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RESEARCH COMMUNICATION
1 Department of Physiological Chemistry I, Biocenter, University of Würzburg, Würzburg 97074, Germany; 2 European Molecular Biology Laboratory, Heidelberg 69117, Germany
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[Keywords: neural patterning; medial floor plate; midkine; pleiotrophin; heparin-binding growth factors; zebrafish]
Received July 1, 2004; revised version accepted February 28, 2005.
). In zebrafish, mutant analyses and lineage tracing have shown that MFP cells originate from a pool of midline precursor cells in the gastrula organizer, the embryonic shield (Appel et al. 1999; Le Douarin and Halpern 2000). Accordingly, the induction of the MFP in zebrafish occurs during gastrulation by shield-derived factors (Tian et al. 2003). Initially induced MFP precursors persist in the tailbud and contribute to the MFP during later embryonic development. The molecular mechanisms and factors that regulate allocation of cells to the MFP in the posteriorly extending trunk after gastrulation, however, remain unclear. Here we describe that midkine-a (mdka) regulates the allocation of MFP precursor cells in zebrafish during neurulation.
Midkine and the related pleiotrophin genes encode secreted heparin-binding growth factors with neurotrophic activity in cell culture assays (for review, see Muramatsu 2002). In mammals, they are widely expressed during embryonic development and have been implicated in processes like outgrowth and survival of neurons, angiogenesis, wound healing, and cancer (for review, see Kadomatsu and Muramatsu 2004). In zebrafish, two midkine genes, mdka and mdkb are expressed in restricted and nonoverlapping patterns during embryonic development (Winkler et al. 2003). Analyzing its expression, regulation, and activity, we show that Mdka expressed in the paraxial mesoderm functions in MFP formation in zebrafish.
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Results and Discussion |
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) and other markers (Fig. 2d,e,g,h; data not shown). While the overall morphology of the embryos at the 14 somite (14 s) stage appeared normal, the width of the MFP increased from one to three cell diameters in anterior trunk regions (data not shown) and was even broader in more posterior regions (Fig. 2e,h). The effect of ubiquitously expressed mdka on the neural tube was confined to the MFP. Lateral floor plate cells expressing nkx2.2b (Schäfer et al. 2005) and the motoneuron domain expressing olig2 (Park et al. 2002) were shifted laterally, but otherwise showed no significant differences in number and size (Fig. 2l-o; see Supplementary Fig. S1). Thus, ectopic mdka specifically promotes MFP formation.
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The observed formation of MFP cells at the expense of the notochord is in agreement with earlier reports on cell fate restrictions in the pool of midline precursor cells (Halpern et al. 1997; Appel et al. 1999; Amacher et al. 2002). Expansion of the MFP in zebrafish (e.g., by ectopically expressed deltaA) coincides with the reduction of the notochord (Appel et al. 1999). Also, mutants deficient for the T-box gene no tail (ntl)/Brachyury lack the notochord, and instead show a wider MFP (Halpern et al. 1997; Amacher et al. 2002). Therefore, to promote the notochord fate, ntl appears to be required to repress MFP. This suggests that several counteracting pathways regulate the decision of midline cell fates. Mdka promotes MFP and acts during tailbud extension, but in contrast to ntl and deltaA is not expressed in the tailbud. Consequently, inactivation of Mdka should therefore lead to a reduction of MFP during post-gastrulation stages.
To inactivate Mdka function we used a morpholino antisense approach (Nasevicius and Ekker 2000). Knock-down of mdka resulted in severe defects in MFP formation (Fig. 3). Analysis of several markers revealed large and irregularly spaced gaps in the MFP (Fig. 3d-f). These were consistently restricted to the trunk region posterior to the hindbrain, supporting the hypothesis that mdka is not involved in MFP formation during early gastrulation. We never obtained a complete lack of MFP in morpholino-injected embryos. This indicates that parallel pathways regulate MFP formation. A similar situation was reported in Delta-Notch-deficient mutant embryos (Appel et al. 1999), while mutants deficient for the nodal-related gene cyc show a complete loss of MFP (Halpern et al. 1997; Tian et al. 2003). The appearance of gaps is best explained by an incomplete redundancy of these pathways in different precursor cell populations. Alternatively, these gaps could reflect an incomplete knock-down of Mdka that results in an uneven allocation of remaining MFP cells during tailbud extension movements.
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; Appel et al. 1999) and that mdka is required for allocation of initially induced progenitor cells, we also noticed defective hypochord development in mdka morpholino-injected embryos. Large gaps were evident in this structure located underneath the notochord (Fig. 3f).
Cell lineage and mutant analyses in chicken, Xenopus, and zebrafish suggested that MFP specification occurs early during gastrulation in the organizer (Halpern et al. 1997; Teillet et al. 1998; Appel et al. 1999; Lopez et al. 2003). In zebrafish, the continuous signaling by nodal-related Cyclops is required for early MFP induction during gastrulation (Tian et al. 2003). In addition, Ntl, Floating head (Flh) and the Delta-Notch pathway are implicated in MFP formation (Halpern et al. 1997; Appel et al. 1999; Amacher et al. 2002). As Mdka promotes MFP at the expense of the notochord, our data provide additional support for this model and open the possibility that Mdka interacts with these pathways during allocation of MFP cells initially induced in the shield. To investigate Mdka's interactions with these factors, we analyzed the effects of mdka overexpression and knockdown in several mutant conditions (Fig. 4).
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). Ectopic expression of mdka in flh-/- mutants partially rescued the MFP defect. On the other hand, knockdown had no influence on the remaining MFP cells, suggesting that endogenous Mdka is not responsible for the appearance of remaining MFP islands in flh-/- mutants (Fig. 4b,c). Mindbomb (mib) mutants are deficient for a RING-type ubiquitin ligase that is essential for Delta/Notch signaling (Itoh et al. 2003). These mutants lack single MFP cells similar to the situation in dlA-/- mutants (Fig. 4f; Appel et al. 1999). Mdka overexpression and knockdown in mib-/- mutants produced identical MFP and notochord phenotypes as in wild-type embryos (Fig. 4h-j). This suggests that the Mdka pathway acts in parallel to and independent of Delta/Notch signaling.
Mutant analyses in zebrafish have shown that continuous Cyc signaling during 60%-90% epiboly is crucial for MFP formation (Tian et al. 2003). One-eyed-pinhead (oep) encodes an essential extracellular cofactor for Cyc signaling (Gritsman et al. 1999). Homozygous oep mutants consequently lack the complete MFP (Fig. 4m; Strähle et al. 1997). Overexpression of mdka in oep-/- mutants rescued the mutant phenotype and resulted in an enlarged MFP (Fig. 4n). To show that mdka is also required for cyc dependent MFP formation, we coinjected cyc RNA together with mdka morpholinos. Overexpression of cyc alone resulted in an expansion of MFP (Fig. 4o; Tian et al. 2003). Coinjection rescued this effect and resulted in a MFP with normal width and sporadic appearance of gaps (Fig. 4p; data not shown), suggesting that mdka is required for the effects of cyc overexpression on MFP expansion. Ntl-/- mutants, as described above, lack a notochord, but have an enlarged MFP (Fig. 4q,r,u). In ntl-/-, mdka knockdown resulted in a nearly complete rescue of the MFP phenotype (Fig. 4s,v). However, no rescue of the notochord was observed, possibly due to defects in the earliest steps of notochord formation. This shows that mdka affects MFP cells even in the absence of a notochord. Taken together, these results indicate that mdka is capable of rescuing defects in early MFP induction.
Intriguingly, mdka overexpression could rescue MFP formation in mutants with defects in early MFP induction, which show reduced number of MFP precursors. This is best explained by ectopic Mdka activity in the tailbud, where it drives remaining precursors into the MFP fate. Inhibition of endogenous Mdka in embryos with expanded MFP (ntl-/- mutants and cyc-overexpressing embryos) reduced the MFP size, showing that allocation of MFP cells is controlled by trunk-derived Mdka. Thus, in addition to known signals from the shield (and possibly the organizer in higher vertebrates), MFP formation is also controlled by signals from outside these regions.
The epistatic analysis opened the possibility that mdka acts either independently or downstream of factors known to induce MFP during gastrulation. To test whether mdka is regulated by these factors at the transcriptional level, we analyzed its expression in mutants of the corresponding pathways (Fig. 5). In zebrafish, deltaA is expressed in the tailbud and regulates MFP formation within the midline precursor cell population (Appel et al. 1999). In delta/notch-deficient mib-/- mutants, mdka transcription was not affected (Fig. 5b). At the four-somite stage, ntl, oep, and flh are all expressed in the notochord and its precursors (Fig. 5c,e,g). In ntl-/- and oep-/-, no effect on mdka expression was observed (Fig. 5d,f). In flh-/-, ectopic mdka expression was evident in the anterior midline mesoderm (Fig. 5h). This, however, does not result from a direct regulation by flh, but rather results from a lack of a notochord in these mutants as notochordal precursors transfate into somitic cells. This consequently leads to the presence of somitic mesoderm at the midline. Despite ectopic mdka expression in the midline of flh-/- mutants, no expanded MFP was observed. Also, mdka knockdown had no effect on MFP formation in flh-/- (Fig. 4c). This could be explained by the rapid transformation of flh-/- midline cells to somitic cells at a stage before Mdka becomes active and can drive these precursors into the MFP fate. Most notably, in all analyzed mutants mdka was not ectopically expressed in the tailbud region, where midline precursor cells are located. Thus, mdka expression appears not to be regulated by factors known as inducers of MFP cells during gastrulation. Our preliminary data suggest that mdka expression is regulated by Shh. Ectopic overexpression of shh results in a repression of mdka transcription in the paraxial mesoderm (data not shown). Importantly, it has been shown that Shh is not involved in MFP formation in zebrafish (Schauerte et al. 1998). Consequently, the MFP in shh-overexpressing embryos is normal (data not shown). It therefore is likely that Shh overexpression does not sufficiently inhibit Mdka activity to interfere with MFP formation.
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We therefore propose a two-phase model for MFP formation in zebrafish (Fig. 5i). In a first Mdka-independent phase during gastrulation, induction of MFP precursors in the embryonic shield is regulated by Ntl, Cyc, Delta/Notch, and others (Halpern et al. 1997; Appel et al. 1999; Amacher et al. 2002; Tian et al. 2003). During neurulation, midline precursor cells in the tailbud are still multipotential and form MFP, as well as the notochord and hypochord. Based on our data, we now postulate a second Mdka-dependent phase of MFP formation that becomes important during this period of development. As the embryo elongates along its anteroposterior axis, initially induced precursors come under influence of Mdka expressed within the paraxial mesoderm in a caudally extending wave. This results in the refinement of a subset of precursor cells and their final allocation into the MFP. Our data have shown that both phases are required for complete formation of MFP. These phases appear strictly separated from each other in a temporal and spatial manner.
Conclusion
Two different models have been used in the past to explain MFP formation in vertebrates (see Strähle et al. 2004). On the one hand, several lines of evidence suggest that MFP is induced in the neural plate during neurulation in chicken and mouse. This is controlled by signals secreted from the underlying notochord, most notably Sonic Hedgehog (Shh) (Placzek et al. 1993; Chiang et al. 1996). Accordingly, floor plate and notochord cells do not share the same origin (Patten et al. 2003; Jeong and Epstein 2003). The alternative model postulates an induction of MFP in the early organizer within a common pool of midline precursor cells that also give rise to the notochord and hypochord (Halpern et al. 1997; Teillet et al. 1998; Appel et al. 1999; Le Douarin and Halpern 2000). Our observations are consistent with an origin of MFP cells from midline precursor cells in zebrafish. In addition to this we show that signals from outside the shield are required for complete MFP formation. This aspect is consistent with the model of trunk-derived signals involved in this process. While these signals are secreted from the notochord in chicken (Placzek et al. 2000), we show an involvement of paraxial mesoderm in zebrafish.
In higher vertebrates, Midkine and the related Pleiotrophin show widespread expression during embryogenesis. In vitro experiments have implicated these growth factors in a variety of processes, including neurite outgrowth and survival, angiogenesis, and tumor growth (for review, see Muramatsu 2002). The exact in vivo function, in particular its role during mammalian MFP formation, however, remains unclear as Midkine knockout mice show no obvious embryonic phenotype (Nakamura et al. 1998). In vitro binding studies identified several candidate receptors for Midkine, including receptor-type protein-tyrosine phosphatase 
and LDL receptor-related proteins (LRPs) (for review, see Kadomatsu and Muramatsu 2004). It will be interesting to analyze in the future whether any of these candidate receptors binds to Mdka in vivo and is dynamically expressed in subsets of midline precursor cells during embryonic development.
The identification of Mdka as a novel factor involved in MFP formation is a step forward on the way to elucidate this process in higher vertebrates and allows us to explain species-specific differences.
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Materials and methods |
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Wild-type fish were reared and embryos obtained as described (Westerfield 1995). The mutant alleles used were floating head (flhn1), mindbomb (mibta52b), no tail (ntlb195), and one-eyed-pinhead (oepm134).
RT-PCR analysis
For analysis of mdka expression, cDNA was prepared from pooled zebrafish embryos at different stages and used for RT-PCR using primers MK3-105 (5'-CACTCTGCATTTGACTTTCCT-3') and MKEST-UP (5'-GTTTCAGTGAGGGAACTTTCG-3'). Actin expression was determined for calibration using the primers actin-up (5'-TTCAACAGCCCTGCCATGTA-3') and actin-down (5'-GCAGCTCATAGCTCTTCTCCAGGGAG-3').
RNA in situ hybridization
One- and two-color whole mount in situ hybridizations were performed as described (Winkler et al. 2003). Stained embryos were manually dissected from the yolk and flat mounted in 100% glycerol for photography. Sections were made manually using razor blades.
Zebrafish embryo injection
Capped messenger RNA was transcribed in vitro as described (Winkler et al. 2003). Fifty picograms mdka and 3 pg cyc RNA were injected into embryos at the one- to two-cell stage. For knockdown experiments, embryos were injected with the morpholino mdka MO (5'-CCGCATTTTGTTTTCTGTGTCGAAA-3') at different doses. The most efficient dose (18-20 ng/nL) was determined and the specificity of the oligo confirmed in control experiments (see Supplementary Fig. S4).
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TopAbstractResults and DiscussionMaterials and methodsAcknowledgmentsReferences |
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Footnotes |
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Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/gad.336305.
3 Corresponding author.
E-MAIL cwinkler{at}biozentrum.uni-wuerzburg.de; FAX 49-931-888 4150. 
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References |
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TopAbstractResults and DiscussionMaterials and methodsAcknowledgmentsReferences |
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