The left-right asymmetry of liver lobation is generated by Pitx2c-mediated asymmetries in the hepatic diverticulum

Abstract

Internal organs exhibit left-right asymmetric sizes, shapes and anatomical positions, but how these different lateralities develop is poorly understood. Here we use the experimentally tractable Xenopus model to uncover the morphogenetic events that drive the left-right asymmetrical lobation of the liver. On the right side of the early hepatic diverticulum, endoderm cells become columnar and apically constricted, forming an expanded epithelial surface and, ultimately, an enlarged right liver lobe. In contrast, the cells on the left side become rounder, and rearrange into a compact, stratified architecture that produces a smaller left lobe. Side-specific gain- and loss-of-function studies reveal that asymmetric expression of the left-right determinant Pitx2c elicits distinct epithelial morphogenesis events in the left side of the diverticulum. Surprisingly, the cellular events induced by Pitx2c during liver development are opposite those induced in other digestive organs, suggesting divergent cellular mechanisms underlie the formation of different lateralities.

Keywords: Endoderm; Left-right asymmetry; Liver; Morphogenesis; Pitx2; Xenopus.

Figure 1. The development of morphological left-right asymmetry in the Xenopus liver

Cartoon illustrations (A, E) demarcate the positions of the left (L; yellow) and right (R; orange) sides of the developing liver in whole embryos (A; ventral view) and dissected gastrointestinal tracts (E; anterior view). The embryonic midline is indicated by the dashed line in A, and the orientation of the dorsal-ventral axis is indicated by the arrow in E. In situ hybridization was performed at the indicated NF stages (33-44) to detect the liver-specific marker hhex in whole embryos (B–C; NF 33-37, ventral views) or fibrinogen in isolated tracts (F–G; NF 42-44, anterior views). Orange and yellow outlines demarcate the borders of the right and left lobes, respectively. For clarity, the stained specimens are also shown without outlines at higher magnification in B′–C′ and F′–G′; brackets define the extent of each lobe, with the arrow indicating the boundary between left and right lobes. At the earliest stages, the entire hhex-expressing region was outlined (B). Later (~NF 35), when the gall bladder (GB) becomes distinguishable as a separate structure, only the liver lobes themselves were measured (C). The right/left ratio of hhex-expressing tissue area (ventral view) was then quantified at successive stages of development (D; NF 27-39). Ratios close to one (red line) indicate symmetry; left-right asymmetry becomes apparent at NF 33, when the ratio becomes significantly greater than one. At later stages (NF 42-44), the right lobe becomes visibly more elongated than the left (F–G), as quantified by gross length to width ratio (H; NF 46). Asterisks indicate p<0.05 (*) or p<0.0001 (**).

Figure 2. The development of cellular-level left-right asymmetry in the Xenopus liver

To determine the contribution of cells from the left and right side of the embryo to the developing liver, fate-mapping was performed by microinjecting mRNA encoding membrane-tethered GFP (mgfp, green) and membrane-tethered mcherry (red) into the left and right dorsovegetal region of the 8-cell embryo, respectively (A). Initially, the right and left labels are distributed symmetrically on opposite sides of the embryonic midline (NF 32; B, B′, C″). However, as morphogenesis proceeds, the distribution of left and right labels (in transverse section, C–C″) becomes asymmetrical. Beginning at NF 33, right side cells occupy a slightly greater proportion of the total diverticular perimeter than left side cells (C″). In the tadpole (D–D′; feeding stage, NF 46), the right and left labels are confined predominately to the right or left lobe of the liver, respectively, indicating that early left and right hepatic progenitors execute independent morphogenetic programs to form distinct left and right lobes. Asterisks indicate p<0.05 (*) or p<0.001 (**). E–H) Transverse sections through the budding liver diverticulum (see cartoons) were immunostained for Integrin (Int, green) to reveal cellular morphology at NF 32 (E), 33/34 (F), 35/36 (G), and 37/38 (H); nuclei were stained with Topro-3 (blue). Fate mapping (A–D) of serial sections was used to approximate the right (R; orange) versus left (L; yellow) sides of the diverticulum (see Methods). Representative sections are pseudo-colored in orange and yellow to illustrate the consensus left and right contributions at each stage. Higher magnification images of the boxed regions in G (I–J) show the shapes of representative right (orange, I) or left (yellow, J) endoderm cells. At NF 32 (E), the endoderm of the liver diverticulum is symmetrically comprised of a pseudostratified epithelium. However, by NF 35/36, the apical/basal width ratio of right side cells has significantly decreased (i.e., indicating increased apical constriction; p<0.01; I, K), while this parameter increases in left side cells (i.e., they become less apically constricted; p<0.01; J, K). Right side cells also acquire a greater length/width ratio (an indication of columnar morphology and cell heightening) than left side cells (I, J, L). Concomitantly, the number of layers of nuclei along the apical to basal axis of the epithelium increases substantially on the left side (p<0.01), while remaining relatively constant on the right (M). Asterisks in K–M indicate stages at which there were significant differences between sides; p<0.05 (*) or p<0.0001 (**). Closed circles in K–M indicate the mean value; error bars = S.E.

Figure 3. Total cell number and proliferation rate are similar on the left and right sides of the developing liver

Transverse sections through the liver at NF 33 (A), 35 (B), 37 (C) and 39 (D) were stained to reveal cell outlines (green, integrin), individual nuclei (blue, Topro-3) and proliferating cells (magenta, Phospho-histone 3). Orange and yellow outlines demarcate the right and left liver diverticulum, respectively, based on fate-mapping (see Methods). No significant difference in the total number of cells (i.e., nuclei counts; E) or mitotic indices (F) were detected between the right and left sides of the liver at any stage. Scale bars= 200 μm.

Figure 4. Asymmetric expression of pitx2 during Xenopus liver morphogenesis

In situ hybridization was performed on whole NF 35 embryos (A–B) or on serial sections (C–D), probing for the liver specific transcription factor, hhex (A, C), or the LR determinant, pitx2 (B, D). Dotted line in A approximates the section planes shown in C and D. The expression of pitx2 is confined to the mesoderm surrounding the left side of the liver diverticulum (arrows in B, D). R=right, L=left. Scale bars= 200μm.

Figure 5. Pitx2c is required for left liver lobe morphology

A Pitx2c morpholino was targeted to the left liver diverticulum, where pitx2c is normally expressed (see Figure 4). Liver morphology was then analyzed in control and Pitx2-deficient embryos at NF 46 using in situ hybridization with a probe for fibrinogen (A–C). In control morpholino injected livers (CoMo, A), the right lobe (R, orange outline) is larger and more elongate than the left (L, yellow outline). In contrast, in Pitx2cMO injected livers (B–C), the left lobe is larger and more elongated, similar to control right side morphology (i.e., the liver exhibits right isomerism). Scale bars= 200 μm. D) Quantification of Normal, Left Isomerism (L-Iso), Right Isomerism (R-Iso), and LR Reversed (Rev) liver phenotypes (see cartoons) in Control and Pitx2cMo embryos. E) As expected in control embryos, the right lobe of the liver has a greater gross length/width ratio than the left lobe (NF 46). In contrast, in embryos injected with Pitx2cMO, the left lobe is larger, similar to a control right lobe. As expected, the dimensions of the right liver lobe are unaffected by the left-targeted Pitx2cMO. Error bars = S.E. Asterisks indicate p<0.05 (*) or p<0.0001 (**); NS, not significant. F–H) Similar results were obtained with CRISPR-Cas9-mediated Pitx2c loss of function. Liver lobe morphology was analyzed at NF 46 in embryos injected with synthetic Cas9 mRNA plus Tyr-gRNA (control; F) or plus pitx2c-gRNA (G–H) using in situ hybridization with a probe for fibrinogen. In control livers (F), the right lobe (R, orange outline) is larger and more elongate than the left (L, yellow outline). In contrast, in livers from pitx2c gRNA injected embryos (G–H), the left lobe is larger and more elongated, similar to control right side morphology (i.e., the liver exhibits right isomerism). Scale bars= 200 μm.

Figure 6. Pitx2c is required for left endoderm cell morphology in the liver diverticulum

Control morpholino (CoMo; A, C, E, G) or Pitx2c morpholino (Pitx2cMo; B, D, F, H) was co-injected with mRNA encoding GFP (mgfp, red, A–B), targeting the left side of the liver diverticulum. C–H) Sections through the liver of injected embryos (NF 35) were immunostained with β-integrin (green) to visualize cell morphology. The boxed regions in C and D are magnified in E–H, as labeled, to highlight right (R, orange; E–F) and left (L, yellow; G–H) cell morphologies. In CoMo-injected embryos, cells on the right (E) are more apically constricted (i.e., they have a lower apical/basal width ratio; I), more elongated (i.e., they have an increased length to width ratio; J) and are arranged in fewer layers (K), compared to cells on the left (G, I–K). In contrast, left side morpholino knockdown of Pitx2c (H) results in a decreased apical/basal width ratio (I), increased length/width ratio (J), and decreased layers of nuclei (K) within the left epithelium such that there is no longer a significant difference between Pitx2cMo-injected left side cells and control right side cells. As expected, the dimensions of the right side cells are unaffected by the left-targeted Pitx2cMo (F, I–K). Scale bars= 100 μm. Error bars= S.E. Asterisks (**) indicate p<0.0001; NS, not significant.

Figure 7. Ectopic right-side Pitx2c induces left liver lobe morphology

Synthetic mRNA encoding dexamethasone (Dex) inducible Pitx2c (Pitx2cGR) was targeted to the mesoderm surrounding the right liver diverticulum (see Figure 8A–B). Liver morphology was then analyzed in un-induced controls (−Dex) and Dex-induced (+Dex) embryos at NF 46 using in situ hybridization with a probe for fibrinogen. In un-induced controls (A), the right (R, orange outline) lobe is larger and more elongate than the left lobe (L, yellow outline). However, in Dex-induced embryos (B–C), the right lobe of the liver is smaller, similar to control left lobe morphology (i.e., the liver exhibits left isomerism; L-Iso). Scale bars= 200 μm. D) Quantification of liver phenotypes (see cartoons) in un-induced (Pitx2cGR −Dex) and induced (Pitx2cGR +Dex) embryos. E) As expected in un-induced embryos (−Dex), the right lobe of the liver has a greater gross length to width ratio than the left lobe (NF 46). However, in embryos with ectopic right-sided Pitx2c activity (+Dex), the right lobe is smaller, with a decreased length to width ratio comparable to that of left lobes. Error bars = S.E. Asterisks indicate p<0.0001 (**); NS, not significant.

Figure 8. Ectopic right-side Pitx2c induces right endoderm cell morphology in the liver diverticulum

Synthetic mRNAs encoding dexamethasone (Dex) inducible Pitx2cGR and mgfp (red, A, B) were targeted to the mesoderm surrounding the right side of the liver diverticulum. C–H) Sections through the liver of injected embryos (NF 35) were immunostained with β-integrin (Int; green) to visualize cell morphology. The boxed regions in C and D are magnified in E–H, as labeled, to highlight right (R, orange; E–F) and left (L, yellow; G–H) cell morphologies. In un-induced controls (−Dex; C, E, G), cells on the right (E) are more apically constricted (i.e., they have a lower apical/basal width ratio; I), more elongated (i.e., they have an increased length to width ratio; J) and are arranged in fewer layers (K), compared to cells on the left (G, I–K). In contrast, induction of right side Pitx2c activity (F) results in an increased apical/basal width ratio (I), decreased length/width ratio (J) and increased layers of nuclei within the right epithelium (K), such that there is no longer a significant difference between right side cells subject to ectopic Pitx2c activity and control left side cells. As expected, the apical constriction of left side cells remains unaffected in these experiments (I); however, ectopic right Pitx2 activity did unexpectedly affect the length to width ratio of left endoderm cells, and the number of cell layers in the left epithelium, such that the value of these parameters became more similar to that normally seen on the right side (J–K). Error bars= SE. Asterisks indicate p<0.0001 (**); NS, not significant. Scale bars= 100 μm.

Figure 9. Model of LR asymmetric liver lobe morphogenesis

During early liver morphogenesis, Pitx2c expression (purple) is confined to the mesoderm surrounding the left side of the liver diverticulum (A–C). Pitx2c activity (arrows) indirectly inhibits apical constriction and cell elongation in the underlying endoderm, and leads to increased cell layering, thus condensing, rather than expanding, the left diverticular epithelium. Consequently, while the right side of the diverticulum gives rise to an enlarged right lobe, the left side produces a smaller, more spherical left counterpart (D).