XB-ART-53052Curr Biol February 20, 2017; 27 (4): 543-548.
Leftward Flow Determines Laterality in Conjoined Twins.
Conjoined twins fused at the thorax display an enigmatic left-right defect: although left twins are normal, laterality is disturbed in one-half of right twins [1-3]. Molecularly, this randomization corresponds to a lack of asymmetric Nodal cascade induction in right twins . We studied leftward flow [5, 6] at the left-right organizer (LRO) [7, 8] in thoracopagus twins in Xenopus, which displayed a duplicated, fused, and ciliated LRO. Cilia were motile and produced a leftward flow from the right LRO margin of the right to the left margin of the left twin. Motility was required for correct laterality in left twins, as knockdown of dynein motor dnah9 prevented Nodal cascade induction. Nodal was rescued by parallel knockdown of the inhibitor dand5 [9, 10] on the left side of the left twin. Lack of Nodal induction in the right twin, despite the presence of flow, was due to insufficient suppression of dand5. Knockdown of dand5 at the center of the fused LRO resulted in asymmetric Nodal cascade induction in the right twin as well. Manipulation of leftward flow and dand5 in a targeted and sided manner induced the Nodal cascade in a predictable manner, in the left twin, the right one, both, or neither. Laterality in conjoined twins thus was determined by cilia-driven leftward fluid flow like in single embryos, which solves a century-old riddle, as the phenomenon was already studied by some of the founders of experimental embryology, including Dareste , Fol and Warynsky , and Spemann and Falkenberg  (reviewed in ).
PubMed ID: 28190730
Article link: Curr Biol
Species referenced: Xenopus
Genes referenced: ctnnb1 dand5 dnah9 foxj1 foxj1.2 lefty nodal nodal1 pitx2 sia1 sia2 sms sox17a sst.1 wnt8a
GO keywords: detection of nodal flow
Morpholinos: dand5 MO1 dand5 MO2 dnah9 MO1 dnah9 MO2 dnah9 MO3
Disease Ontology terms: visceral heterotaxy
Phenotypes: Xla Wt + ctnnb1 (Fig 1 B)
Article Images: [+] show captions
|Figure 1. Leftward Flow at the Fused LRO in βcat-Induced Conjoined Twins. (A) Development of the LRO at the Xenopus gastrocoel roof: the superficial mesoderm (SM) involutes during gastrulation to give rise to the GRP during early neurulation, which is shown in a dorsal explant in a ventral view on the right (blue). (B and C) Endogenous (closed arrowhead) and induced (open arrowhead) SM marked by foxj1 expression in a singleton (B) and a conjoined twin (C; ∗, injected side). Note that the two SMs are clearly separated. (D–G) Ventral views of dorsal explants of singleton embryos (D and F) and conjoined twins (E and G). GRP tissues are highlighted by the absence of sox17a (D and E) and the presence of dnah9 transcripts (F and G). Closed arrowhead, endogenous GRP; open arrowhead, induced GRP. Note that the GRPs are posteriorly fused in the twins (E and G). (H) Scanning electron micrograph of representative twinned GRP (blue). (I–K) Compilation of cilia polarization in endogenous (I), fused central (J), and induced (K) parts of three twinned GRPs (n, number of cells). (L and M) Leftward flow at twinned GRPs. (L) Tracks of fluorescent beads. (M) Directionality of flow. (N) Schematic representation. a, anterior; ab, absent; bc, blastocoel; bp, blastopore; c, central; d, dorsal; e, endogenous twin; gc, gastrocoel; i, induced twin; l, left; lec, lateral endodermal cells; no, notochord; p, posterior; r, right; so, somite; st., stage; v, ventral. ∗, induced twin. See also Figures S1 and S2 and Movies S1 and S2.|
|Figure 2. Leftward Flow Is Required for nodal1 Expression in Conjoined Twins (A) Injection scheme and predicted nodal1 expression in CoMO- or dnah9MO-injected embryos following βcat-induced twinning on the left (iL) or right (iR) side. (B–D) Left-sided nodal1 expression (closed arrowhead) in CoMO-injected twins (B) was absent (open arrowhead) in dnah9-injected specimens upon twinning on the right (C) but present (closed arrowhead) when the induced twin was placed on the left of the endogenous embryo (D). (E) Summary of nodal1 expression in the induced twin (green, present; red, absent; n, number of analyzed specimens). ∗∗∗p <0.001; ns, not significant. (F–L) nodal1 (F and G) and dand5 (H–L) expression in twinned GRPs of flow (F and H) and post-flow (G and I–L) embryos. Arrowheads, non-reduced dand5; open arrowheads, reduced dand5. Note that dand5 was clearly reduced on the left side of the left embryo (open arrowhead) but only partially in the left half of the fused central domain. ∗, induced twin. See also Movie S3.|
|Figure 3. Knockdown of dand5 on the Right Side of the Fused Central Domain Induces nodal1 in Right Twins (A) Injection scheme: CoMO or dand5MO were targeted to the left side of the GRP and twinning was induced on the left side, such that dand5MO was delivered to the right side of the fused center of the twinned GRP. (B–E) nodal1 (B and C) and pitx2c expression (D and E) in CoMO- (B and D) and dand5MO-injected (C and E) specimens. Green arrowheads mark the presence and red arrowheads mark the absence of marker gene expression.|
|Figure 4. Flow-Mediated Repression of dand5 Determines Laterality in Conjoined Twins (A–E) Injection scheme (A) and anticipated nodal1 expression patterns (B–E) in (1) CoMO- (B and C) or dand5MO- (D and E) injected primary embryos; with (2) twinning induced on the left (iL; D and E) or right side (iR; B and C); and with (3) intact or ablated leftward flow following buffer (green; B and D) or methylcellulose (MC, red; C and E) injection into the gastrocoel before the onset of flow (stage 14). (B–E) Representative specimens. (F) Summary of nodal1 expression patterns (n, number of analyzed specimens). Closed and open arrowheads mark presence and absence of nodal1 gene expression, respectively. (G) Model of flow-mediated symmetry breakage in conjoined twins. For details, see the main text. ∗, induced twin; l, left; r, right. See also Figure S3.|
|Figure S1 (Related to Figure 1) Nodal cascade expression and organ situs in ßcat-induced conjoined twins (A) Injection scheme, observed Nodal cascade expression and heart situs in twinned embryos. ßcat mRNA injection into the left (L) or right (R) blastomere of the 4-cell embryo induced twinning on the L (iL) or R (iR) side of the primary embryo. (B-D) Representative specimens displaying nodal1 (B), lefty2 (C) and pitx2c (D) in the left LPM of the left twin. (E-G) Heart looping in ßcat-induced conjoined twins. Representative stage 45 tadpoles displaying situs solitus in the left and situs inversus in the right twin (E, blowup in F), or situs solitus on both sides (G). (H-J) Summary of nodal1 and pitx2c expression patterns in the left LPM of the left twin in iL and iR specimens of ßcat- (H), wnt8a- (I) and sia1-induced (J) conjoined twins, as well as compilation of heart situs data. Note that the Nodal cascade was only induced on the left side of the left twin, irrespective of the placement of the induced twin. Note also that the heart situs was normal in left twins and randomized in right ones, again irrespective of the placement of the induced twin. LT, left twin; RT, right twin.|
|Figure S2 (Related to Figure 1) Ciliation in a representative twinned GRP (A) Scheme of fused GRP in ßcat-induced conjoined twin. (B) Scanning electron micrograph of fused GRP. a, anterior; l, left; p, posterior; r, right. (C-E) Blow-ups of central regions of endogenous (C) and induced (D) GRP as well as of fused region in center (E). Cilia highlighted in yellow. (C’-E’) Evaluation of ciliation and cilia polarization. Green, cells with polarized cilia; yellow, cells with central cilia; red, cells without cilia. Blue dots, base of cilia. Note that cells with polarized cilia predominate in central regions while the fused center is characterized by cells lacking or displaying nonpolarized cilia.|
|Figure S3 (Related to Figure 4) Pitx2 expression in a conjoined twinned mouse embryo. This specimen of about 10-12 somites arose spontaneously in a litter isolated from a wildtype mouse. Note that Pitx2 mRNA was only found in the left LPM of the left twin. (A) Dorsal view. (B) labeling of axial structures. (C) Line drawing of twinned specimens with labeled Pitx2 expression (blue) and labeling of axial structures. a, anterior; l, left; LPM, lateral plate mesoderm; nt, neural tube; p, posterior; r, right; som, somite.|
|Graphical abstract: Organ asymmetry is normal in left and randomized in right conjoined twins. Tisler et al. report that cilia-driven leftward flow determines laterality in conjoined twins like in singletons. The right twin is randomized because flow is insufficient to repress the Nodal inhibitor dand5 in the center domain between the twins.|