The ventral to dorsal BMP activity gradient in the early zebrafish embryo is determined by graded expression of BMP ligands

Abstract

In the early zebrafish embryo, a ventral to dorsal gradient of bone morphogenetic protein (BMP) activity is established, which is essential for the specification of cell fates along this axis. To visualise and mechanistically determine how this BMP activity gradient forms, we have used a transgenic zebrafish line that expresses monomeric red fluorescent protein (mRFP) under the control of well-characterised BMP responsive elements. We demonstrate that mRFP expression in this line faithfully reports BMP and GDF signalling at both early and late stages of development. Taking advantage of the unstable nature of mRFP transcripts, we use in situ hybridisation to reveal the dynamic spatio-temporal pattern of BMP activity and establish the timing and sequence of events that lead to the formation of the BMP activity gradient. We show that the BMP transcriptional activity gradient is established between 30% and 40% epiboly stages and that it is preceded by graded mRNA expression of the BMP ligands. Both Dharma and FGF signalling contribute to graded bmp transcription during these early stages and it is subsequently maintained through autocrine BMP signalling. We show that BMP2B protein is also expressed in a gradient as early as blastula stages, but do not find any evidence of diffusion of this BMP to generate the BMP transcriptional activity gradient. Thus, in contrast to diffusion/transport-based models of BMP gradient formation in Drosophila, our results indicate that the establishment of the BMP activity gradient in early zebrafish embryos is determined by graded expression of the BMP ligands.

Tumor Suppressor Lzap Suppresses Wnt/β-Catenin Signaling to Promote Zebrafish Embryonic Ventral Cell Fates via the Suppression of Inhibitory Phosphorylation of Glycogen Synthase Kinase 3

“…In zebrafish embryos, bmp2b and bmp7a are initially ubiquitously expressed, and dorsal determinants (Wnt/␤-catenin signaling) are actively induced to stimulate chd expression, thus creating BMP gradients at a later stage (30% epiboly stage) (6,43,44). To investigate how the BMP signaling gradient was affected in lzap-knockdown embryos, we further analyzed expression of bmp2b and chd at the sphere stage (4 hpf).…”

“…5, c, d, and h). In contrast, phosphorylated p-ERK, downstream of active Fgf signaling (6,46), was strongly detected in nearly every cell of lzap morphants, as compared with the dorsally restricted p-ERK signals of wildtype embryos (Fig. 5, e and f).…”

“…The dorsal determinant Wnt/␤-catenin signaling is known to activate transcription of fgfs and bozozok/dharma, which act in parallel to control chd expression in the prospective dorsal region during the midblastula transition stage (approximately 1 h before the sphere stage) ( 6,7,45). We then examined Wnt/␤-catenin signaling activity in embryos injected with lzap5Ј-UTR MO by analyzing expression of its direct target, axin2 (which acts as a negative feedback regulator (15), at the sphere stage (Fig.…”

“…In zebrafish embryos, wnt3a, which is co-expressed with wnt8a at the gastrula margin (4), is also able to activate the maternal Wnt/␤-catenin pathway (3). Downstream targets of ␤-catenin include bozozok/dharma and fibroblast growth factors (fgfs), which may act in parallel to activate transcripts involved in dorsal cell fate specification (5)(6)(7). For example, Chordin is a BMP 5 antagonist involved in the establishment of the ventral to dorsal BMP signaling gradient (8).…”

“…lzap homozygous knock-out mice are embryonic lethal. 6 In addition, lzap is maternally deposited and ubiquitously expressed in zebrafish embryos during early development, and knockdown of lzap results in slower cell division and delayed epibolic movement (the process by which blastomeres move from the animal pole to the vegetal pole to cover the yolk) (18). These results indicate that LZAP controls several cellular processes during development; nevertheless, it is less clear how its dysregulation causes developmental disorders and cancer formation.…”