Amphioxus Sp5 is a member of a conserved Specificity Protein complement and is modulated by Wnt/β-catenin signalling

Abstract

A cluster of three Specificity Protein (Sp) genes (Sp1-4, Sp5 and Sp6-9) is thought to be ancestral in both chordates and the wider Eumetazoa. Sp5 and Sp6-9 gene groups are associated with embryonic growth zones, such as tailbuds, and are both Wnt/β-catenin signalling pathway members and targets. Currently, there are conflicting reports as to the number and identity of Sp genes in the cephalochordates, the sister group to the vertebrates and urochordates. We confirm the SP complement of Branchiostoma belcheri and Branchiostoma lanceolatum, as well as their genomic arrangement, protein domain structure and residue frequency. We assay Sp5 expression in B. lanceolatum embryos, and determine its response to pharmacologically increased β-catenin signalling. Branchiostoma possesses three Sp genes, located on the same genomic scaffold. Phylogenetic and domain structure analyses are consistent with their identification as SP1-4, SP5 and SP6-9, although SP1-4 contains a novel glutamine-rich N-terminal region. SP5 is expressed in axial mesoderm and neurectoderm, and marks the cerebral vesicle and presumptive pharynx. Early exposure to increased β-catenin caused ubiquitous SP5 expression in late gastrula, while later treatment at gastrula stages reduced SP5 expression in the posterior growth zone during axis elongation. Amphioxus possess a typical invertebrate eumetazoan SP complement, and SP5 expression in embryos is well conserved with vertebrate homologues. Its expression in the tailbud, a posterior growth zone, is consistent with expression seen in other bilaterians. Branchiostoma SP5 shows a dynamic response to Wnt/β-catenin signalling.

Developmental cell-cell communication pathways in the cephalochordate amphioxus: actors and functions

“…This is accompanied by a downregulation of Sp5 in the same domain, an unexpected result given that Sp5 was shown to be activated downstream of canonical Wnt/b-catenin signalling (Fujimura et al, 2007, Weidinger et al, 2005. The authors propose that this may be the result of a negative auto-regulatory feedback loop, as other pharmacological treatments result in Sp5 upregulation (Dailey et al, 2017), highlighting the importance of developmental stage and duration of signal modulation in evaluating the role of any given signalling pathway.…”

“…Moreover, the results suggest that there may be only limited cross-talk between RA and Wnt/beta-catenin in amphioxus, in contrast to the situation in vertebrates (Onai et al, 2009). Chemical inhibition using two specific GSK3b inhibitors has identified possible downstream targets of Wnt/b-catenin signalling both during gastrulation and axis extension (Dailey et al, 2017). Continuous treatment with 1-Azakenpaullone at 10mM from late gastrula onwards results in expansion of the posterior Brachyury2 (Bra2) domain by mid-neurula, as expected if the latter were a bona fide target of Wnt/b-catenin signalling, as well as a lack of anterior structures including a pharynx in larvae.…”

Evolution of vertebrate spinal cord patterning

“…Hox14 is also responsive to RA even though it is only expressed in the posterior notochord, gut, and cerebral vesicle at larval stage. 133,134 Several Wnt genes show tailbud expression, 46,135 though only limited manipulation of Wnt signaling has been conducted in amphioxus to date 136 and we do not know whether Wnt regulates Hox. FGF inhibition alone does not seem to have an effect on Hox1 expression but when coupled with an RA antagonist there is no Hox1 expression in the neural plate.…”

β-catenin dependent axial patterning in Cnidaria and Bilateria uses similar regulatory logic

“…3A). We found 25 such genes, and two of them attracted our attention as members of the Sp family of the Krüppel-like transcription factors, which are important regulators of axial patterning and mediators of -catenin-signaling in Bilateria (18)(19)(20)(21)(22)(23) and in Hydra (24). Nematostella has representatives of all three Sp families present in Bilateria.…”

“…Nematostella has representatives of all three Sp families present in Bilateria. Like in chordates (20,23) and in Hydra (24), Nematostella Sp5 is activated by -catenin signaling. It is a "saturating" gene expressed in the domain similar to that of Axin-like and Tcf (fig.…”