Department of Biological Sciences, Vanderbilt University, Nashville, TN 37232, USA
*Author for correspondence (e-mail: b.appel{at}vanderbilt.edu)
Accepted 15 March 2002
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SUMMARY |
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Key words: Delta, Notch, Notochord, Hypochord, Midline, Fate map, Zebrafish, Gastrulation
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INTRODUCTION |
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In zebrafish, development of midline cells requires the Delta ligand-Notch receptor signaling system. Specifically, embryos with a mutation of deltaA (dla), one of four known zebrafish Delta homologs, have a deficit of floor-plate and hypochord cells and excess notochord cells; by contrast, overexpression of dla blocked notochord development and produced excess floor-plate and hypochord cells (Appel et al., 1999). Fate mapping had shown that notochord and floor-plate precursors are close together in the zebrafish organizer (Shih and Fraser, 1995
; Melby et al., 1996
) and functional analyses of no tail (ntl), which encodes a T-box transcription factor homologous to mouse Brachyury, had indicated that Ntl mediates a choice between notochord and floor-plate fates (Halpern et al., 1997
). As gastrula stage zebrafish embryos express Delta and Notch genes (Bierkamp and Campos-Ortega, 1993
; Dornseifer et al., 1997
; Westin and Lardelli, 1997
; Haddon et al., 1998
; Appel et al., 1999
), we hypothesized that Delta-Notch signaling during gastrulation regulates specification of zebrafish midline precursors for notochord, floor-plate and hypochord fates by regulating ntl expression.
We present tests of hypochord specification. By fate mapping, we found that hypochord precursors arise from the edge of the shield, which is the zebrafish gastrula organizer. These cells are closely associated with cells that become either notochord or slow muscle but not floor plate. Presumptive hypochord precursors express notch5 and her4, a Notch target gene, during gastrulation. Initially, all her4-expressing cells express ntl, which marks midline precursors, but later they do not. This raises the possibility that hypochord precursors emerge from the ntl-positive midline precursor population. Cells that express her4 are close to cells of the paraxial mesoderm, which express deltaC (dlc) and deltaD (dld), and loss-of-function experiments show that dla, dld and dla, which gastrulating embryos uniformly express, are redundantly required for her4 expression and hypochord development. Finally, we show that unregulated Notch activity inhibits ntl expression. We propose that cells at the lateral edges of the midline precursor domain can develop either as notochord or hypochord, and that hypochord development is induced by Delta ligands expressed by neighboring paraxial mesoderm cells.
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MATERIALS AND METHODS |
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Cell labeling
We injected one- to four-cell stage zebrafish embryos with a 2% solution of DMNB-caged fluorescein dextran (Molecular Probes) in 1x Danieau solution. Injected embryos were maintained in the dark at 28.5°C in embryo medium until shield stage. Embryos were dechorionated with watchmakers forceps and mounted in 2% methyl cellulose on bridged slides with the dorsal side of the embryos facing upwards. The dye was photoactivated in three to five cells using 5-10 second pulses of 365 nm light (Serbedzija et al., 1998) generated by a Photonics Micropoint Laser System focused using a 40x objective mounted on a Zeiss Axiskop. Dye activation was confirmed using epifluorescence optics. Labeled embryos were maintained at 28.5°C until 24 hpf, at which time the fates of the labeled cells were determined by examining living embryos with a compound microscope. Images were obtained using a Hammamatsu Orca CCD camera. Embryos selected for histochemical analysis were fixed in 4% paraformaldehyde.
In situ RNA hybridization and immunohistochemistry
Previously described probes include those for notch5 (Westin and Lardelli, 1997), notch1a (Bierkamp and Campos-Ortega, 1993
), her4 (Takke et al., 1999
), myod (Weinberg et al., 1996
), ntl (Schulte-Merker et al., 1994
), dld and dlc (Haddon et al., 1998
), col2a1 (Yan et al., 1995
), and isl1 (Appel et al., 1995
). In situ RNA hybridization was performed essentially as described (Hauptmann and Gerster, 2000
; Jowett, 2001
). For double labeling, best results were obtained by staining with BCIP/NBT (Roche Diagnostics) first, followed by staining with BCIP/INT (Roche Diagnostics). To convert the fluorescent signal in photoactivated embryos to a blue precipitate, the embryos were incubated with alkaline phosphatase-conjugated anti-fluorescein antibody (Roche Diagnostics) at 1:10,000 dilution followed by staining with BCIP/NBT. Slow muscle cells were detected by incubating embryos with monoclonal F59 antibody (gift of Frank Stockdale) at 1:10 dilution, followed by incubation with Alexa Fluor 568 goat anti-mouse IgG conjugate (Molecular Probes) at 1:200 dilution. Myc expression was detected using monoclonal anti-Myc IgG antibody (Oncogene Research Products) at 2 µg/ml, followed by goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) at 1:500 dilution, then streptavidin-conjugated peroxidase (Jackson ImmunoResearch Laboratories) at 1:1000. Peroxidase activity was used to produce a brown precipitate in the presence of Fast DAB (Sigma).
Embryos for sectioning were embedded in 1.5% agar/5% sucrose and frozen in 2-methyl-butane chilled by liquid nitrogen. Sections (10 µm) were obtained using a cryostat microtome.
Whole-mount embryos were cleared in methanol, mounted in 75% glycerol and photographed with a Spot digital camera (Diagnostic Instruments) mounted on a compound microscope. Images of sectioned material were also obtained with the Spot camera.
Antisense morpholino oligonucleotide, DNA and RNA injections
Morpholino antisense oligonucleotides (Gene Tools, LLC.) for dla (5'-CTTCTCTTTTCGCCGACTGATTCAT-3') and dlc (5'-AGCACGTAATAAAACACGAGCCAT-3') were resuspended in water. Working dilutions of 2.5-5.0 mg/ml were made using 1xDanieau solution and approximately 5 nl injected into one- to two-cell stage embryos collected from matings of wild-type or aeiAR33/+ fish using a pressure injector (Applied Scientific Instruments, Inc.). Injected embryos were fixed at yolk-plug closure, three-somite stage or 24 hpf in 4% paraformaldehyde, and were processed for in situ RNA hybridization. Synthetic mRNA encoding the intracellular domain of Xenopus laevis Notch1 fused to the Myc tag (NICDMT) (Wettstein et al., 1997) was produced from a construct kindly provided by Chris Kintner using the mMessage Machine kit from Ambion. Approximately 5 nl mRNA at 100 ng/µl was injected into a single cell of eight-cell stage embryos. To make a construct for heat-shock induction of NICDMT, we moved coding sequence from our mRNA expression plasmid and fused it to the zebrafish heat shock 70 promoter (Shoji et al., 1998
) in pBluescript and injected approximately 5 nl of the resulting plasmid at 50 µg/µl in the blastodisc of one-cell zygotes. Injected embryos were raised at 28.5°C until shield stage, and subjected to three cycles of 37°C for 15 minutes followed by 28.5°C for 45 minutes. After the end of heat shock cycles, the embryos were raised at 28.5°C until yolk plug closure stage and fixed in 4% paraformaldehyde.
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RESULTS |
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Presumptive hypochord precursors express notch5 and her4
Cells bordering the posterior dorsal midline of mid and late gastrula stage embryos express notch5 and her4 (Westin and Lardelli, 1997; Takke et al., 1999
) (Fig. 2A-D). The distribution of these cells is similar to the distribution of hypochord precursors as they move towards the dorsal midline (see above). To investigate the identity of cells that express notch5 and her4, we examined sectioned material from embryos hybridized with RNA probes. Sagittal and transverse sections of 9.5 hpf embryos at the end of the gastrula stage showed that deep mesendodermal cells, close to the yolk, express notch5 and her4 (Fig. 2E-G,I-L). her4- or notch5-positive cells are next to adaxial cells, marked by myod expression (Fig. 2F,G). We never observed cells that express both notch5 and myod or her4 and myod. Cells that express her4 and notch5 also were closely associated with ntl-positive midline precursors (Fig. 2H-K). Posteriorly, all her4-positive cells express ntl (Fig. 2I). More anteriorly some her4-positive cells express ntl but others do not (Fig. 2J). Similarly, some notch5-positive cells express ntl, whereas others do not (Fig. 2K). The similarity of her4 and notch5 RNA distribution indicated that the same cells might express these genes, which we confirmed by double labeling (Fig. 2L).
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The coincident expression of notch5 and her4 is consistent with the possibility that Notch5 activity promotes her4 expression. However, we detect her4 RNA at the same time or, perhaps, slightly earlier than notch5 RNA. Midline cells express at least two other notch genes, notch1a and notch1b (Bierkamp and Campos-Ortega, 1993; Westin and Lardelli, 1997
) (Fig. 2N). This observation opens the possibility that multiple Notch receptors promote her4 transcription in presumptive hypochord precursors.
Taken together, our data indicate that hypochord precursors arise at the lateral edges of the ntl-expressing midline precursor domain and that similarly positioned notch5/her4-positive presumptive hypochord precursors downregulate ntl expression. These observations raise the possibility that hypochord cells originate as ntl-expressing midline precursors.
deltaD and deltaC-expressing cells border presumptive hypochord precursors
Gastrulation stage zebrafish embryos express three of four known Delta genes. During mid and late gastrulation stages, embryos express dla uniformly, except for a few cells near the dorsal midline margin that transiently express dla at elevated levels (Appel et al., 1999). By contrast, paraxial mesoderm, but not axial mesoderm, strongly expresses dlc and dld (Dornseifer et al., 1997
; Haddon et al., 1998
) (Fig. 3A-F). Double labeling experiments showed that dlc and dld-expressing cells are adjacent to ntl-positive midline precursors (Fig. 3G,H and data not shown). Additionally, we found that dlc-positive cells are next to her4-expressing cells (Fig. 3I). This raises the possibility that her4 expression is induced by DeltaC and DeltaD ligands expressed by neighboring paraxial mesoderm cells.
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To test the specificity of our MO effects we examined primary neurogenesis. Neural plate cells express dla and dld but not dlc (Dornseifer et al., 1997; Appel and Eisen, 1998
; Haddon et al., 1998
). dladx2 mutant embryos have a large excess of primary neurons, perhaps because of the dominant negative nature of the allele (Appel et al., 1999
; Appel et al., 2001
). aeiAR33 mutant embryos have only a small increase in the number of primary neurons (Holley et al., 2000
) (Fig. 4I). Twenty-four percent of embryos (22/90 embryos) produced by an intercross of aeiAR33/+ adults and injected with approximately 25 ng dlc MO had primary neuron phenotypes similar to uninjected aeiAR33 homozygous mutant embryos (Fig. 4J). Thus, reduction of dlc function did not appear to enhance the neural phenotype produced by loss of dld function, indicating that the dlc MO did not interfere, nonspecifically, with neurogenesis. By contrast, we found that injection of dla MO produced excess primary neurons in a dose-dependent manner when injected into wild-type embryos (data not shown). Injection of approximately 5 ng dla MO, which did not cause a strong neural phenotype in wild-type embryos, greatly enhanced the neural phenotype of aeiAR33 mutant embryos (17/74 embryos; 23%) (Fig. 4K). As the dla and dlc MO phenotypes correlate with the dla and dlc expression patterns, we conclude that the MO effects are specific to their intended targets.
Notch signaling represses ntl expression by midline precursors
One interpretation of our results is that Delta signaling, primarily from paraxial mesoderm, to midline precursors expressing Notch receptors inhibits ntl expression and promotes her4 expression, consequently instructing these cells to develop as hypochord. We tested this by expressing a constitutively active form of Notch. To perform these experiments, we found it necessary to limit the number of cells that expressed constitutively active Notch so that embryos would develop fairly normally. First, we confirmed and extended a previous observation that Notch activity promotes her4 expression (Takke et al., 1999) by using a heat shock promoter (Shoji et al., 1998
) to express during gastrulation a constitutively active form of X. laevis Notch1 (Wettstein et al., 1997
) fused to the Myc tag (NICDMT). This resulted in embryos that had ligand-independent Notch activity in some but not all cells. Of 57 injected embryos, 44 had numerous cells that expressed NICDMT, identified by staining for the fused Myc epitope. Many of these cells expressed her4 (data not shown). Analysis of sectioned embryos revealed that NICDMT-positive cells ectopically expressed her4 (Fig. 5A). We never saw ectopic her4-expressing cells that did not express NICDMT, indicating that Notch induction of her4 expression is cell autonomous. Next, we injected one cell of eight-cell stage embryos with synthetic mRNA encoding NICDMT, raised the embryos to the end of the gastrula stage and probed for ntl expression. Twenty-two of 116 (19%) injected embryos had midline cells that expressed NICDMT; the ntl expression domain was reduced in each of these indicating that unregulated Notch activity in midline precursor cells inhibits ntl expression (Fig. 5B,C). Tissue sections revealed that NICDMT-positive midline cells did not express ntl, whereas neighboring cells did (Fig. 5D), indicating that Notch activity cell autonomously inhibits ntl expression. Together, these observations show that Notch activity can inhibit ntl expression and promote her4 expression, consistent with our hypothesis that Delta-Notch signaling induces a subset of midline precursors to develop as hypochord.
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DISCUSSION |
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Fate mapping of axolotl and histological analyses of frog and fish embryos indicated that hypochord is derived from endoderm (Lofberg and Collazo, 1997; Cleaver et al., 2000
; Eriksson and Lofberg, 2000
). However, labeled hypochord cells did not appear in a fate map of zebrafish endoderm (Warga and Nusslein-Volhard, 1999
). In addition, mutations of one-eyed-pinhead and casanova, which are required for endoderm development (Schier et al., 1997
; Alexander et al., 1999
), do not result in loss of trunk hypochord (Alexander et al., 1999
) (and data not shown). Our data showing hypochord precursors are closely associated with precursors of other mesodermal cell types at the onset of gastrulation indicate that zebrafish hypochord originates from mesoderm and not endoderm.
Distinct cell populations express Delta and Notch genes
Previous publications showed that cells that form two columns on either side of the midline of late-gastrulation stage embryos transcribe notch5 and her4 (Westin and Lardelli, 1997; Takke et al., 1999
). We demonstrated here that the same cells expressed notch5 and her4, and that they were deep within the mesoderm, close to the yolk. The distribution of notch5- and her4-expressing cells during late gastrula stage was similar to distribution of photoactivated cells that went on to produce hypochord. After gastrula stage, notch5- and her4-expressing cells were ventral to notochord, in the final position of hypochord. We also found that cells that expressed notch5 and her4 during late gastrulation were next to adaxial cells, the slow muscle precursors. This is consistent with our fate-mapping results that hypochord-containing polyclones often contained slow muscle cells. notch5- and her4-expressing cells also were close to ntl-positive midline precursors, again consistent with our observations that polyclones often contained both hypochord and notochord cells. Thus, notch5- and her4-expressing cells are probably hypochord precursors.
We showed previously that gastrulating embryos express dla uniformly, except for transient upregulation at the dorsal midline (Appel et al., 1999). Thus, the pattern of dla transcription does not predict the pattern of hypochord precursor specification. We now show that paraxial mesoderm, which includes adaxial cells adjacent to midline precursors, expresses dlc and dld. Thus, adaxial cells could be the primary source of Delta signals that induce hypochord development.
Delta function is required for hypochord specification
Results from our loss-of-function experiments support the idea that dlc and dld signaling from adaxial cells induce hypochord development and also indicate that dla may contribute to hypochord specification. Embryos that lack dld function, by mutation, or have reduced dlc function, by MO injection, have incompletely penetrant and variably expressive hypochord phenotypes. By contrast, all embryos that lack both dlc and dlc functions have very few hypochord cells, indicating that dld and dlc are functionally redundant for hypochord specification. We showed previously that embryos having the dominant negative dladx2 mutant allele have fewer hypochord cells (Appel et al., 1999). This mutant allele, which is predicted to substitute tyrosine for a conserved cysteine in the second EGF repeat of the extracellular domain, may produce DeltaA protein that can interfere with the functions of DeltaD and DeltaC, perhaps by forming heterodimers. As gastrulating embryos uniformly express dla transcripts, mutant DeltaA protein could interfere with the hypochord-inducing functions of DeltaD and DeltaC. However, we found here that reduction of dla function by antisense morpholino oligonucleotide injection also reduced the number of hypochord cells in wild-type embryos and enhanced the penetrance and expressivity of the aei/dld mutant phenotype. This apparent requirement for dla activity raises the question of whether a localized source of Notch ligand can fully account for the pattern of hypochord specification. However, dla is not sufficient to specify hypochord in the absence of dlc and dld. Thus, we propose that high levels of Delta proteins produced by adaxial cells are required to induce midline precursors to develop as hypochord (Fig. 6).
How might Delta signals induce hypochord development? One key might be regulation of ntl expression. ntl mutant embryos lack notochord and rostral hypochord and have excess floor plate (Rissi et al., 1995; Halpern et al., 1997
). Halpern et al. (Halpern et al., 1997
) proposed that ntl regulates a midline precursor fate decision by promoting notochord and inhibiting floor-plate development. We propose that modulation of ntl expression within midline precursors by Delta-Notch signaling is required for hypochord development. In our model, ntl promotes formation of a population of midline precursors that have the potential to develop either as notochord or hypochord. Activation of Notch in a subset of precursors by Delta ligands expressed by neighboring paraxial mesoderm cells induces her4 and represses ntl expression. Consistent with this, we show here that constitutive Notch activity can cell-autonomously drive ectopic her4 expression. In the absence of Notch activity, her4 expression is not induced, as we demonstrated here, and excess midline cells express ntl, as we showed previously (Appel et al., 1999
). Thus, Notch activity diverts midline precursors from notochord to hypochord fate.
As her4 is a member of the hairy-Enhancer of split gene family, which generally encode transcription repressors (reviewed by Fisher and Caudy, 1998), Notch inhibition of ntl expression could occur via direct action of Her4 on ntl regulatory elements. Notably, in the tunicate Ciona intestinalis, Notch activity promotes expression of the Brachyury gene, apparently by direct binding of Supressor of Hairless (Corbo et al., 1997
; Corbo et al., 1998
). Direct comparison of the regulatory DNA that controls expression of Brachyury homologs in tunicates and zebrafish may provide interesting insights to the evolution of the embryonic midline.
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ACKNOWLEDGMENTS |
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