XB-ART-53809Dev Biol. September 1, 2017; 429 (1): 213-224.
Wbp2nl has a developmental role in establishing neural and non-neural ectodermal fates.
PubMed ID: 28663133
PMC ID: PMC5554722
Article link: Dev Biol.
Grant support: R01 DE022065 NIDCR NIH HHS, U54 HD090257 NICHD NIH HHS , R01 DE022065 NIDCR NIH HHS, U54 HD090257 NICHD NIH HHS
Genes referenced: a2m chrd.1 foxd3 foxd4l1.1 foxi1 irx1 myc pax3 six1 sox11 sox17b.1 sox2 t wbp2 wbp2nl xk81a1 yap1 yes1 zic1 zic2
Morpholinos referenced: wbp2nl MO1 wbp2nl MO2
Article Images: [+] show captions
|Fig. 3. Increasing Wbp2nl levels alters ectodermal gene expression. (A) Increased Wbp2nl causes ectopic expression of epidermal keratin (K81) in the neural ectoderm (ne) at gastrula stages (left) but not at neural plate (np) stages (right). Pink nuclei identify the cells that are expressing excess Wbp2nl and the black arrow indicated the posterior extent of the clone. A-D, dorsal views with animal to top. (B) Increased Wbp2nl does not induce ectopic foxi1 in the ne. (C) Increased Wbp2nl causes ectopic expression of foxd3 in the neural plate (np). (D) Increased Wbp2nl causes ectopic expression of zic2 in the neural plate. (E-G) Increased Wbp2nl does not cause ectopic neural plate expression of pax3, zic1 or six1 (dorsal views with animal to top). In fact, zic1 neural plate expression is reduced on the injected side (F, right image, anterior view, dorsal to top). (H) Increased Wbp2nl does not alter foxd4l1 neural ectoderm expression. Vegetal-dorsal view. (I) Neural ectoderm expression of sox2 in the gastrula is reduced compared to lateral regions (black arrows) that do not contain Wbp2nl-expressing cells (pink nuclei). Dorsal view, animal to top. (J) Both the neural plate (red bar) and placode (red arrows) domains of irx1 are reduced by increased Wbp2nl compared to control side (black bar). Dorsal view, anterior to top. (K) Both the neural plate and placode (red arrows) expression of sox11 are reduced by increased Wbp2nl. Anterior view, dorsal to top. (L) The placode (red arrows) domain of six1 is reduced by increased Wbp2nl, compared to control uninjected side (black arrows). Side views, dorsal to top. (M) The placode (red arrows) domain of sox11 is reduced by increased Wbp2nl compared to control uninjected side (black arrows). Note reduced neural plate (np) staining as well. Side views, dorsal to top. Frequencies of the phenotypes and sample sizes (n) are given in each panel.|
|Fig. 4. Altered Wbp2nl levels affect mesoderm but not endoderm. (A) At gastrula stages, sox17 expression surrounding the ventral blastopore on the MO-injected side of an embryo (left image, vegetal view) is not discernably different from control side. Increased Wbp2nl in the animal pole ectoderm (right image, animal view; clone marked by pink nuclei and red arrow) does not ectopically induce sox17. (B) At gastrula stages, edd expression surrounding the ventral blastopore on the MO-injected side (left image, vegetal view) is not discernably different from control side. Increased Wbp2nl in the neural ectoderm (right image, dorsal view; clone marked by pink nuclei and red arrow) does not ectopically induce edd. (C) At gastrula stages, mesoderm expression of bra encircles the blastopore in uninjected control embryos (left image, vegetal view); knock-down of Wbp2nl causes a loss of bra expression (left middle image, vegetal view, red arrow). In contrast, at neural plate stages (right middle image, vegetal view), we detect a posterior expansion (red bar) of bra expression on the knock-down side. At gastrula stages, increased Wbp2nl (right image, dorsal view; clone marked by pink nuclei between red arrows) does not alter endogenous bra expression nor ectopically induce it. (D) At gastrula stages, mesoderm expression of chd is reduced by Wbp2nl knock-down (red arrow, left image, vegetal view; dashed line indicates dorsal midline). In contrast, at neural plate stages, chd is expanded (compare uninjected control pattern [left middle image] to MO-injected pattern [right middle image]; dorsal views with anterior to the top). Red bar shows a wider domain and red arrows denote ectopic expression. At gastrula stages, increased Wbp2nl (right image, dorsal view, anterior to top; dashed line indicates dorsal midline) also reduces endogenous chd expression (red arrow). Frequencies of the phenotypes and sample sizes (n) are given in each panel.|
|Fig. 5. BMP signaling underlies Wbp2nl-induced ectopic expression phenotypes. (A) The ectopic expression of K81, foxd3 and zic2 are minimized by co-expression of Chd, a BMP antagonist. Red arrows point to weak posterior ectopic gene expression observed in those few embryos that were positively scored. (B) Immunostaining for phosphorylated SMAD 1/5/8, an indicator of BMP signaling when located in the nucleus. Only embryos in which ventral cells, which are subject to high levels of endogenous BMP signaling, showed nuclear staining were analyzed (b). No embryo injected only with lineage tracer (blue) showed nuclear staining in the neural plate (a), whereas a majority of embryos injected with lineage tracer plus wbp2nl mRNA showed nuclear staining (c). (C) Expression of Wbp2nl in the ventral epidermis causes ectopic expression of two neural crest genes (foxd3, zic2). Pink nuclei (lineage tracer) indicate the Wbp2nl containing cells. Ventral views, anterior to the top. (D) Ventral animal blastomeres were dissected from the 16-cell stage embryo and cultured in simple salt medium. Those dissected from uninjected control embryos never expressed foxd3 or zic2, whereas those that express Wbp2nl (red nuclei) always expressed these genes. Frequencies of the phenotypes and sample sizes (n) are given in each panel.|
|Supplementary material Supplemental Fig. 1: Phylogenetic Tree for Wbp2 and Wbp2nl orthologues. Tree analysis shows that the Wbp/Wbp2nl proteins separate into two clades across several vertebrate taxa (Homo = human; mus = mouse; anolis and alligator = reptiles; Falco = falcon (bird); Danio = zebrafish; X. Laev and X. trop = amphibian). Mammalian WBP2NL (human, mouse) do not tightly cluster with the homologous proteins of the other vertebrates. Numbers on branches are Bootstrap Values. Protein identifiers are: Human_Wbp2nl - AAH22546.1, Mus_Wbp2nl - NP_083342.1, Alligator_Wbp2 - XP_014372734.1, Falco_Wbp2 - XP_014142228.1, Anolis_Wbp2 - XP_014142228.1, Mus_Wbp2 - AAH55058.1, Homo_Wbp2 - AAH10616.1, X.laev._Wbp2 - NP_001083140.1, X.trop._Wbp2 - AAH88817.1, Danio_Wbp2 - AAH95106.1, X.laev._Wbp2nl - NP_001088037.1, X.trop._Wbp2nl = NP_001107397.1, Anolis_Wbp2nl - XP_008108972.1, Falco_Wbp2nl - XP_014137125.1, Alligator_Wbp2nl - XP_006027702.1, Danio_Wbp2nl - NP_956004.1.|
|Fig. S4. Xenopus Wbp2nl (colored amino acids) contains several predicted sites of phosphorylation (underlined) many of which are contained within motifs (black letters with phosphorylation site in bold) characteristic of a number of kinases (listed on left margin).|