Isthmin1, a secreted signaling protein, acts downstream of diverse embryonic patterning centers in development

Abstract

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.

Advances in research of biological functions of Isthmin-1

“…Notably, sqt and boz upregulate ISM1 expression even when β-catenin is blocked, which, combined with previous studies reporting that β-catenin is a transient signaling molecule, suggests that the Wnt/β-catenin pathway may not be required for ISM1 activation (Kesavan et al 2021). However, in contrast to these early observations, late ISM1 expression appears to be independent of nodal signaling (Kesavan et al 2021), suggesting that ISM1 expression is temporally controlled by a different signaling system.…”

“…It has been confirmed that ISM1 and Fgf-8 genes are expressed at very similar sites, with partially overlapping or co-expressing expression domains at the anterior margin of the neural plate, MHB, gill arch, posterior mesencephalon, and ear capsule in African clawed frogs (Pera et al 2002 ). The expression domains of ISM1 in zebrafish embryos include the dorsal edge of the embryo, the dorsal hypoderm, the posterior medial part of the mesoderm, the MHB, and the notochord; these ISM1 expression locations are very similar to those of Fgf-8 (Kesavan et al 2021 ). This also reaffirms the conserved expression of ISM1 across species and the possible important role of ISM1 in neurological development during the embryonic stage.…”

“…Pera et al (Pera et al 2002 ) observed that ISM1 was expressed in several sites in frog embryos, including the ventral ectodermal lip, proximal mesoderm, neural folds, notochord, MHB, isthmus, and hind mesencephalon.Additionally, Ism1 is expressed in the branchial arches. A correlation study of the timing and location of ISM1 expression in zebrafish found that it was expressed in the MHB and hind trunk regions during the late prometaphase/early segmentation stage and thereafter only in the tail bud region and notochord; the expression level in the notochord increased at 22 h post fertilization (hpf), decreased in the tail bud at 30 hpf, decreased in the notochord at 48 hpf, and disappeared at 72 hpf (Kesavan et al 2021 ). Interestingly, deletion of ISM1 did not affect embryonic morphological changes during early development of zebrafish embryos, which may be triggered by genetic compensation phenomena through mutations in other genes (El-Brolosy et al 2019 ; Kesavan et al 2021 ).…”

“…Studies have shown that ISM1 is directly and indirectly controlled by β-catenin. Injection of the nodal signaling gene, sqt , and the homologous frame transcription factor, bozozok ( boz ), mRNA during zebrafish gastrula embryogenesis resulted in ectopic expression of ISM1 in the embryonic marginal region, as well as upregulation of ISM1 expression, suggesting that both genes may be upstream of ISM1 (Kesavan et al 2021 ). Notably, sqt and boz upregulate ISM1 expression even when β-catenin is blocked, which, combined with previous studies reporting that β-catenin is a transient signaling molecule, suggests that the Wnt/β-catenin pathway may not be required for ISM1 activation (Kesavan et al 2021 ).…”

“…Injection of the nodal signaling gene, sqt , and the homologous frame transcription factor, bozozok ( boz ), mRNA during zebrafish gastrula embryogenesis resulted in ectopic expression of ISM1 in the embryonic marginal region, as well as upregulation of ISM1 expression, suggesting that both genes may be upstream of ISM1 (Kesavan et al 2021 ). Notably, sqt and boz upregulate ISM1 expression even when β-catenin is blocked, which, combined with previous studies reporting that β-catenin is a transient signaling molecule, suggests that the Wnt/β-catenin pathway may not be required for ISM1 activation (Kesavan et al 2021 ). However, in contrast to these early observations, late ISM1 expression appears to be independent of nodal signaling (Kesavan et al 2021 ), suggesting that ISM1 expression is temporally controlled by a different signaling system.…”