mauthner cells; reticulospinal neurons; lateral inhibition; Notch. INTRODUCTION Mutagenesis screens in zebrafish have identified mutants that affect both neurogenesis and somitogenesis (Jiang et al., 1996; Schier et al., 1996; van Eeden et al., 1996). Two mutants, mindbomb (mib) and after eight (aei) encoding deltad (Holley et al., 2000), show varying degrees of nervous system hyperplasia. mib has an over-abundance of early developing neurons, including Mauthner cells, whereas aei/deltad has a more subtle neurogenic phenotype only reported to affect primary sensory neurons (Schier et al., 1996; Jiang et al., 1996; Holley et al., 2000). Furthermore, mutations in both of these genes cause defects in somite boundary formation (van Eeden et al., 1996; Jiang et al., 1996, 2000; Durbin et al., 2000; Holley et al., 2000). 1 To whom correspondence should be addressed at: Neurobiotechnology Center/Dept. of Neuroscience, Ohio State University, 115 Rightmire Hall, 1060 Carmack Road, Columbus, OH Fax: beattie.24@osu.edu. Mutations in two other genes, beamter (bea) and deadly seven (des) have somite defects similar to mib and aei/ deltad (van Eeden et al., 1996; Durbin et al., 2000; Jiang et al., 2000), but nervous system abnormalities have not been described for these mutants. In frogs, fish, and mice, perturbing genes in the Notch Delta signaling pathway leads to defects both in neurogenesis and somitogenesis. In Xenopus laevis, the Delta homologue, X-Delta-1, is expressed in the nervous system and interfering with its activity results in an overproduction of early developing neurons (Chitnis et al., 1995). Overexpressing RNA encoding X-Delta-1 or a constitutively activated form of Xenopus Notch has the opposite effect causing a decrease in the number of early developing neurons (Chitnis et al., 1995). Overexpression of a dominantnegative form of another Delta homolog, X-Delta-2, normally expressed in the presomitic mesoderm, leads to a defect in somitogenesis (Jen et al., 1997). Overexpressing zebrafish delta genes also causes neurogenic defects (Haddon et al., 1998; Appel et al., 1998) and overexpression of /01 $35.00 Copyright 2001 by Academic Press All rights of reproduction in any form reserved.

2 Zebrafish deadly seven Functions in Neurogenesis 307 FIG. 1. Somite boundary formation is aberrant in des b420 mutants. Lateral views of live 18-h wild-type (A C) and mutant (D F) embryos at the level of anterior trunk (segments 2 5; A, D), mid-trunk (segments 8 12; B, E), and tail (segments 14 18; C, F). Black arrowheads denote normal somite boundaries and white arrowheads denote incomplete somite boundaries. In this and all subsequent lateral views, anterior is to the left and dorsal is to the top. Scale bar, 25 m. deltad results in defects in somite boundary formation (Dornseifer et al., 1997; Takke et al., 1999). Consistent with this, aei/deltad mutants have a neurogenic phenotype and defects in somitogenesis (van Eeden et al., 1996; Holley et al., 2000). Both mouse Notch1 and a mouse Delta homologue, Dll1, are expressed in paraxial mesoderm. Disruption of either of these genes, as well as genes required for Notch glycosylation and processing, results in abnormal somite formation (Conlon et al., 1995; Oka et al., 1995; de Angelis et al., 1997; Wong et al., 1997; Evard et al., 1998; Kusumi et al., 1998; Zhang and Gridley, 1998). A neurogenic defect is also observed when Notch1, its downstream effector RBP- JK, or Presenilin-1, a gene involved in Notch processing, are mutated in mice (de la Pompa et al., 1997; Handler et al., 2000; Donoviel et al., 1999). Thus, there is overwhelming evidence that disrupting the Notch Delta signaling pathway perturbs both neurogenesis and somitogenesis in vertebrates. We have isolated a mutation with a defect in somite formation closely resembling that of known zebrafish and mouse Notch Delta pathway mutants. In this report, we demonstrate that the mutation we isolated is an allele of the zebrafish mutant des (des b420 ). We expand on the previously described motor axon defect (van Eeden et al., 1996) by analyzing individual primary motor axons. By creating genetic mosaics, we show that the motor axon defect in des b420 mutants is non-cell autonomous and is likely due to aberrant somite and myotome formation. We also find that the normally segmental pattern of neural crest cell migration is lost and neural crest-derived dorsal root ganglion (DRG) neurons are misplaced and fail to coalesce into ganglia. Furthermore, analysis of the primary nervous system of des b420 mutants reveals a restricted neurogenic defect that only affects a subset of neuronal cell types, including the identified, hindbrain interneuron, the Mauthner cell. We show that overexpressing RNA encoding an activated form of Xenopus Notch (Notch-ICD; Coffman et al., 1993; Chitnis et al., 1995) rescues the Mauthner cell phenotype suggesting that des acts in the Notch Delta signaling pathway at or upstream of the level of the Notch receptor. The specificity of the des neurogenic phenotype, with respect to the subsets of neurons affected, implies that different Notch Delta pathway components may exhibit temporal or regional specificity during zebrafish development. MATERIALS AND METHODS Fish Adult zebrafish and embryos were maintained essentially as described in Westerfield (1995) and staged by hours (h) or days (d) TABLE 1 Analysis of Aberrant Somites in des b420 Mutants Age (h) (n 10) Abnormal somites Beginning at somite number Note. Somites were analyzed along the entire rostrocaudal axis with the most rostral somite being number 1. A somite was considered normal if both the anterior and posterior boundaries were complete. n 10 embryos analyzed for each time point. Numbers are represented as mean 95% confidence interval.

3 308 Gray et al. FIG. 2. Somite and myotome gene expression is perturbed in des b420 mutants. Dorsal views (anterior to the top) of whole-mount RNA in situ hybridization of her1 (A, D; 13 h), myod (B, E; 15 h), and mespa (C, F; 13 h) in wild-type (A C) and mutant (D F) embryos. Black arrowheads denote bands of gene expression in wild-type embryos and the corresponding region in mutants. Arrow in (E) designates normal myod expression in anterior somites. Scale bar, 150 m (A, C, D, F), 50 m (B, E). postfertilization at approximately 28.5 C as in Kimmel (1995). Mutant embryos (*AB background) were collected from pairwise matings of heterozygous adults and identified based on somite morphology. In Situ Hybridization and Immunohistochemistry Staged embryos were processed for whole-mount in situ hybridization as described by Thisse et al. (1993). Antisense digoxigenin islet1, islet2 (Korzh et al., 1993; Inoue et al., 1994; Appel et al., 1995), mesp-a (Sawada et al., 2000; Durbin et al., 2000), crestin (Rubenstein et al., 2000), and dopachrome tautomerase/ tyrosinase-related protein 2 (dct; Kelsh and Eisen, 2000) riboprobes were synthesized from plasmids linearized with EcoRI and transcribed with T7 polymerase. valentino/kriesler (val) was synthesized from a plasmid linearized with PstI and transcribed with Sp6 (Moens et al., 1998). myod was synthesized from a plasmid linearized with XbaI and transcribed with T7 (Weinberg et al., 1996) and her1 was synthesized from a plasmid linearized with XhoI and transcribed with T3 (Muller et al., 1996). For znp1 (Melancon et al., 1997), monoclonal antibody 16A11 (anti-hu; Henion et al., 1996), acetylated tubulin (Sigma), anti-islet 1 (Developmental Studies Hybridoma Bank; Korzh et al., 1993), zrf-1 (Trevarrow et al., 1990), and 3A10 (Hatta, 1992) immunohistochemistry, embryos were fixed in 4% paraformaldehyde overnight at 4 C, then washed in PBS and preincubated in phosphate buffered saline with 0.5% Triton X-100, 1% bovine serum albumin, 1% dimethysulfoxide, and 2.5% goat serum (PBDT). Antibodies were diluted in PBDT and incubated overnight at 4 C. For antineurofilament (RMO44) antibody labeling, 48-h embryos were fixed in 2% trichloroacetic acid and processed as described by Pöpperl et al., For visualization under transmitted light, the Clonal PAP system (Sternberger Monoclonals Inc.) with 3,3 diaminobenzidine (DAB) as substrate was used whereas anti-mouse Oregon Green (Molecular Probes) was used for fluorescent detection. For cross-sectional analysis of DRG and enteric neurons, embryos were fixed at 5 d, embedded in 1.5% agar/5% sucrose and sectioned on a cryostat at 16 m. Anti-Hu was added for 2hat room temperature followed by incubation in anti-mouse Oregon Green for 1 h at room temperature. Embryos and sections were analyzed with a Zeiss Axioplan microscope and photographed with Kodak Ektachrome 64T film or digitally imaged by using a Photometrics SPOT camera. BrdU Incorporation and in Situ Hybridization Embryos collected from pairwise matings of heterozygous des b420 fish were dechorinated at 6 14 h with 2 mg/ml pronase (Sigma) and incubated in 10 mm BrdU (Roche)/10% DMSO in embryo medium (Westerfield, 1995). Embryos were soaked for 45 min in BrdU starting at 7, 9, 11, and 14 h. After incorporation, embryos were washed once quickly followed by two 15-min washes in embryo medium. To process for val RNA in situ hybridization, BrdUtreated embryos remained in embryo medium until 19 h. Afterward, they were fixed for h in 4% paraformaldehyde at room temperature followed by in situ hybridization with a digoxigenin val riboprobe. Following the color reaction with NBT/BCIP and washing in PBSt (1 PBS and 0.5% Tween), embryos were incu-

4 Zebrafish deadly seven Functions in Neurogenesis 309 FIG. 3. Motor nerves are disorganized in des b420 mutants. Lateral views of whole-mount embryos labeled with the znp1 monoclonal antibody at 24 (A, C) and 48 h (B, D) in wild-type (A, B) and mutant (C, D) embryos. Arrowheads point to one axon (A, C) or nerve (B, D). Scale bar, 50 m. bated in 2 N HCl for 1 h at 37 C then processed for BrdU detection using anti-brdu (Roche; 1/100) followed with a rhodamineconjugated secondary antibody. Following antibody labeling, embryos were embedded in 1.5% agar/5% sucrose and sectioned on a cryostat at 16 m. Single Cell Labels and Transplants Individual motoneurons were labeled with rhodamine dextran ( MW; Molecular Probes) as described (Eisen et al., 1989; Beattie et al., 2000). Embryos were mounted in 1.2% agar on a microslide. After labeling, embryos were removed from the agar and placed in embryo medium (Westerfield, 1995) containing 50 units penicillin and 5 g streptomycin at 28.5 C. Labeled cells were visualized with a Zeiss Axioskop. Images were captured with a Photometrics SPOT camera and were colorized by using Photoshop (Adobe). CaP transplants were performed essentially as described (Eisen, 1991; Beattie et al., 2000). Briefly, 16-h donor embryos labeled at the 1- to 4-cell stage with rhodamine dextran ( MW; Molecular Probes) were mounted side by side with unlabeled host embryos in 1.2% agar on a microslide. Individual CaP motoneurons were transplanted from labeled donors to unlabeled host spinal hemisegments from which the native CaP and VaP (Eisen et al., 1990) motoneurons had been removed. Transplanted cells were visualized with a Zeiss Universal Compound Microscope equipped with a Dark Invader low light level camera. Images were captured by using AxoVideo (Axon Instruments) and colorized with Photoshop (Adobe). RNA Injections and Analysis of Injected Embryos To make Xenopus Notch-ICD myc-tagged RNA, plasmid DNA was linearized with NotI (Chitnis et al., 1995). Transcription was performed by using Sp6 mmessage mmachine Kit (Ambion). After transcription, RNA was phenol/chloroform extracted and concentrated using Microcon YM-50 microconcentrator filter devices (Amicon). RNA quality was assayed by formaldehyde gel electrophoresis. The RNA was diluted in 1% phenol red and water to 0.8 pg/pl and was pressure injected into 1- to 2-cell stage embryos. The amount of injected RNA was approximately pg. All Notch-ICD-injected embryos, except those that were severely deformed and/or dying, were fixed at 28 h in 4% formaldehyde and processed for antibody labeling using a 3A10 monoclonal antibody (Developmental Studies Hybridoma Bank) and a polyclonal anti-c-myc antibody (Santa Cruz Biotechnology). Antibody labeling was visualized with an anti-mouse Oregon Green (Molecular Probes) and anti-rabbit Cy3 (Jackson ImmunoResearch) under an Axioplan compound microscope. Only embryos with myc expression in the head and hindbrain were analyzed for 3A10 staining. RESULTS In a mutagenesis screen designed to identify genes involved in motor axon guidance (Beattie et al., 1999), we isolated a mutation, b420, that had abnormal motor axons (see below) and that also caused defects in somite formation similar to those described for fused-somite-type mutants; a class including fused somites (fss), bea, des, aei, and mib (van Eeden et al., 1996, 1998). To determine whether b420 was an allele of any of these mutations, we performed complementation tests by pairwise matings between heterozygous b420 and heterozygous mib a52b, bea tm98, aei tr233, or des tp37 embryos. In crosses between heterozygous b420 and des tp37, approximately 25% of the embryos displayed the mutant phenotype indicating that these were mutations in the same gene (2 crosses; 403 embryos). b420 crossed with other mutants yielded only wild-type embryos. Consistent with zebrafish nomenclature (Mullins, 1995), we named

5 310 Gray et al. b420, des b420. Unlike the other 10 alleles isolated (van Eeden et al., 1996), and another allele described here, des b638, des b420 is a late embryonic lethal; larvae fail to form swim bladders and die at 9 10 days postfertilization (d). Somitogenesis Is Disrupted in des b420 Mutants While the first approximately 6 somites appeared normal at 18 h in des b420 mutants, the remainder formed incomplete somite boundaries (Fig. 1, Table 1). The number of correctly formed somites increased slightly over time with somite boundaries present for the first 8 or so somites at 48 h, suggesting that 2 3 somite boundaries may recover (Table 1). To examine the des b420 somite defect in more detail, we analyzed expression of a number of genes by RNA in situ hybridization. All of these genes were expressed in patterns consistent with those previously described for des mutants (Fig. 2; van Eeden et al., 1996; Durbin et al., 2000; Holley et al., 2000; Jiang et al., 2000; Sawada et al., 2000). In particular, the normal bands of her1 expression in the unsegmented mesoderm (Muller et al., 1996) were not distinct (Figs. 2A and 2D). myod, which is normally expressed in the posterior region of developing myotomes (Weinberg et al., 1996), was expressed throughout myotomes (Figs. 2B and 2E). However, myod expression was normal in rostral myotomes of des b420 mutants where somite boundaries were present (arrow in Fig. 2E). Genes expressed in the anterior portion of somites were also perturbed. For example, mespa, normally expressed in the anterior region of presumptive somites (Durbin et al., 2000; Sawada et al., 2000) lacked this restricted expression pattern in des b420 mutants (Figs. 2C and 2F). These data, as discussed previously (Durbin et al., 2000; Sawada et al., 2000), indicate that cells with anterior and posterior fates are not segregated within des mutant somites, but are mixed together resulting in no clear distinction between anterior and posterior somite and myotome regions. Spinal Motor Axons Are Disorganized in des b420 Mutants des b420 was originally identified by its motor axon defect (Beattie et al., 1999). znp1 antibody labeling at 24 and 48 h revealed that primary motor axons and motor nerves branched aberrantly and lacked their stereotyped morphology in segments posterior to somite 7 (Fig. 3). In the rostral trunk where somite and myotome formation was normal in des b420 mutants, motor axons were also normal (data not shown). To characterize axonal morphology in more detail, we labeled individual caudal and middle primary motoneurons (CaP and MiP) in spinal cord hemisegments corresponding to myotomes 8 10 where somite formation was aberrant, at 22 h with vital fluorescent dye, and visualized their axonal projections. We found that axons of both ventrally (CaP) and dorsally (MiP) projecting primary motor neurons had abnormalities including excessive branching, short axons and bifurcating axons (Fig. 4). To determine whether the defect in motor axons was cell-autonomous, we created genetic mosaics between wild-type and des b420 mutant embryos. Single CaP motoneurons were transplanted from labeled donor embryos (either wild-type or mutant) into unlabeled host embryos (either wild-type or mutant) before axogenesis and followed over time in living embryos to determine axonal morphology. In control transplants, consisting of wild-type CaPs transplanted into wild-type host embryos, the transplanted CaP always exhibited a normal morphology (Fig. 5A). When wild-type CaP motoneurons were transplanted into des b420 mutant hosts, however, they had an abnormal axonal morphology (Fig. 5B). CaP motoneurons from des b420 mutants transplanted into wild-type host embryos exhibited normal CaP axonal morphology (Fig. 5C). These experiments demonstrate that des function is not required in motoneurons for the development of normal axonal trajectories. Previous studies have shown that disrupting somitogenesis alters formation of motor axons (Eisen and Pike, 1991) and that anterioposterior myotome patterning may affect motor axon guidance (Bernhardt et al., 1998; Roos et al., 1999). Therefore, the most likely cause of the motor axon phenotype in des b420 mutants is disrupted anterioposterior myotome patterning. Trunk Neural Crest Migration Is Selectively Disrupted in des b420 Mutants In higher vertebrates, mesoderm patterning influences neural crest migration. In chick embryos, for example, neural crest cells migrate exclusively along the rostral sclerotome (Keynes and Stern, 1984; Rickmann et al., 1985; Bronner-Fraser, 1986; Teillet et al., 1987; Loring and Erickson, 1987). Ablation of the entire somite or experimental manipulations that result in rostral-only or caudal-only sclerotome results in disrupted neural crest migration and dorsal root ganglion (DRG) formation (Kalcheim and Teillet, 1989). In zebrafish, neural crest cells migrate ventromedially (referred to as the medial pathway) between somite and neural tube and ventrolaterally between myotome and skin (referred to as the lateral pathway) (Raible et al., 1992). Neural crest cells migrating along the medial pathway give rise to DRG neurons, sympathetic neurons, enteric neurons, glial cells, and pigment cells, whereas those migrating on the lateral pathway, only give rise to pigment cells (Raible and Eisen, 1994). Since somite and myotome patterning are disrupted in des mutants, we asked whether neural crest migration was also affected. crestin mrna is expressed in migrating neural crest cells (Rubinstein et al., 2000), but is much more strongly expressed in cells migrating along the medial pathway; pigment cells migrating along the lateral pathway begin to differentiate as they migrate (Raible et al., 1992) and concomitantly downregulate crestin expression (Luo et al., 2001). Analysis of crestin expression in des b420 mutants revealed that in the first approximately 6 7 somites, the pattern of crestin expression along the medial pathway was

6 Zebrafish deadly seven Functions in Neurogenesis 311 comparable to that seen in wild-type embryos (data not shown). crestin-expressing cells in segments posterior to somite 7, however, lacked the striking segmental pattern observed in wild-type embryos (Figs. 6A and 6B). We also examined the gene, dct, which is expressed in differentiating melanocytes as they migrate along both the medial and lateral pathways (Kelsh et al., 2000). In wild-type embryos, dct-expressing cells on the medial pathway are segmentally patterned, however this pattern is more subtle than that observed with crestin (Figs. 6A and 6E; Lou et al., 2000; Kelsh et al., 2000). We found that the pattern of dctexpressing cells was similar between wild-type and mutant embryos at 28 h on the medial pathway, although the segmental arrangement of dct-expressing cells in mutants was slightly less regular (Figs. 6E and 6F). We also found that dct-expressing cells along the lateral pathway in des b420 mutants looked comparable to wild-type dct-expressing cells along the lateral pathway (data not shown). To determine if neural crest derivatives were affected, we examined mutants at 3 4 d. Approximately, 35% of DRG neurons failed to coalesce into ganglia resulting in misplaced neurons (Figs. 6C and 6D; see also Fig. 9). We did not see any defect in cervical sympathetic ganglion or enteric neurons. These latter cell populations originate from the vagal/anterior trunk crest and migrate along the anterior 1 5 somites (Epstein et al., 1994; Raible et al., 1992) which are unaffected in des b420 mutants. When we analyzed the pattern of all three pigment cell types, melanophores, iridophores, and xanthophores at 3 5 days in mutant embryos, they looked indistinguishable from the pattern in wild-type embryos (Figs. 6G and 6H and data not shown). Due to the lack of molecular markers for glial cells in zebrafish, we cannot determine whether this population is affected in des b420 mutants. These data suggest that there are non-pigment neural crest cell precursors along the medial pathway in des b420 mutants whose segmental pattern of migration is disrupted. Since neural crest cells that give rise to DRG neurons migrate along the medial pathway, it is possible that this migration defect leads to the failure of the DRG to form correctly at 3 d. Neural crest migration along the lateral pathway appears unaffected. des b420 Mutants Have a Neurogenic Phenotype Two other fused somite-type zebrafish mutants, mib and aei/deltad, have somite defects similar to des, but also exhibit neurogenic phenotypes (Jiang et al., 1996; Schier et al., 1996; Holley et al., 2000). mib mutants exhibit a strong neurogenic phenotype, resulting in supernumerary primary neurons throughout the CNS and periphery and a concomitant reduction in secondary motoneurons, melanocytes, and eye and hindbrain radial glial cells (Schier et al., 1996; Jiang et al., 1996). A thorough characterization of the neurogenic phenotype in aei/deltad mutants has not been reported; however, they do have increased numbers of spinal primary sensory neurons (Holley et al., 2000). To determine whether des b420 mutants also had a neurogenic defect, we examined numerous cell types within the nervous system. Primary motoneurons. We used RNA in situ hybridization with anti-sense islet1 and islet2 probes to examine spinal motoneurons (Appel et al., 1995; Inoue et al., 1994). At 20 h, we found a subtle, but statistically significant increase in the number of motoneurons expressing islet2 in des b420 mutants (Fig. 7). islet2 expression is usually limited to 1 2 ventral cells per spinal hemisegment, corresponding to CaP and VaP motoneurons (Appel et al., 1995). In wild-type embryos, only 4% of the hemisegments had more than two cells in this location whereas in des b420 mutants approximately 20% of the hemisegments contained more than two ventral, islet2-expressing cells. This result was corroborated using islet1, which is expressed in MiP and RoP motoneurons at this stage of development (Appel et al., 1995). Again, we saw a small but statistically significant increase in islet1-expressing cells in the ventral spinal cord of des b420 mutants (Fig. 7C). Reticulospinal neurons. The embryonic zebrafish hindbrain contains a set of individually identified, primary reticulospinal interneurons that have characteristic cell body positions within rhombomeres 1 7 and stereotyped axonal projections that descend within the spinal cord (Metcalfe et al., 1986; Mendelson, 1986). Previous experiments demonstrated that perturbing Delta function in zebrafish by expression of wild-type or dominant negative forms of X-Delta-1 altered the number of Mauthner neurons, a conspicuous reticulospinal neuron present in hindbrain rhombomere 4 (Haddon et al., 1998), suggesting that Notch-Delta signaling determines Mauthner cell number. We visualized the primary reticulospinal neurons in des b420 mutants both by retrograde labeling (data not shown) and using a neurofilament monoclonal antibody (RMO44; Lee et al., 1987). We observed a dramatic increase in Mauthner cells present in rhombomere 4 and a significant increase in RoL2 cells present in rhombomere 2 and MiD3cm cells present in rhombomere 6 (Fig. 8). Other reticulospinal neurons, such as those present in oddnumbered rhombomeres 3, 5, and 7, and the T-interneurons in the caudal hindbrain were unaffected in des b420 (Table 2, and data not shown). We also examined reticulospinal neurons in two other des alleles; tp37 (Jiang et al., 1996; and b638, this report). We found that all alleles had an increase in Mauthner cells, RoL2 cells, and MiD3cm cells. b420 mutant embryos, however, had the greatest increase in Mauthner and MiD3cm cells, while tp37 mutant embryos were the least affected (Table 3). Indeed, the difference in Mauthner cell number between these two alleles was statistically significant (P 0.001; Table 3). Therefore, b420 may be a stronger allele compared to tp37 and b638. We examined other cells in des b420 mutants and found them to be unaffected (Table 2). Many of these cell types are increased or decreased in number in mib mutants (Schier et al., 1996; Jiang et al., 1996). Therefore, des mutant embryos exhibit a restricted neurogenic phenotype with only particular populations of neurons affected.

7 312 Gray et al. FIG. 4. CaP and MiP motor axons display abnormalities in des b420 mutants. Lateral view of pseudocolor images of live CaP (A, B) and MiP (C, D) motoneurons labeled with rhodamine dextran and visualized at approximately 26 h in wild-type (A, C) and des b420 mutant (B, D) embryos. Cell bodies reside in the spinal cord and white lines denote the dorsal and ventral aspects of the notochord (A, B) and the dorsal aspect of the notochord (C, D). Scale bar, 10 m. FIG. 5. des b420 is non-cell autonomous for CaP motoneurons. Single CaP motoneurons were removed from rhodamine dextran labeled donors at approximately 16 h, a time before axogenesis, and transplanted into stage-matched, unlabeled host embryos from which native CaP and VaP motoneurons had been removed. (A) Wild-type CaP transplanted into wild-type host (n 4). (B) Wild-type CaP transplanted into a des b420 mutant host (n 4). (C) des b420 mutant CaP transplanted into wild-type host (n 4). White lines denote dorsal and ventral aspects of the notochord. Scale bar, 10 m.

8 Zebrafish deadly seven Functions in Neurogenesis 313 FIG. 6. Migrating neural crest and neural crest derivatives are differentially affected in des b420 mutants. Lateral view of mid-trunk (segments 9 14) neural crest on the medial pathway as revealed by crestin expression (arrowheads) at 24 h in wild-type (A) and mutant (B) embryos. Cross section view of Hu-positive DRG neurons (arrowheads) in 72-h wild-type (C) and mutant (D) embryos. Lateral view of mid-trunk (segments 9 14) dct-expressing cells in wild-type (E) and mutant (F) embryos along the medial pathway. Pigment cells positioned in the lateral stripe in 3 d wild-type (G) and mutant (H) embryos. Scale bar, 50 m (A, B); 30 m (C F); 75 m (G, H). Reciprocal Effects on Primary and Secondary Sensory Neurons in des Mutants Rohon Beard (RB) cells are primary sensory neurons present in the dorsal spinal cord (Lamborghini, 1980; Metcalfe et al., 1990) which, like the neural crest cells that differentiate into DRG neurons, are derived from the lateral edge of the neural plate. It has been suggested that RB cells and neural crest form an equivalence group and their fates are determined by Notch Delta signaling during develop-

9 314 Gray et al. TABLE 2 Cell Types Unaffected in des b420 Mutants Cell type Age Probe wt b420 Trigeminal ganglion neurons 26 h anti-islet1 antibody Ear sensory hair cells a 28 h anti-acetylated tubulin antibody Statoacoustic neurons 24 h anti-islet1 antibody Hindbrain radial glial b 38 h Zrf1 antibody Hindbrain T-cells 48 h anti-neurofilament antibody Enteric neurons c 5 d anti-hu antibody Trunk melanocytes d 24 h dct mrna Trunk iridophores e 4 d NA Note. Cell types were analyzed at specific stages (Age) using either RNA in situ hybridization or immunohistochemistry (Probe). Under these conditions, we found no significant difference between the number of cells in mutant and in wild-type embryos. Cells were counted and reported as mean 95% confidence interval in 10 embryos/larvae except for hindbrain T cells (n 14) and statoacoustic neurons (n 7). a Ear sensory hair cells were counted in both the anterior and posterior maculae. b Hindbrain radial glial fiber bundles are present in pairs between rhombomeres. The number of individual glial fibers in each bundles was not quantitated. c Hu-positive neurons encircling the gut were counted in five cross sections per larvae from the mid-trunk region. d Trunk melanocytes were counted on one side of each embryo along the entire length of the trunk. e Trunk iridophores were counted on one side of each embryo along the entire length of the trunk using indirect light. ment (Cornell and Eisen, 2000). To determine whether des b420 mutants had a defect in this process, we examined RB and DRG neurons in mutant embryos. RNA in situ hybridization with an islet2 antisense probe at 20 h revealed that RB cells were decreased in des b420 mutants (Figs. 9A, 9B and 9E). To ensure that this reduction was not allele specific, we examined RB cell number in two other alleles, tp37 and b638, and found a similar reduction in RB cell number (see Fig. 9 legend for cell counts). Using the anti-hu antibody (Henion et al., 1996), we examined DRG neurons at 72 h. We found that in addition to the previously described defect in DRG ganglion formation, there was an increase in the number of DRG neurons in mutant embryos compared to wild types (Figs. 9C 9E). To determine whether this increase was specific for DRG neurons, we examined other trunk neural crest derivatives. We found no difference in the number of trunk melanocytes, iridophores, or enteric neurons (Table 2). There was also no obvious difference in the number of xanthophores, and cervical sympathetic ganglion neurons; however, these cells cannot be readily counted. Thus, the only trunk neural crest derivative that appears to be present in excess in des b420 mutants is DRG neurons. Supernumerary Mauthner Cells Are Born at the Same Stage in des b420 Mutants and Wild Types There are at least two scenarios for how supernumerary neurons could arise during development; a defect in cellcycle regulation or a defect in lateral inhibition. If excess Mauthner cells in des b420 mutants were produced by a defect in cell-cycle regulation, we would predict that cells would be generated over a protracted period of time. Alternatively, if the des mutant phenotype were caused by a defect in lateral inhibition, we would predict that all of the Mauthner cells might be born at the same time, since lateral inhibition occurs within a cluster of cells with equivalent developmental potential (see Greenwald, 1998). Thus, a disruption in lateral inhibition would result in the entire cluster of cells acquiring the same fate at presumably the same time. To differentiate between these two possibilities, we examined the birthdate of Mauthner cells in des b420 mutants using BrdU incorporation followed by RNA in situ hybridization with an anti-sense probe to val (Moens et al., 1998) to identify Mauthner cells. Wild-type Mauthner cells are born at approximately 7.5 h (Mendelson, 1986); the cell cycle at this time is 1 3 h (Kimmel et al., 1994). We performed BrdU incorporation at 7, 9, 11, and 14 h for 45 min followed by val RNA in situ hybridization at 19 h. If a Mauthner cell precursor was still dividing at any of these times, then we would expect the Mauthner cells it generated to be both BrdU and val positive. A Mauthner cell that has completed its last S-phase will only be val positive. In wild-type embryos, val-expressing Mauthner cells incorporated BrdU at 7 and 9 h and did not incorporate BrdU at 11 and 14 h (Fig. 10). All Mauthner cells examined in des b420 mutants incorporated BrdU at 7 h (Fig. 10). At 9 h, 78% were both val and BrdU positive and at 11 and 14 h, all val-positive cells were BrdU-negative. Thus, Mauthner cells in des b420 mutants were not generated over a protracted time period, but underwent their final cell division at a time coincident with the production of Mauthner cells in wild-type embryos. These data are consistent with the hypothesis that supernumerary Mauthner cells in des b420 mutants result from a defect in lateral inhibition.

10 Zebrafish deadly seven Functions in Neurogenesis 315 TABLE 3 Two Other des Alleles Have Supernumerary Reticulospinal Neurons Embryos (n) Mauthner RoL2 MiD3cm wt (19) b420 (14) b638 (16) tp37 (24) Note. Embryos were labeled at 48 h with the anti-neurofilament antibody, RMO44, and axons originating from cells on both sides of the midline were counted. Numbers are reported as mean 95% confidence interval. Differences in the mean cell counts of Mauthner cells, RoL2 cells, and MiD3cm cells of the three alleles compared to wild-type embryos were statistically significant (P 0.01 P 0.001). The difference in the means between the number of Mauthner cells in b420 and tp37 was also statistically significant (P 0.001). All other differences between the three alleles were not statistically significant. Activated Notch Rescues the Mauthner Cell Phenotype in des b420 Mutants To further investigate whether the neurogenic defect in des b420 mutants is caused by a disruption in lateral inhibition, we asked if an activated form of Notch could rescue the mutant phenotype. When a constitutively active form of Xenopus Notch lacking its extracellular domain was expressed in Xenopus embryos, it inhibited primary neurogenesis (Notch ICD or Notch E; Coffman et al., 1993; Chitnis et al., 1995). Injecting RNA encoding Notch E into zebrafish embryos also blocked primary neurogenesis (Haddon et al., 1998). We focused our analysis on Mauthner cells, as they are the most dramatically affected neuronal cell-type in des b420 mutants. In wild-type embryos, there is one Mauthner cell on both the right and left sides of rhombomere 4. Even though we analyzed embryos that expressed the myc-tagged Notch-ICD RNA throughout the brain, we analyzed each side separately since exogenous RNA may be distributed differently between the two sides. When embryos from heterozygous des b420 matings were mock-injected with phenol red (a tracer for injections) at the 1- to 2-cell stage, the expected ratio of mutants and wild types was observed with approximately 75% of the sides possessing one Mauthner cell and the remaining 25% of the sides having between 4 and 9 Mauthner cells (Fig. 11, Table 4). When embryos from wild-type matings were injected with RNA encoding Notch-ICD, we found that 34.8% of the sides had zero Mauthner cells whereas 65.2% of the sides had the expected single Mauthner cell (Fig. 11, Table 4). When embryos from heterozygous des b420 matings were injected with RNA encoding Notch-ICD, only 5.5% of the sides had more than 4 Mauthner cells while 19.6% had 2 or 3 Mauthner cells (compare to 0% of the sides having this number in mockinjected embryos from heterozygous des b420 matings). Moreover, 12.4% of the sides had no Mauthner cells (Table 4). Although we were not able to determine the genotype of injected embryos, approximately 17% had 2 or more Mauthner cells on one side and zero or 1 Mauthner cell on the other side (Fig. 11D). Since wild-type embryos never have more than 1 and mutants always have more than 3 Mauthner cells per side (Figs. 11A and 11B), these embryos are likely homozygous mutants that have a decreased number of Mauthner cells due to the presence of activated Notch. Therefore, using an activated form of Notch, we were able to decrease the number of Mauthner cells in des b420 mutant embryos supporting the idea that des acts in the Notch Delta signaling pathway, at the level of or upstream of, the Notch receptor. DISCUSSION Here, we describe the characterization of a mutant allele of the zebrafish des gene. In agreement with other work (van Eeden et al., 1996; Durbin et al., 2000; Sawada et al., 2000), our studies show that des is critical for anterioposterior patterning within the somite and myotome. Our genetic mosaic experiments support the idea that this mispatterning affects motor axon outgrowth. The normal segmented pattern of neural crest migration is aberrant for some populations of neural crest cells migrating along the medial pathway. Moreover, DRG are disorganized suggestive of a correlation between these two defects. Our finding that des mutants have a restricted neurogenic phenotype suggests that des functions to regulate the number of neurons in specific regions of the central and peripheral nervous system. Spatial localization of des expression or temporal control of des expression during development could generate this specificity. To investigate whether des functions in lateral inhibition via the Notch Delta pathway, we determined when the supernumerary Mauthner cells in des mutants were born and whether activated Notch could rescue the Mauthner cell phenotype. Data from both of these experiments support the hypothesis that des functions in subsets of cells where it is involved in Notch Delta signaling during Mauthner cell-fate specification. Although des b420 is lethal, all the other alleles of des are viable (van Eeden et al., 1996), including the b638 allele also used in this study. All of these mutations, except the b638 allele, were induced by ENU mutagenesis of spermatogonial stem cells, they are likely point mutations or small deletions (Knapik, 2000). b638 was induced by ENU mutagenesis of postmeiotic sperm (Riley and Grunwald, 1995). Since the phenotypes of the different alleles are similar, it is possible that another gene is affected in des b420 which causes the lethality. Motor Axon Outgrowth and Neural Crest Migration in des b420 Mutants In mouse and chick, anterioposterior patterning of the sclerotomal compartment of the somite functions to pat-

11 316 Gray et al. FIG. 7. des b420 mutants have excess primary motoneurons. CaP and VaP motoneurons (arrowheads) as revealed by islet2 RNA in situ hybridization at 20 h in a wild-type (A) and mutant (B) embryo. (C) Counts of islet1 and islet2 expressing cells at 20 h in spinal hemisegments 5 9; n 10 embryos for each point. For these and all subsequent histograms the mean 95% confidence interval is plotted. Significance was determined by Student s t test with *, P ; **, P Scale bar, 25 m. FIG. 8. Supernumerary reticulospinal neurons are present in des b420 mutants. Dorsal view of a confocal image (anterior to the top) of a RMO44 labeled wild-type (A) and des b420 mutant (B) embryo. Somata of affected cells are labeled in (A) and arrowheads point to their corresponding axons. Note the increased number of axons in the mutant embryo. (C) Cell counts of affected reticulospinal neurons. Cells on both sides of the midline were analyzed for wild-type (n 14) and mutant (n 14) embryos. See Fig. 7 legend for details regarding statistics. Mth, Mauthner cell. Scale bar, 20 m. tern motor nerves and neural crest (Keynes and Stern, 1984; Rickmann et al., 1985; Bronner-Fraser, 1986; Teillet et al., 1987; Loring and Erickson, 1987; Krull and Koblar, 2000). In contrast, zebrafish sclerotome does not appear important for motor nerves or neural crest migration, rather the myotome appears to function in this capacity (Morin- Kensicki and Eisen, 1997). Myotome is the largest component of the zebrafish somite (Kimmel et al., 1995) and gene expression patterns have revealed anterioposterior patterning within this tissue (reviewed in Stickney et al., 2000). Motor axons extend along developing myotomes during the first day of development and Bernhardt et al. (1998) have shown that the CaP axon extends along the anterior region of the medial myotome immediately adjacent to the border between anterior and posterior myotome. Because somitic anterioposterior polarity is severely disturbed in des mutants, we were not surprised to see a pathfinding defect. We extend this observation using genetic mosaic analysis to demonstrate that the motor axon defect in CaP is non-cell autonomous. Because motor axons extend directly out of the spinal cord and onto the myotome, it is likely that the motor axon defect is caused by the lack of distinction

12 Zebrafish deadly seven Functions in Neurogenesis 317 FIG. 9. Analysis of trunk sensory neurons in des b420 mutants. Dorsal view (anterior to the left) of a 20-h wild-type (A) and mutant (B) embryo showing islet2 expressing RB neurons. Arrowheads denote the absence of RB neurons in the spinal cord of des b420 mutants. All three des alleles examined had a similar decrease in RB cells (b , b , and tp compared to wild type ). Lateral view of 72-h wild-type (C) and mutant (D) embryo showing Hu-expressing DRG neurons (arrowheads). (E) islet2-expressing RB neurons and Hu-expressing DRG neurons were counted in hemisegments 5 9; n 10 embryos for RB counts and n 6 embryos for DRG counts. See Fig. 7 legend for details regarding statistics. Scale bar, 20 m. between anterior and posterior myotome regions. However, we have not ruled out the possibility that other tissues are involved. Zebrafish neural crest cells also migrate along the somite and developing myotome (Raible et al., 1992). The neural crest defect in des b420 mutants is intriguing as certain populations of neural crest are affected while others are not. For example, streams of crestin-expressing cells migrating along the medial pathway lack their segmental pattern. DRG neurons, which are derived from neural crest cells that migrate along the medial pathway (Raible and Eisen, 1994), are misplaced in mutant embryos and often fail to coalesce into ganglia. However, dct-expressing cells migrating along the medial pathway apparently retain their

13 318 Gray et al. FIG. 10. Supernumerary Mauthner cells in des b420 mutants are formed at the same time as Mauthner cells in wild-type embryos. Numerical representation of Mauthner cell birthdays in wild-type and des b420 mutants. At 7 h, all Mauthner cell precursors examined in wild-type (n 13) and des b420 (n 64) were undergoing DNA synthesis. At 9 h, all wild-type (n 16) and 78% of mutant (n 46) Mauthner cell precursors were still dividing. At 11 and 14 h, all Mauthner cells in both wild-type (n 16, n 15) and mutant (n 65, n 87) embryos had undergone their last S-phase. val-expressing cells (n) in 10 embryos were examined and the mean 95% confidence interval were plotted. Due to cross sectional analysis, not all Mauthner cells present in each embryo were analyzed. segmental migration pattern and their derivatives, melanocytes, are unaffected in mutant embryos. One interpretation of these data is that neural crest cells migrating along the medial pathway that give rise to DRG neurons are affected, either cell autonomously or non-cell autonomously, in des b420 mutants whereas cells that give rise to melanocytes are unaffected. This hypothesis is supported by data showing that there are populations of fate-restricted neural crest cells (Raible and Eisen, 1994; Henion and Weston, 1997; Ma et al., 1998) and some zebrafish trunk neural crest cells become lineage-restricted before they reach their final location (Raible and Eisen, 1994). Neural crest specified to become DRG neurons may be more sensitive to the disruption of somitic mesoderm anterioposterior patterning than neural crest cells specified to become melanocytes. Even without evoking fate restriction, it is possible that there is differential sensitivity to disrupted anterioposterior pattern by unique progeny of multipotent stem cells (see Anderson, 1997). des Regulates Neuronal Cell Number Unlike the zebrafish mutant, mib, which contains a dramatic excess of neurons throughout the nervous system (Jiang et al., 1996; Schier et al., 1996), des b420 has a different neurogenic profile. In the hindbrain, only the number of Mauthner cells is increased dramatically. Two other hindbrain reticulospinal neurons, RoL2 and MiD3cm show a modest increase in number, whereas other reticulospinal neurons appear unaffected. In the des b420 mutant trunk, there is also an increase in the number of primary motoneurons and DRG neurons and a decrease in the number of RB neurons. Our finding that only certain subpopulations of neurons are regulated by des is intriguing. If des is acting in the Notch Delta signaling pathway, we speculate that it only acts in a subpopulation of cells. Analysis of zebrafish notch and delta genes reveal both overlapping and unique patterns of expression throughout the nervous system and mesoderm (Bierkamp and Campos-Ortega, 1993; Dornseifer et al., 1997; Westin and Lardelli, 1997; Appel and Eisen, 1998; Haddon et al., 1998; Smithers et al., 2000). Thus, mutations in these genes or in their specific downstream components could cause a restricted neurogenic phenotype like that seen in des mutants. Eventual cloning of the des gene and localization of its product will clarify how des function is spatiotemporally regulated. des Regulates the Number of Trunk Sensory Neurons The relationship between RB cells and neural crest has recently been investigated by examining deltaa mutant embryos. Cornell and Eisen (2000) found that in deltaa mutants there were supernumerary RB cells and a concomitant decrease in trunk neural crest derivatives, including DRG neurons and melanocytes. These results parallel what was found when a dominant negative form of delta, that apparently reduces function of all Delta proteins, was expressed in embryos (Haddon et al., 1998; Appel and Eisen, 1998). Moreover, aei/deltad mutants also have supernumerary RB cells (Holley et al., 2000). These findings indicate that the function of DeltaA and DeltaD is to limit the number of cells that take on the RB cell fate. We also found a reciprocal relationship between RB cells and DRG neurons in des b420 mutants; but surprisingly, it was in the opposite direction. Throughout the dorsal spinal cord we found a sporadic decrease in RB cells; a curious finding because other neurogenic mutants have increased numbers of RB cells (Holley et al., 2000; Cornell and Eisen, 2000). Raible and Eisen (1996) showed that interactions, such as lateral inhibition, among neural crest cells influence cell fate. Moreover, in chick DRG, Morrison et al. (2000) showed that activated Notch causes a decrease in the number of DRG neurons and an increase in glial cells whereas inhibiting Notch function enhanced neurogenesis. Thus, because there is precedent for Notch Delta signaling functioning in cell fate determination among neural crest cells, it is not surprising that decreasing des, which acts in the Notch Delta pathway, also affects cell fate determination within the neural crest. Since RB cells and neural crest arise from an equivalence group (Cornell and Eisen, 2000), a change in the neural crest cell population could certainly have an effect on RB cell number.

14 Zebrafish deadly seven Functions in Neurogenesis 319 FIG. 11. Notch rescues the des b420 Mauthner cell defect. Embryos from wild-type or heterozygous des b420 matings were injected at the 1- to 2-cell stage with RNA encoding Notch-ICD-myc (Coffman et al., 1993; Chitnis et al., 1995). Embryos were fixed at 28 h and processed for 3A10 antibody labeling to visualize Mauthner cells. (A) Mock-injected wild-type embryos have one Mauthner cell on either side of hindbrain rhombomere 4. (B) Approximately 25% of mock-injected embryos from a heterozygous des b420 mating have supernumerary Mauthner cells. Injecting RNA encoding Notch-ICD decreases the number of Mauthner cells in wild-type (C) and des b420 mutant (D) embryos. The Number of Mauthner Cells Is Controlled by des-mediated Lateral Inhibition Previous studies suggest that within the hindbrain, more than one cell can take on the Mauthner cell fate (reviewed in Kimmel and Model, 1978) supporting the idea that lateral inhibition within an equivalence group regulates the number of Mauthner cells. More recent experiments demonstrate a direct role for Notch Delta mediated lateral inhibition in Mauthner cell generation. Haddon et al. (1998) blocked Notch Delta signaling by over-expressing a construct encoding a dominant negative form of delta (X- Delta1 dn ) and found extra Mauthner cells in the hindbrain. Conversely, over-expressing full-length delta (X-Delta1) resulted in a reduction in Mauthner cell number. In this study, we find that injecting RNA encoding an activated form of Notch into embryos at the 1- to 2-cell stage, causes a decrease in Mauthner cells in both wild-type embryos and embryos from heterozygous des b420 matings. Our interpretation of these results is that des is a gene that functions in lateral inhibition either epistatic to, or at the level of, Notch and Delta. Cells expressing Notch-ICD fail to take on the primary, Mauthner cell fate and instead take on the secondary, non-mauthner cell fate. At this time, we do not know what these cells become. We were never able to completely eliminate Mauthner cells in embryos from des b420 matings; however, in 17% of wild-type embryos we did eliminate both Mauthner cells. When Xenopus X-Delta1 was overexpressed, 27% of injected wild-type zebrafish lacked both Mauthner cells (Haddon et al., 1998). The discrepancy in these numbers may be due to the effectiveness of the gene in suppressing Mauthner cell-fate specification or the concentration of RNA. We did not analyze embryos that were overtly aberrant suggestive of overly high concentrations of injected RNA. Analysis of