May 15, 2012;
Connexin26-mediated transfer of laterality cues in Xenopus.
-driven leftward flow of extracellular fluid breaks bilateral symmetry in the dorsal midline of the neurula
stage vertebrate embryo
. The left
signaling cascade in the lateral
) is key to asymmetric morphogenesis and placement of organs during subsequent development. The nature of the initial asymmetric cue(s) as well as the transfer of information from the midline to the left
side has remained elusive. Gap junctional communication has been previously involved in Xenopus left
(LR) development, however a function at cleavage
stages was inferred from inhibitor experiments. Here we show by heptanol-mediated block of connexin function that flow stages during neurulation represent the critical time window. Flow in Xenopus occurs at the gastrocoel
roof plate (GRP), a ciliated sheath of cells of mesodermal fate transiently positioned within the dorsal epithelial lining of the forming archenteron
. We reasoned that endodermal cells immediately adjacent to the GRP are important for transfer of asymmetry. A systematic screen identified two connexin genes, Cx26
, which were co-expressed in these lateral
endodermal cells. Gain- and loss-of-function experiments pinpointed Cx26
as the critical connexin for LR development, while Cx32
had no effect on laterality. Importantly, GRP morphology, ciliation and flow were not affected in Cx26
morphants. Our results demonstrate a decisive role of Cx26
in the transfer of laterality cues from the GRP to the left LPM
, providing a novel access to the identification of the initial asymmetric signal generated by flow.
[+] show captions
Fig. 1. Neurula stage embryos require GJC for LR development.
(A) Scheme depicting type and time course of treatments. (B) HepOH treatments of neurula stage embryos resulted in absence of Pitx2c expression in the left LPM of 2-day tadpoles. (B) Summary of experimental data. (C,D) Representative wildtype (C) and HepOH-treated (D) embryos displaying left-asymmetric (C) or absent (D) expression of Pitx2c in the left LPM. (E) Consequences of treatments for situs development in the 3-day tadpole. (E) Summary of results. (F,G) Characteristic untreated (F) and HepOH-incubated (G) tadpoles displaying normal organ situs (F) or situs inversion (G), as determined by the direction of heart looping (outlined by dashed yellow line) and gut coiling (outlined in black). Note that ethanol and undecanol were inefficient. Note also that early treatments before gastrulation (≤ stage 9) and late injections (stage 19/20) were less striking or without effect. a, anterior; an, animal; d, dorsal; EtOH, ethanol; HepOH, HepOH; ht, heterotaxia; n.s., not significant; p, posterior; si, situs inversus; st., stage; UnOH, undecanol; v, ventral; veg, vegetal; wt, wildtype; *, significant (p<0.05); **, highly significant (p<0.01); *** very highly significant (p<0.001). Numbers in brackets represent number of analyzed specimens. Statistical significances were calculated using Pearson's chi square test.
Fig 2. Cx26 and Cx32 are co-expressed in the epithelial lining of the gastrocoel next to the GRP.
Temporal and spatial expression patterns were determined by WMISH of staged embryos. (A) Cx26. (A) In the early gastrula embryo Cx26 expression around the blastopore is enhanced on the dorsal side (arrowhead). (A′) Histological section shows Cx26 staining in the involuting endodermal cells. (B,C) In the stage 17 neurula embryo Cx26 mRNA is found in the endoderm lining the gastrocoel. (B) Dorsal explant. Outline of GRP, which is free of staining, indicated by dashed line. (C) Sectioning (C′) of whole-mount embryo (C) shows Cx26 expression all around the gastrocoel with the marked exception of the GRP, demonstrated in blowup shown in (C′). (D) Cx26 expression at stage 35. Transversal section (D′) shows staining in the pronephros, the liver diverticulum and in the dorsal half of the endoderm. (E,F) Cx32. (E) Expression in posterior half of stage 18 neurula embryo. Section in (E′) demonstrates staining in two open ring-like domains representing the limit of the endoderm, compassing the endodermal yolk cells. Blowup in (E′) shows that the GRP is free of Cx32 expression. (F) Cx32 expression at stage 30. Transversal hemisection in (F′) reveals mRNA staining in the pronephros, hatching gland and throughout the endoderm. a, anterior; bc, blastocoel; bp, blastopore; d, dorsal; eym, endodermal yolk mass; gc, gastrocoel; l, left; ld, liver diverticulum; no, notochord; nt, neural tube; p, posterior; pn, pronephros; r, right;s, somite; st., stage; v, ventral. The position of the GRP is indicated by dashed lines in (B) and (E). Red arrowheads mark the position of the blastopore in (A,A′). Lines in (C,D,F) indicate planes of histological sections in (C′,D′,F′).
Fig 3. Cx26 and Cx32 form heteromeric connexons in Xenopus embryos.
Fusion constructs (Cx26-eGFP and Cx32-RFP) were co-injected into the animal region of single blastomeres at the 4-8 cell stage and cultured through blastula-gastrula stages. Fluorescent fusion proteins were co-expressed in ectodermal cells of the animal cap region. Expression was found in vesicles (white arrowheads) and the plasma membrane (blue arrowheads). (A) Cell boundaries. Positions highlighted in (B) are indicated by arrowheads. (B) Cx26-eGFP staining. (C) Cx32-RFP staining. (D) Overlay of frames. Scale bar represents 10 .
Fig 4. Overexpression of Cx26 but not Cx32 impairs LR development.
Full-length expression constructs were injected alone or in combination into single left or right blastomeres of the 4-8 cell embryos, and Pitx2c expression was analyzed in the 2-day tadpole. (A,A′) Injection scheme and cell lineage at stage 17. (A) Injections were targeted to the C2 lineage, i.e. cells which during flow stages (st. 17; A′) end up in the marginal cells of the GRP (somitic GRP; sGRP) and the adjacent lateral endodermal crest cells (LEC). (B) Summary of results. Note that right-sided injections did not alter Pitx2c expression. Note also that left-sided Cx26 overexpression prevented Pitx2c induction in the left LPM with very high statistical significance, while Cx32 had no effect on laterality, whether applied alone or in combination with Cx26. Numbers in brackets represent number of embryos analyzed. Statistical significances were calculated using Pearson's chi square test.
Fig 5. Cx26 but not Cx32 is required for LR development.
Lineage and side-specific knockdown of Cx26 and Cx32 alone or in combination as specified, followed by Pitx2c expression analysis in morphant tadpoles. Injections were targeted to the C2 lineage, i.e. the somitic GRP cells and the adjacent endodermal cells, or to the C3 lineage, i.e. more lateral endodermal cells (Fig. 4A,A′). Note that Cx32 knockdown did not affect Pitx2c expression, and that Cx26 was only required on the left side. Note also two non-overlapping Cx26-MOs, which targeted the 5′-UTR and the start AUG, respectively, gave qualitatively identical results, confirming the specificity of the knockdown. Note further that targeting to the endodermal cells adjacent to the GRP (C2 lineage) resulted in highly significant disruption of Pitx2c expression, while effects were not significant in lateral endodermal cells (C3 lineage). Numbers represent number of embryos analyzed. Statistical significances were calculated using Pearson's chi square test.
Fig 6. GRP morphology and ciliation are unaffected in Cx26 morphants.
SEM-analysis of GRP ciliation and morphology in stage 17 dorsal explants of morphants injected with Cx26-MO or Co-MO into left blastomeres at the 4-cell stage. (A) Representative dorsal explant of Cx26 morphant specimen revealed normal cilia length, number of ciliated cells and cilia polarization. (B) Quantification of mean ciliation rates (B) and mean cilia lengths (C) of GRP cilia in a defined area (A′). (D) Cilia polarization (assessed in the same areas). Note that a not significant (n.s.) slight tendency towards fewer and shorter cilia was observed on the injected side in both Cx26 and co-MO morphants, but that differences between Cx26 morphants and co-MO injected specimens were not significant. Numbers in brackets indicate number of analyzed dorsal explants or cilia. Statistical significances were calculated using Student's t-test.