1 Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200, USA
2 Institute of Neuroscience, University of Oregon, Eugene, OR 97403-1254, USA
Present address: Howard Hughes Medical Institute, Division of Basic Science, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA
*Author for correspondence (e-mail: amacher{at}uclink4.berkeley.edu)
Accepted 22 April 2002
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: VegT, Brachyury, Genetic mosaic, Fate map, Genetic redundancy, Genetic synergy, Zebrafish
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Zebrafish cell lineage analyses indicate that some ntl cells located in a region of the dorsal organizer from which notochord cells would originate in wild-type embryos become mesenchymal cells that lie beneath the spinal cord (Halpern et al., 1993; Melby et al., 1996
). Additionally, ntl embryos have a wider medial floor plate (MFP), a ventral row of midline spinal cord cells that is usually only one cell wide (Strähle et al., 1996
; Odenthal et al., 1996
; Halpern et al., 1997
). This observation prompted the idea that some ntl dorsal organizer cells in the wild-type notochord domain may adopt a floor plate fate (Halpern et al., 1997
). Consistent with both ideas, mouse and zebrafish chimera analysis has shown that Brachyury/ntl mutant cells form disorganized mesodermal patches near wild-type notochord and show a propensity to form floor plate (Halpern et al., 1993
; Wilson et al., 1995
).
At least three additional T-box genes, Eomesodermin, Tbx6 and VegT, are involved in mesodermal fate specification. Eomesodermin (Eomes) was first identified in X. laevis and is implicated in mesoderm development (Ryan et al., 1996). Mouse Eomes mutant embryos arrest soon after implantation, and tetraploid chimera analyses demonstrates that Eomes is required in the embryo for mesoderm formation (Russ et al., 2000
). Eomes mutant cells can occasionally adopt mesodermal fates in chimeras, suggesting that Eomes may function to recruit cells into the primitive streak (Russ et al., 2000
). In the mouse embryo, Tbx6 is expressed in the primitive streak, the paraxial mesoderm, and the tail bud (Chapman et al., 1996
). In Tbx6 mutant embryos, posterior paraxial mesoderm develops as neural tissue, suggesting that Tbx6 is required in paraxial mesoderm to block neural development (Chapman and Papaioannou, 1998
). The zebrafish tbx6 gene, although not considered the true ortholog of mouse Tbx6 (see Ruvinsky et al., 1998
) is expressed very similarly to the mouse gene (Hug et al., 1997
). A third gene, X. laevis VegT, was isolated independently by several groups (Zhang and King, 1996
; Lustig et al., 1996
; Stennard et al., 1996
; Horb and Thomsen, 1997
). Maternal VegT transcripts are localized vegetally in X. laevis oocytes; VegT is zygotically expressed in the presumptive mesoderm of the marginal zone and is restricted to the lateral and ventral mesoderm by late gastrulation (reviewed by Smith, 1999
). Depletion experiments show that VegT function is required for endoderm and mesoderm development by regulating TGFß family signaling molecules, with evidence for a cell-autonomous, dose-sensitive VegT requirement as well (Zhang et al., 1998a
; Clements et al., 1999
; Kofron et al., 1999
; Kavka and Green, 2000
; Xanthos et al., 2001
).
The zebrafish spadetail (spt) gene, originally identified by mutation, is likely a VegT ortholog (Griffin et al., 1998). Although zygotic spt is expressed similarly to X. laevis VegT, the two genes probably have functional differences, since spt/VegT transcripts are maternally provided in frogs but not fish. Later, the gene is expressed in lateral mesoderm in both fish and frogs and in prechordal plate mesoderm in fish, but not frogs (Griffin et al., 1998
; Ruvinsky et al., 1998
). The X. laevis VegT expression pattern reflects expression of two differentially spliced forms: one is provided maternally, whereas the other is expressed zygotically in a pattern similar to zebrafish spt (Stennard et al., 1999
). Homozygous spt mutant embryos lack trunk somites and later are deficient in trunk muscle; additional analyses demonstrate that spt is required in trunk somitic precursors for convergence movements and for muscle cell fate decisions (Kimmel et al., 1989
; Ho and Kane, 1990
; Amacher and Kimmel, 1998
; Yamamoto et al., 1998
). Other mesodermal derivatives, such as blood, pronephros, and pectoral fin, are variably deficient in spt embryos (Kimmel et al., 1989
; Solnica-Krezel et al., 1996
; Thompson et al., 1998
).
The phenotypes of ntl and spt embryos are less severe than mutations in or depletion of their mouse and frog counterparts. This could be due to partial functional redundancy among zebrafish T-box genes. For example, tbx-c shows expression overlap with ntl (Dheen et al., 1999) and tbx6 is expressed very similarly to spt (Hug et al., 1997
; Griffin et al., 1998
; Ruvinsky et al., 1998
).
Previous work suggested that spt and ntl genes function synergistically, as expression of zebrafish tbx6 is completely abolished in spt;ntl embryos, a more severe effect than if the mutations were merely additive (Griffin et al., 1998). Here, we confirm that spt and ntl function together in mesoderm patterning. Using double mutant and genetic mosaic analyses, we demonstrate that spt and ntl are cell-autonomously required for development of all trunk and tail mesoderm. Although most dorsal-ventral patterning in the spt;ntl neural tube is relatively normal, the posterior medial floor plate (MFP) is completely absent. The lack of posterior MFP is the most striking synergistic interaction we observe, especially since mutations in ntl or spt appear to enhance MFP development and can suppress MFP defects in some mutant backgrounds. Currently, there is some controversy about the origin and timing of floor plate development (see Le Douarin and Halpern, 2000
; Placzek et al., 2000
). The fate mapping data we present support the idea that floor plate originates from a midline precursor population and that ntl function is required during early gastrulation in cells that normally make notochord to repress floor plate and promote notochord fate. However, the genetic mosaic analysis we present suggests that the lack of posterior MFP in spt;ntl embryos results from loss of an inducing signal from mesoderm, suggesting that a requirement for non cell-autonomous factors as well. We discuss the possible mesodermal signals and cell interactions that may be involved and the role that T-box genes play in development of posterior mesoderm and medial floor plate.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mutant alleles used were sptb104, cyclopsb16 (cycb16) and ntlb195. The molecular lesions in each of these alleles are described as definite or likely null alleles: sptb104 (Griffin et al., 1998) (this paper); cycb16 (Rebagliati et al., 1998
; Sampath et al., 1998
); ntlb195 (Schulte-Merker et al., 1994b
). spt/tbx16 maps to LG 8 (S. L. A., unpublished data), cyc/znr1/ndr2 maps to LG 12 (Talbot et al., 1998
; Sampath et al., 1998
; Rebagliati et al., 1998
), and ntl maps to LG 19 (Postlethwait et al., 1994
).
Carriers doubly heterozygous for sptb104 and cycb16 mutations and for sptb104 and ntlb195 mutations were obtained by crossing fish heterozygous for one mutation to fish heterozygous for the other and raising their progeny. Doubly heterozygous fish were intercrossed to produce homozygous double mutant embryos. At 24 hours postfertilization (hpf), the progeny of two doubly heterozygous carriers can be sorted into four phenotypic classes in the 9:3:3:1 Mendelian ratio expected for two independently assorting mutations. Progeny from spt;cyc doubly heterozygous mutant carriers were obtained in a ratio of 8.84 : 3.04 : 3.13 : 0.98 (WT : cyc : spt : spt;cyc, n=1534, 2=0.91, P>0.80). Progeny from spt;ntl doubly heterozygous mutant carriers were obtained in a ratio of 8.99 : 3.03 : 2.97 : 1.01 (WT : ntl : spt : spt;ntl, n=1972,
2=0.10, P>0.95). Approximately two-thirds of the spt embryos from the latter intercross had a more severe phenotype (reduced posterior notochord and floor plate, severe muscle reduction, more necrotic cell accumulation in tail) that we suspected was due to heterozygosity for the ntl mutation. To test this idea, we crossed fish that were heterozygous for the spt mutation (but homozygous for the wild-type ntl allele) to fish that were doubly heterozygous for both the spt and ntl mutations. Approximately one-quarter of the progeny had the spt phenotype, with half of those having more severe phenotypic disturbances (WT : spt : spt severe=2.94 : 0.52 : 0.54; n=884;
2=1.3, P>0.70). In these experiments, it did not matter whether the male or female was the doubly heterozygous carrier. The severe phenotype will be described in detail elsewhere (L. Goering and D. J. Grunwald, personal communication).
Antibody generation and immunohistochemistry
Polyclonal anti-Spt antibodies were generated by immunizing mice with a purified 6-histidine-tagged fusion protein containing Spt amino acids 215-382, produced using the pQE expression system (QIAGEN). Whole-mount antibody staining was performed on paraformaldehyde-fixed embryos following in situ hybridization or incubation at 65°C overnight in buffer containing 50% formamide, 2x SSC, 0.1% Tween 20, pH 6.0. Embryos were incubated for 4 hours in antibody staining buffer (PBS containing 1% DMSO, 0.1% Triton X-100, 2% normal goat serum, 2 mg/ml BSA), followed by overnight incubation in a 1:1000 dilution of mouse anti-Spt polyclonal antiserum in antibody staining buffer. Following incubation with horseradish peroxidase (HRP)-conjugated secondary antibody and extensive washes in wash buffer (PBS containing 1% DMSO, 0.1% Triton X-100), protein localization was visualized using the Vectastain ABC HRP kit as recommended (Vector Laboratories).
For co-localization of Spt and Ntl proteins, fixed embryos were embedded in agarose and cryostat sectioned as previously described (Westerfield, 1995). Sectioned embryos were stained with a 1:600 dilution of mouse anti-Spt polyclonal antiserum and a 1:5,000 dilution of rabbit anti-Ntl polyclonal antiserum (Schulte-Merker et al., 1992
). Following washes, sections were incubated simultaneously with a 1:200 dilution each of goat anti-rabbit Alexa Fluor 594 and goat anti-mouse Alexa Fluor 488 (Molecular Probes). Sectioned embryos were examined using a Zeiss Axiophot microscope or a Zeiss LSM laser scanning confocal microscope.
In situ hybridization and histological sectioning
Embryos were processed for whole-mount in situ hybridization as described by Thisse et al. (Thisse et al., 1993), with the modifications described by Melby et al. (Melby et al., 1997
). Digoxigenin-labeled RNA probes were synthesized from the following plasmid templates: no tail (ntl) (Schulte-Merker et al., 1992
), spadetail (spt) (Griffin et al., 1998
), myoD (Weinberg et al., 1996
), pax2 (Krauss et al., 1991
), sonic hedgehog (shh) (Krauss et al., 1993
), islet1 (Appel et al., 1995
), her1 (Müller et al., 1996
), tropomyosin (Thisse et al., 1993
), and krox20 (Oxtoby and Jowett, 1993
). Following probe detection, embryos were dehydrated, cleared and mounted either between coverslips or on bridged slides (Melby et al., 1997
). For preparation of sections after whole-mount in situ hybridization, embryos were dehydrated and embedded in Epon and sectioned as described (Westerfield, 1995
). For the sections shown in Fig. 2E-H, embryos were first fixed in Bouins solution and processed as above. Embryos were photographed on a Zeiss Universal microscope using a 35 mm camera or on a Zeiss Axioplan II using a Zeiss Axiocam digital camera.
|
Caged fluorescein fate-mapping
Embryos for fate mapping experiments were generated by injecting 1% lysine-fixable caged fluorescein (Molecular Probes) in 0.2 M KCl at the 1-4 cell stage. Caged fluorescein was injected into embryos from a cross of ntlb195 heterozygotes or into embryos co-injected with antisense ntl morpholino-modified oligomer (ntl-MO, kind gift from Steve Ekker). ntl-MO was injected at a concentration of 0.5 mg/ml in 0.1 M KCl containing 0.25% Phenol Red, basically as described by Nasevicius and Ekker (Nasevicius and Ekker, 2000), except that 1% caged fluorescein was injected simultaneously.
Fluorescein was uncaged with pulses of a 375 nm nitrogen laser (Micropoint Laser System, Photonic Instruments) as described previously (Gritsman et al., 2000). The beam was focused through a 50x Leitz water immersion objective on a Zeiss standard microscope. Early gastrula stage embryos (shield stage to 60% epiboly) were positioned shield-up in 0.2% agarose in embryo medium supplemented with 10 mM Hepes, and dorsal-up positioning was verified by the presence of forerunner cells just beyond the shield on the yolk syncytial layer (YSL) surface (Melby et al., 1996
). To activate fluorescein in a patch of about 20 notochord-domain cells, the laser beam was successively focused on 4 adjacent cells, at and just one cell behind the blastoderm margin, and about 3 cells deep (the shield is about 6 cell-diameters deep at this stage). Firing the laser at each position yields a string of several brightly fluorescing cells along the path of the beam (including a single surface EVL cell) and a few dimly fluorescing neighboring cells.
Uncaged fluorescein label was detected either by fluorescence using a Zeiss Axiophot microscope or by a more sensitive procedure using anti-fluorescein Fab antibody conjugated to alkaline phosphatase (Boehringer Mannheim). Addition of anti-fluorescein antibody and subsequent detection of alkaline phosphatase activity was done essentially as described previously (Cornell and Eisen, 2000; Gritsman et al., 2000
), except that digestion by Proteinase K (Boehringer Mannheim; 10 µg/ml, 4 minutes) was performed before BSA blocking.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
spt and ntl are required together for trunk and tail mesoderm development
To examine the functional consequence of removing both ntl and spt T-box genes, we constructed the spt;ntl double mutant (Fig. 2). Anterior morphology appears normal in spt;ntl embryos; however, there is an extreme deficit of mesodermal cells in the trunk and tail (Fig. 2A-D). Some deficiencies were expected based upon single mutant phenotypes. For example, ntl embryos lack a tail and a differentiated notochord (Halpern et al., 1993), whereas spt embryos are deficient in ventrolateral mesoderm (Kimmel et al., 1989
) (compare Fig. 2A with 2B,C). Muscle development is affected in both mutants to varying extents (Kimmel et al., 1989
; Halpern et al., 1993
). About two-thirds of the spt embryos display reductions in notochord, floor plate and tail muscle due to heterozygosity at the ntl locus (data not shown), which we confirmed by observing the same severe phenotype in crosses of spt;ntl heterozygous fish to spt heterozygous fish (see Materials and Methods). Interestingly, spt;ntl embryos lack the characteristic spt spade tail (Fig. 2D). The spt spade cells are trunk somitic precursors that fail to migrate properly during gastrulation and express ntl (Ho and Kane, 1990
) (Fig. 1F). Thus, the abnormal accumulation of cells in the spt tail bud requires ntl function. Histological sections of wild-type and mutant embryos at 30 hpf reveal that the trunk region of spt;ntl embryos consists of a spinal cord covered by epidermis (Fig. 2E-H). Trunk mesodermal cell types, such as muscle and pronephros, that are present to various extents in both single mutant embryos (Fig. 2F,G), are absent in spt;ntl embryos (Fig. 2H).
Posterior mesodermal development, but not mesodermal induction, is blocked in spt;ntl embryos
Panmesodermal expression
Widespread expression of ntl/Brachyury is an immediate early response to mesoderm-inducing signals (Smith et al., 1991). Embryos carrying the ntlb195 mutation do not produce functional protein and antibodies fail to detect Ntl protein in ntl and spt;ntl embryos (see Fig. 3J,L). However, ntlb195 mutants produce ntl mRNA. During midgastrula stages, we find that ntl is expressed in epiblast cells at the blastoderm margin in wild-type, spt, ntl and spt;ntl embryos (Fig. 3A-D), suggesting that spt;ntl cells respond normally to early mesoderm-inducing signals.
|
Myogenic cells and differentiated muscle
During normal development, muscle precursors express myoD several hours before completion of the gastrula period (Weinberg et al., 1996). In contrast, myoD expression is not initiated in either spt or ntl single mutant embryos until the end of the gastrula period, although myoD expression partially recovers in both single mutants during segmentation stages (Weinberg et al., 1996
). In ntl embryos, myoD is expressed in the posterior border of somites as they form, but only very weakly in adaxial cells of the presomitic mesoderm, whereas its expression is variable in spt embryos in cells that flank the developing midline (Fig. 3J,K) (Weinberg et al., 1996
). In contrast, in spt;ntl embryos, myoD-expressing cells are never detected posterior to the head at any stage (Fig. 3L). Although differentiated posterior muscle (visualized by tropomyosin expression) is not observed in spt;ntl embryos (compare Fig. 3M-O with P), head musculature and a small heart are present (data not shown).
Pronephric precursors and embryonic pronephric tubules
During segmentation stages, pax2.1 is expressed at the midbrain-hindbrain boundary, in developing otic placodes, and in presumptive pronephros (Krauss et al., 1991). Cells expressing pax2.1 are found in all these domains in wild-type, spt, and ntl embryos (Fig. 3I-K), but are missing at the edge of the lateral plate mesoderm (pronephros) in spt;ntl embryos (Fig. 3L). The absence of early pax2.1 staining in the presumptive pronephros correlates well with the absence of pronephric tubules in older spt;ntl embryos (Fig. 2H).
Taken together, the expression analyses suggest that spt and ntl together are not required for initial mesoderm induction, but are required and are partially redundant for further development of mesodermal cell types. Because mesodermally derived signals have been implicated in neural patterning, we next investigated the anterior-posterior and dorsal-ventral patterning of the spt;ntl neural tube.
Neither spt nor ntl function is required for most dorsal-ventral spinal cord patterning
General anterior-posterior patterning of the spt;ntl neural tube, as revealed by markers of midbrain, hindbrain and primary motoneurons (pax2.1 and krox20 in Fig. 3; islet1 in Fig. 4), appears normal. To characterize dorsal-ventral neural tube patterning, we examined several markers that are differentially expressed along the dorsal-ventral axis (Fig. 4). The genes msxb and pax3 (Ekker et al., 1997; Seo et al., 1998
) are expressed in the normal dorsal territory in spt;ntl spinal cord at 24 hpf (data not shown). In the intermediate neural tube of spt;ntl embryos, pax2.1-expressing interneurons (Krauss et al., 1991
) are present, but reduced in number, and histological sections reveal that they are sometimes medially displaced (Fig. 4A-D). Early expression of islet1 is in dorsally located Rohon-Beard sensory neurons and in ventrally located primary motoneurons (Inoue et al., 1994
; Tokumoto et al., 1995
; Appel et al., 1995
). At 13-14 hpf (8- to 10-somite stage), both types of islet1-expressing cells appear present in single and double mutant embryos based upon their position (Fig. 4E-H; sections in Fig. 4I-L). However, fewer ventral islet1-expressing cells are observed in spt;ntl embryos, and histological sections reveal that they sometimes are located in midline positions (Fig. 4L).
|
spt and ntl together are required for trunk and tail medial floor plate formation
The most ventral neural tube tissue is the floor plate, and in zebrafish, the floor plate is composed of a single midline row of medial floor plate (MFP) cells flanked by lateral floor plate (LFP) cells (Odenthal and Nusslein-Volhard, 1998). A wide medial floor plate (MFP) forms in ntl embryos (Fig. 5B) (Odenthal et al., 1996
; Strähle et al., 1996
; Halpern et al., 1997
). Similarly, the spt posterior MFP is often more than one-cell wide, and frequently, cells expressing MFP markers are observed ventral to the notochord (Fig. 5C; Fig. 6C) (Amacher and Kimmel, 1998
). In contrast to both single mutant phenotypes, the spt;ntl MFP is severely truncated and extends only slightly posterior of the hindbrain (Fig. 5D). The single and double mutant floor plate phenotypes observed using shh as a marker are also observed with other MFP markers, including tiggy-winkle hedgehog (Ekker et al., 1995
),
-collagen2 (Yan et al., 1995
), and axial/HNF3ß (Strähle et al., 1993
) at the 20-somite stage (data not shown). Thus, although posterior MFP develops in excess in spt and ntl embryos, posterior MFP formation is abolished in spt;ntl embryos.
|
|
spt and ntl together are required cell-autonomously in mesodermal cells, but neither gene is required in medial floor plate cells for their fate
To determine whether spt and ntl are required cell-autonomously in mesoderm and/or floor plate, we created genetic mosaic embryos. We transplanted blastula cells from a rhodamine-labeled donor embryo derived from an intercross of two heterozygous spt;ntl carriers, together with blastula cells from a fluorescein-labeled wild-type donor, into the presumptive mesoderm region of a wild-type host embryo (Fig. 7; see Materials and Methods). Because transplantations were performed before mutant phenotypes are distinguishable, the donor genotype was retrospectively determined by examining each donor embryo at later stages when the morphological phenotype is obvious (see Fig. 2). When wild-type donor cells adopted mesoderm or floor plate fates, we assessed whether mutant donor cells co-transplanted into the same fate map position could also adopt those fates. In control transplants (n=72), where both fluorescein- and rhodamine-labeled donor cells were wild type, we observed near-perfect overlap in cell fates adopted by cells from both donors. In over 97% of the cases in which rhodamine-labeled wild-type donor cells formed mesoderm or floor plate, the fluorescein-labeled cells also contributed cells to the same tissue. When spt or ntl mutant cells were transplanted into wild-type hosts, we observed that they could adopt floor plate and some mesodermal fates (Fig. 7C-E, data not shown). ntl cells fail to form notochord in wild-type host embryos (Fig. 7D) (Halpern et al., 1993), whereas spt cells fail to form trunk muscle and instead contribute to various tail derivatives (Fig. 7E) (Ho and Kane, 1990
). In striking contrast to control and single mutant results, we never observed spt;ntl cells adopting mesodermal fates in a wild-type environment (n=12). The co-transplanted wild-type cells adopted either mesodermal fates only (4/12) or mixed mesodermal and neural fates (7/12). In one case, the co-transplanted wild-type cells contributed only to neural tissue. In every case, the co-transplanted spt;ntl cells never adopted mesodermal fates, but instead adopted ectodermal fates (Fig. 7F).
|
Origin of the floor plate in ntl single mutants
We and others have suggested that ntl gene function is required to promote notochord and repress floor plate development in midline cells (see Halpern et al., 1997). If loss of ntl function leads to excess floor plate at the expense of notochord, one would predict that the ectopic ntl floor plate cells would arise from a domain of the gastrula fate map normally corresponding to notochord. To determine the origin of floor plate cells in ntl embryos, we injected embryos with caged fluorescein-dextran during early cleavage stages and then uncaged the fluorophore in a small population of approximately 20 dorsal organizer cells during early gastrulation (Fig. 8A; see Materials and Methods). Uncaged fluorescein label was detected by fluorescence microscopy and/or using anti-fluorescein antibody. Because the fluorophore was uncaged in approximately 3 tiers of cells, we expected to find labeled cells in derivatives of the enveloping layer (EVL) and of the deep layer (DEL). EVL cells do not involute or undergo convergence movements, but instead stay on the surface and differentiate as periderm (Kimmel et al., 1990
). In contrast, deep layer (DEL) cells located at the blastoderm margin adopt mesodermal and endodermal fates, depending upon developmental stage (Kimmel et al., 1990
; Melby et al., 1996
). In control wild-type embryos (n=6), the fluorophore was detected in large populations of labeled notochord cells and a dorsal patch of labeled cells in the enveloping layer (EVL). In three of the six wild-type embryos, labeled DEL derivatives were restricted to the notochord (Fig. 8B,D). In two embryos (those uncaged at the earliest stage), labeled DEL derivatives also included hatching gland (like notochord, hatching gland derives from the shield margin) (Melby et al., 1996
). In one embryo, labeled DEL derivatives included a small number of labeled floor plate cells along with a larger population of notochord cells. A strikingly different distribution of labeled progeny was observed when fluorescein was uncaged in the same domain in embryos depleted of ntl function (by injecting ntl-MO; see Materials and Methods). ntl-depleted embryos (n=8), all contained large numbers of labeled floor plate cells and a dorsal patch of labeled EVL (Fig. 8C,E). In seven of the eight ntl-depleted embryos, DEL labeling was restricted to the floor plate; in one, a few mesenchymal cells underlying the floor plate were also labeled.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The lack of posterior mesoderm in spt;ntl embryos is very similar to the phenotype of zebrafish and frog embryos in which FGF signaling has been disrupted (Amaya et al., 1991; Griffin et al., 1995
; Griffin et al., 1998
). To date, several zebrafish FGF genes (fgf8, fgf3, gfgf, fgf4) have been isolated that are expressed (at least transiently) in mesodermal precursors (Furthauer et al., 1997
; Reifers et al., 1998
; Furthauer et al., 2001
; Phillips et al., 2001
) (B. W. D. and C. B. K., unpublished data). Gene expression analyses in ntl, spt and fgf8/ace single mutants and compound heterozygotes indicate that zebrafish T-box genes and fgf8 are involved in a regulatory loop (B. W. D. and C. B. K., unpublished data), similar to the auto-regulatory loop described for X. laevis Brachyury and eFGF (Issacs et al., 1994
; Schulte-Merker and Smith, 1995
; Casey et al., 1998
). The X. laevis TGFß family member Derriére is involved in mesoendoderm development and appears to function in posterior regions of the embryo (Sun et al., 1999
). It has been proposed that Derriére, zygotic VegT and Brachyury operate in an FGF-dependent regulatory loop in the early gastrula to specify posterior mesoderm development (Sun et al., 1999
). A zebrafish derriére homolog has not yet been described, but may prove to be an important spt and/or ntl target gene.
The floor plate paradox why is there excess floor plate in spt and ntl single mutant embryos, but lack of posterior medial floor plate in spt;ntl double mutant embryos?
We propose that spt and ntl function at two different times during posterior MFP development, just as we suggest they have early and late functions during posterior mesoderm development. An early requirement for spt or ntl promotes MFP fate, but at later times, spt and ntl each function to restrict MFP fate. Thus, spt;ntl double mutant embryos lack posterior MFP, because an early promoting influence (both spt and ntl function) is missing. In contrast, single mutant embryos have excess MFP, because the early promoting influence (spt or ntl function) is present, but a later repressive influence (spt or ntl function) that restricts MFP number, is lacking. First, we consider the role of spt and ntl in promoting MFP fate, and then in the sections that follow, we detail the possible roles of each T-box gene in restricting MFP fate.
How do spt and ntl function to promote MFP fate?
We show that spt;ntl embryos lack posterior MFP (Fig. 5), but that spt;ntl cells can form posterior MFP when placed into a wild-type environment (Fig. 7). The simplest explanation for the spt;ntl MFP defect is that a mesodermal derivative important for posterior MFP induction is missing in spt;ntl embryos. Alternatively, embryos lacking spt and ntl function may fail to generate a midline precursor population (a cell population normally giving rise to both notochord and floor plate), yet individual spt;ntl cells can be recruited into the midline precursor population and adopt a floor plate fate if nudged along by wild-type cells in a genetic mosaic. One way we are distinguishing between these two possibilities is by transplanting wild-type cells into spt;ntl embryos to ask if wild-type cells or tissues can restore spt;ntl MFP development. To date, we have observed large stretches of posterior spt;ntl MFP in two hosts transplanted with wild-type cells (S. L. A., unpublished observations). In both cases, the wild-type cells were located in presumptive midline mesodermal derivatives at midbrain and hindbrain levels of the head, suggesting that signals from anterior mesoderm (the prechordal plate or nearby tissues) can induce posterior MFP. Thus, we favor the first possibility, that a mesodermally derived signal is required to induce MFP fate. (If the second possibility were true, we would predict that spt;ntl host MFP cells would be co-mingled with wild-type donor MFP cells in the same region.)
Are there prechordal plate mesoderm defects in spt;ntl double mutant embryos? We note that anterior mesoderm (e.g., hatching gland and head muscle) is present in spt;ntl embryos. However, spt is expressed in prechordal plate mesoderm (Griffin et al., 1998; Ruvinsky et al., 1998
) and ntl may be transiently expressed there (judged by the early gastrula co-expression of ntl and goosecoid, a marker of anterior mesoderm) (Schulte-Merker et al., 1994a
), suggesting that spt or ntl expression in anterior mesoderm may be required to generate a posterior MFP-inducing signal or cell interaction.
Floor plate induction is differentially regulated along the anterior/posterior axis
In chick and mouse, Sonic hedgehog (Shh) is a potent floor plate-inducing molecule (Dodd et al., 1998; Placzek et al., 2000
). In zebrafish, MFP induction requires Nodal signaling, but not Shh signaling, whereas LFP formation requires Shh signaling (Sampath et al., 1998
; Rebagliati et al., 1998
; Zhang et al., 1998b
; Schauerte et al., 1998
; Karlstrom et al., 1999
; Pogoda et al., 2000
; Sirotkin et al., 2000
; Odenthal et al., 2000
; Chen et al., 2001
; Etheridge et al., 2001
; Lewis and Eisen, 2001
; Varga et al., 2001
). One Nodal signal, Cyclops, is required in the prechordal plate mesoderm, but not in the notochord, for MFP induction along the entire axis (Sampath et al., 1998
). Furthermore, the EGF-CFC Nodal cofactor One-eyed pinhead (Oep) (Gritsman et al., 1999
) is required in floor plate cells for MFP fate, presumably for reception of Nodal signals (Strähle et al., 1997
; Shinya et al., 1999
). Because spt;ntl embryos lack posterior MFP, one might suspect that Nodal signaling is disrupted. However, there are important differences between spt;ntl embryos and embryos deficient in Nodal signaling. Nodal signaling mutants completely lack MFP and have severe anterior defects including cyclopia and prechordal plate mesoderm deficiencies (Sampath et al., 1998
; Rebagliati et al., 1998
; Zhang et al., 1998b
; Pogoda et al., 2000
; Sirotkin et al., 2000
). In contrast, spt;ntl embryos only lack posterior MFP (Fig. 5) and anterior development is morphologically normal, suggesting that Nodal signaling, at least anteriorly, is not disrupted (Fig. 2). Characterization of the spt;ntl phenotype demonstrates that anterior versus posterior MFP formation is differentially regulated along the anterior-posterior axis. Whether novel, non-Nodal signals are involved in posterior MFP induction is an open question.
ntl functions to repress floor plate fate in midline precursor cells
A function for ntl in repressing floor plate fate was first proposed because posterior MFP forms in ntl;cyc embryos, whereas cyc single mutant embryos lack MFP (Halpern et al., 1997). Although lack of ntl function bypasses the cyc requirement for posterior MFP development (Halpern et al., 1997
), it only partially bypasses the requirement for the Nodal cofactor Oep (Schier et al., 1997
; Strähle et al., 1997
). Together, these data suggest that cells lacking ntl function may be diverted to a MFP fate, but that Nodal signals are likely required for the transfating event. Therefore, ntl appears to have a dual role during floor plate formation; in addition to promoting MFP fate (as revealed by analyses of the spt;ntl mutant), ntl also functions to repress MFP development.
Recent studies in chick and zebrafish have suggested that a pool of precursor cells in the organizer region (the gastrula embryonic shield or the chordoneural hinge of later stage embryos) contains both notochord and floor plate precursors (see Le Douarin and Halpern, 2000). Segregation of notochord and floor plate fates occurs while cells are still in the organizer or chordoneural hinge, long before differentiation begins (see Le Douarin and Halpern, 2000
). We provide fate mapping data to support the idea that notochord and floor plate precursors segregate early into distinct populations within the organizer (Fig. 8). A role for zebrafish ntl in notochord versus floor plate fate choice was first hypothesized by Halpern et al. (Halpern et al., 1997
) (see also Le Douarin and Halpern, 2000
). Here, we demonstrate that ntl cells in the organizer domain that would form notochord in wild-type embryos, adopt a floor plate fate instead (Fig. 8), clearly establishing a role for ntl in repressing floor plate fate. Cell fate choice in this domain may also be mediated by Notch-Delta signaling, since overexpression of zebrafish deltaA (dlA) results in excess floor plate at the expense of notochord (Appel et al., 1999
). Conversely, inhibition of Delta-Notch signaling leads to excess notochord at the expense of floor plate (Appel et al., 1999
). These observations led to the proposal that Notch activity represses notochord fate, allowing cells to respond to factors that specify the alternate floor plate midline fate. Considering the opposing roles of ntl and deltaA in notochord and floor plate development, it will be interesting to examine the epistatic relationship of these two genes in midline cell fate selection.
What is the role of spt in midline cell fate choice?
We show that posterior MFP is slightly expanded and sometimes forms in ectopic postions in spt embryos (Figs 5, 6) (Amacher and Kimmel, 1998). Additionally, we show that loss of spt function partially suppresses the MFP defect of cyc mutant embryos (Fig. 6) and the MFP and notochord defects of flh single mutant embryos (Amacher and Kimmel, 1998
). Together, these results suggest a normal role for spt in repressing posterior midline fates (notochord and floor plate) in addition to its well-established positive role in trunk somitic precursor cell movements and cell fate. A repressive role is further strengthened by the observation that non-midline spt cells can produce notochord in response to ectopic fgf4 expression. When fgf4 mRNA is injected into wild-type embryos, notochord expands laterally, but when fgf4 mRNA is injected into spt embryos, notochord gene expression encompasses the entire embryo (B. W. D. and C. B. K., unpublished results). Thus, spt function may be required to limit cell number in the midline precursor cell population (i.e., Spt-positive cells are not responsive to dorsalizing signals). Indeed, a significant amount of MFP forms in spt;cyc embryos when compared to almost complete lack of MFP in cyc embryos (Fig. 6). In spt;cyc embryos, a probable source of early acting Nodal is Squint (Feldman et al., 1998
), a molecule shown to act as a morphogen over considerable distance (Chen and Schier, 2001
). Another possible explanation for expanded MFP in spt embryos and MFP presence in spt;cyc embryos is that slower convergence of spt midline cells (Thisse et al., 1995
; Warga and Nüsslein-Volhard, 1998
) allows MFP-inducing signals to be sent over wider distances or for longer times. Whether the expanded MFP in spt embryos is explained by increased midline precursor cell number, or broader or prolonged signaling, our genetic mosaic data (Fig. 7) suggest that the signal itself (not the ability to respond to signal) is diminished when both spt and ntl functions are lacking.
Future directions
Zebrafish embryos lacking function of two T-box genes, spt and ntl, lack all mesoderm and MFP in the trunk and tail. The downstream target genes that mediate spt- and ntl-dependent signaling function are unknown, but intriguing possibilities are FGFs and TGFß family members (Nodals and Derrière). Additionally, our work suggests that some targets of spt and ntl must function within mesodermal cells, since the genetic mosaic data demonstrate that spt;ntl cells cannot adopt mesodermal fates in the trunk and tail even when surrounded by wild-type cells. The identification of such target genes, as well as a posterior MFP-inducing signal, are important goals for the future.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Amacher, S. L. and Kimmel, C. B. (1998). Promoting notochord fate and repressing muscle development in zebrafish axial mesoderm. Development 125, 1397-1406.
Amaya, E., Musci, T. J. and Kirschner, M. W. (1991). Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos. Cell 66, 257-270.[Medline]
Appel, B., Korzh, V., Glasgow, E., Thor, S., Edlund, T., Dawid, I. B. and Eisen, J. S. (1995). Motoneuron fate specification revealed by patterned LIM homeobox gene expression in embryonic zebrafish. Development 121, 4117-4125.
Appel, B., Fritz, A., Westerfield, M., Grunwald, D. J., Eisen, J. S. and Riley, B. B. (1999). Delta-mediated specification of midline cell fates in zebrafish embryos. Curr. Biol. 9, 247-256.[Medline]
Casey, E. S., OReilly, M. J., Conlon, F. L. and Smith, J. C. (1998). The T-box transcription factor Brachyury regulates expression of eFGF through binding to a non-palindromic response element. Development 125, 3887-3894.
Chapman, D. L., Agulnik, I., Hancock, S., Silver, L. M. and Papaioannou, V. E. (1996). Tbx6, a mouse T-box gene implicated in paraxial mesoderm formation at gastrulation. Dev. Biol. 180, 534-542.[Medline]
Chapman, D. L. and Papaioannou, V. E. (1998). Three neural tubes in mouse embryos with mutations in the T-box gene Tbx6. Nature 391, 695-697.[Medline]
Chen, W., Burgess, S. and Hopkins, N. (2001). Analysis of the zebrafish smoothened mutant reveals conserved and divergent function of hedgehog activity. Development 128, 2385-2396.
Chen, Y. and Schier, A. F. (2001). The zebrafish Nodal signal Squint functions as a morphogen. Nature 411, 607-610.[Medline]
Clements, D., Friday, R. V. and Woodland, H. R. (1999). Mode of action of VegT in mesoderm and endoderm formation. Development 126, 4903-4911.
Conlon, F. L., Fairclough, L., Price, B. M. J., Casey, E. S. and Smith, J. C. (2001). Determinants of T box protein specificity. Development 128, 3749-3758.
Conlon, F. L., Sedgwick, S. G., Weston, K. M. and Smith, J. C. (1996). Inhibition of Xbra transcription activation causes defects in mesodermal patterning and reveals autoregulation of Xbra in dorsal mesoderm. Development 122, 2427-2435.
Cornell, R. A. and Eisen, J. S. (2000). Delta signalling mediates segregation of neural crest and spinal sensory neurons from zebrafish lateral neural plate. Development 127, 2873-2882.
Cunliffe, V. and Smith, J. C. (1992). Ectopic mesoderm formation in Xenopus embryos caused by widespread expression of a Brachyury homologue. Nature 358, 427-430.[Medline]
Dheen, T., Sleptsova-Friedrich, I., Xu, Y., Clark, M., Lehrach, H., Gong, Z. and Korzh, V. (1999). Zebrafish tbx-c functions during formation of midline structures. Development 126, 2703-2713.
Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue chez la souris noveau-née et sur lexistence dun caractère (facteur) héréditaire "non-viable". C. R. Seanc. Soc. Biol. 97, 114-116.
Dodd, J., Jessell, T. M. and Placzek, M. (1998). The when and where of floor plate induction. Science 282, 1654-1657.
Ekker, M., Akimenko, M. A, Allende, M. L., Smith, R., Drouin, G., Langille, R. M., Weinberg, E. S. and Westerfield, M. (1997). Relationships among msx gene structure and function in zebrafish and other vertebrates. Mol. Biol. Evol. 14, 1008-1022.[Abstract]
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]
Etheridge, L. A., Wu, T., Liang, J. O., Ekker, S. C. and Halpern, M. E. (2001). Floor plate develops upon depletion of Tiggy-winkle and Sonic hedgehog. Genesis 30, 164-169.[Medline]
Feldman, B., Gates, M. A., Egan, E. S., Dougan, S. T., Rennebeck, G., Sirotkin, H. I., Schier, A. F. and Talbot, W. S. (1998). Zebrafish organizer development and germ-layer formation require nodal-related signals. Nature 395, 181-185.[Medline]
Furthauer, M., Thisse, C. and Thisse, B. (1997). A role for FGF-8 in the dorsoventral patterning of the zebrafish gastrula. Development 124, 4253-4264.
Furthauer, M., Reifers, F., Brand, M., Thisse, B. and Thisse, C. (2001). spouty4 acts in vivo as a feedback-induced antagonist of FGF signaling in zebrafish. Development 128, 2175-2186.
Griffin, K., Patient, R. and Holder, N. (1995). Analysis of FGF function in normal and no tail zebrafish embryos reveals separate mechanisms for formation of the trunk and the tail. Development 121, 2983-2994.
Griffin, K. J. P., Amacher, S. L., Kimmel, C. B. and Kimelman, D. (1998). Molecular identification of spadetail: regulation of zebrafish trunk and tail mesoderm by T-box genes. Development 125, 3379-3388.
Gritsman, K., Talbot, W. S. and Schier, A. (2000). Nodal signalling patterns the organizer. Development 127, 921-932.
Gritsman, K., Zhang, J., Cheng, S., Heckscher, E., Talbot, W. S. and Schier, A. F. (1999). The EGF-CFC protein One-Eyed Pinhead is essential for Nodal signaling. Cell 97, 121-132.[Medline]
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]
Halpern, M. E., Hatta, K., Amacher, S. L., Talbot, W. S., Yan, Y.-L., Thisse, B., Thisse, C., Postlethwait, J. H. and Kimmel, C. B. (1997). Genetic interactions in zebrafish midline development. Dev. Biol. 187, 154-170.[Medline]
Hatta, K., Kimmel, C. B., Ho, R. K. and Walker, C. (1991) The cyclops mutation blocks specification of the floor plate of the zebrafish central nervous system. Nature 350, 339-341.[Medline]
Herrmann, B. G., Labeit, S., Poustka, A., King, T. R. and Lehrach, H. (1990). Cloning of the T gene required in mesoderm formation in the mouse. Nature 343, 617-622.[Medline]
Ho, R. K. and Kane, D. A. (1990). Cell-autonomous action of zebrafish spt-1 mutation in specific mesodermal precursors. Nature 348, 728-730.[Medline]
Horb, M. E. and Thomsen, G. H. (1997). A vegetally localized T-box transcription factor in Xenopus eggs specifies mesoderm and endoderm and is essential for embryonic mesoderm formation. Development 124, 1689-1698.
Hug, B., Walter, V. and Grunwald, D. J. (1997). tbx6, a Brachyury-related gene expressed by ventral mesendodermal precursors in the zebrafish embryo. Dev. Biol. 183, 61-73.[Medline]
Inoue, A., Takahashi, M., Hatta, K., Hotta, Y. and Okamoto, H. (1994). Developmental regulation of islet-1 mRNA expression during neuronal differentiation in embryonic zebrafish. Dev. Dyn. 199, 1-11.[Medline]
Issacs, H. V., Pownall, M. E. and Slack, J. M. W. (1994). EFGF regulates Xbra expression during Xenopus gastrulation. EMBO J. 13, 4469-4481.[Abstract]
Jessell, T. M. (2000). Neuronal specification in the spinal cord: inductive signals and transcriptional codes. Nature Rev. Genet. 1, 20-29.[Medline]
Karlstrom, R. O., Talbot, W. S. and Schier, A. F. (1999). Comparative synteny cloning of zebrafish you-too: mutations in the Hedgehog target gli2 affect ventral forebrain patterning. Genes Dev. 13, 388-393.
Kavka, A. I. and Green, J. B. A. (2000). Evidence for dual mechanisms of mesoderm establishment in Xenopus embryos. Dev. Dyn. 219, 77-83.[Medline]
Kimmel, C. B., Kane, D. A., Walker, C., Warga, R. M. and Rothman, M. B. (1989). A mutation that changes cell movement and cell fate in the zebrafish embryo. Nature 337, 358-362.[Medline]
Kimmel, C. B., Warga, R. M. and Schilling, T. F. (1990). Origin and organization of the zebrafish fate map. Development 108, 581-594.[Abstract]
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]
Kispert, A. and Herrmann, B. G. (1993). The Brachyury gene encodes a novel DNA binding protein. EMBO J. 12, 3211-3220.[Abstract]
Kofron, M., Deme, T., Xanthos, J., Lohr, J., Sun, B., Sive, H., Osada, S.-I., Wright, C., Wylie, C. and Heasman, J. (1999). Mesoderm induction in Xenopus is a zygotic event regulated by maternal VegT via TGFß growth factors. Development 126, 5759-5770.
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]
Krauss, S., Johansen, T., Korzh, V. and Fjose, A. (1991). Expression of the zebrafish paired box gene pax[zf-b] during early neurogenesis. Development 113, 1193-1206.[Abstract]
Le Douarin, N. M. and Halpern, M. E. (2000). Origin and specification of the neural tube floor plate: insights from the chick and zebrafish. Curr. Opin. Neurobiol. 10, 23-30.[Medline]
Lewis, K. E. and Eisen, J. S. (2001). Hedgehog signaling is required for primary motoneuron induction in zebrafish. Development 128, 3485-3495.
Lustig, K. D., Kroll, K. L., Sun, E. E. and Kirschner, M. W. (1996). Expression cloning of a Xenopus T-related gene (Xombi) involved in mesodermal patterning and blastopore lip formation. Development 122, 4001-4012.
Melby, A. E., Warga, R. M. and Kimmel, C. B. (1996). Specification of cell fates at the dorsal margin of the zebrafish gastrula. Development 122, 2225-2237.
Melby, A. M., Kimelman, D. and Kimmel, C. B. (1997). Spatial regulation of floating head expression in the developing notochord. Dev. Dyn. 209, 156-165.[Medline]
Müller, C. W. and Herrmann, B. G. (1997). Crystallographic structure of the T domain-DNA complex of the Brachyury transcription factor. Nature 389, 884-888.[Medline]
Müller, M., v. Weizsäcker, E. and Campos-Ortega, J. A. (1996). Expression domains of a zebrafish homologue of the Drosophila pair-rule gene hairy correspond to primordia of alternating somites. Development 122, 2071-2078.
Nasevicius, A. and Ekker, S. C. (2000). Effective targeted gene knockdown in zebrafish. Nature Genetics 26, 216-220.[Medline]
Odenthal, J., Haffter, P., Vogelsang, E., Brand, M., van Eeden, F. J. M., Furutani-Seiki, M., Granato, M., Hammerschmidt, M., Heisenberg, C.-P., Jiang, Y.-J., Kane, D. A., Kelsh, R. N., Mullins, M. C., Warga, R. M., Allende, M. L., Weinberg, E. S. and Nüsslein-Volhard, C. (1996). Mutations affecting the formation of the notochord in the zebrafish, Danio rerio. Development 123, 103-115.
Odenthal, J. and Nüsslein-Volhard, C. (1998). fork head domain genes in zebrafish. Dev. Genes Evol. 208, 245-258.[Medline]
Odenthal, J., van Eeden, F. J. M., Haffter, P., Ingham, P. W. and Nüsslein-Volhard, C. (2000). Two distinct cell populations in the floor plate of the zebrafish are induced by different pathways. Dev. Biol. 219, 350-363.[Medline]
OReilly, M.-A., Smith, J. C. and Cunliffe, V. (1995). Patterning of the mesoderm in Xenopus: dose-dependent and synergistic effects of Brachyury and Pintallavis. Development 121, 1351-1359.
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]
Phillips, B. T., Bolding, K. and Riley, B. B. (2001). Zebrafish fgf3 and fgf8 encode redundant functions required for otic placode induction. Dev. Biol. 235, 351-365.[Medline]
Placzek, M., Dodd, J. and Jessell, T. M. (2000). The case for floor plate induction by the notochord. Curr. Opin. Neurobiol. 10, 15-22.[Medline]
Pogoda, H. M., Solnica-Krezel, L., Driever, W. and Meyer, D. (2000). The zebrafish forkhead transcription factor FoxH1/Fast1 is a modulator of nodal signaling required for organizer formation. Curr. Biol. 10, 1041-1049.[Medline]
Postlethwait, J. H., Johnson, S. L., Midson, C. N., Talbot, W. S., Gates, M., Ballinger, E. W., Africa, D., Andrews, R., Carl, T., Eisen, J. S., Horne, S., Kimmel, C. B., Hutchinson, M., Johnson, M. and Rodriguez, A. (1994). A genetic linkage map for the zebrafish. Science 264, 699-703.[Medline]
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.
Reifers, F., Bohli, H., Walsh, E. C., Crossley, P. H., Stainier, D. Y. and Brand, M. (1998). Fgf8 is mutated in zebrafish acerebellar (ace) mutants and is required for maintenance of midbrain-hindbrain boundary development and somitogenesis. Development 125, 2381-2395.
Russ, A. P., Wattler, S., Colledge, W. H., Aparicio, S. A., Carlton, M. B., Pearce, J. J., Barton, S. C., Surani, M. A., Ryan, K., Nehls, M. C., Wilson, V. and Evans, M. J. (2000). Eomesodermin is required for mouse trophoblast development and mesoderm formation. Nature 404, 95-99.[Medline]
Ruvinsky, I., Silver, L. M. and Ho, R. K. (1998). Characterization of the zebrafish tbx16 gene and evolution of the vertebrate T-box family. Dev. Genes Evol. 208, 94-99.[Medline]
Ryan, K., Garrett, N., Mitchell, A. and Gurdon, J. B. (1996). Eomesodermin, a key early gene in Xenopus mesoderm differentiation. Cell 87, 989-1000.[Medline]
Sampath, K., Rubinstein, A. L., Cheng, A. M. S., Liang, J. O., Fekany, K., Solnica-Krezel, L., Zorzh, V., Halpern, M. E. and Wright, C. V. E. (1998). Induction of the zebrafish ventral brain and floorplate requires cyclops/nodal signalling. Nature 395, 185-189.[Medline]
Schauerte, H. E., van Eeden, F. J. M., Fricke, C., Odenthal, J., Strähle, U. and Haffter, P. (1998). Sonic hedgehog is not required for the induction of medial floor plate cells in the zebrafish. Development 125, 2983-2993.
Schier, A. F., Neuhauss, S. C. F., Helde, K. A., Talbot, W. S. and Driever, W. (1997). The one-eyed pinhead gene functions in mesoderm and endoderm formation in zebrafish and interacts with no tail. Development 124, 327-342.
Schulte-Merker, S. and Smith, J. C. (1995) Mesoderm formation in response to Brachyury requires FGF signalling. Curr. Biol. 5, 62-67.[Medline]
Schulte-Merker, S., Hammerschmidt, M., Beuchle, D., Cho, K. W., de Robertis, E. M. and Nüsslein-Volhard, C. (1994a). Expression of zebrafish goosecoid and no tail gene products in wild-type and mutant no tail embryos. Development 120, 843-852.
Schulte-Merker, S., van Eeden, F. J. M., Halpern, M. E., Kimmel, C. B. and Nüsslein-Volhard, C. (1994b). no tail (ntl) is the zebrafish homologue of the mouse T (Brachyury) gene. Development 120, 1009-1015.
Schulte-Merker, S., Ho, R. K., Herrmann, B. G. and Nüsslein-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.
Seo, H. C., Saetre, B. O., Havik, B., Ellingsen, S. and Fjose, A. (1998). The zebrafish Pax3 and Pax7 homologues are highly conserved, encode multiple isoforms and show dynamic segment-like expression in the developing brain. Mech. Dev. 70, 49-63.[Medline]
Shinya, M., Furutani-Seiki, M., Kuroiwa, A. and Takeda, H. (1999). Mosaic analysis with oep mutant reveals a repressive interaction between floor-plate and non-floor-plate mutant cells in the zebrafish neural tube. Dev. Growth Differ. 41, 135-142.[Medline]
Sirotkin, H. I., Gates, M. A., Kelly, P. D., Schier, A. F. and Talbot, W. S. (2000). fast1 is required for the development of dorsal axial structures in zebrafish. Curr. Biol. 10, 1051-1054.[Medline]
Smith, J. C., Price, B. M. J., Green, J. B. A., Weigel, D. and Herrmann, B. G. (1991). Expression of a Xenopus homolog of Brachyury (T) is an immediate-early response to mesoderm induction. Cell 67, 79-87.[Medline]
Smith, J. C. (1999). T-box genes: what they do and how they do it. Trends Genet. 15, 154-158.[Medline]
Solnica-Krezel, L., Stemple, D. L., Mountcastle-Shah, E., Rangini, Z., Neuhauss, S. C., Malicki, J., Schier, A. F., Stainier, D. Y., Zwartkruis, F., Abdelilah, S. and Driever, W. (1996). Mutations affecting cell fates and cellular rearrangements during gastrulation in zebrafish. Development 123, 67-80.
Stennard, F., Carnac, G. and Gurdon, J. B. (1996). The Xenopus T-box gene, Antipodean, encodes a vegetally localised maternal mRNA and can trigger mesoderm formation. Development 122, 4179-4188.
Stennard, F., Zorn, A. M., Ryan, K., Garrett, N. and Gurdon, J. B. (1999). Differential expression of VegT and Antipodean protein isoforms in Xenopus. Mech. Dev. 86, 87-98.[Medline]
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]
Strähle, U, Blader, P. and Ingham, P. W. (1996). Expression of axial and sonic hedgehog in wildtype and midline defective zebrafish embryos. Int. J. Dev. Biol. 40, 929-940.[Medline]
Strähle, U., Jesuthasan, S., Blader, P., Garcia-Villalba, P., Hatta, K. and Ingham, P. W. (1997). one-eyed pinhead is required for development of the ventral midline of the zebrafish (Danio rerio) neural tube. Genes Funct. 1, 131-148.[Medline]
Sun, B. I., Bush, S. M., Collins-Racie, L. A., LaVallie, E. R., DiBlasio-Smith, E. A., Wolfman, N. M., McCoy, J. M. and Sive, H. L. (1999). derrière: a TGF-ß family member required for posterior development in Xenopus. Development 126, 1467-1482.
Tada, M. and Smith, J. C. (2001). T-targets: clues to understanding the functions of T-box proteins. Dev. Growth Differ. 43, 1-11.[Medline]
Talbot, W. S., Egan, E. S., Gates, M. A., Walker, C., Ullmann, B., Neuhauss, S. C. F., Kimmel, C. B. and Postlethwait, J. H. (1998). Genetic analysis of chromosomal rearrangements in the cyclops region of the zebrafish genome. Genetics 148, 373-380.
Thisse, C., Thisse, B. and Postlethwait, J. H. (1995). Expression of snail2, a second member of the zebrafish Snail family, in cephalic mesendoderm and presumptive neural crest of wild-type and spadetail mutant embryos. Dev. Biol. 172, 86-99.[Medline]
Thisse, C., Thisse, B., Schilling, T. F. and Postlethwait, J. H. (1993). Structure of the zebrafish snail1 gene and its expression in wild-type, spadetail and no tail mutant embryos. Development 119, 1203-1215.
Thompson, M. A., Ransom, D. G., Pratt, S. J., MacLennan, H., Keiran, M. W., Detrich, H. W., III, Vail, B., Huber, T. L., Paw, B., Brownlie, A. J., Oates, A. C., Fritz, A., Gates, M. A., Amores, A., Bahary, N., Talbot, W. S., Her, H., Beier, D. R., Postlethwait, J. H. and Zon, L. I. (1998). The cloche and spadetail genes differentially affect hematopoiesis and vasculogenesis. Dev. Biol. 197, 248-269.[Medline]
Tokumoto, M., Gong, Z., Tsubokawa, T., Hew, C. L., Uyemura, K., Hotta, Y. and Okamoto, H. (1995). Molecular heterogeneity among primary motoneurons and within myotomes revealed by the differential mRNA expression of novel islet-1 homologs in embryonic zebrafish. Dev. Biol. 171, 578-589.[Medline]
Varga, Z. M., Amores, A., Lewis, K. E., Yan, Y.-L., Postlethwait, J. H., Eisen, J. S. and Westerfield, M. (2001). Zebrafish smoothened functions in ventral neural tube specification and axon tract formation. Development 128, 3497-3509.
Warga, R. M. and Nüsslein-Volhard, C. (1998). spadetail-dependent cell compaction of the dorsal zebrafish blastula. Dev. Biol. 203, 116-121.[Medline]
Weinberg, E. S., Allende, M. L., Kelly, C. S., Abdelhamid, A., Murakami, T., Andermann, P., Doerre, O. G., Grunwald, D. J. and Riggleman, B. (1996). Developmental regulation of zebrafish MyoD in wild-type, no tail, and spadetail embryos. Development 122, 271-280.
Westerfield, M. (1995). The Zebrafish Book. A guide for the laboratory use of zebrafish (Danio rerio). 3rd edition. Eugene: University of Oregon Press.
Wilson, V., Manson, L. Skarnes, W. C. and Beddington, R. S. P. (1995). The T gene is necessary for normal mesodermal morphogenetic cell movements during gastrulation. Development 121, 877-886.
Xanthos, J. B., Kofron, M., Wylie, C. and Heasman, J. (2001). Maternal VegT is the initiator of a molecular network specifying endoderm in Xenopus laevis. Development 128, 167-180.
Yamamoto, A., Amacher, S. L., Kim, S.-H., Geissert, D., Kimmel, C. B. and DeRobertis, E. M. (1998). Zebrafish paraxial protocadherin is a downstream target of spadetail involved in morphogenesis of gastrula mesoderm. Development 125, 3389-3387.
Yan, Y.-L., Hatta, K., Riggleman, B. and Postlethwait, J. H. (1995). Expression of a type II collagen gene in the zebrafish embryonic axis. Dev. Dyn. 203, 363-376.[Medline]
Zhang, J. and King, M. L. (1996). Xenopus VegT RNA is localized to the vegetal cortex during oogenesis and encodes a novel T-box transcription factor involved in mesodermal patterning. Development 122, 4119-4129.
Zhang, J., Houston, D. W., King, M. L., Payne, C., Wylie, C. and Heasman, J. (1998a). The role of maternal VegT in establishing the primary germ layers in Xenopus embryos. Cell 94, 515-524.[Medline]
Zhang, J., Talbot, W. S. and Schier, A. F. (1998b). Positional cloning identifies zebrafish one-eyed pinhead as a permissive EGF-related ligand required during gastrulation. Cell 92, 241-251.[Medline]