Abstract: Individual cell shape changes are essential for epithelial morphogenesis. A transcriptional network for epithelial cell shape change is emerging in Drosophila, but this area remains largely unexplored in vertebrates. The distinction is important as so far, key downstream effectors of cell shape change in Drosophila appear not to be conserved. Rather, Shroom3 has emerged as a central effector of epithelial morphogenesis in vertebrates, driving both actin- and microtubule-based cell shape changes. To date, the morphogenetic role of Shroom3 has been explored only in the neural epithelium, so the broad expression of this gene raises two important questions: what are the requirements for Shroom3 in non-neural tissues and what factors control Shroom3 transcription? Here, we show in Xenopus that Shroom3 is essential for cell shape changes and morphogenesis in the developing vertebrate gut and that Shroom3 transcription in the gut requires the Pitx1 transcription factor. Moreover, we show that Pitx proteins directly activate Shroom3 transcription, and we identify Pitx-responsive regulatory elements in the genomic DNA upstream of Shroom3. Finally, we show that ectopic expression of Pitx proteins is sufficient to induce Shroom3-dependent cytoskeletal reorganization and epithelial cell shape change. These data demonstrate new breadth to the requirements for Shroom3 in morphogenesis, and they also provide a cell-biological basis for the role of Pitx transcription factors in morphogenesis. More generally, these results provide a foundation for deciphering the transcriptional network that underlies epithelial cell shape change in developing vertebrates.
Abstract: During digestive organogenesis, the primitive gut tube (PGT) undergoes dramatic elongation and forms a lumen lined by a single-layer of epithelium. In Xenopus, endoderm cells in the core of the PGT rearrange during gut elongation, but the morphogenetic mechanisms controlling their reorganization are undetermined. Here, we define the dynamic changes in endoderm cell shape, polarity, and tissue architecture that underlie Xenopus gut morphogenesis. Gut endoderm cells intercalate radially, between their anterior and posterior neighbors, transforming the nearly solid endoderm core into a single layer of epithelium while concomitantly eliciting "radially convergent" extension within the gut walls. Inhibition of Rho/ROCK/Myosin II activity prevents endoderm rearrangements and consequently perturbs both gut elongation and digestive epithelial morphogenesis. Our results suggest that the cellular and molecular events driving tissue elongation in the PGT are mechanistically analogous to those that function during gastrulation, but occur within a novel cylindrical geometry to generate an epithelial-lined tube.
Abstract: Few studies provide empirical evidence for phenotypic plasticity's role in the evolution of novel traits. One way to do so is to test whether latent plasticity is present in an ancestor that can be refined, enhanced, or diminished by selection in derived taxa (through "genetic accommodation"), thereby producing novel traits. Here, we evaluated whether gut plasticity preceded and promoted the evolution of a novel feeding strategy in spadefoot toad tadpoles. We studied Scaphiopus couchii, whose tadpoles develop an elongate gut and consume only detritus, and two derived species, Spea multiplicata and Sp. bombifrons, whose tadpoles also express a novel, short-gut phenotype in response to a novel resource (anostracan shrimp). Consistent with the expectations of plasticity-mediated trait evolution, we found that shrimp induced a range of phenotypes in Scaphiopus that were not produced with detritus. This plasticity was either suppressed or exaggerated in Spea depending on whether the induced phenotypes were adaptive. Moreover, in contrast to its effects on morphology, shrimp induced little or no functional plasticity, as assessed by gut cell proliferation, in Scaphiopus. Shrimp did, however, induce substantial proliferation in Sp. bombifrons, the species that consumes the most shrimp and that produces the short-gut phenotype the most frequently. Thus, if Spea had ancestral morphological plasticity in response to a novel diet, their shrimp-induced short-gut morphology may have undergone subsequent genetic accommodation that improved its functionality. Hence, diet-induced phenotypic plasticity may have preceded and even promoted the evolution of a novel phenotype.
Abstract: The looping events that establish left-right asymmetries in the vertebrate gut tube are poorly understood. Retinoic acid signaling is known to impact left-right development in multiple embryonic contexts, although its role in asymmetric digestive organ morphogenesis is unknown. Here, we show that the genes for retinaldehyde dehydrogenase (RALDH2) and a retinoic acid hydroxylase (CYP26A1) are expressed in complementary patterns in the Xenopus gut during looping. A late-stage chemical genetic assessment reveals that agonists and antagonists of retinoid signaling generate abnormal gut looping topologies, digestive organ heterotaxias, and intestinal malrotations. Accessory organ deformities commonly associated with intestinal malrotation in humans, such as annular pancreas, pancreas divisum, and extrahepatic biliary tree malformations, are also induced by distinct retinoid receptor agonists. Thus, late-stage retinoic acid signaling is likely to play a critical role in asymmetric gut tube morphogenesis and may underlie the etiology of several clinically relevant defects in the digestive system.
Abstract: The left-right asymmetry of the vertebrate heart is evident in the topology of the heart loop, and in the dissimilar morphology of the left and right chambers. How left-right asymmetric gene expression patterns influence the development of these features is not understood, since the individual roles of the left and right sides of the embryo in heart looping or chamber morphogenesis have not been specifically defined. To this end, we have constructed a bilateral heart-specific fate map of the left and right contributions to the developing heart in the Xenopus embryo. Both the left and right sides contribute to the conoventricular segment of the heart loop; however, the left side contributes to the inner curvature and ventral face of the loop while the right side contributes to the outer curvature and dorsal aspect. In contrast, the left atrium is derived mainly from the original left side of the embryo, while the right atrium is derived primarily from the right side. A comparison of our fate map with the domain of expression of the left-right gene, Pitx2, in the left lateral plate mesoderm, reveals that this Pitx2-expressing region is fated to form the inner curvature of the heart loop, the left atrioventricular canal, and the dorsal aspect of the left atrium. We discuss the implications of these results for the role of left-right asymmetric gene expression in heart looping and chamber morphogenesis.
Abstract: The morphogenetic mechanisms by which developing organs become left-right asymmetric entities are unknown. To investigate this issue, we compared the roles of the left and right sides of the Xenopus embryo during the development of anatomic asymmetries in the digestive system. Although both sides contribute equivalently to each of the individual digestive organs, during the initial looping of the primitive gut tube, the left side assumes concave topologies where the right side becomes convex. Of interest, the concave surfaces of the gut tube correlate with expression of the LR gene, Pitx2, and ectopic Pitx2 mRNA induces ectopic concavities in a localized manner. A morphometric comparison of the prospective concave and convex surfaces of the gut tube reveals striking disparities in their rate of elongation but no significant differences in cell proliferation. These results provide insight into the nature of symmetry-breaking morphogenetic events during left-right asymmetric organ development.
Abstract: Although the gut is homologous among different vertebrates, morphological differences exist between different species. The most obvious variation in the guts of extant vertebrates appears in the stomach. To investigate the evolution of this structure, we compared the histology of the stomach and gastrointestinal tract in amphibian (Xenopus laevis), avian (Gallus gallus), and mammalian (Mus musculus) organisms, and defined the expression patterns of several genes within the developing guts of these lineages. In all three groups, we find that the anterior portion of the stomach has a similar glandular histology as well as a common embryonic expression of the secreted factors Wnt5a and BMP-4. Likewise, within the amniote lineages, the posterior nonglandular stomach and pyloric sphincter regions are also comparable in both histological and molecular phenotypes. The posterior stomach expresses Six2, BMPR1B, and Barx1, whereas the pyloric sphincter expresses Nkx2.5. Although the adult Xenopus stomach exhibits both glandular and aglandular regions and a distinct pyloric sphincter similar to that of the amniotic vertebrates, the histology of the Xenopus tadpole gut shows less distinct variation in differentiation in this region, which is most likely a derived condition. The molecular signature of the embryonic Xenopus gut correlates with the more derived morphology of the larval phase. We conclude that the global patterning of the gut is remarkably similar among the different vertebrate lineages. The distinct compartments of gene expression that we find in the gut be necessary for the unique morphological specializations that distinguish the stomachs from terrestrial vertebrates.
Abstract: Left-right (LR) asymmetry is a fascinating problem in embryonic morphogenesis. Recently, a pathway of genes has been identified which is involved in LR patterning in vertebrates. Although this work characterizes the interactions of several asymmetrically-expressed genes, it is still entirely unclear how such asymmetric expression is set up in the first place. There are two promising molecular candidates which may play a role is such a process: the motor protein dynein, and the gap junction protein connexin-43 (Cx43). We present two models, significantly supported by previous findings, which hypothesize that (a) dynein asymmetrically localizes LR determinants in individual cells to establish cell-autonomous LR biasing, and (b) asymmetric activity of Cx43 gap junctions within key cells sets up electric potentials in multicellular fields, thus establishing large-scale LR asymmetry.
Abstract: Vertebrates appear bilaterally symmetrical but have considerable left-right (LR) asymmetry in the anatomy and placement of internal organs such as the heart. Although a number of asymmetrically expressed genes are known to affect LR patterning, both the initial source of asymmetry and the mechanism that correctly orients the LR axis remain controversial. In this study, we show that the induction of dorsal organizing centers in the embryo can orient LR asymmetry. Ectopic organizing centers were induced by microinjection of mRNA encoding a variety of body axis duplicating proteins, including members of the Wnt signal transduction pathway. The ectopic and primary body axes form side-by-side conjoined twins, with the secondary axis developing as either the left or right sibling. In all cases, correct LR asymmetry was observed in the left twin, regardless of whether it was derived from the primary axis or induced de novo by injection of Xwnt-8, beta-catenin, or Siamois mRNA. In contrast, the right twin was generally unbiased, regardless of the origin of the left body axis, as seen in many instances of experimentally induced and spontaneous conjoined twins. An unanticipated exception was that right twins induced by beta-catenin and Siamois, two downstream effectors of Wnt signaling, exhibited predominately normal heart looping, even when they formed the right twin. Taken together, these results indicate that LR asymmetry is locally oriented as a consequence of Wnt signaling through beta-catenin and Siamois. We discuss the possibility that signals upstream of beta-catenin and Siamois might be required in order for a right sibling to be randomized.
Abstract: Heart induction in Xenopus has been thought to be dependent primarily on the interaction of the heart primordia with the Spemann organizer. We demonstrate, however, that signals derived from the deep dorsoanterior endoderm during early gastrulation are also essential for heart formation. The presence of deep endoderm dramatically enhances heart formation in explants of heart primordia, both in the presence and absence of organizer. Likewise, extirpation of the entire endoderm can decrease the frequency of heart formation in embryos that retain organizer activity. Finally, we show that the combined presence of both endoderm and organizer is necessary and sufficient to induce heart in ventral mesoderm explants that would not otherwise form heart tissue. Xenopus heart induction, therefore, may be a multistep process requiring separate dorsalization and cardiogenic signalling events. This is the first demonstration of a heart-inducing role for the endoderm in Xenopus, indicating that the mechanism of heart formation may be similar in most vertebrates.
