Anteroposterior axis patterning by early canonical Wnt signaling during hemichordate development

The Wnt family of secreted proteins has been proposed to play a conserved role in early specification of the bilaterian anteroposterior (A/P) axis. This hypothesis is based predominantly on data from vertebrate embryogenesis as well as planarian regeneration and homeostasis, indicating that canonical Wnt (cWnt) signaling endows cells with positional information along the A/P axis. Outside of these phyla, there is strong support for a conserved role of cWnt signaling in the repression of anterior fates, but little comparative support for a conserved role in promotion of posterior fates. We further test the hypothesis by investigating the role of cWnt signaling during early patterning along the A/P axis of the hemichordate Saccoglossus kowalevskii. We have cloned and investigated the expression of the complete Wnt ligand and Frizzled receptor complement of S. kowalevskii during early development along with many secreted Wnt modifiers. Eleven of the 13 Wnt ligands are ectodermally expressed in overlapping domains, predominantly in the posterior, and Wnt antagonists are localized predominantly to the anterior ectoderm in a pattern reminiscent of their distribution in vertebrate embryos. Overexpression and knockdown experiments, in combination with embryological manipulations, establish the importance of cWnt signaling for repression of anterior fates and activation of mid-axial ectodermal fates during the early development of S. kowalevskii. However, surprisingly, terminal posterior fates, defined by posterior Hox genes, are unresponsive to manipulation of cWnt levels during the early establishment of the A/P axis at late blastula and early gastrula. We establish experimental support for a conserved role of Wnt signaling in the early specification of the A/P axis during deuterostome body plan diversification, and further build support for an ancestral role of this pathway in early evolution of the bilaterian A/P axis. We find strong support for a role of cWnt in suppression of anterior fates and promotion of mid-axial fates, but we find no evidence that cWnt signaling plays a role in the early specification of the most posterior axial fates in S. kowalevskii. This posterior autonomy may be a conserved feature of early deuterostome axis specification

Phenotypic and molecular characterisation of CDK13-related congenital heart defects, dysmorphic facial features and intellectual developmental disorders

Background: De novo missense variants in CDK13 have been described as the cause of syndromic congenital heart defects in seven individuals ascertained from a large congenital cardiovascular malformations cohort. We aimed to further define the phenotypic and molecular spectrum of this newly described disorder. Methods: To minimise ascertainment bias, we recruited nine additional individuals with CDK13 pathogenic variants from clinical and research exome laboratory sequencing cohorts. Each individual underwent dysmorphology exam and comprehensive medical history review. Results: We demonstrate greater than expected phenotypic heterogeneity, including 33% (3/9) of individuals without structural heart disease on echocardiogram. There was a high penetrance for a unique constellation of facial dysmorphism and global developmental delay, as well as less frequently seen renal and sacral anomalies. Two individuals had novel CDK13 variants (p.Asn842Asp, p.Lys734Glu), while the remaining seven unrelated individuals had a recurrent, previously published p.Asn842Ser variant. Summary of all variants published to date demonstrates apparent restriction of pathogenic variants to the protein kinase domain with clustering in the ATP and magnesium binding sites. Conclusions: Here we provide detailed phenotypic and molecular characterisation of individuals with pathogenic variants in CDK13 and propose management guidelines based upon the estimated prevalence of anomalies identified. Keywords: CDK13, CHDFIDD, De novo variant, Neurodevelopmental disorders, Agenesis of the corpus callosum, Hypertelorism, Developmental delay, Cyclin-dependent kinase, Undiagnosed Diseases Networ

Anteroposterior axis patterning by early canonical Wnt signaling during hemichordate development

The Wnt family of secreted proteins has been proposed to play a conserved role in early specification of the bilaterian anteroposterior (A/P) axis. This hypothesis is based predominantly on data from vertebrate embryogenesis as well as planarian regeneration and homeostasis, indicating that canonical Wnt (cWnt) signaling endows cells with positional information along the A/P axis. Outside of these phyla, there is strong support for a conserved role of cWnt signaling in the repression of anterior fates, but little comparative support for a conserved role in promotion of posterior fates. We further test the hypothesis by investigating the role of cWnt signaling during early patterning along the A/P axis of the hemichordate Saccoglossus kowalevskii. We have cloned and investigated the expression of the complete Wnt ligand and Frizzled receptor complement of S. kowalevskii during early development along with many secreted Wnt modifiers. Eleven of the 13 Wnt ligands are ectodermally expressed in overlapping domains, predominantly in the posterior, and Wnt antagonists are localized predominantly to the anterior ectoderm in a pattern reminiscent of their distribution in vertebrate embryos. Overexpression and knockdown experiments, in combination with embryological manipulations, establish the importance of cWnt signaling for repression of anterior fates and activation of mid-axial ectodermal fates during the early development of S. kowalevskii. However, surprisingly, terminal posterior fates, defined by posterior Hox genes, are unresponsive to manipulation of cWnt levels during the early establishment of the A/P axis at late blastula and early gastrula. We establish experimental support for a conserved role of Wnt signaling in the early specification of the A/P axis during deuterostome body plan diversification, and further build support for an ancestral role of this pathway in early evolution of the bilaterian A/P axis. We find strong support for a role of cWnt in suppression of anterior fates and promotion of mid-axial fates, but we find no evidence that cWnt signaling plays a role in the early specification of the most posterior axial fates in S. kowalevskii. This posterior autonomy may be a conserved feature of early deuterostome axis specification

Summary of Wnts, Wnt antagonists, and Fzs receptor expression.

<p>Wnts are expressed in nested domains posteriorly, whereas <i>sfrps</i> are expressed in the anteriormost ectoderm at the blastula and gastrula stages. <i>dkk1/2/4</i> is expressed in three discrete domains of gastrula ectoderm. At juvenile stages, Sfrps are expressed in the very anterior ectoderm (apical tuft), and <i>sfrp1/5</i> is also expressed in the entire proboscis mesoderm, whereas <i>dkk1/2/4</i> is broadly expressed in the anterior ectoderm. Wnts are expressed in three discrete ectodermal domains: the base of the proboscis, the anterior trunk (over the first gill slit), and the posterior-most ectoderm. In addition, <i>wnt9</i> and <i>wntA</i> are expressed in posterior internal tissues. Fz genes are expressed in nested domains along the ectoderm. Territories are color coded: endomesoderm (grey), posterior ectoderm (dark blue), intermediate ectoderm (medium blue) and anterior ectoderm (light blue). Fz, frizzled; Sfrp, secreted frizzled-related protein.</p

Blocking cWnt pathway anteriorizes the embryo.

<p>(i), Overexpression of <i>sfrp1/5</i> and <i>dkk1/2/4</i> produces embryos with enlarged proboscis and reduced trunk. Control embryos (A, C, E, G, I and K). Embryos injected with <i>sfrp1/5</i> mRNA (B and D), <i>dkk1/2/4</i> mRNA (F and H), and <i>fz5/8</i> siRNA (J and L). In situ hybridization for the most apical marker <i>foxQ2-1</i> (E, F, I and J), the proboscis markers <i>six3</i> (A, B, G and G) and <i>rx</i> (K and L), and the anterior trunk marker <i>engrailed</i> (C and D). 48 h embryos (C, D, G-L). Seventy-two h embryos (A, B, E and F). Anterior to the top left, ventral to the bottom left. Numbers indicate embryos with the displayed phenotyped over the number of analyzed embryos. (ii), Knock down of β-catenin extends anterior-most fate only into anterior ectoderm. siRNA against β-catenin was injected at 4-cell stage into single blastomeres with a rhodamine tracer (red cell in diagrams, which then develop into a full quadrant of the embryo at gastrula, also shown in the model in red). Expression by in situ hybridization at 28 h of the apical marker <i>foxQ2-1</i> (A-C), the midtrunk marker <i>msx</i> (D-F), and the posterior marker <i>hox9/10</i> (G-I). Control embryos are shown in (A,D and G), and G represents an injection control. Panels B, E, and H all show the expression of markers genes following injection of β-catenin siRNA. Panels C, F, and I also show the fluorescent tracer showing the lineage of the injected blastomeres at gastrulation. siRNA, short interfering RNA.</p

Activation of the cWnt pathway leads to anterior truncation.

<p>(A-D), Treatment of embryos with the GSK3β inhibitor 1-azakenpaullone leads to a loss of proboscis at 5 μM (Aii, Bii, Cii, Dii) and to a loss of both proboscis and collar at 10 μM (Aiii, Biii, Ciii, Diii). DMSO-treated control embryos (Ai, Bi, Ci, Di). In situ hybridization for ectodermal markers of the anterior collar <i>barH</i> (A), anterior trunk <i>engrailed</i> (B), trunk <i>msx</i> (C), and posterior trunk <i>hox9/10</i> (D). Embryos at two and a half (C-D) and five (A-B) days of development. Earlier sampling at gastrula stage shows no morphological change but significant transformation of markers <i>sfrp1/5</i> (Eii) and <i>otx</i> (Fii), but no change in <i>hox9/10</i> (Gii) at 10 μM 1-azakenpaullone. DMSO control embryos (Ei, Fi, Gi). Anterior to the top, ventral to the left. (H-I), Overexpression of <i>Wnt3</i> by mRNA injection produces virtually identical phenotypes: loss of proboscis (Hii and Iii) or loss of proboscis and collar (Hiii and Iiii), depending on the strength of the phenotype. In situ hybridization for ectodermal markers of the anterior trunk <i>engrailed</i> (H) and posterior trunk <i>hox11/13c</i> (I) at three days of development (numbers indicate embryos with the displayed phenotyped over the number of analyzed embryos). Anterior to the top left, ventral to bottom left. C, control embryo; cWnt, canonical Wnt; DMSO, dimethyl sulfoxide; GSK3β, glycogen synthase kinase 3 beta.</p

RT-PCR analysis of <i>Wnt</i> and <i>Fz</i> genes expression during early development.

<p>Embryos were harvested at six different stages: oocytes, 16- to 32-cell cleavage stage embryos, late blastula, mid-gastrula, at 48 hpf, and 72 hpf. The first panel shows levels of all 13 <i>Wnt</i> genes; the second panel shows the positive control <i>actin</i> and a negative control. The third panel shows the levels of the four <i>Fz</i> receptor genes. The transcript amounts are comparable across all three panels. <i>Fz</i>, frizzled; hpf, h postfertilization; RT-PCR, reverse transcription PCR.</p

Expression of Wnt genes with blastoporal localization.

<p>Whole mount in situ hybridization of Wnt genes with early expression domains around the blastopore. All data are presented as optical sections following clearing in Murray Clear. Developmental staging is from blastula to 72 h of development. All embryos are oriented with anterior or animal (in the case of blastula) to the top left of the panel and posterior or vegetal to the bottom right of the panel. Right column, ventral is to the bottom left. Unless otherwise noted, expression is ectodermal. (A), Schematic representation of optical section through embryos showing the main regions of the embryo representing the major divisions and landmarks of the body plan at 72 hpf (Ai), blastula stages (Aii), gastrula stages (Aiii), 48 h (Aiv), 60 h (Av), and 72 h (Avi) of development with endomesoderm (gray), posterior ectoderm (dark blue), midfate ectoderm (medium blue), and anterior ectoderm (light blue) precursors. (B), Expression of <i>wnt3</i>. No expression at blastula (Bi), gastrula side view (Bii), at 48 hpf frontal view (Biii), at 60 hpf in side view (Biv), and at 72 hpf in side view (Bv). (C), Expression of <i>wnt1</i> at blastula (Ci), at gastrula stage (Cii), at 48 h frontal section (Ciii), at 60 h side view (Civ), and at 72 h side view (Cv). (D), Expression of <i>wnt4</i>, (Di) at blastula, (Dii) at gastrula, (Diii) at 36 h side view, (Div) at 60 h side view, and (Dv) at 72 h side view. (E), Expression of <i>wnt6</i>, (Ei) at blastula, (Eii) at gastrula, (Eiii) at 48 h side view, (Eiv) at 60 h side view, and (Ev) at 72 h side view. (F), Expression of <i>wnt16</i>, (Fi) at blastula, (Fii) at gastrula, (Fiii) at 48 h side view. The white arrow indicates expression in the ventral posterior endoderm. (Fiv) Expression at 60 h side view, and (Fv) at 72 h side view. hpf, hours postfertilization.</p