Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH COMMUNICATION activity in cell culture assays (for review, see Muramatsu Medial floor plate formation 2002). In mammals, they are widely expressed during em- in zebrafish consists of two bryonic development and have been implicated in pro- cesses like outgrowth and survival of neurons, angiogen- phases and requires esis, wound healing, and cancer (for review, see Kadomatsu and Muramatsu 2004). In zebrafish, two midkine genes, trunk-derived Midkine-a mdka and mdkb are expressed in restricted and nonover- Matthias Schäfer,1 Martina Rembold,2 lapping patterns during embryonic development (Winkler et al. 2003). Analyzing its expression, regulation, and ac- Joachim Wittbrodt,2 Manfred Schartl,1 and 1,3 tivity, we show that Mdka expressed in the paraxial meso- Christoph Winkler derm functions in MFP formation in zebrafish. 1Department of Physiological Chemistry I, Biocenter, University of Würzburg, Würzburg 97074, Germany; Results and Discussion 2European Molecular Biology Laboratory, Heidelberg 69117, Germany mdka transcription is first detectable by RT–PCR during late gastrulation (at 75% epiboly; 8 h post-fertilization; The medial floor plate (MFP) organizes the specification hpf) (Fig. 1a). At the beginning of neurulation (10 hpf), of neurons and outgrowth of axons in the ventral spinal when the tailbud starts to extend caudally to form the cord of vertebrates. We show that the growth factor Mid- trunk and tail, mdka transcription is confined to the kine-a, expressed in the paraxial mesoderm, is required rostral paraxial mesoderm that is still unsegmented at for formation of the MFP in zebrafish. Our epistatic this stage (Fig. 1b). Later, its expression moves as a wave analyses demonstrate that development of MFP com- from anterior to posterior in this region (Fig. 1c–h). The prises two independent sequential phases. Following ini- caudal front of the mdka wave in the paraxial mesoderm tial MFP induction in the gastrula organizer, Midkine-a progresses in parallel to the formation of the neural keel regulates allocation of MFP cells during subsequent de- that emerges from the converging neural plate (Fig. 1i,j). velopment. Thus in zebrafish, trunk-derived signals are At the same point, the morphologically distinct MFP required for complete MFP formation from a common starts to form, which is marked by the restriction of pool of organizer-derived midline precursor cells. broad tiggy-winkle hedgehog (twhh) expression to a row of single cells (Fig. 1k). Therefore, expression of mdka Supplemental material is available at http://www.genesdev.org. coincides temporally and spatially with the appearance Received July 1, 2004; revised version accepted February 28, of a morphologically recognizable floor plate. After 13 2005. hpf, mdka expression is also found dorsally in the ante- rior neural tube (Fig. 1l; data not shown). The medial floor plate (MFP) is the ventral-most struc- We first addressed the function of mdka during MFP ture in the vertebrate neural tube with important orga- formation by overexpressing Mdka through mRNA in- nizer functions during specification and differentiation jection into zebrafish embryos (Fig. 2). This resulted in a of neurons and glia. Its origin and the timing of the speci- strong expansion of the MFP as defined by the expression fication are matters of ongoing debates (see Strähle et al. of sonic hedgehog (shh) (Krauss et al. 1993) and other 2004). In zebrafish, mutant analyses and lineage tracing markers (Fig. 2d,e,g,h; data not shown). While the overall have shown that MFP cells originate from a pool of mid- morphology of the embryos at the 14 somite (14 s) stage line precursor cells in the gastrula organizer, the embry- appeared normal, the width of the MFP increased from onic shield (Appel et al. 1999; Le Douarin and Halpern one to three cell diameters in anterior trunk regions (data 2000). Accordingly, the induction of the MFP in ze- not shown) and was even broader in more posterior re- brafish occurs during gastrulation by shield-derived fac- tors (Tian et al. 2003). Initially induced MFP precursors gions (Fig. 2e,h). The effect of ubiquitously expressed persist in the tailbud and contribute to the MFP during mdka on the neural tube was confined to the MFP. Lat- later embryonic development. The molecular mecha- eral floor plate cells expressing nkx2.2b (Schäfer et al. nisms and factors that regulate allocation of cells to the 2005) and the motoneuron domain expressing olig2 (Park MFP in the posteriorly extending trunk after gastrula- et al. 2002) were shifted laterally, but otherwise showed tion, however, remain unclear. Here we describe that no significant differences in number and size (Fig. 2l–o; midkine-a (mdka) regulates the allocation of MFP pre- see Supplementary Fig. S1). Thus, ectopic mdka specifi- cursor cells in zebrafish during neurulation. cally promotes MFP formation. Midkine pleiotrophin and the related genes encode se- The significant increase in MFP cell number could creted heparin-binding growth factors with neurotrophic simply be due to enhanced cell proliferation in this structure. However, immunostaining for phospho his- [Keywords: neural patterning; medial floor plate; midkine; pleiotrophin; tone H3 and BrdU labeling to detect cells in M and S heparin-binding growth factors; zebrafish] phases, respectively, showed no significant difference in 3Corresponding author. proliferation rates between mdka-injected and control E-MAIL [email protected]; FAX 49-931-888 4150. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ embryos (Supplementary Fig. S2). We also excluded the gad.336305. possibility of ingression of MFP precursors from neigh- GENES & DEVELOPMENT 19:897–902 © 2005 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/05; www.genesdev.org 897 Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Schäferetal. 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 ecto- pically expressed deltaA) coincides with the reduction of the notochord (Appel et al. 1999). Also, mutants defi- cient 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 regu- late 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. Conse- Figure 1. Mdka expression during zebrafish embryogenesis. (a)RT– PCR analysis: mdka transcription is first detected at 8 hpf (h); actin expression was used for calibration. −RT control using actin primers without addition of reverse transcriptase. (b–h) RNA in situ hybrid- ization on 10–19-h embryos showing advancing mdka transcription in the paraxial mesoderm; horizontal bars mark the position of the first somite. (g) Double staining with myoD at 17 h shows progres- sive loss of mdka transcription in anterior somites. (i) mdka is ex- pressed at the six-somite (6s; 12 hpf) stage in cells of the unseg- mented paraxial mesoderm when convergence of the neural plate has just started. (j,k) The front of advancing mdka expression (j) demarcates the position of twhh restriction in MFP precursors (k)as indicated by arrowheads that also show level of transverse section in i.(l) At later stages, mdka is expressed in paraxial mesoderm and also the anterior neural keel, but excluded from a ring of cells sur- rounding the shh-positive notochord and MFP (8s stage, shh labeled in red). In dorsal views, anterior is to the top in b–h,j,k. Bars: j,10µm;l, 50 µm. boring trunk areas by using confocal time-lapse analyses (data not shown). Instead, ectopic mdka is likely to affect the specification of midline precursor cells that give rise Figure 2. Mdka overexpression enlarges the MFP at the expense of to MFP and the notochord. This was evident by a highly the notochord. Lateral views (a,c) and transverse sections (b, poste- significant reduction in notochord size observed in rior trunk) of noninjected 14s control embryos. (d–f) In embryos mdka RNA-injected embryos with increased MFP (Fig. injected with 50 pg mdka RNA, the MFP is enlarged (expanded shh in MFP, n = 165/191, 86%; f-spondin,inn = 36/46, 76%; col2a1, 2d–f,k; Supplementary Fig. S3). More than 50% of em- n = 134/145, 92% [data not shown]) and the notochord is reduced bryos showed a complete block of notochord formation (reduced shh in the notochord in n = 20/38, 53%; col2a1 in n = 27/ in the posterior trunk (Fig. 2g–i). Ectopic mdka had no 88, 31%; and ntl in n = 11/45, 24%). (g–i) Severely affected embryo effect on axial mesoderm formation during early gastru- with complete block of notochord formation in the posterior trunk (lacking shh expression in n = 16/38, 42%; col2a1 in n = 41/88, 46%, lation (n = 43/43; 100% of embryos with normal expres- and ntl in n = 30/45, 67%). White lines in a, d, and g indicate border sion of cyclops [cyc] as an early axial mesoderm marker; between the notochord and MFP. Sections in b, e, and h are at data not shown) indicating that Mdka does not affect similar positions in the posterior trunk. (j) Endogenous mdka tran- scription in the paraxial mesoderm extends up to the tailbud. (k) early cell specification in the shield. Rather, it impairs mdka overexpression resulted in a significant decrease of notochord notochord development later during neurulation.