Gata6 Promotes GLI3 Repressor Activities in the Limb

Gli3 is a major regulator of Hedgehog signaling during limb development. In the anterior mesenchyme, GLI3 is proteolytically processed into GLI3R, a truncated repressor form that inhibits Hedgehog signaling. Although numerous studies have identified mechanisms that regulate Gli3 function in vitro, it is not completely understood how Gli3 function is regulated in vivo. In this study, we show a novel mechanism of regulation of GLI3R activities in limb buds by Gata6, a member of the GATA transcription factor family. We show that conditional inactivation of Gata6 prior to limb outgrowth by the Tcre deleter causes preaxial polydactyly, the formation of an anterior extra digit, in hindlimbs. A recent study suggested that Gata6 represses Shh transcription in hindlimb buds. However, we found that ectopic Hedgehog signaling precedes ectopic Shh expression. In conjunction, we observed Gata6 and Gli3 genetically interact, and compound heterozygous mutants develop preaxial polydactyly without ectopic Shh expression, indicating an additional prior mechanism to prevent polydactyly. These results support the idea that Gata6 possesses dual roles during limb development: enhancement of Gli3 repressor function to repress Hedgehog signaling in the anterior limb bud, and negative regulation of Shh expression. Our in vitro and in vivo studies identified that GATA6 physically interacts with GLI3R to facilitate nuclear localization of GLI3R and repressor activities of GLI3R. Both the genetic and biochemical data elucidates a novel mechanism by Gata6 to regulate GLI3R activities in the anterior limb progenitor cells to prevent polydactyly and attain proper development of the mammalian autopod

Noise-resistant developmental reproducibility in vertebrate somite formation

いいかげんに働く細胞たちが協調してからだを作る仕組みを解明 --リズムを刻む体内時計によるノイズキャンセル機構--. 京都大学プレスリリース. 2019-02-06.The reproducibility of embryonic development is remarkable, although molecular processes are intrinsically stochastic at the single-cell level. How the multicellular system resists the inevitable noise to acquire developmental reproducibility constitutes a fundamental question in developmental biology. Toward this end, we focused on vertebrate somitogenesis as a representative system, because somites are repeatedly reproduced within a single embryo whereas such reproducibility is lost in segmentation clock gene-deficient embryos. However, the effect of noise on developmental reproducibility has not been fully investigated, because of the technical difficulty in manipulating the noise intensity in experiments. In this study, we developed a computational model of ERK-mediated somitogenesis, in which bistable ERK activity is regulated by an FGF gradient, cell-cell communication, and the segmentation clock, subject to the intrinsic noise. The model simulation generated our previous in vivo observation that the ERK activity was distributed in a step-like gradient in the presomitic mesoderm, and its boundary was posteriorly shifted by the clock in a stepwise manner, leading to regular somite formation. Here, we showed that this somite regularity was robustly maintained against the noise. Removing the clock from the model predicted that the stepwise shift of the ERK activity occurs at irregular timing with irregular distance owing to the noise, resulting in somite size variation. This model prediction was recently confirmed by live imaging of ERK activity in zebrafish embryos. Through theoretical analysis, we presented a mechanism by which the clock reduces the inherent somite irregularity observed in clock-deficient embryos. Therefore, this study indicates a novel role of the segmentation clock in noise-resistant developmental reproducibility

ERK Activity Dynamics during Zebrafish Embryonic Development

During vertebrate development, extracellular signal-regulated kinase (ERK) is activated by growth factors such as fibroblast growth factor (FGF), and it regulates the formation of tissues/organs including eyes, brains, somites, limbs, and inner ears. However, an experimental system to monitor ERK activity dynamics in the entire body of the vertebrate embryo is lacking. We recently studied ERK activity dynamics in the pre-somitic mesoderm of living zebrafish embryos injected with mRNAs encoding a Förster resonance energy transfer (FRET)-based ERK biosensor. In this study, transgenic zebrafish stably and ubiquitously expressing the ERK biosensor were generated to monitor ERK activity dynamics throughout embryonic development. The system allowed the identification of ERK activation domains in embryos from the late blastula to the late segmentation stage, consistent with immunostaining patterns obtained using anti-phosphorylated ERK antibody. A spatiotemporal map of ERK activity in the entire body during zebrafish embryogenesis was generated, and previously unidentified activation dynamics and ERK domains were identified. The proposed system is the first reported method to monitor ERK activity dynamics during vertebrate embryogenesis, providing insight into the role of ERK activity in normal and abnormal development in living vertebrate embryos

LIVE IMAGING OF ERK ACTIVITY STEPWISE PATTERNING DURING SOMITOGENESIS

Periodical segmentation of the anterior extremity of the presomitic mesoderm (PSM) generates metameric structure of somites during vertebrate development. During somite segmentation in zebrafish, msep determines a future somite boundary at position B-2 within the PSM. However, heat shock experiments suggest that an earlier future of somite boundary exists at B-5, but the molecular signature of this boundary remains unidentified. Our recent study demonstrated that fibroblast growth factor (FGF) gradient is converted into an ON-OFF boundary of downstream Erk activity, which corresponds to the future B-5 somite boundary. Moreover, we also revealed that the segmentation clock is required for a stepwise posterior shift of the Erk activity boundary during each segmentation. To clarify this evidence, here we perform time-lapse imaging of Erk activity in living embryos using a FRET biosensor. We focused on FRET signals within the PSM to observe spatial and temporal changes of Erk activity. Consistent with the data from fixed embryos, we observed ON-OFF boundary of Erk activity within the PSM and the position stepwisely shifted to posterior during somite formation. In order to test the contribution of the segmentation clock to the stepwise movements of the ON-OFF boundary of Erk activity, we disrupted the segmentation clock in zebrafish embryos by knocking down two segmentation clock genes, her1 and her7. Double knockdown of her1 and her7 resulted in the failure of the shift of the Erk ON-OFF boundary during somite segmentation, leading to segmentation defects of somites. These results strongly suggest that the clock-dependent stepwise movement of Erk activity is a key mechanism to generate the perfect repetitive structure of somites.Keywords: Live imaging, Erk, stepwise, somitogenesi

Live Imaging Of ERK Activity Stepwise Patterning During Somitogenesis

Periodical segmentation of the anterior extremity of the presomitic mesoderm (PSM) generates metameric structure of somites during vertebrate development. During somite segmentation in zebrafish, msep determines a future somite boundary at position B-2 within the PSM. However, heat shock experiments suggest that an earlier future of somite boundary exists at B-5, but the molecular signature of this boundary remains unidentified. Our recent study demonstrated that fibroblast growth factor (FGF) gradient is converted into an ON-OFF boundary of downstream Erk activity, which corresponds to the future B-5 somite boundary. Moreover, we also revealed that the segmentation clock is required for a stepwise posterior shift of the Erk activity boundary during each segmentation. To clarify this evidence, here we perform time-lapse imaging of Erk activity in living embryos using a FRET biosensor. We focused on FRET signals within the PSM to observe spatial and temporal changes of Erk activity. Consistent with the data from fixed embryos, we observed ON-OFF boundary of Erk activity within the PSM and the position stepwisely shifted to posterior during somite formation. In order to test the contribution of the segmentation clock to the stepwise movements of the ON-OFF boundary of Erk activity, we disrupted the segmentation clock in zebrafish embryos by knocking down two segmentation clock genes, her1 and her7. Double knockdown of her1 and her7 resulted in the failure of the shift of the Erk ON-OFF boundary during somite segmentation, leading to segmentation defects of somites. These results strongly suggest that the clock-dependent stepwise movement of Erk activity is a key mechanism to generate the perfect repetitive structure of somites

Time-lapse observation of stepwise regression of Erk activity in zebrafish presomitic mesoderm

Abstract During somite segmentation, clock genes oscillate within the posterior presomitic mesoderm (PSM). The temporal information ties up with the posteriorly moving FGF gradient, leading to the formation of a presumptive somite within the PSM. We previously investigated Erk activity downstream of FGF signaling by collecting stained zebrafish embryos, and discovered that the steep gradient of Erk activity was generated in the PSM, and the Erk activity border regularly shifted in a stepwise manner. However, since these interpretations come from static analyses, we needed to firmly confirm them by applying an analysis that has higher spatiotemporal resolutions. Here we developed a live imaging system for Erk activity in zebrafish embryos, using a Förster resonance energy transfer (FRET)-based Erk biosensor. With this system, we firmly showed that Erk activity exhibits stepwise regression within the PSM. Although our static analyses could not detect the stepwise pattern of Erk activity in clock-deficient embryos, our system revealed that, in clock-deficient embryos, the stepwise regression of Erk activity occurs at an irregular timing, eventually leading to formation of irregularly-sized somites. Therefore, our system overcame the limitation of static analyses and revealed that clock-dependent spatiotemporal regulation of Erk is required for proper somitogenesis in zebrafish