Biphasic wnt8a expression is achieved through interactions of multiple regulatory inputs

Abstract

Background: Vertebrate axis development depends upon wnt8a transcription in a dynamic pool of mesoderm progenitors at the posterior pole of the gastrulating embryo. The transcriptional mechanisms controlling wnt8a expression are not understood, but previous studies identified two phases of wnt8a expression in zebrafish: Nodal-dependent activation during early gastrulation (phase I) and No tail (Ntl)-dependent regulation from mid gastrula stages (phase II). Results: We identified two upstream cis-regulatory regions, proximal and distal, each of which possesses a promoter. The proximal regulatory region contains a margin-specific enhancer that is required for both the Nodal and Ntl responses. Phase I expression requires Nodal activation of the margin enhancer in combination with the transcription factor Zbtb4 and the distal regulatory region. Phase II expression requires Ntl regulation of the margin enhancer in the context of the proximal regulatory region. An additional mechanism is required to ensure the transition from phase I to phase II regulation. Analysis of stickleback wnt8a suggests this mechanism of regulation may be conserved. Conclusions: The seemingly simple wnt8a expression pattern reflects complex interactions of multiple regulatory inputs.

A cargo model of yolk syncytial nuclear migration during zebrafish epiboly

“…Rassf1 has previously been shown to stabilize interphase microtubules (Liu et al, 2003;Dallol et al, 2004). A YSL enhancer from the wnt8a gene was used to express zebrafish Rassf1 in the YSL by plasmid injection (Narayanan and Lekven, 2012). Embryos were injected as described above to label microtubules and nuclei and co-injected with the rassf1 plasmid.…”

“…The rassf1 insert was amplified using forward primer: 5′-ACGGGATCCACCATGGCAA-AATGTGAGCTCAT-3′; and reverse primer: 5′-CCGTCTAGAGGTTC-AGCCAGGCTTGCTGAAGT-3′, digested with BamH1 and XbaI and ligated into BamH1/XbaI-digested pCS2+. Gibson cloning (New England Biolabs) was used to insert rassf1 into a ClaI-digested plasmid containing the YSL-enhancer from the wnt8a gene (Narayanan and Lekven, 2012) to generate FP2-rassf1. The Gibson forward primer was: 5′-GGTCACTC-ACGCAACAATACAAGCTACTTGTTCTTTTTG-3′; and Gibson reverse primer: 5′-CATGTCTGGATCATCATTACGTAATACGACTCACTA-TAG-3′.…”

Divergent Wnt8a Gene Expression in Teleosts

“…Like in other teleosts [17]–[19], the two medaka Wnt8a paralogs are organized in a tandem arrangement in the genome. We isolated the full length coding sequences of medaka Wnt8a1 (formerly named Wnt8-like ) [16] and Wnt8a2 .…”

“…In teleosts, the genome duplication resulted in the generation of an additional Wnt8a paralog in close genomic proximity and in tandem to the first [17]. This arrangement appears very conserved amongst teleosts [17]–[19]. Interestingly, besides a transcript for the second Wnt8a paralog, a bicistronic Wnt8a transcript encoding both Wnt8a proteins has been identified in zebrafish [17].…”

Analysis of the wnt1 regulatory chromosomal landscape

“…Whether simultaneous or alternating, consistent and prolonged proximity of enhancer and promoter sequences appear to be associated with active transcription (Chen et al, 2018). The identification of critical enhancers for wnt1 and wnt10b in zebrafish will provide the basis for future studies to dissect enhancer-promoter interactions in their TAD architecture in this important vertebrate developmental context, and may provide a model to understand enhancer-promoter dynamics in other complex Wnt loci (Narayanan and Lekven, 2012;Ramel et al, 2004).…”