A transgenic wnt8a:PAC reporter reveals biphasic regulation of vertebrate mesoderm development

Abstract

Vertebrate wnt8a links anteroposterior and dorsoventral axis patterning, but the regulation of wnt8a expression and its relationship to mesoderm induction and maintenance pathways is unclear. To address this, we have generated zebrafish transgenic for a modified genomic PAC clone that expresses EGFP from the wnt8a locus. The EGFP reporter transgene is expressed in a pattern nearly identical to wnt8a, including maternal deposition, expression in the ventrolateral mesoderm and in the yolk syncytial layer. Loss of function studies show that wnt8a expression is under biphasic control by Nodal and No Tail/Brachyury, whereby early phase expression is Nodal-dependent but late phase expression is Ntl/Bra dependent. EGFP fluorescence persists in cells that transcribe the reporter, thus comprising a tracer for ventrolaterally derived mesodermal lineages. We use this property to show that wnt8a expression marks Nodal-independent tail mesoderm formation and that Ntl/Bra predominantly regulates wnt8a in paraxial mesoderm progenitors. Developmental Dynamics 240:898-907,

Biphasic wnt8a expression is achieved through interactions of multiple regulatory inputs

“…We previously generated a wnt8aPA-C:EGFP transgenic line in which bright EGFP labeling of several mesodermally derived organs, including somites, YSL, pronephros, heart, and blood, is observed despite the absence of transcripts at that stage (Narayanan et al, 2011). Thus, EGFP fluorescence in these tissues at 24 hpf, especially in somites and YSL, is a convenient assay to determine whether reporter constructs are active in the precursors of these tissues.…”

“…We recently demonstrated that zebrafish wnt8a.1 is regulated in a biphasic manner via cis-elements within an 80-KB genomic interval (Narayanan et al, 2011). This biphasic regulation correlates with the transition from Nodal-dependent mesoderm induction to Ntl-dependent posterior growth.…”

“…Zebrafish wnt8a transcripts are deposited maternally, decline by $2.5 hr post fertilization (hpf, 128-256 cell stage) and again increase at the midblastula transition (Kelly et al, 1995). In situ hybridization reveals wnt8a transcripts at the ventrolateral embryonic margin and in the yolk syncytial layer (YSL) starting at 30% epiboly (Kelly et al, 1995;Ho et al, 1999;Narayanan et al, 2011). Expression of wnt8a expands up to the dorsal organizer and is maintained in the ventrolateral margin during gastrulation.…”

“…Expression of wnt8a expands up to the dorsal organizer and is maintained in the ventrolateral margin during gastrulation. After gastrulation, wnt8a is expressed in the tailbud, presomitic mesoderm and prospective pronephros (Kelly et al, 1995;Narayanan et al, 2011).…”

“…Consistent with Nodal being an upstream regulator, zebrafish wnt8a expression is reduced in cyc;sqt double mutants, while ectopic Nodal expression can induce wnt8a (Erter et al, 2001;Agathon et al, 2003;Narayanan et al, 2011). Whether this is a direct or indirect interaction is unknown.…”