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Integration of left-right Pitx2 transcription and Wnt signaling drives asymmetric gut morphogenesis via Daam2.

ABSTRACT: A critical aspect of gut morphogenesis is initiation of a leftward tilt, and failure to do so leads to gut malrotation and volvulus. The direction of tilt is specified by asymmetric cell behaviors within the dorsal mesentery (DM), which suspends the gut tube, and is downstream of Pitx2, the key transcription factor responsible for the transfer of left-right (L-R) information from early gastrulation to morphogenesis. Although Pitx2 is a master regulator of L-R organ development, its cellular targets that drive asymmetric morphogenesis are not known. Using laser microdissection and targeted gene misexpression in the chicken DM, we show that Pitx2-specific effectors mediate Wnt signaling to activate the formin Daam2, a key Wnt effector and itself a Pitx2 target, linking actin dynamics to cadherin-based junctions to ultimately generate asymmetric cell behaviors. Our work highlights how integration of two conserved cascades may be the ultimate force through which Pitx2 sculpts L-R organs.

SUBMITTER: Welsh IC

PROVIDER: S-EPMC3965270 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Integration of left-right Pitx2 transcription and Wnt signaling drives asymmetric gut morphogenesis via Daam2.

Welsh Ian C IC Thomsen Michael M Gludish David W DW Alfonso-Parra Catalina C Bai Yan Y Martin James F JF Kurpios Natasza A NA

Developmental cell 20130901 6

A critical aspect of gut morphogenesis is initiation of a leftward tilt, and failure to do so leads to gut malrotation and volvulus. The direction of tilt is specified by asymmetric cell behaviors within the dorsal mesentery (DM), which suspends the gut tube, and is downstream of Pitx2, the key transcription factor responsible for the transfer of left-right (L-R) information from early gastrulation to morphogenesis. Although Pitx2 is a master regulator of L-R organ development, its cellular targ ...[more]

PMID: 24091014

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Project description:BackgroundAlthough vertebrates are bilaterally symmetric organisms, their internal organs are distributed asymmetrically along a left-right axis. Disruption of left-right axis asymmetric patterning often occurs in human genetic disorders. In zebrafish embryos, Kupffer's vesicle, like the mouse node, breaks symmetry by inducing asymmetric expression of the Nodal-related gene, spaw, in the left lateral plate mesoderm (LPM). Spaw then stimulates transcription of itself and downstream genes, including lft1, lft2, and pitx2, specifically in the left side of the diencephalon, heart and LPM. This developmental step is essential to establish subsequent asymmetric organ positioning. In this study, we evaluated the role of krüppel-like factor 8 (klf8) in regulating left-right asymmetric patterning in zebrafish embryos.MethodsZebrafish klf8 expression was disrupted by both morpholino antisense oligomer-mediated knockdown and a CRISPR-Cas9 system. Whole-mount in situ hybridization was conducted to evaluate gene expression patterns of Nodal signalling components and the positions of heart and visceral organs. Dorsal forerunner cell number was evaluated in Tg(sox17:gfp) embryos and the length and number of cilia in Kupffer's vesicle were analyzed by immunocytochemistry using an acetylated tubulin antibody.ResultsHeart jogging, looping and visceral organ positioning were all defective in zebrafish klf8 morphants. At the 18-22 s stages, klf8 morphants showed reduced expression of genes encoding Nodal signalling components (spaw, lft1, lft2, and pitx2) in the left LPM, diencephalon, and heart. Co-injection of klf8 mRNA with klf8 morpholino partially rescued spaw expression. Furthermore, klf8 but not klf8?zf overexpressing embryos showed dysregulated bilateral expression of Nodal signalling components at late somite stages. At the 10s stage, klf8 morphants exhibited reductions in length and number of cilia in Kupffer's vesicle, while at 75% epiboly, fewer dorsal forerunner cells were observed. Interestingly, klf8 mutant embryos, generated by a CRISPR-Cas9 system, showed bilateral spaw expression in the LPM at late somite stages. This observation may be partly attributed to compensatory upregulation of klf12b, because klf12b knockdown reduced the percentage of klf8 mutants exhibiting bilateral spaw expression.ConclusionsOur results demonstrate that zebrafish Klf8 regulates left-right asymmetric patterning by modulating both Kupffer's vesicle morphogenesis and spaw expression in the left LPM.

Project description:BackgroundLeft-right (LR) asymmetry is an essential feature of bilateral animals. Studies in vertebrates show that LR asymmetry formation comprises three major steps: symmetry breaking, asymmetric gene expression, and LR morphogenesis. Although much progress has been made in the first two events, mechanisms underlying asymmetric morphogenesis remain largely unknown due to the complex developmental processes deployed by vertebrate organs.ResultsWe here addressed this question by studying Pitx gene function in the basal chordate amphioxus whose asymmetric organogenesis, unlike that in vertebrates, occurs essentially in situ and does not rely on cell migration. Pitx null mutation in amphioxus causes loss of all left-sided organs and incomplete ectopic formation of all right-sided organs on the left side, whereas Pitx partial loss-of-function leads to milder phenotypes with only some LR organs lost or ectopically formed. At the N1 to N3 stages, Pitx expression is gradually expanded from the dorsal anterior domain to surrounding regions. This leads to activation of genes like Lhx3 and/or Prop1 and Pit, which are essential for left-side organs, and downregulation of genes like Hex and/or Nkx2.1 and FoxE4, which are required for right-side organs to form ectopically on the left side. In Pitx mutants, the left-side expressed genes are not activated, while the right-side genes fail to decrease expression on the left side. In contrast, in embryos overexpressing Pitx genes, the left-side genes are induced ectopically on the right side, and the right-side genes are inhibited. Several Pitx binding sites are identified in the upstream sequences of the left-side and right-side genes which are essential for activation of the former and repression of the latter by Pitx.ConclusionsOur results demonstrate that (1) Pitx is a major (although not the only) determinant of asymmetric morphogenesis in amphioxus, (2) the development of different LR organs have distinct requirements for Pitx activity, and (3) Pitx controls amphioxus LR morphogenesis probably through inducing left-side organs and inhibiting right-side organs directly. These findings show much more dependence of LR organogenesis on Pitx in amphioxus than in vertebrates. They also provide insight into the molecular developmental mechanism of some vertebrate LR organs like the lungs and atria, since they show a right-isomerism phenotype in Pitx2 knockout mice like right-sided organs in Pitx mutant amphioxus. Our results also explain why some organs like the adenohypophysis are asymmetrically located in amphioxus but symmetrically positioned in vertebrates.

Dataset Information

Integration of left-right Pitx2 transcription and Wnt signaling drives asymmetric gut morphogenesis via Daam2.

Publications

Integration of left-right Pitx2 transcription and Wnt signaling drives asymmetric gut morphogenesis via Daam2.

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