Medial floor plate formation in zebrafish consists of two phases and requires trunk-derived Midkine-a

Abstract

The medial floor plate (MFP) is the ventral-most structure in the vertebrate neural tube with important organizer functions during specification and differentiation of neurons and glia. Its origin and the timing of the specification are matters of ongoing debates (see Strähle et al. 2004). 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. Results and Discussionmdka transcription is first detectable by RT-PCR during late gastrulation (at 75% epiboly; 8 h post-fertilization; hpf) (Fig. 1a). At the beginning of neurulation (10 hpf), when the tailbud starts to extend caudally to form the trunk and tail, mdka transcription is confined to the rostral paraxial mesoderm that is still unsegmented at this stage (Fig. 1b). Later, its expression moves as a wave from anterior to posterior in this region (Fig. 1c-h). The caudal front of the mdka wave in the paraxial mesoderm progresses in parallel to the formation of the neural keel that emerges from the converging neural plate (Fig. 1i,j). At the same point, the morphologically distinct MFP starts to form, which is marked by the restriction of broad tiggy-winkle hedgehog (twhh) expression to a row of single cells (Fig. 1k). Therefore, expression of mdka coincides temporally and spatially with the appearance of a morphologically recognizable floor plate. After 13 hpf, mdka expression is also found dorsally in the anterior neural tube ( Fig. 1l; data not shown).We first addressed the function of mdka during MFP formation by overexpressing Mdka through mRNA injection into zebrafish embryos (Fig. 2). This resulted in a strong expansion of the MFP as defined by the expression of sonic hedgehog (shh) (Kra...

Midkine‐b regulates cell specification at the neural plate border in zebrafish

“…In contrast to this, we could show that the two duplicated midkine genes in zebrafish show divergent activities (Winkler et al, 2003). On one hand, Mdka regulates formation of the medial floor plate (Schafer et al, 2005). On the other hand, Mdkb is required for the formation of neural crest cells and sensory neurons (this study).…”

“…Zebrafish contains two duplicated midkine genes, mdka, and mdkb, with mostly nonoverlapping expression patterns and divergent activities during embryogenesis (Winkler et al, 2003). While Mdka functions in the formation of the medial floor plate in the ventral neural tube (Schafer et al, 2005), we have earlier shown that Mdkb is involved in neural plate patterning during gastrulation (Winkler and Moon, 2001). Using a dominant-negative approach, we initially reported that Mdkb is required for the formation of a subset of premigratory ncc expressing foxd3 (Winkler and Moon, 2001).…”

Pituitary-Interrenal Interaction in Zebrafish Interrenal Organ Development

“…To test for morpholino efficiency, a GFP-based approach was used as described earlier (50). Briefly, the 25-nucleotide target sequence for the mc2r morpholino was cloned in front of GFP into the CS2ϩGFP expression vector using annealed oligos containing BamHI overhangs.…”

Mice doubly deficient in the midkine and pleiotrophin genes exhibit deficits in the expression of β-tectorin gene and in auditory response

“…33,34 In a recent study in zebra fish, which have two species of MK due to gene duplication, it was established that a form of MK plays essential roles in the formation of the medial floor plate, one of the most ventral neural tube structures. 35 The factors are also expressed in specific regions during development of the sensory organs, suggesting roles in the developmental process. 20,21 MK and PTN have 45% sequence identity at the protein level, and share many of the in vitro activities.…”