Project description:A longstanding challenge in regeneration biology is to understand the role of developmental mechanisms in restoring lost or damaged tissues and organs. As these body structures were built during embryogenesis, it is not surprising that a number of developmental mechanisms are also active during regeneration. However, it remains unclear whether developmental mechanisms act similarly or differently during regeneration as compared to development. Since regeneration is studied in the context of mature, differentiated tissues, it is difficult to evaluate comparative studies with developmental processes due to the latter's highly proliferative environment. We have taken a more direct approach to study regeneration in a developmental context (regrowth). Xenopus laevis, the African clawed frog, is a well-established model for both embryology and regeneration studies, especially for the eye. Xenopus eye development is well-defined. Xenopus is also an established model for retinal and lens regeneration studies. Previously, we demonstrated that Xenopus tailbud embryo can successfully regrow a functional eye that is morphologically indistinguishable from an age-matched control eye. In this study, we assessed the temporal regulation of retinal differentiation and patterning restoration during eye regrowth. Our findings showed that during regrowth, cellular patterning and retinal layer formation was delayed by approximately 1 day but was restored by 3 days when compared to eye development. An assessment of the differentiation of ganglion cells, photoreceptor cells, and Müller glia indicated that the retinal birth order generated during regrowth was consistent with that observed for eye development. Thus, retina differentiation and patterning during regrowth is similar to endogenous eye development. We used this eye regrowth model to assess the role of known mechanisms in development versus regrowth. Loss-of-function studies showed that Pax6 was required for both eye development and regrowth whereas apoptosis was only required for regrowth. Together, these results revealed that the mechanisms required for both development and regrowth can be distinguished from regrowth-specific ones. Our study highlights this developmental model of eye regrowth as a robust platform to systematically and efficiently define the molecular mechanisms that are required for regeneration versus development.
Project description:Gaze stabilization relies on bilateral mirror-symmetric vestibular endorgans, central circuits, and extraocular motor effectors. Embryonic removal of one inner ear before the formation of these structures was used to evaluate the extent to which motor outputs in the presence of a singular inner ear can develop. Near-congenital one-eared tadpoles subjected to separate or combinatorial visuo-vestibular motion stimulation exhibited comparable eye movements, though smaller in gain to controls, whereas isolated visuo-motor responses were unaltered. Surprisingly, vestibulo-ocular reflexes were robust during off-direction motion toward the missing ear in most cases and often attenuated during on-direction motion. This bidirectional plasticity of signal encoding appears to occur at the expense of vestibular reflexes during motion in the normally preferential activation direction of the singular ear. Consequently, formation of central vestibulo-motor circuits in one-eared animals likely relies on multi-neuronal homeostatic strategies, including enhanced afferent fiber activity in the attempt to adjust bilateral sensorimotor transformations.
Project description:We have isolated the Xenopus orthologue of the T-box gene, Tbx20, and characterized its developmental expression profile. We show that Tbx20 is one of the earliest markers of heart tissue in Xenopus, and is expressed throughout all cardiac tissue during later stages of development. In addition, we also observe expression in the cement gland, the jugular vein, the lung bud, the cloacal aperture, rhombomeres 2, 4, 6 and 8, and in a subset of motor neurons.
Project description:SoxC genes are involved in many developmental processes such as cardiac, lymphoid, and bone development. The SoxC gene family is represented by Sox4, Sox11, and Sox12. Loss of either Sox4 or Sox11 function is lethal during mouse embryogenesis. Here, we demonstrate that sox4 and sox11 are strongly expressed in the developing eye, heart as well as brain in Xenopus laevis. Morpholino oligonucleotide mediated knock-down approaches in anterior neural tissue revealed that interference with either Sox4 or Sox11 function affects eye development. A detailed analysis demonstrated strong effects on eye size and retinal lamination. Neural induction was unaffected upon Sox4 or Sox11 MO injection and early eye field differentiation and cell proliferation were only mildly affected. Depletion of both genes, however, led independently to a significant increase in cell apoptosis in the eye. In summary, Sox4 and Sox11 are required for Xenopus visual system development.
Project description:SUMO proteins are small ubiquitin-related modifiers. All SUMOs are synthesized as propeptides that are post-translationally cleaved prior to conjugation. After processing, SUMOs become covalently conjugated to cellular targets through a pathway that is similar to ubiquitination. Ubiquitin like protein proteases/Sentrin specific proteases (Ulp/SENPs) mediate both processing and deconjugation of SUMOs. The action of Ulp/SENPs makes SUMOylation a highly dynamic post-translational modification. To investigate how Ulp/SENPs are regulated in a developmental context, we isolated and characterized all Ulp/SENPs in Xenopus laevis. Xenopus possess homologues of mammalian SENP3, 5, 6 and 7. All of these enzymes reacted with HA-tagged vinyl sulfone derivatives of SUMO-2 (HA-SU2-VS) but not SUMO-1 (HA-SU1-VS), suggesting that they act primarily on SUMO-2 and -3. In contrast, Xenopus possess a single member of the SENP1/SENP2 subfamily of Ulp/SENPs, most closely related to mammalian SENP1. Xenopus SENP1 reacted with HA-SU1-VS and HA-SU2-VS, suggesting that it acts on all SUMO paralogues. We analyzed the mRNA and protein levels for each of the Ulp/SENPs through development; we found that they show distinct patterns of expression that may involve both transcriptional and post-transcriptional regulation. Finally, we have characterized the developmental function of the most abundant Ulp/SENP found within Xenopus eggs, SENP3. Depletion of SENP3 using morpholino antisense oligonucleotides (morpholinos) caused accumulation of high molecular weight SUMO-2/3 conjugated species, defects in developing embryos and changes in the expression of some genes regulated by the transforming growth factor beta (TGF-beta) pathway. These findings collectively indicate that SUMO proteases are both highly regulated and essential for normal development.
Project description:Uncovering the molecular mechanisms of eye development is crucial for understanding the embryonic morphogenesis of complex structures, as well as for the establishment of novel biomedical approaches to address birth defects and injuries of the visual system. Here, we characterize change in transmembrane voltage potential (V(mem)) as a novel biophysical signal for eye induction in Xenopus laevis. During normal embryogenesis, a striking hyperpolarization demarcates a specific cluster of cells in the anterior neural field. Depolarizing the dorsal lineages in which these cells reside results in malformed eyes. Manipulating V(mem) of non-eye cells induces well-formed ectopic eyes that are morphologically and histologically similar to endogenous eyes. Remarkably, such ectopic eyes can be induced far outside the anterior neural field. A Ca(2+) channel-dependent pathway transduces the V(mem) signal and regulates patterning of eye field transcription factors. These data reveal a new, instructive role for membrane voltage during embryogenesis and demonstrate that V(mem) is a crucial upstream signal in eye development. Learning to control bioelectric initiators of organogenesis offers significant insight into birth defects that affect the eye and might have significant implications for regenerative approaches to ocular diseases.
Project description:It has been suggested that Jmjd6 plays an important role in gene regulation through its demethylation or hydroxylation activity on histone and transcription factors. In addition, Jmjd6 has been shown to regulate RNA splicing by interaction with splicing factors. In this study, we demonstrated that Jmjd6a is expressed in developing Xenopus laevis eye during optic vesicle formation and retinal layer differentiation stages. Knockdown of Jmjd6a by an antisense morpholino resulted in eye malformation including a deformed retinal layer and no lens formation. We further found down-regulation of gene expression related to eye development such as Rx1, Otx2, and Pax6 in Jmjd6a morpholino injected embryos. Jmjd6 interacts with splicing factor U2AF25 and GSK3β RNA in the anterior region of Xenopus embryos. Knockdown of Jmjd6a led to deletion of GSK3β RNA exon 1 and 2, which resulted in generation of N'-terminal truncated GSK3β protein. This event further caused decreased phosphorylation of β-catenin and subsequently increased β-catenin stability. Therefore, our result may suggest that Jmjd6a plays an important role in Xenopus eye development through regulation of GSK3β RNA splicing and canonical Wnt/β-catenin signaling.
Project description:Retinal progenitor cells (RPCs) are a multipotent and highly proliferative population that give rise to all retinal cell types during organogenesis. Defining their molecular signature is a key step towards identifying suitable approaches to treat visual impairments. Here, we performed RNA sequencing of whole eyes from Xenopus at three embryonic stages and used differential expression analysis to define the transcriptomic profiles of optic tissues containing proliferating and differentiating RPCs during retinogenesis. Gene Ontology and KEGG pathway analyses showed that genes associated with developmental pathways (including Wnt and Hedgehog signaling) were upregulated during the period of active RPC proliferation in early retinal development (Nieuwkoop Faber st. 24 and 27). Developing eyes had dynamic expression profiles and shifted to enrichment for metabolic processes and phototransduction during RPC progeny specification and differentiation (st. 35). Furthermore, conserved adult eye regeneration genes were also expressed during early retinal development, including sox2, pax6, nrl, and Notch signaling components. The eye transcriptomic profiles presented here span RPC proliferation to retinogenesis and include regrowth-competent stages. Thus, our dataset provides a rich resource to uncover molecular regulators of RPC activity and will allow future studies to address regulators of RPC proliferation during eye repair and regrowth.
Project description:Thyroid hormone receptors generally activate transcription of target genes in the presence of thyroid hormone (T(3)) and repress their transcription in its absence. Here, we investigated the role of unliganded thyroid hormone receptor (TR) during vertebrate development using an amphibian model. Previous studies led to the hypothesis that before production of endogenous T(3), the presence of unliganded receptor is essential for premetamorphic tadpole growth. To test this hypothesis, we generated a Xenopus laevis TR beta mutant construct ineffective for gene repression owing to impaired corepressor NCoR recruitment. Overexpression by germinal transgenesis of the mutant receptor leads to lethality during early development with numerous defects in cranio-facial and eye development. These effects correlate with TR expression profiles at these early stages. Molecular analysis of transgenic mutants reveals perturbed expression of genes involved in eye development. Finally, treatment with iopanoic acid or NH-3, modulators of thyroid hormone action, leads to abnormal eye development. In conclusion, the data reveal a role of unliganded TR in eye development.
Project description:Interest in cortical excitability-the ability of the cell cortex to generate traveling waves of protein activity-has grown considerably over the past 20 years. Attributing biological functions to cortical excitability requires an understanding of the natural behavior of excitable waves and the ability to accurately quantify wave properties. Here we have investigated and quantified the onset of cortical excitability in Xenopus laevis eggs and embryos and the changes in cortical excitability throughout early development. We found that cortical excitability begins to manifest shortly after egg activation. Further, we identified a close relationship between wave properties-such as wave frequency and amplitude-and cell cycle progression as well as cell size. Finally, we identified quantitative differences between cortical excitability in the cleavage furrow relative to nonfurrow cortical excitability and showed that these wave regimes are mutually exclusive.