Regulation of Nodal signaling propagation by receptor interactions and positive feedback

Abstract

During vertebrate embryogenesis, the germ layers are patterned by secreted Nodal signals. In the classical model, Nodals elicit signaling by binding to a complex comprising Type I/II Activin receptors (Acvr) and the co-receptor Tdgf1. However, it is currently unclear whether receptor binding can also affect the distribution of Nodals themselves through the embryo, and it is unknown which of the putative Acvr paralogs mediate Nodal signaling in zebrafish. Here, we characterize three Type I (Acvr1) and four Type II (Acvr2) homologs and show that - except for Acvr1c - all receptor-encoding transcripts are maternally deposited and present during zebrafish embryogenesis. We generated mutants and used them together with combinatorial morpholino knockdown and CRISPR F0 knockout (KO) approaches to assess compound loss-of-function phenotypes. We discovered that the Acvr2 homologs function partly redundantly and partially independently of Nodal to pattern the early zebrafish embryo, whereas the Type I receptors Acvr1b-a and Acvr1b-b redundantly act as major mediators of Nodal signaling. By combining quantitative analyses with expression manipulations, we found that feedback-regulated Type I receptors and co-receptors can directly influence the diffusion and distribution of Nodals, providing a mechanism for the spatial restriction of Nodal signaling during germ layer patterning.

Single-molecule tracking of Nodal and Lefty in live zebrafish embryos supports hindered diffusion model

“…Interestingly, while we also observed labeled Nodals and Leftys in the cytoplasm (excluded in the present analysis) in addition to their extracellular localization, we only rarely (in approximately 3 out of 100 movies) observed events where single molecules clearly passed the membrane and entered the cytoplasm. This indicates that internalization of Nodals/Leftys is a rare event, as opposed to the prolonged binding of Nodals on the cell surface, which provides a possible explanation for the hour-long half-lives of Nodals/Leftys in living zebrafish embryos 25 , 41 , 88 . Rare internalization also contrasts with the transcytosis mechanism described for the TGF-β superfamily ligand Dpp in Drosophila , in which repeated rounds of exocytosis and endocytosis lead to morphogen dispersal 72 .…”

“…Nodals are well known to bind to the EGF-CFC co-receptor Oep 36 , 66 , which is essential for Nodal signaling 67 . Furthermore, the signaling range and distribution of Squint was shown to be extended in the absence of oep 40 , 41 , but a direct effect of Oep on Nodal dispersal at the nanometer-to-micrometer scale has not yet been directly demonstrated. To test whether immobile Squint in our experiments was due to binding to Oep, we co-injected 0.3 pg, 3 pg and 30 pg of Oep-encoding mRNA together with the Squint-HaloTag construct and compared the diffusion properties of Squint in conditions of oep overexpression with those of endogenous Oep levels.…”

“…Second, effective diffusion coefficients on a tissue level across a cube of approximately 8 × 8 × 8 cells measured by fluorescence recovery after photobleaching (FRAP) were found to be much lower for Nodals than for Leftys (Cyclops < Squint < Lefty1 < Lefty2) 25 , 37 – 39 . Third, it has been shown that Nodals bind their receptors with nanomolar affinity 14 , and manipulating the levels of the co-receptor Oep modulated the Nodal signaling range and distribution 40 , 41 . However, it remains unclear whether and how such treatments affect Nodal and Lefty movement, how effective diffusivity through a tissue emerges from interactions at the molecular scale, how binding on the cell surface contributes to Nodal and Lefty movement, and how tissue geometry affects morphogen spreading.…”

Gastrulation-stage gene expression inNipbl+/-mouse embryos foreshadows the development of syndromic birth defects

“…6D, data S25, data S26, and data S27). Within the mesoderm, two-to six-fold upregulation of genes involved in Nodal signaling, including Tdgf1, Eomes, Lefty2, Lhx1 , was observed ( 63–66 ) . At the same time, three clusters of genes were robustly downregulated: those associated with primitive erythropoiesis, including Gata1 and Klf1 (2-3 fold) ( 67, 68 ), Hox genes such as Hoxb8, Hoxb5, and Hoxa5 (2-fold), and genes associated with Notch signaling, including Hey2 and Nr2f2 (2-fold) ( 69, 70 ) (Fig.…”

Gastrulation-stage gene expression in Nipbl +/− mouse embryos foreshadows the development of syndromic birth defects

“…8E). E7.5 Nanog Dox+ embryos overexpressed genes linked with the Nodal signaling pathway (Tdgf1, Eomes, Fgf5, Lhx1, Foxd3, Lefty2, and Nodal) (96)(97)(98)(99)(114)(115)(116) and those integral to maintaining pluripotency (Nanog and Pou5f1) (Fig. 8E) (112).…”