1 Albert-Ludwigs-Universität Freiburg, Institut für Biologie I, Hauptstrasse 1, D-79104 Freiburg i. Br., Germany
2 Max Delbrueck Center for Molecular Medicine, Robert-Roessle Strasse 10, D-13125 Berlin, Germany
3 Department of Organismal Biology and Anatomy, University of Chicago, 1027 E. 57th St, Chicago, IL 60637, USA
*Author for correspondence (e-mail: driever{at}uni-freiburg.de)
Accepted 26 December 2001
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SUMMARY |
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Key words: Rhombomere, Hox genes, ephA4, kreisler, Krox20, krx20, rtk1, spg, valentino, Danio rerio
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INTRODUCTION |
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Members of the vertebrate Hox gene family are expressed in partially overlapping domains in the embryonic hindbrain with anterior expression borders that coincide with distinct segmental boundaries (reviewed by McGinnis and Krumlauf, 1992). The results of targeted gene inactivation of individual Hox genes in mouse revealed that Hox genes are involved in conferring rhombomeric identity (Goddard et al., 1996
; Studer et al., 1996
), and suggested that at least some Hox genes may play a role in the segmentation process itself (Carpenter et al., 1993
; Dollé et al., 1993
; Mark et al., 1993
; Gavalas et al., 1997
).
Krox20 (Egr2) (Wilkinson et al., 1989) and kreisler (kr/Mafb) (Cordes and Barsh, 1994
) have been shown to be direct transcriptional regulators of distinct Hox genes in the hindbrain. Krox20 is activated before morphologically visible segmentation in the r3 and r5 primordia (Wilkinson et al., 1989
; Oxtoby and Jowett, 1993
) and directly regulates the expression of Hoxa2 (Nonchev et al., 1996
) and Hoxb2 (Sham et al., 1993
) in these rhombomeres. Targeted inactivation of Krox20 leads to progressive disappearance of r3 and r5 during development (Schneider-Maunoury, 1993
; Swiatek and Gridley, 1993
; Schneider-Maunoury et al., 1997
). kr is required for specification of r5 and r6 (Frohman et al., 1993
; Cordes and Barsh, 1994
; McKay et al., 1994
) and has been shown to be a direct regulator of Hoxa3 and Hoxb3 in r5 (Manzanares et al., 1997
; Manzanares et al., 1999
). In contrast to our knowledge of Hox gene regulation, however, relatively little is known about the control of Krox20 and kr expression.
Another family of regulatory genes with members expressed in the developing hindbrain is the POU gene family (Ryan and Rosenfeld, 1997). POU genes are expressed in the zebrafish CNS in distinct but overlapping domains during embryonic development (Takeda et al., 1994
; Hauptmann and Gerster, 1995
; Hauptmann and Gerster, 1996
; Spaniol et al., 1996
; Hauptmann and Gerster, 2000a
). Zebrafish spiel ohne grenzen (spg) mutants were isolated based on their failure to develop a normal mid/hindbrain boundary (Schier et al., 1996
) and we have identified pou2 (Takeda et al., 1994
; Hauptmann and Gerster, 1995
) as the gene affected in these mutants (Belting et al., 2001
). Here, we provide evidence for the involvement of spg/pou2 in regional patterning of the hindbrain primordium. Segment-specific expression of hox genes, krx20, ephA4 and val is altered in spg mutants, indicating reduction of r1, r3, r5 and r6 territories. Since both the size and shape of rhombomeric territories are altered in strong spg mutants from the beginning of segmentation, pou2 appears to function in an early step of hindbrain regionalization to ensure the establishment of normal rhombomere precursor territories. Our findings suggest that spg/pou2 acts before krx20 (egr2) and val in hindbrain segmentation.
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MATERIALS AND METHODS |
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Retrograde labeling of reticulospinal neurons
Retrograde labeling of reticulospinal neurons was performed essentially as described previously (Moens et al., 1996). Three-dimensional images of tetra-methyl-rhodamine dextran-labeled reticulospinal neurons were reconstructed using a confocal Zeiss LSM 510 laser scanning microscope, and were depth-coded in color using the LSM 510 3D imaging software.
Whole-mount in situ hybridization and immunohistochemistry
Standard methods for one- and two-color whole-mount in situ hybridization (WISH) were used (Hauptmann and Gerster, 1994; Hauptmann and Gerster, 2000b
). Cryosections were prepared following WISH. Control embryos, denoted wild-type in the figures, are phenotypically wild-type siblings (spg/+ or +/+) of mutant embryos shown in the same experiment.
The wild-type expression patterns of the genes analyzed in this study and the cDNAs used for generation of probes have been described in the references provided. The gene previously described as hoxa1 (Alexandre et al., 1996) has recently been assigned to the hoxbb cluster (Amores et al., 1998
), and is therefore termed hoxb1b. The gene previously termed hoxb1 (Prince et al., 1998
) has been assigned to the hoxba cluster and is therefore termed hoxb1a. As there are no duplicates of the other hox genes used as probes in this study, the a or b assignment for duplicated clusters (Amores et al., 1998
) has been omitted from their names. For identification of Mauthner cells, we used the monoclonal antibody 3A10 (Hatta, 1992
).
Microinjection of mRNA
pou2 cDNA (Hauptmann and Gerster, 1995) was subcloned into the pCS2+ vector and transcribed using the Sp6 Message Machine kit (Ambion). About 20 pg in vitro synthesized mRNA was microinjected into one-cell stage embryos using pressure driven manual microinjectors. In some experiments co-injection of lacZ mRNA was used to monitor the distribution of overexpressed proteins. After whole-mount in situ hybridization and photographic documentation, spgm793 homozygous mutant embryos were identified by PCR-genotyping as described previously (Belting et al., 2001
).
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RESULTS |
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In wild-type embryos at the 22-somite stage, r2 through r6 are recognized as an anteroposterior series of similar-sized bulges, with the otic vesicle lying lateral to r5 (Fig. 1A). In spgm216 embryos, the r3 and, to a lesser extent, the r5 bulges are reduced in size, while those of r2 and r4 are enlarged (Fig. 1B). In spgm793 mutants, most rhombomere boundaries are not discernible (Fig. 1C). In addition, the otic vesicle is shorter anteroposteriorly, resulting in a circular rather than an oval shape (Fig. 1B,C).
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To determine whether the segmental pattern of neuronal differentiation in the hindbrain is also altered in spg mutants, we assayed the segmental distribution of specific subsets of hindbrain neurons. In wild-type embryos at 1 dpf, lim1-positive neurons are arranged in two transverse stripes in each rhombomere (Fig. 1G) (Toyama and Dawid, 1997). In spgm216 mutants, only a single lim1-positive domain is detected in each of r1, r3, and r5 (Fig. 1H), consistent with reduction of these rhombomeres. In spgm793 mutants, individual lim1 expression domains cannot easily be assigned to specific segments. In addition, ectopic lim1-positive neurons are found in aberrant dorsal locations within the preotic hindbrain (Fig. 1I).
In wild-type embryos at 5 dpf, a series of defined reticulospinal neurons can be visualized by retrograde labeling and each neuron recognized based on cell size, position, and projections (Fig. 1J) (Kimmel et al., 1982). In spgm793 mutants, reticulospinal neurons cannot be visually identified (Fig. 1L). Spherical neuronal cell bodies are observed, but these exhibit no segment-specific morphology. Accordingly, Mauthner cells, primary reticulospinal neurons that form in r4 (Kimmel et al., 1981
), are not detected in spgm793 mutants by a Mauthner cell specific antibody (3A10; data not shown). Weak spgm216 mutants show relatively normal primary reticulospinal neurons in rhombomeres 2-5 (Fig. 1K). Analysis of 15 spgm216 mutants revealed that expressivity of the phenotype ranges in severity from the embryo shown in Fig. 1K to neuronal disorganization similar to that of spgm793 mutants (Fig. 1L). Duplicated (Fig. 1K) or lost Mauthner cells are occasionally observed in spgm216 mutants (in 2 and 3, respectively, of 15 embryos). Our results show that disruption of hindbrain segmentation in spg mutants is apparent at the cellular level and affects various hindbrain neurons.
Altered hox gene expression in spg mutants
To investigate hindbrain segmental patterning in spg mutants, we assayed hox (Prince et al., 1998) and krx20 (Oxtoby and Jowett, 1993
) expression at the 10-somite stage. As each rhombomere is characterized by a distinct combination of hox gene and krx20 expression, we were able to identify r2 to r7 individually in wild-type, spgm216 and spgm793 embryos (Fig. 2A-W). Our results are summarized in schematic drawings (Fig. 2X,Y,Z).
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In spgm793 mutants, the krx20 expression domains in r3 and r5 are even smaller than in spgm216 mutants, and are split into bilateral patches that are displaced laterally and develop at variable dorsoventral positions (Fig. 2I,L). Both the hoxb1a domain (Fig. 2I1,I2) and the low-level expression domain of hoxb2 (Fig. 2O1,O2), corresponding to r4, are variably enlarged, and extend medially between the lateralized krx20 domains. These changes in gene expression indicate that medial regions normally fated to form r3 and r5 may have acquired some aspects of r4 identity. The lateral displacement of r3 and r5, as assayed by krx20 expression, appears to lead to the medial juxtaposition of r2, r4, and r6 (see schematic drawing in Fig. 2Z). In spgm793 mutants, high-level hoxa2 expression indicative of r2 directly juxtaposes low-level hoxa2 expression corresponding to r4 (Fig. 2C). Similarly, hoxb1a and hoxb2 expression marking r4 abuts strong hoxa2 expression in r2 (compare Fig. 2I,O with 2C). Thus, r2 and r4 territories appear to directly juxtapose in spgm793 mutants. Elevated hoxb3 expression between the lateralized r5 patches likely indicates r6 identity (Fig. 2R1), and directly adjoins the posterior side of r4 (compare Fig. 2R1 and 2I1/2O1). Similarly, the anterior expression limit of hoxd3, which normally correlates with the r5/r6 boundary (Fig. 2S), extends anteriorly between the lateralized r5 patches (Fig. 2T), reaching the r4 domain (compare Fig. 2T with 2I1/2O1). Thus, r4 and r6 are also juxtaposed in spgm793 mutants. In the most strongly affected spgm793 mutants, elevated hoxb3 expression is seen only laterally, indicating medial disruption of both r5 and r6 (Fig. 2R2). Expression of hoxb4, which has an anterior boundary within r7 at this stage, is not obviously altered in spgm793 mutant embryos (Fig. 2W).
Taken together, our analysis shows that in all spg mutants, each rhombomere expresses the normal complement of hox genes, indicating normal segmental identity. Differences in the spatial extent of individual hox and krx20 expression domains, however, show that hindbrain segmentation is altered.
spg is required for formation of normal krx20 and val expression domains
Krox-20/krx20 (Oxtoby and Jowett, 1993; Schneider-Maunoury, 1993
; Swiatek and Gridley, 1993
) and kr/val (Cordes and Barsh, 1994
; Moens et al., 1998
) are thought to be required for development of r3/r5 and r5/r6, respectively. In wild-type embryos, krx20 expression is first detected during late gastrula stages (100% epiboly) in bilateral stripes in the r3 primordium (Fig. 3A) and slightly later in that of r5 (Oxtoby and Jowett, 1993
). At about the same time, val expression begins in bilateral stripes corresponding to the r5 and r6 primordia (Fig. 3C) (Moens et al., 1998
). The initially bilateral expression domains of krx20 (Fig. 3E,I) and val (Fig. 3G,K) fuse at the midline during early somitogenesis stages. In spgm793 mutants, the initial activation of krx20 (Fig. 3B1, and slightly later Fig. 3B2) and val (Fig. 3D1,D2) is affected and occurs only in reduced bilateral expression domains, each composed of dispersed groups of cells. These bilateral domains do not properly extend towards the midline. During early segmentation stages, the krx20- and val-positive cells form lateral cell clusters (Fig. 3F,H,J,L), which may be connected by a very thin band of labeled cells (Fig. 3L). The krx20- and val-positive cell clusters in r5 and r5/r6, respectively, increase in size over time (compare Fig. 3F,H with 3J,L), but still remain smaller than the wild-type domains of the corresponding stage (Fig. 2J,L). krx20 expression in r3 is more severely affected and remains restricted to very tiny cell clusters that only slightly increase in size (Fig. 3F,J). Taken together, these data show that spg/pou2 is required for two aspects of the establishment of normal krx20 and val expression. First, the initial expression of these genes is reduced in spgm793 mutants, and second, the condensation of the initially bilateral expression domains across the midline is impaired.
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Segmental expression of ephA4 is altered in spg mutants
In mouse, Krox20 has been shown to directly activate the expression of Epha4 in r3 and r5 (Theil et al., 1998). We therefore examined the expression of ephA4 (efna4/rtk1) (Xu et al., 1995
) in spg mutant embryos. Shortly after the onset of krx20 expression, ephA4 expression begins in r3 (from tailbud stage) and r5 (from the 3-somite stage onwards; Fig. 5A) (Xu et al., 1995
). In spg mutants, alterations in ephA4 expression are evident during early somitogenesis. By the 10-somite stage, ephA4 expression in r3 and r5 is slightly reduced in anteroposterior extent in spgm216 mutants (Fig. 5B), while absent medially in spgm793 embryos (Fig. 5C). At 1 dpf, ephA4 is also expressed in a thin domain in r1 (Fig. 5D-I). In spg mutants, in addition to persistent alterations in r3 and r5, ephA4 expression is also absent medially in r1 (Fig. 5H,I). The r1 ephA4 domain is at an oblique angle, indicating a reduction of r1 on the ventral side in spg mutants (Fig. 5E,F).
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Midline gene expression in spg mutants appears normal
In spgm793 mutants, alterations in mid- and hindbrain gene expression are most severe in the medial neural plate. In this region, other genes expressed in wild-type axial mesendoderm and/or ventral CNS may mediate the spg mutant phenotype. We therefore investigated whether alterations in midline gene expression can be detected between 80% epiboly and the 2-somite stage, the developmental time period when defects in the mid- and hindbrain primordia of spg mutants are first observed. Midline expression of cyclops/znr1/ndr2 (Rebagliati et al., 1998; Sampath et al., 1998
), taram-a (Renucci et al., 1996
), lefty1/antivin (Bisgrove et al., 1999
; Thisse and Thisse, 1999
), shh (Krauss et al., 1993
), twhh (Ekker et al., 1995
), ehh (Currie and Ingham, 1996
), chordin (Schulte-Merker et al., 1997
), noggin1 (Fürthauer et al., 1999
), ntl (Schulte-Merker et al., 1992
), flh (Talbot et al., 1995
), fkd1/axial (Strähle et al., 1993
), fkd2 and fkd4 (Odenthal and Nüsslein-Volhard, 1998
) was present in all analyzed spgm793 mutant embryos (each gene analyzed at one or two time points; data not shown). At the beginning of somitogenesis, the expression levels of twhh in the prechordal plate appeared more variable in spgm793 mutants than in wild-type embryos. However, the twhh domain is anterior to the segmentation defects observed in the hindbrain. Taken together, this suggests that pou2 is not required for normal activation of midline expression of these genes in or beneath the hindbrain primordium.
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DISCUSSION |
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In spgm216 mutants, the even-numbered rhombomeres, r2 and r4 are expanded along the anteroposterior axis, whereas odd-numbered ones, r1, r3 and, to a lesser extent, r5 are shortened (Figs 2Y, 5E,H). This suggests that spg/pou2 is required for normal anteroposterior hindbrain patterning and may play an essential role in defining the anteroposterior spatial extents of individual rhombomeric domains.In addition to abnormal rhombomere sizes, in spgm793 mutants r1, r3, r5, and in strongest mutants also r6 are disrupted in the midline of the neural plate (Fig. 2Z). The medial disruptions of rhombomere precursor territories in spgm793 mutants may result from enhanced deficiencies in anteroposterior patterning. These may cause increased thinning of prospective r3 and r5 territories, such that the bilateral rhombomeric primordia fail to fuse across the midline of the neural plate, allowing even-numbered rhombomeres to juxtapose. Since cells of the same parity can mix with each other relatively freely (Guthrie and Lumsden, 1991; Guthrie et al., 1993
), the juxtaposition of r2, r4, and r6 in spgm793 mutants could lead to irreversible fusion of these even-numbered segments. As a consequence, the krx20-expressing cell patches of r3 and r5 identity may be excluded from the fused r2/r4/r6 territory and pushed towards the lateral edges of the hindbrain. Consistent with this idea, lateral protrusions of r3 and r5 from the neural keel are often observed in spgm793 mutants (Fig. 2I1,I2,L).
Alternatively, the medial rhombomeric disruptions may point to a specific requirement for spg/pou2 along the midline of the neural plate. Likewise, expression of pax2.1 in the prospective posterior midbrain is also most strongly affected in the medial neural plate of spgm793 mutants (Fig. 6B) (Belting et al., 2001). This raises the question of whether spg/pou2 may be involved in mediolateral patterning of both the midbrain and hindbrain primordia. Vertical signaling from the axial mesendoderm has previously been implicated in patterning the medial (later ventral) neural primordium (Placzek et al., 1993
). However, expression of ntl (Schulte-Merker et al., 1992
) and flh (Talbot et al., 1995
), both essential for zebrafish notochord development (Halpern et al., 1993
; Talbot et al., 1995
), is not altered in spgm793 mutants, suggesting normal specification of the notochord precursor underlying the hindbrain primordium. Further, pou2 is not expressed in the axial hypoblast and mesendoderm. Thus, if pou2 acts in mediolateral patterning, it acts only within the neuroectoderm. For example, pou2 may be involved in interpreting mesendoderm derived midline signals. Nodal and Sonic hedgehog signals have been implicated in dorsoventral patterning of the neural tube at all axial levels (Rubenstein and Beachy, 1998
; Schier and Shen, 2000
). In spgm793 mutants, we found some variability in the expression of twhh, while the expression of other Hh homologs and Nodal pathway genes was not altered. However, homozygous cyclops mutants display normal expression of pax2.1 and krx20 in the mid- and hindbrain primordia, respectively, despite elimination of twhh and reduction of shh midline expression (Krauss et al., 1993
; Ekker et al., 1995
; Sirotkin et al., 2000
). This suggests that the mediolateral abnormalities in mid- and hindbrain gene expression in spgm793 mutants are probably not caused by defective Hh or Nodal midline signaling.
Other developmental mechanisms could also account for the reduction of rhombomere precursor territories in spg mutants. Examination of cell death using the terminal nuclear transferase assay did not reveal specific patterns of dying cells in spgm793 mutants in the mid-/hindbrain region during gastrulation. Increased cell death scattered throughout the neural keel in spgm793 mutants was detected from the 2-somite stage onwards (data not shown). This, however, could not cause the earlier and selective reduction of distinct rhombomere precursor territories observed. Between 70% epiboly (7.7 hpf) and the 3-somite stage (11 hpf), deep cells that form all embryonic lineages undergo only one cell cycle (cycle 14, around 8.2 hpf) (Kane et al., 1992), so changes in cell proliferation could not cause the observed patterning defects. The reduction of odd-numbered rhombomeres may result from reduced recruitment of progenitor cells into the hindbrain territory. However, the expression domains of gbx1 and fgf8, which prefigure the anterior hindbrain territory including prospective r3, are of similar size in wild-type and spgm793 mutant embryos during late gastrula stages (Belting et al., 2001
).
Impaired hindbrain segmental boundary formation in spg mutants
Transplantation experiments in chick have shown that segmental boundaries do not usually form between even-numbered rhombomeres. The results from these experiments suggest that boundary formation in the hindbrain requires the juxtaposition of alternating odd- and even-numbered rhombomeres as these may have distinct cell adhesion properties (Guthrie and Lumsden, 1991; Guthrie et al., 1993
). Recent studies in zebrafish suggest that the differences in adhesive and repulsive properties between odd- and even-numbered rhombomeres may be mediated through the bi-directional signaling between EphA4 receptors (expressed in r3 and r5) and EphrinB proteins (located in r2, r4, and r6) (Xu et al., 1995
; Mellitzer et al., 1999
; Xu et al., 1999
; Cooke et al., 2001
). In spgm793 mutants, subsequent to alterations in krx20 expression in r3 and r5, ephA4 expression is absent medially and restricted to lateral cell clusters. Thus, interactions of EphA4 receptors with Ephrin B proteins cannot occur in the medial hindbrain. Medial disruption of r3 and r5 territories brings even-numbered rhombomeres 2, 4 and 6 into direct opposition. The observed alterations might then prevent the formation of segmental boundaries. Consistent with this idea, spgm793 mutants have no obvious morphological inter-rhombomeric boundaries. Accordingly, pax6.1 expression, normally enriched along rhombomere boundaries, is not observed in spgm793 mutants. In contrast, rhombomere boundaries are formed in spgm216 mutants, as r3 and r5 territories, albeit reduced, separate even-numbered rhombomeres.
With the juxtaposition of even-numbered rhombomeres seen in strong spgm793 mutants, loss of the normal restriction of cell movement between segmental compartments might be expected (Fraser et al., 1990; Guthrie et al., 1993
). In contrast, we observed that hox gene expression borders at the r2/r4 and r4/r6 interfaces are still relatively sharp in spgm793 mutants (Fig. 2C,I,O,R1). This could be due to reprogramming of hox expression when cells of adjacent segments intermingle. Alternatively, this could indicate that residual r2/r4 and r4/r6 boundaries may have formed or been maintained that are sufficient to restrict extensive intermingling between cells of adjacent segments. In support of this idea, Krox20 mutant mice show signs of segmental boundary formation in the ventral hindbrain despite the complete disappearance of r3 and r5 during development (Schneider-Maunoury et al., 1997
).
spg/pou2 is required in a permissive manner for segmental krx20 and val expression
Krox20 and kreisler play important roles in hindbrain segmentation (Schneider-Maunoury, 1993; Swiatek and Gridley, 1993
; Cordes and Barsh, 1994
; Schneider-Maunoury et al., 1997
). The regulatory pathways that lead to their restricted expression in specific rhombomeres are, however, still elusive. We have shown that pou2 expression in the hindbrain primordium precedes the activation of krx20 and val, and overlaps with their expression domains. In spg mutants, the expression of krx20 and val is reduced from the earliest time of detection, while injection of pou2 mRNA can restore normal expression. These data indicate that pou2 is required for the initial establishment of normal segmental expression of krx20 and val in the zebrafish hindbrain primordium. As widespread Pou2 overexpression is not sufficient to induce ectopic krx20 or val expression, Pou2 protein need not be strictly localized to limit its activity, suggesting that positional information is provided by other sources. Thus, Pou2 may function as an essential but permissive cofactor that, directly or indirectly, enables other transcription factors to activate krx20 and val. However, pou2-independent activation of krx20 and val may occur in the lateral neural plate, as both genes are still activated at lateral positions in spgm793 mutants. Further, pou2 appears not to be required for later maintenance of krx20 and val expression, as hindbrain expression of pou2 ceases during early somitogenesis (Hauptmann and Gerster, 1995
).
pou2 is an essential component of the regulatory cascade controlling hindbrain segmentation
A pre-segmental stage of hindbrain regionalization likely involves the retinoid pathway (Gale et al., 1999; Gavalas and Krumlauf, 2000
; Niederreither et al., 2000
). During this stage, the hindbrain primordium is subdivided into an anterior (r1-r3) and a posterior (r4-r7) domain, which requires retinoid signaling to develop posterior characteristics. It has been shown that pou2 hindbrain expression can be influenced by application of retinoic acid in a similar manner as other segmentation genes such as krx20 (Hauptmann and Gerster, 1995
). We suggest that pou2 may function in subdividing the pre-defined anterior and posterior hindbrain domains into single rhombomere territories.
Our data show that the establishment of normal segmental krx20 and val domains is affected in spg mutants. This suggests that pou2 is required for the initial specification of r3, r5 and r6. In contrast, analysis of Krox20 mouse mutants revealed that Krox20 is required for maintenance, but not for establishment, of r3 and r5 (Schneider-Maunoury, 1993; Swiatek and Gridley, 1993
; Schneider-Maunoury et al., 1997
). Analysis of zebrafish val mutants suggests that val functions in the later subdivision of an r5/r6 protosegment into definitive r5 and r6 (Moens et al., 1996
; Moens et al., 1998
). Thus, pou2 is required for the initial specification of distinct segmental territories, while krx20 and val appear to be required only subsequently. Taken together, this strongly suggests that pou2 acts before krx20 and val during hindbrain segmentation.
Similar to that of krx20 and val, the initial expression of hoxa2 in r2 and r3, hoxb2 in r3 and r5, and hoxb3 in r5 and r6 is affected in spgm793 mutants by the 1- to 2-somite stage. Thus, pou2 appears to be required for the proper establishment of the initial segmental expression domains of hoxa2, hoxb2 and hoxb3. In mouse, Krox20 directly regulates the expression of Hoxa2 and Hoxb2 in r3 and r5 (Sham et al., 1993; Nonchev et al., 1996
), and Hoxb3 is a direct target of kreisler in r5 (Manzanares et al., 1997
). Therefore, the alterations in hox gene expression in zebrafish spgm793 mutants may be secondary to those in krx20 and val in r3/r5 and r5/r6, respectively. Accordingly, hoxb3 is down-regulated in r5 and r6 in zebrafish val mutants (Prince et al., 1998
). spgm793 mutants may also have krx20 and val independent alterations in hox gene expression as initial hoxa2 expression is disrupted in r2. In contrast, the initial hindbrain expression of early-acting hoxb1b and hoxb1a is normal in spg mutants, suggesting that these genes are activated independently of spg/pou2.
In addition to regulating Hox gene expression, Krox20 also directly activates the expression of Epha4 in r3 and r5 in mouse (Theil et al., 1998). In spgm793 mutants, we observed that ephA4 expression in r3 and r5 is restricted to the reduced krx20 expression domains. Thus, the abnormal rhombomere boundaries may be caused by disturbance of a regulatory pathway in which pou2 is required directly or indirectly for krx20 expression, which in turn controls ephA4 expression. Furthermore, the disruption of val expression in r5 and r6 of spgm793 mutants may also contribute to defective rhombomere boundary formation, since inactivation of val leads to lack of rhombomere boundaries caudal to the r3/r4 boundary (Moens et al., 1996
; Moens et al., 1998
) and disruption of normal Eph/Ephrin signaling (Cooke et al., 2001
). Taken together, the loss of morphological rhombomere boundaries in spgm793 mutants is likely a consequence of the early disruption of krx20 expression in r3 and r5, and val expression in r5 and r6.
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ACKNOWLEDGMENTS |
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REFERENCES |
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Alexandre, D., Clarke, J. D. W., Oxtoby, E., Yan, Y. L., Jowett, T. and Holder, N. (1996). Ectopic expression of Hoxa-1 in the zebrafish alters the fate of the mandibular arch neural crest and phenocopies a retinoic acid-induced phenotype. Development 122, 735-746.
Amores, A., Force, A., Yan, Y., Joly, L., Amemiya, C., Fritz, A., Ho, R. K., Langeland, J., Prince, V., Wang, Y. et al. (1998). Zebrafish hox clusters and vertebrate genome evolution. Science 282, 1711-1714.
Belting, H.-G., Hauptmann, G., Meyer, D., Abdelilah-Seyfried, S., Chitnis, A., Eschbach, C., Söll, I., Thisse, C., Thisse, B., Artinger, K. B. et al. (2001). spiel ohne grenzen/pou2 is required during establishment of the zebrafish midbrain-hindbrain boundary organizer. Development 128, 4165-4176.
Birgbauer, E. and Fraser, S. C. (1994). Violation of cell lineage restriction compartments in the chick hindbrain. Development 120, 1347-1356.
Bisgrove, B. W., Essner, J. J. and Yost, H. J. (1999). Regulation of midline development by antagonism of lefty and nodal signaling. Development 126, 3253-3262.
Carpenter, E. M., Goddard, J. M., Chisaka, O., Manley, N. R. and Capecchi, M. R. (1993). Loss of Hox-A1 (Hox-1.6) function results in the reorganization of the murine hindbrain. Development 118, 1063-1075.
Clarke, J. D. W. and Lumsden, A. (1993). Segmental repetition of neuronal phenotype sets in the chick embryo hindbrain. Development 118, 151-162.
Cooke, J. E., Moens, C. B., Roth, L. W. A., Durbin, L., Shiomi, K., Brennan, C., Kimmel, C. B., Wilson, S. W. and Holder, N. (2001). Eph signalling functions downstream of Val to regulate cell sorting and boundary formation in the caudal hindbrain. Development 128, 571-580.
Cordes, S. P. and Barsh, G. S. (1994). The mouse segmentation gene kr encodes a novel basic domain-leucine zipper transcription factor. Cell 79, 1025-1034.[Medline]
Currie, P. D. and Ingham, P. W. (1996). Induction of a specific muscle cell type by a hedgehog-like protein in zebrafish. Nature 382, 452-455.[Medline]
Dollé, P., Lufkin, T., Krumlauf, R., Mark, M., Duboule, D. and Chambon, P. (1993). Local alterations of Krox-20 and Hox gene expression in the hindbrain suggest lack of rhombomeres 4 and 5 in homozygote null Hoxa-1 (Hox-1.6) mutant embryos. Proc. Natl. Acad. Sci. USA 90, 7666-7670.
Ekker, S. C., Ungar, A. R., Greenstein, P., von Kessler, D. P., Porter, J. A., Moon, R. T. and Beachy, P. A. (1995). Patterning activities of vertebrate hedgehog proteins in the developing eye and brain. Curr. Biol. 5, 944-955.[Medline]
Fraser, S., Keynes, R. and Lumsden, A. (1990). Segmentation in the chick embryo hindbrain is defined by cell lineage restrictions. Nature 344, 431-435.[Medline]
Frohman, M. A., Martin, G. R., Cordes, S. P., Halamek, L. P. and Barsh, G. S. (1993). Altered rhombomere-specific gene expression and hyoid bone differentiation in the mouse segmentation mutant, kreisler (kr). Development 117, 925-936.
Fürthauer, M., Thisse, B. and Thisse, C. (1999). Three different noggin genes antagonize the activity of bone morphogenetic proteins in the zebrafish embryo. Dev. Biol. 214, 181-196.[Medline]
Gale, E., Zile, M. and Maden, M. (1999). Hindbrain respecification in the retinoid-deficient quail. Mech. Dev. 89, 43-54.[Medline]
Gavalas, A., Davenne, M., Lumsden, A., Chambon, P. and Rijli, F. M. (1997). Role of Hoxa-2 in axon pathfinding and rostral hindbrain patterning. Development 124, 3693-702.
Gavalas, A. and Krumlauf, R. (2000). Retinoid signalling and hindbrain patterning. Curr. Opin. Genet. Dev. 10, 380-386.[Medline]
Gilland, E. and Baker, R. (1993). Conservation of neuroepithelial and mesodermal segments in the embryonic vertebrate head. Acta. Anat. 148, 110-123.[Medline]
Goddard, J. M., Rossel, M., Manley, N. R. and Capecchi, M. R. (1996). Mice with targeted disruption of Hoxb-1 fail to form the motor nucleus of the V11th nerve. Development 122, 3217-3228.
Guthrie, S. and Lumsden, A. (1991). Formation and regeneration of rhombomere boundaries in the developing chick hindbrain. Development 112, 221-229.[Abstract]
Guthrie, S., Prince, V. and Lumsden, A. (1993). Selective dispersal of avian rhombomere cells in orthotopic and heterotopic grafts. Development 118, 527-538.
Halpern, M. E., Ho, R. K., Walker, C. and Kimmel, C. B. (1993). Induction of muscle pioneers and floor plate is distinguished by the zebrafish no tail mutation. Cell 75, 99-111.[Medline]
Hanneman, E., Trevarrow, B., Metcalfe, W. K., Kimmel, C. B. and Westerfield, M. (1988). Segmental pattern of development of the hindbrain and spinal cord of the zebrafish embryo. Development 103, 49-58.[Abstract]
Hatta, K. (1992). Role of the floor plate in axonal patterning in the zebrafish CNS. Neuron 9, 629-642.[Medline]
Hauptmann, G. and Gerster, T. (1994). Two-color whole-mount in situ hybridization to vertebrate and Drosophila embryos. Trends Genet. 10, 266.[Medline]
Hauptmann, G. and Gerster, T. (1995). Pou-2 a zebrafish gene active during cleavage stages and in the early hindbrain. Mech. Dev. 51, 127-138.[Medline]
Hauptmann, G. and Gerster, T. (1996). Complex expression of the zp-50 pou gene in the embryonic zebrafish brain is altered by overexpression of sonic hedgehog. Development 122, 1769-1780.
Hauptmann, G. and Gerster, T. (2000a). Combinatorial expression of zebrafish Brn-1 and Brn-2-related POU genes in the embryonic brain, pronephric primordium, and pharyngeal arches. Dev. Dyn. 218, 345-358.[Medline]
Hauptmann, G. and Gerster, T. (2000b). Multicolor whole-mount in situ hybridization. In Developmental Biology Protocols Vol. III; Methods in Molecular Biology, vol. 137 (ed. R. S. Tuan and C. W. Lo), pp. 139-148. Totowa: Humana Press.
Kane, D. A., Warga, R. M. and Kimmel, C. B. (1992). Mitotic domains in the early embryo of the zebrafish. Nature 360, 735-737.[Medline]
Kimmel, C. B., Sessions, S. K. and Kimmel, R. J. (1981). Morphogenesis and synaptogenesis of the zebrafish Mauthner neuron. J. Comp. Neurol. 198, 101-120.[Medline]
Kimmel, C. B., Powell, S. L. and Metcalfe, W. K. (1982). Brain neurons which project to the spinal cord in young larvae of the zebrafish. J. Comp. Neurol. 205, 112-127.[Medline]
Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B. and Schilling, T. F. (1995). Stages of embryonic development of the zebrafish. Dev. Dyn. 203, 253-310.[Medline]
Krauss, S., Concordet, J. P. and Ingham, P. W. (1993). A functionally conserved homolog of the Drosophila segment polarity gene hh is expressed in tissues with polarizing activity in zebrafish embryos. Cell 75, 1431-1444.[Medline]
Lumsden, A. and Keynes, R. (1989). Segmental patterns of neuronal development in the chick hindbrain. Nature 337, 424-428.[Medline]
Lumsden, A. and Guthrie, S. (1991). Alternating patterns of cell surface properties and neural crest cell migration during segmentation of the chick hindbrain. Development Supplement 2, 9-15.
Manzanares, M., Cordes, S., Kwan, C.-T., Sham, M. H., Barsh, G. S. and Krumlauf, R. (1997). Segmental regulation of Hoxb-3 by kreisler. Nature 387, 191-195.[Medline]
Manzanares, M., Cordes, S., Ariza-McNaughton, L., Sadl, V., Maruthainar, K., Barsh, G. and Krumlauf, R. (1999). Conserved and distinct roles of kreisler in regulation of the paralogous Hoxa3 and Hoxb3 genes. Development 126, 759-769.
Mark, M., Lufkin, T., Vonesch, J. L., Ruberte, E., Olivo, J. C., Dolle, P., Gorry, P., Lumsden, A. and Chambon, P. (1993). Two rhombomeres are altered in Hoxa-1 mutant mice. Development 119, 319-338.
McGinnis, W. and Krumlauf, R. (1992). Homeobox genes and axial patterning. Cell 68, 283-302.[Medline]
McKay, I. J., Muchamore, I., Krumlauf, R., Maden, M., Lumsden, A. and Lewis, J. (1994). The kreisler mouse: a hindbrain segmentation mutant that lacks two rhombomeres. Development 120, 2199-2211.
Mellitzer, G., Xu, Q. and Wilkinson, D. G. (1999). Eph receptors and ephrins restrict cell intermingling and communication. Nature 400, 77-80.[Medline]
Moens, C. B., Yan, Y.-L., Appel, B., Force, A. G. and Kimmel, C. B. (1996). valentino: a zebrafish gene required for normal hindbrain segmentation. Development 122, 3981-3990.
Moens, C. B., Cordes, S. P., Giorgianni, M. W., Barsh, G. S. and Kimmel, C. B. (1998). Equivalence in the genetic control of hindbrain segmentation in fish and mouse. Development 125, 381-391.
Niederreither, K., Vermot, J., Schuhbaur, B., Chambon, P. and Dollé, P. (2000). Retinoic acid synthesis and hindbrain patterning in the mouse embryo. Development 127, 75-85.
Nonchev, S., Vesque, C., Maconochie, M., Seitanidou, T., Ariza-McNaughton, L., Frain, M., Marshall, H., Sham, M. H., Krumlauf, R. and Charnay, P. (1996). Segmental expression of Hoxa-2 in the hindbrain is directly regulated by Krox-20. Development 122, 543-554.
Odenthal, J. and Nüsslein-Volhard, C. (1998). fork head domain genes in zebrafish. Dev. Genes Evol. 208, 245-258.[Medline]
Oxtoby, E. and Jowett, T. (1993). Cloning of the zebrafish krox-20 gene (krx-20) and its expression during hindbrain development. Nucl. Acids Res. 21, 1087-1095.[Abstract]
Placzek, M., Jessell, T. M. and Dodd, J. (1993). Induction of floor plate differentiation by contact-dependent, homeogenetic signals. Development 117, 205-218.
Prince, V. E., Moens, C. B., Kimmel, C. B. and Ho, R. K. (1998). Zebrafish hox genes: expression in the hindbrain region of wild-type and mutants of the segmentation gene, valentino. Development 125, 393-406.
Püschel, A. W., Gruss, P. and Westerfield, M. (1992). Sequence and expression pattern of pax-6 are highly conserved between zebrafish and mice. Development 114, 643-651.[Abstract]
Rebagliati, M. R., Toyama, R., Haffter, P. and Dawid, I. B. (1998). cyclops encodes a nodal-related factor involved in midline signaling. Proc. Natl. Acad. Sci. USA 95, 9932-9937.
Renucci, A., Lemarchandel, V. and Rosa, F. (1996). An activated form of type I serine/threonine kinase receptor TARAM-A reveals a specific signalling pathway involved in fish head organiser formation. Development 122, 3735-3743.
Rubenstein, J. L. R. and Beachy, P. A. (1998). Patterning of the embryonic forebrain. Curr. Opin. Neurobiol. 8, 18-26.[Medline]
Ryan, A. K. and Rosenfeld, M. G. (1997). POU domain family values: flexibility, partnerships, and developmental codes. Genes Dev. 11, 1207-1225.[Medline]
Sampath, K., Rubinstein, A. L., Cheng, A. M., Liang, J. O., Fekany, K., Solnica-Krezel, L., Korzh, V., Halpern, M. E. and Wright, C. V. (1998). Induction of the zebrafish ventral brain and floorplate requires cyclops/nodal signalling. Nature 395, 185-189.[Medline]
Schier, A. F., Neuhauss, S. C., Harvey, M., Malicki, J., Solnica-Krezel, L., Stainier, D. Y., Zwartkruis, F., Abdelilah, S., Stemple, D. L., Rangini, Z. et al. (1996). Mutations affecting the development of the embryonic zebrafish brain. Development 123, 165-178.
Schier, A. F. and Shen, M. M. (2000). Nodal signalling in vertebrate development. Nature 403, 385-389.[Medline]
Schilling, T. F. and Kimmel, C. B. (1994). Segment and cell type lineage restrictions during pharyngeal arch development in the zebrafish embryo. Development 120, 483-494.
Schneider-Maunoury, S., Seitanidou, T., Charnay, P. and Lumsden, A. (1997). Segmental and neuronal architecture of the hindbrain of Krox-20 mouse mutants. Development 124, 1215-1226.
Schneider-Maunoury, S., Gilardi-Hebenstreit, P. and Charnay, P. (1998). How to build a vertebrate hindbrain. Lessons from genetics. C.R. Acad. Sci. Paris, Sciences de la vie/Life Sciences 321, 819-834.
Schneider-Maunoury, S., Topilko, P., Seitanidou, T., Levi, G., Cohen-Tannoudji, M., Pournin, S., Babinet, C. and Charnay, P. (1993). Disruption of Krox-20 results in alteration of rhombomeres 3 and 5 in the developing hindbrain. Cell 75, 1199-1214.[Medline]
Schulte-Merker, S., Ho, R. K., Herrmann, B. G. and Nusslein-Volhard, C. (1992). The protein product of the zebrafish homologue of the mouse T gene is expressed in nuclei of the germ ring and the notochord of the early embryo. Development 116, 1021-1032.
Schulte-Merker, S., Lee, K. J., McMahon, A. P. and Hammerschmidt, M. (1997). The zebrafish organizer requires chordino. Nature 387, 862-863.[Medline]
Sechrist, J., Serbedzija, G. N., Scherson, T., Fraser, S. E. and Bronner-Fraser, M. (1993). Segmental migration of the hindbrain neural crest does not arise from its segmental generation. Development 118, 691-703.
Sham, M. H., Vesque, C., Nonchev, S., Marshall, H., Frain, M., Das Gupta, R., Whiting, J., Wikinson, D., Charnay, P. and Krumlauf, R. (1993). The zinc finger gene Krox20 regulates HoxB2 (Hox2.8) during hindbrain segmentation. Cell 72, 183-196.[Medline]
Sirotkin, H. I., Dougan, S. T., Schier, A. F. and Talbot, W. S. (2000). bozozok and squint act in parallel to specify dorsal mesoderm and anterior neuroectoderm in zebrafish. Development 127, 2583-2592.
Spaniol, P., Bornmann, C., Hauptmann, G. and Gerster, T. (1996). Class III POU genes of zebrafish are predominantly expressed in the central nervous system. Nucl. Acids Res. 24, 4874-4881.
Strähle, U., Blader, P., Henrique, D. and Ingham, P. W. (1993). Axial, a zebrafish gene expressed along the developing body axis, shows altered expression in cyclops mutant embryos. Genes Dev. 7, 1436-1446.[Abstract]
Studer, M., Lumsden, A., Ariza-McNaughton, L., Bradley, A. and Krumlauf, R. (1996). Altered segmental identity and abnormal migration of motor neurons in mice lacking Hoxb-1. Nature 384, 630-634.[Medline]
Swiatek, P. J. and Gridley, T. (1993). Perinatal lethality and defects in hindbrain development in mice homozygous for a targeted mutation of the zinc finger gene Krox20. Genes Dev. 7, 2071-2084.[Abstract]
Takeda, H., Matsuzaki, T., Oki, T., Miyagawa, T. and Amanuma, H. (1994). A novel POU domain gene, zebrafish pou2: expression and roles of two alternatively spliced twin products in early development. Genes Dev. 8, 45-59.[Abstract]
Talbot, W. S., Trevarrow, B., Halpern, M. E., Melby, A. E., Farr, G., Postlethwait, J. H., Jowett, T., Kimmel, C. B. and Kimelman, D. (1995). A homeobox gene essential for zebrafish notochord development. Nature 378, 150-157.[Medline]
Theil, T., Frain, M., Gilardi-Hebenstreit, P., Flenniken, A., Charnay, P. and Wilkinson, D. G. (1998). Segmental expression of the EphA4 (Sek-1) receptor tyrosine kinase in the hindbrain is under direct transcriptional control of Krox-20. Development 125, 443-452.
Thisse, C. and Thisse, B. (1999). Antivin, a novel and divergent member of the TGFß superfamily, negatively regulates mesoderm induction. Development 126, 229-240.
Toyama, R. and Dawid, I. B. (1997). lim6, a novel LIM homeobox gene in the zebrafish: comparison of its expression pattern with lim1. Dev. Dyn. 209, 406-417.[Medline]
Trevarrow, B., Marks, D. L. and Kimmel, C. B. (1990). Organization of hindbrain segments in the zebrafish embryo. Neuron 4, 669-679.[Medline]
Westerfield, M. (1994). The Zebrafish Book. Eugene: University of Oregon Press.
Wilkinson, D. G., Bhatt, S., Chavrier, P., Bravo, R. and Charnay, P. (1989). Segment-specific expression of a zinc-finger gene in the developing nervous system of the mouse. Nature 337, 461-464.[Medline]
Xu, Q., Mellitzer, G., Robinson, V. and Wilkinson, D. G. (1999). In vivo cell sorting in complementary segmental domains mediated by Eph receptors and ephrins. Nature 399, 267-271.[Medline]
Xu, Q. L., Alldus, G., Holder, N. and Wilkinson, D. G. (1995). Expression of truncated Sek-1 receptor tyrosine kinase disrupts the segmental restriction of gene expression in the Xenopus and zebrafish hindbrain. Development 121, 4005-4016.