Search

Morphogen gradients impart positional information to cells in a homogenous tissue field. Fgf8a, a highly conserved growth factor, has been proposed to act as a morphogen during zebrafish gastrulation. However, technical limitations have so far prevented direct visualization of the endogenous Fgf8a gradient and confirmation of its morphogenic activity. Here, we monitor Fgf8a propagation in the developing neural plate using a CRISPR/Cas9-mediated EGFP knock-in at the endogenous fgf8a locus. By combining sensitive imaging with single-molecule fluorescence correlation spectroscopy, we demonstrate that Fgf8a, which is produced at the embryonic margin, propagates by diffusion through the extracellular space and forms a graded distribution towards the animal pole. Overlaying the Fgf8a gradient curve with expression profiles of its downstream targets determines the precise input-output relationship of Fgf8a-mediated patterning. Manipulation of the extracellular Fgf8a levels alters the signaling outcome, thus establishing Fgf8a as a bona fide morphogen during zebrafish gastrulation. Furthermore, by hindering Fgf8a diffusion, we demonstrate that extracellular diffusion of the protein from the source is crucial for it to achieve its morphogenic potential., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Background: Heparan sulfate proteoglycan (HSPG) expression is found in many animal tissues and regulates growth factor signaling such as of Fibroblast growth factors (Fgf), Wingless/Int (Wnt) and Hedgehog (HH). Glypicans, which are GPI (glycosylphosphatidylinositol)‐anchored proteins, and transmembrane‐anchored syndecans represent two major HSPG protein families whose involvement in development and disease has been demonstrated. Their participation in regenerative processes both of the central nervous system and of regenerating limbs is well documented. However, whether HSPG are expressed in regenerating zebrafish fins, is currently unknown. Results: Here, we carried out a systematic screen of glypican and syndecan mRNA expression in regenerating zebrafish fins during the outgrowth phase. We find that 8 of the 10 zebrafish glypicans and the three known zebrafish syndecans show specific expression at 3 days post amputation. Expression is found in different domains of the regenerate, including the distal and lateral basal layers of the wound epidermis, the distal most blastema and more proximal blastema regions. Conclusions: HSPG expression is prevalent in regenerating zebrafish fins. Further research is needed to delineate the function of glypican and syndecan action during zebrafish fin regeneration. Key Findings: A systematic screen on HSPG expression in regenerating zebrafish fins during the outgrowth phase was carried outMost HSPG are expressed in regenerating zebrafish finsHSPG expression is found in the wound epidermis, and different parts of the regeneration blastemaThe prevalence of HSPG expression suggests a functional contribution to fin regeneration [ABSTRACT FROM AUTHOR]

Extracellular signals play essential roles during embryonic patterning by providing positional information in a concentration-dependent manner, and many such signals, like Wnt, fibroblast growth factor (FGF), Hedgehog (Hh), and retinoic acid, act by being secreted into the extracellular space, thereby triggering receptor-mediated responses in other cells. Isthmin1 (ism1) is a secreted protein whose gene expression pattern coincides with that of early dorsal determinants, nodal ligand genes like sqt and cyc, and with fgf8 during various phases of zebrafish development. Ism1 functions in early embryonic patterning and development are poorly understood; however, it has recently been shown to interact with nodal pathway genes to control organ asymmetry in chicken. Here, we show that misexpression of ism1 deletion constructs disrupts embryonic patterning in zebrafish and exhibits genetic interactions with both Fgf and nodal signaling. Unlike Fgf and nodal pathway mutants, CRISPR/Cas9-engineered ism1 mutants did not show obvious developmental defects. Further, in vivo single molecule fluorescence correlation spectroscopy (FCCS) showed that Ism1 diffuses freely in the extra-cellular space, with a diffusion coefficient similar to that of Fgf8a; however, our measurements do not support direct molecular interactions between Ism1 and either nodal ligands or Fgf8a in the developing zebrafish embryo. Together, data from gain- and loss-of-function experiments suggest that zebrafish Ism1 plays a complex role in regulating extracellular signals during early embryonic development. [ABSTRACT FROM AUTHOR]

Forty years ago, it was proposed that during embryonic development and organogenesis, morphogen gradients provide positional information to the individual cells within a tissue leading to specific fate decisions. Recently, much insight has been gained into how such morphogen gradients are formed and maintained; however, which cellular mechanisms govern their interpretation within target tissues remains debated. Here we used in vivo fluorescence correlation spectroscopy and automated image analysis to assess the role of endocytic sorting dynamics on fibroblast growth factor 8 (Fgf8) morphogen gradient interpretation. By interfering with the function of the ubiquitin ligase Cbl, we found an expanded range of Fgf target gene expression and a delay of Fgf8 lysosomal transport. However, the extracellular Fgf8 morphogen gradient remained unchanged, indicating that the observed signalling changes are due to altered gradient interpretation. We propose that regulation of morphogen signalling activity through endocytic sorting allows fast feedback-induced changes in gradient interpretation during the establishment of complex patterns. [ABSTRACT FROM AUTHOR]