p18ahub mutants exhibit delayed progression of epiboly and severe dorsalization.

Late blastula stage WT embryo (A), and age-matched p18ahub embryo (B). WT embryo at an early gastrula stage (C) with the shield (organizer) on the dorsal side (arrow). A p18ahub embryo at an early gastrula stage (D) displaying apparent radial hyperconvergence with no definitive dorsal side. (A,B) lateral views; (C,D) animal pole views. WT (E) and p18ahub (F) embryos at 24 hours post fertilization (hpf) (lateral views). (G) Pie chart depicting proportion of embryos with indicated phenotypes at 24 hpf sampled over a total of 2308 embryos. (H and I), in situ hybridization on 3–5-somite stage WT (H, n = 15) and age-matched p18ahub (I, n = 13) embryos for six3 (s) expression in presumptive forebrain, pax2.1 expression in the midbrain-hindbrain boundary (p) and pronephros (p*), krox20 (k) in hindbrain rhombomeres 3 and 5, and myod (m) in paraxial mesoderm; dorsal view in (H), lateral view in (I), anterior to top. (J and K), 10-somite stage WT (J, n = 17, dorsal view), and age-matched p18ahub embryos (K, n = 18, anterior view) processed for krox20 in situ hybridization. (L–Q) in situ hybridization on mid gastrula stage WT and equivalent stage p18ahub embryos shown for: otx2, WT (L, n = 18) and p18ahub (M, n = 10); cyp26a, WT (N, n = 16) and p18ahub (O, n = 15); and hoxb1b, WT (P, n = 22) and p18ahub (Q, n = 21). Lateral views, anterior at top, dorsal to right.

Reduced bmp expression but intact BMP signal transduction in p18ahub mutants.

(A–H) In situ hybridization on early gastrula stage embryos or equivalent, except (C) and (D) are mid gastrula stage or equivalent. Embryos were stage-matched. Animal pole views, dorsal to the right for all. Expression of bmp ligand gene in WT embryos (A, n = 32, C, n = 43, and E, n = 21) and p18ahub embryos (B, n = 30, D, n = 18, and F, n = 16). Expression of the BMP antagonist chordin (chd) was confined to the dorsal side of WT embryos (G, n = 21) but was expanded around the entire margin of p18ahub embryos (H, n = 20) by early gastrulation. (I–N) Pie charts indicate fractions of embryos at 24 hpf that displayed the indicated phenotypes (categories described in the text). Number of embryos is shown at the center of each chart. p18ahub embryos (I, O, uninjected) that were injected with 20 pg bmp2b mRNA were rescued to WT or ventralized (K, P, +bmp2b mRNA) similarly to WT embryos (J, +bmp2b mRNA). p18ahub mutant embryos (L, uninjected) depleted of Chordin, Noggin1, and Follistatin-like 2b proteins by MO injection were similarly ventralized (N, +MOcnf) to WT embryos (M, +MOcnf).

Dorsal organizer gene expression is expanded in p18ahub mutants

In situ hybridization using goosecoid (gsc) probe (A–D). Animal pole views, dorsal to right for (A) and (B); dorsal view, animal pole up in (C) and (D). WT embryos at early gastrula (A, n = 27) and mid gastrula stages (C, n = 41). p18ahub embryos (B, n = 33 and D, n = 10) displayed radially expanded gsc expression at the same stages. The embryo in (D) is tilted toward the viewer to show radial gsc expression. WT (E, n = 18) and p18ahub (F, n = 18) blastula stage embryos showed no obvious differences in the number or distribution of β-catenin immunopositive nuclei (arrows). (G–J) In situ hybridization for bozozok (boz), lateral views, dorsal to right; insets show animal pole views. WT and p18ahub embryos showed normal boz expression at late blastula (G, n = 19 and H, n = 18) and early gastrula stages (I, n = 14 and J, n = 16, respectively). As described in the text, epiboly progression was delayed in p18ahub embryos. At 6 hpf WT embryos form a morphologically apparent organizer, indicated by a thickening of the blastoderm on the dorsal side of the embryo and boz expression within the hypoblast. In contrast, at 6 hpf p18ahub embryos still appeared to be in a late blastula stage, although their boz expression was similar to WT.

The Wnt8a pathway is repressed but mechanistically intact in p18ahub embryos.

(A–J) In situ hybridization for Wnt8a pathway components or downstream genes. Animal pole views, dorsal at right, except (C, D) are lateral views, dorsal at right, insets show animal pole views. At an early gastrula stage in WT (A, n = 14) wnt8a was expressed around the margin but was absent from the axial mesoderm (arrows). In p18ahub embryos at a late blastula stage (B, n = 20) wnt8a expression was absent from the presumptive dorsal half of the embryo. The expression of vox was reduced in late blastula p18ahub embryos (D, 11/13 embryos) compared to WT embryos (C, n = 18). By early gastrulation, WT embryos (E, n = 20) showed prominent ventrolateral vox expression, while vox was nearly absent from equivalent stage p18ahub embryos (F, n = 12). ved was expressed similarly to vox in WT (G, n = 10) and was also nearly undetectable in p18ahub embryos (H, n = 10) at early gastrulation. eve1 in WT (I, n = 17) marks developing ventroposterior mesoderm and was nearly absent from p18ahub embryos by early gastrulation (J, n = 12). Splice blocking wnt8a MOs variably dorsalized WT embryos (K). (L) Injection of MO against chd ventralized both WT and p18ahub embryos (compare to p18ahub uninjected, K). (M) Simultaneous reduction of Chd and Wnt8a causes dorsalization in p18ahub embryos, indicating that endogenous Wnt8a signal transduction is intact and functional in p18ahub embryos that are depleted of Chd. Representative embryos at 1 dpf are shown in N–Q.

Excessive axial mesoderm at the expense of ventrolateral mesoderm in p18ahub embryos.

WT (A, n = 19) and stage-matched p18ahub (B, n = 20) embryos at a mid blastula stage, exhibited similar nodal related 1 (ndr1) expression. WT (C, n = 14) and age-matched p18ahub (D, n = 13) embryos at 6 hpf after synchronized matings displayed similar lefty1 (lft1) expression, although blastoderm involution was not evident in the mutants. (A–D) are animal pole views, dorsal to right. (E–L) are mid gastrula stage, except (G and H) are 3–5 somite stage or equivalent. Dorsal views, animal to top, except (H–H3) are animal pole views. WT embryos (E, n = 6) displayed marginal and axial ntl expression. In contrast, some p18ahub embryos displayed only axial ntl expression distributed circumferentially (F, n = 16), while others displayed broadened axial and reduced ventrolateral ntl expression (F inset, n = 8). In WT embryos (G, n = 22) ntl was expressed in the notochord and tail bud mesenchyme. In equivalent stage p18ahub embryos, (H–H3, n = 21), ntl expression was observed in multiple notochords terminating in smaller tail bud-like domains. Consistent with ectopic axial ntl expression, the expression of floating head (flh), a marker of notochord precursors, confined dorsally in WT embryos (I, n = 20), was expressed around the entire embryonic margin in p18ahub embryos (J, n = 16). In WT (K, n = 18) sox17 is expressed in endodermal precursor cells and in a single cluster of dorsal forerunner cells (arrow). In p18ahub embryos (L, n = 16) sox17 expression indicates the presence of ectopic clusters of dorsal forerunner cells (arrows).

Lefty misexpression suppresses patterning defects of p18ahub embryos.

(A) Pie charts indicate fractions of embryos at 24 hpf with the indicated phenotypes. Number of embryos is at the center of each chart. Labels across indicate the amount of lefty1 (lft1) mRNA injected. Top row shows uninjected p18ahub control clutch for each experiment, middle row indicates p18ahub embryos injected with mRNA, and bottom row is WT embryos injected with mRNA. +1× lft mRNA corresponds to 1 pg mRNA. (B and C) In situ hybridization for lft1 in a mid gastrula stage WT (B, n = 12) and p18ahub (C, n = 13) embryos; lateral views, dorsal facing.

p18ahub affects the Integrator Complex Subunit 6 (Ints6).

(A) p18ahub was narrowed to an approximately 1.35 Mb interval between SSLP marker z7120 and an SSLP marker that we generated in BAC CR545476.14 (Zv9) (right marker) by examining meiotic recombination between markers flanking the mutation. The recombinants found in the number of meioses examined at each marker is shown and the positions of markers are approximate. (B) p18ahub is a T to A transition in an exon of ints6 that converts valine 375 to an aspartate. An alignment of several Ints6 proteins was generated using ClustalW and reveals that V375 is nearly invariant among widely divergent species. (C and D) In situ hybridization for ints6 transcripts on WT embryos at (C) 128-cell (n = 8) and (D) late blastula stage (n = 19); lateral views, animal to top. (E–G) Rescue experiments: pie charts show fractions of embryos evaluated at 1 dpf with indicated phenotypes. Numbers of embryos are shown in the center of each chart. (H) Uninjected p18ahub mutant embryos and (I) mutant embryos rescued by injection of 50 pg ints6 mRNA, shown at 1 dpf.