Frog Oocytes Research Articles

Wnt-5a/Ca2+ pathway modulates endogenous current and oocyte structure of Xenopus laevis

Wnt signaling plays an essential role in cellular processes like development, maturation, and function maintenance. Xenopus laevis oocytes are a suitable model to study not only the development but also the function of different receptors expressed in their membranes, like those receptors expressed in the central nervous system (CNS) including Frizzled 7. Here, using frog oocytes and recordings of endogenous membrane currents in a two-electrode path configuration along with morphological observations, we evaluated the role of the non-canonical Wnt-5a ligand in oocytes. We found that acute application of Wnt-5a generated changes in endogenous calcium-dependent currents, entry oscillatory current, the membrane's outward current, and induced membrane depolarization. The incubation of oocytes with Wnt-5a caused a reduction of the membrane potential, potassium outward current, and protected the ATP current in the epithelium/theca removed (ETR) model. The oocytes exposed to Wnt-5a showed increased viability and an increase in the percentage of the germinal vesicle breakdown (GVBD), at a higher level than the control with progesterone. Altogether, our results suggest that Wnt-5a modulates different aspects of oocyte structure and generates calcium-dependent endogenous current alteration and GVDB process with a change in membrane potential at different concentrations and times of the exposition. These results help to understand the cellular effect of Wnt-5a and present the use of Xenopus oocytes to explore the mechanism that could impact the activation of Wnt signaling.

An aquaporin and an aquaglyceroporin have roles in low temperature adaptation of mosquitoes (Anopheles sinensis).

Mosquitoes (Anopheles sinensis), widely geographically distributed in Asia including China, are the primary vector of the malaria parasite Plasmodium vivax and other parasitic diseases such as Malayan filariasis. An. sinensis can survive through low winter temperatures. Aquaporin channels are found in all life forms, where they facilitate environmental adaptation by allowing rapid trans-cellular movement of water (classical aquaporins) or water and solutes such as glycerol (aquaglyceroporins). Here, we identified and characterized 2 aquaporin (AQP) homologs in An. sinensis: AsAQP2 (An. sinensis aquaglyceroporin) and AsAQP4 (An. sinensis aquaporin). When expressed in frog (Xenopus laevis) oocytes, AsAQP2 transported water, glycerol, and urea; AsAQP4 transported only water. Water permeation through AsAQP2 and AsAQP4 was inhibited by mercuric chloride. AsAQP2 expression was slightly higher in adult female mosquitoes than in males, and AsAQP4 expression was significantly higher in adult males. The 2 AsAQPs were highly expressed in Malpighian tubules and midgut. AsAQP2 and AsAQP4 expression was up-regulated by blood feeding compared with sugar feeding. At freezing point (0°C), the AsAQP4 expression level increased and An. sinensis survival time reduced compared with those at normal temperature (26°C). At low temperature (8°C), the AsAQP2 and AsAQP4 expression levels decreased and survival time was significantly longer compared with those at 26°C. These results suggest that AsAQP2 and AsAQP4 have roles in water homeostasis during blood digestion and in low temperature adaptation of A. sinensis. Together, our results show that the 2 AQPs are important for mosquito diuresis after blood feeding and when exposed to low temperatures.

Control of poly(A)-tail length and translation in vertebrate oocytes and early embryos

During oocyte maturation and early embryogenesis, changes in mRNA poly(A)-tail lengths strongly influence translation, but how these tail-length changes are orchestrated has been unclear. Here, we performed tail-length and translational profiling of mRNA reporter libraries (each with millions of 3' UTR sequence variants) in frog oocytes and embryos and in fish embryos. Contrasting to previously proposed cytoplasmic polyadenylation elements (CPEs), we found that a shorter element, UUUUA, together with the polyadenylation signal (PAS), specify cytoplasmic polyadenylation, and we identified contextual features that modulate the activity of both elements. In maturing oocytes, this tail lengthening occurs against a backdrop of global deadenylation and the action of C-rich elements that specify tail-length-independent translational repression. In embryos, cytoplasmic polyadenylation becomes more permissive, and additional elements specify waves of stage-specific deadenylation. Together, these findings largely explain the complex tapestry of tail-length changes observed in early frog and fish development, with strong evidence of conservation in both mice and humans.

Parameter Identifiability in PDE Models of Fluorescence Recovery After Photobleaching.

Identifying unique parameters for mathematical models describing biological data can be challenging and often impossible. Parameter identifiability for partial differential equations models in cell biology is especially difficult given that many established in vivo measurements of protein dynamics average out the spatial dimensions. Here, we are motivated by recent experiments on the binding dynamics of the RNA-binding protein PTBP3 in RNP granules of frog oocytes based on fluorescence recovery after photobleaching (FRAP) measurements. FRAP is a widely-used experimental technique for probing protein dynamics in living cells, and is often modeled using simple reaction-diffusion models of the protein dynamics. We show that current methods of structural and practical parameter identifiability provide limited insights into identifiability of kinetic parameters for these PDE models and spatially-averaged FRAP data. We thus propose a pipeline for assessing parameter identifiability and for learning parameter combinations based on re-parametrization and profile likelihoods analysis. We show that this method is able to recover parameter combinations for synthetic FRAP datasets and investigate its application to real experimental data.

Deep transcriptome profiling reveals limited conservation of A-to-I RNA editing in Xenopus

BackgroundXenopus has served as a valuable model system for biomedical research over the past decades. Notably, ADAR was first detected in frog oocytes and embryos as an activity that unwinds RNA duplexes. However, the scope of A-to-I RNA editing by the ADAR enzymes in Xenopus remains underexplored.ResultsHere, we identify millions of editing events in Xenopus with high accuracy and systematically map the editome across developmental stages, adult organs, and species. We report diverse spatiotemporal patterns of editing with deamination activity highest in early embryogenesis before zygotic genome activation and in the ovary. Strikingly, editing events are poorly conserved across different Xenopus species. Even sites that are detected in both X. laevis and X. tropicalis show largely divergent editing levels or developmental profiles. In protein-coding regions, only a small subset of sites that are found mostly in the brain are well conserved between frogs and mammals.ConclusionsCollectively, our work provides fresh insights into ADAR activity in vertebrates and suggest that species-specific editing may play a role in each animal’s unique physiology or environmental adaptation.

SWEET11b transports both sugar and cytokinin in developing barley grains.

Even though Sugars Will Eventually be Exported Transporters (SWEETs) have been found in every sequenced plant genome, a comprehensive understanding of their functionality is lacking. In this study, we focused on the SWEET family of barley (Hordeum vulgare). A radiotracer assay revealed that expressing HvSWEET11b in African clawed frog (Xenopus laevis) oocytes facilitated the bidirectional transfer of not only just sucrose and glucose, but also cytokinin. Barley plants harboring a loss-of-function mutation of HvSWEET11b could not set viable grains, while the distribution of sucrose and cytokinin was altered in developing grains of plants in which the gene was knocked down. Sucrose allocation within transgenic grains was disrupted, which is consistent with the changes to the cytokinin gradient across grains, as visualized by magnetic resonance imaging and Fourier transform infrared spectroscopy microimaging. Decreasing HvSWEET11b expression in developing grains reduced overall grain size, sink strength, the number of endopolyploid endosperm cells, and the contents of starch and protein. The control exerted by HvSWEET11b over sugars and cytokinins likely predetermines their synergy, resulting in adjustments to the grain's biochemistry and transcriptome.

Embryonic poly(A)-binding protein interacts with translation-related proteins and undergoes phosphorylation on the serine, threonine, and tyrosine residues in the mouse oocytes and early embryos.

Expression of the embryonic poly(A)-binding protein (EPAB) in frog, mouse, and human oocytes and early-stage embryos is maintained at high levels until embryonic genome activation (EGA) after which a significant decrease occurs in EPAB levels. Studies on the vertebrate oocytes and early embryos revealed that EPAB plays key roles in the translational regulation, stabilization, and protection of maternal mRNAs during oocyte maturation and early embryogenesis. However, it remains elusive whether EPAB interacts with other cellular proteins and undergoes phosphorylation to perform these roles. For this purpose, we identified a group of Epab-interacting proteins and its phosphorylation status in mouse germinal vesicle (GV)- and metaphase II (MII)-stage oocytes, and in 1-cell, 2-cell, and 4-cell preimplantation embryos. In the oocytes and early preimplantation embryos, Epab-interacting proteins were found to play roles in the translation and transcription processes, intracellular signaling and transport, maintenance of structural integrity, metabolism, posttranslational modifications, and chromatin remodeling. Moreover, we discovered that Epab undergoes phosphorylation on the serine, threonine, and tyrosine residues, which are localized in the RNA recognition motifs 2, 3, and 4 or C-terminal. Conclusively, these findings suggest that Epab not only functions in the translational control of maternal mRNAs through binding to their poly(A) tails but also participates in various cellular events through interacting with certain group proteins. Most likely, Epab undergoes a dynamic phosphorylation during the oocyte maturation and the early embryo development to carry out these functions.

Cell death in isolated Xenopus laevis (African clawed frog) oocytes is a temperature-dependent process.

After isolation of Xenopus laevis oocytes from adult frogs, the cells deteriorate over a few days in vitro under incubation conditions proposed by most protocols. As we have shown in our previous study, changes in storage conditions significantly increase viability, mainly due to cooling to 5-8 °C. Aim of this study was to elucidate the influence of apoptosis on deterioration during aging. Batches of defolliculated stage V-VI oocytes were stored in salt solutions (MBS-based) with or without apoptosis and maturation inhibitors (metamizole, theophylline, ibuprofen, fenbufen) and an additional energy source (glucose-6-phosphate) either cooled (5 or 8 °C) or at 18 °C. Throughout the storage period of up to 4 weeks, oocytes from each condition were injected at different days post-isolation with RNA for expressing GABA receptors (Drosophila RDL) or potassium channels (human KCNQ1). After 2‑3 days expression at 18°C, oocytes were optically checked for deterioration and current responses recorded by the two-electrode voltage clamp technique. No significant influence was found for solutions containing inhibitors, survival times being 16.1 ± 1.2 days on average. Viability differed between batches in cooled oocytes, from 7-25 days of incubation, with a declining number of viable oocytes during a run. Caspase-3 activity was increased significantly in uncooled oocytes over a few days, but the active protein remains to be detected via Western blotting. Our results indicate a temperature-dependent mechanism involved in aging-related deterioration that may differ from apoptosis.

Decreased heat sensitivity of lungfish TRPV1 revealed by the heterologous expression system

In this study, we focused on TRPV1 of African lungfish, Protopterus annectens. During drought at high temperature, African lungfish can survive by undergoing into aestivation in mud cocoons. Therefore, lungfish is considered to have some specialized heat-sensor, TRPV1, for heat tolerance. Further, lungfish which shares similarities with fishes and amphibians, is one of important species for investigating the fish-tetrapod transition. Since fish TRPV1 and tetrapod TRPV1 have some differences, character of lungfish TRPV1 attracts attention. Here, we first cloned TRPV1 paralogue from lungfish, Protopterus annectens (lfTRPV1) and determined the chemical and thermal sensitivities of lfTRPV1 by two-electrode voltage clamp method using frog oocytes. We detected activation of lfTRPV1 by acid and 2-APB, but capsaicin-induced activation was not observed. The sensitivity to acid of lfTRPV1 was similar to that of rat TRPV1 (rTRPV1), but the 2-APB sensitivity of lfTRPV1 was relatively weaker than rTRPV1. Heat stimulation up to 44 °C did not activate lfTRPV1 and the heat-activation was not detected even on acid condition of pH6. This dramatically decreased heat-sensitivity of TRPV1 may contribute the heat tolerance of African lungfish. Moreover, this might be the property of ancient tetrapod-type TRPV1 gene.

Cyclin B3 implements timely vertebrate oocyte arrest for fertilization.

To ensure successful offspring ploidy, vertebrate oocytes must halt the cell cycle in meiosis II until sperm entry. Emi2 is essential to keep oocytes arrested until fertilization. However, how this arrest is implemented exclusively in meiosis II and not prematurely in meiosis I has until now remained enigmatic. Using mouse and frog oocytes, we show here that cyclin B3, an understudied B-type cyclin, is essential to keep Emi2 levels low in meiosis I. Direct phosphorylation of Emi2 at an evolutionarily highly conserved site by Cdk1/cyclin B3 targets Emi2 for degradation. In contrast, Cdk1/cyclin B1 is inefficient in Emi2 phosphorylation, and this provides a molecular explanation for the requirement of different B-type cyclins for oocyte maturation. Cyclin B3 degradation at exit from meiosis I enables Emi2 accumulation and thus timely arrest in meiosis II. Our findings illuminate the evolutionarily conserved mechanisms that control oocyte arrest for fertilization at the correct cell-cycle stage, which is essential for embryo viability.

Michel Bornens (1938–2022), a visionary scientist

Michel Bornens (1938–2022), a visionary scientist

Dynamic surface patterns on cells.

Dynamic pattern formations are commonly observed in multicellular systems, such as cardiac tissue and slime molds, and modeled using reaction-diffusion systems. Recent experiments have revealed dynamic patterns in the concentration profile of various cortical proteins at a much smaller scale, namely, embryos at their single-cell stage. Spiral waves of Rho and F-actin proteins have been reported in Xenopus frog and starfish oocytes [Bement et al., Nat. Cell Biol. 17, 1471 (2015)], while a pulsatile pattern of Rho and myosin proteins has been found in C. elegans embryo [Nishikawa et al., eLife 6, e30537 (2017)]. Here, we propose that these two seemingly distinct dynamic patterns are signatures of a single reaction-diffusion network involving active-Rho, inactive-Rho, actin, and myosin. We show that a small variation in the concentration of other ancillary proteins can give rise to different dynamical states from the same chemical network.

Anesthetic Pharmacology of the Mint Extracts L-Carvone and Methyl Salicylate

Introduction: Hydrocarbons with sufficient water solubility allosterically modulate anesthetic-sensitive ion channels. Mint extracts L-carvone and methyl salicylate water solubility exceeds modulation cutoff values for γ-amino butyric acid type A (GABAA) receptors, N-methyl-D-aspartate (NMDA) receptors, and type-2 voltage-gated sodium (Nav1.2) channels. We hypothesized that mint extracts modulate these channels at concentrations that anesthetize rats. Methods: Channels were expressed separately in frog oocytes and studied using 2-electrode voltage clamp techniques at drug concentrations up to 10 mM. Normalized current effects were fit to Hill equations. Mint compounds were formulated in a lipid emulsion and administered IV to rats. When unresponsive to the tail clamp, rats were exsanguinated, and plasma drug concentrations were measured. Results: Both mint compounds caused concentration-dependent inhibition of all channels except for methyl salicylate which inhibited GABAA receptors at low concentrations and potentiated at high concentrations. Plasma drug concentrations in anesthetized rats were 7.9 mM for L-carvone and 2.7 mM for methyl salicylate. This corresponded to ≥53% NMDA receptor inhibition and ≥78% Nav1.2 channel inhibition by both compounds and 30% potentiation of GABAA receptors by methyl salicylate. Conclusion: L-Carvone and methyl salicylate allosterically modulate cell receptor targets important to molecular actions of conventional anesthetics at concentrations that also induce general anesthesia in rats.

Sperm associated antigen 7 is activated by T3 during Xenopus tropicalis metamorphosis via a thyroid hormone response element within the first intron.

Thyroid hormone (T3) affects many diverse physiological processes such as metabolism, organogenesis, and growth. The two highly related frog species, diploid Xenopus tropicalis and pseudo tetraploid Xenopus laevis, have been used as models for analyzing the effects of T3 during vertebrate development. T3 regulates T3-inducible gene transcription through T3 receptor (TR)-binding to T3-response elements (TREs). We have previously identified sperm associated antigen 7 (spag7) as a candidate T3 target gene that is potentially involved in adult stem cell development and/or proliferation during intestinal metamorphosis. To investigate whether T3 regulates spag7 directly at the transcriptional level via TR, we first conducted qRT-PCR to analyze its expression during natural and T3-induced metamorphosis and found that spag7 was up-regulated during natural metamorphosis in the intestine, tail, brain and hindlimb, peaking at the climax of metamorphosis in all those organs, and upon T3 treatment of premetamorphic tadpoles. Next, we demonstrated that an intronic TRE in spag7, first identified through bioinformatic analysis, could bind to TR in vitro and in vivo during metamorphosis. A dual luciferase assay utilizing a reconstituted frog oocyte transcription system showed that the TRE could mediate promoter activation by liganded TR. These results indicate that spag7 expression is directly regulated by T3 through the TRE in the first intron during metamorphosis, implicating a role for spag7 early during T3-regulated tissue remodeling and resorption.

Multiscale, multi-perspective imaging assisted robotic microinjection of 3D biological structures.

Microinjection is a widely used technique employed by biologists with applications in transgenesis, cryopreservation, mutagenesis, labeling/dye injection and in-vitro fertilization. However, microinjection is an extremely laborious manual procedure, which makes it a critical bottleneck in the field and thus ripe for automation. Here, we present a computer-guided robot that automates the targeted microinjection of Drosophila melanogaster and zebrafish (Danio rerio) embryos, two important model organisms in biological research. The robot uses a series of cameras to image an agar plate containing embryos at multiple magnifications and perspectives. This imaging is combined with machine learning and computer vision algorithms to pinpoint a location on the embryo for targeted microinjection with microscale precision. We demonstrate the utility of this microinjection robot to successfully microinject Drosophila melanogaster and zebrafish embryos. Results obtained indicate that the robotic microinjection approach can significantly increase the throughput of microinjection as compared to manual microinjection while maintaining survival rates comparable to human operators. In the future, this robotic platform can be used to perform high throughput microinjection experiments and can be extended to automatically microinject a host of organisms such as roundworms (Caenorhabditis elegans), mosquito (Culicidae) embryos, sea urchins (Echinoidea) and frog (Xenopus) oocytes.

Rice aquaporin OsPIP2;2 is a water-transporting facilitator in relevance to drought-tolerant responses.

In rice (Oryza sativa), the PLASMA MEMBRANE INTRINSIC PROTEIN (PIP) family of aquaporin has 11 members, OsPIP1;1 to OsPIP1;3, and OsPIP2;1 to OsPIP2;8, which are hypothesized to facilitate the transport of H2O and other small compounds across cell membranes. To date, however, only OsPIP1;2, OsPIP2;1, and OsPIP2;4 have been demonstrated for substrate selectivity in their source plant (rice). In this study, OsPIP2;2 was characterized as the most efficient facilitator of H2O transport across cell membranes in comparison with the other 10 OsPIPs. In concomitant tests of all OsPIPs, four genes (OsPIP1;3, OsPIP2;1, OsPIP2;2, and OsPIP2;4) were induced to express in leaves of rice plants following a physiological drought stress, while OsPIP2;2 was expressed to the highest level. After de novo expression in frog oocytes and yeast cells, the four OsPIP proteins were localized to the plasma membranes in trimer and tetramer and displayed the activity to increase the membrane permeability to H2O. In comparison, OsPIP2;2 was most supportive to H2O import to oocytes and yeast cells. After de novo expression in tobacco protoplasts, OsPIP2;2 exceeded OsPIP1;3, OsPIP2;1, and OsPIP2;4 to support H2O transport across the plasma membranes. OsPIP2;2‐mediated H2O transport was accompanied by drought‐tolerant responses, including increases in concentrations of proline and polyamines, both of which are physiological markers of drought tolerance. In rice protoplasts, H2O transport and drought‐tolerant responses, which included expression of marker genes of drought tolerance pathway, were considerably enhanced by OsPIP2;2 overexpression but strongly inhibited by the gene silencing. Furthermore, OsPIP2;2 played a role in maintenance of the cell membrane integrity and effectively protected rice cells from electrolyte leakage caused by the physiological drought stress. These results suggest that OsPIP2;2 is a predominant facilitator of H2O transport in relevance to drought tolerance in the plant.

Modulation of Intracellular ROS and Senescence-Associated Phenotypes of Xenopus Oocytes and Eggs by Selective Antioxidants.

Aging of oocytes and eggs diminishes their reproductive and developmental potential. It has been demonstrated previously that reactive oxygen species (ROS) contribute to accelerated aging of various cells. In the present study, we measured intracellular levels of ROS and investigated effects of several selective antioxidants (AOXs) on the viability and functional activity of aging oocytes and eggs of the African clawed frog Xenopus laevis. The fluorescent cell-permeable dye DCFDA, which is widely employed for ROS detection in cultured mammalian cells, was used to monitor ROS levels in the fresh and bench-aged oocytes and eggs by an optimized protocol. It was found that intracellular ROS contents were increased in frog oocytes and eggs aged for 48 h. It was further demonstrated using selective cell-permeable AOXs targeting different ROS-generating mechanisms, that the major source of ROS in Xenopus oocytes and eggs is the plasma membrane NADPH oxidase, and that mitochondrial generation contributes to the intracellular ROS content to a lesser extent. Targeted inhibition of NADPH oxidase with a natural organic compound apocynin reduced ROS levels significantly in Xenopus oocytes and eggs, maintained their normal phenotype and supported their functional competence. To our knowledge this is the first report concerning beneficial effects of apocynin on the isolated gamete cells, such as oocytes and eggs.

YSL3-mediated copper distribution is required for fertility, seed size and protein accumulation in Brachypodium

Addressing the looming global food security crisis requires the development of high-yielding crops. In agricultural soils, deficiency in the micronutrient copper significantly decreases grain yield in wheat (Triticum aestivum), a globally important crop. In cereals, grain yield is determined by inflorescence architecture, flower fertility, grain size, and weight. Whether copper is involved in these processes, and how it is delivered to the reproductive organs is not well understood. We show that copper deficiency alters not only the grain set but also flower development in both wheat and its recognized model, Brachypodium distachyon. We then show that the Brachypodium yellow stripe-like 3 (YSL3) transporter localizes to the phloem, transports copper in frog (Xenopus laevis) oocytes, and facilitates copper delivery to reproductive organs and grains. Failure to deliver copper, but not iron, zinc, or manganese to these structures in the ysl3 CRISPR-Cas9 mutant results in delayed flowering, altered inflorescence architecture, reduced floret fertility, grain size, weight, and protein accumulation. These defects are rescued by copper supplementation and are complemented by YSL3 cDNA. This knowledge will help to devise sustainable approaches for improving grain yield in regions where soil quality is a major obstacle for crop production. Copper distribution by a phloem-localized transporter is essential for the transition to flowering, inflorescence architecture, floret fertility, size, weight, and protein accumulation in seeds.

‘Proximity frequencies’ a new parameter to evaluate the profile of GABAAR modulators

We reported the synthesis of new 8-methoxypyrazolo[1,5-a]quinazolines bearing an amide fragment at the 3-position. The final compounds, as aromatic (2a-i) and 4,5-dihydro derivatives (3a-i), have been evaluated in vitrofor their ability to modulate the chlorine current on recombinant GABAA receptors of the α1β2γ2L type (expressed in frog oocytes of the Xenopus laevis species). From electrophysiological test two groups of compounds emerged: positive modulators agonist (2e, h, i and 3e, h) and null modulators antagonist (2a, b, d, f, g and 3a-d, f, g) of GABAA subtype receptor. Using a set of compounds (new derivatives, known products and GABAA subtype receptor ligands from our library) we identify the amino acids at the α+/γ− interface, which could be involved in the agonist or antagonist profile, using the ‘Proximity Frequencies’, namely the frequencies with which a ligand intercepts two or more binding-site amino acids during the molecular dynamic simulation. The linear discriminant analysis (LDA) evidences that the combination of amino acids αVAL203- γTHR142 and αTYR 160- γTYR 58 allowed to collocate 70.6% of agonists and 72.7% of antagonists in their respective class.

Thyroid hormone directly activates mitochondrial fission process 1 (Mtfp1) gene transcription during adult intestinal stem cell development and proliferation in Xenopus tropicalis

Thyroid hormone (T3) regulates vertebrate development via T3 receptors (TRs). T3 level peaks during postembryonic development, a period around birth in mammals or metamorphosis in anurans. Anuran metamorphosis offers many advantages for studying T3 and TR function in vivo largely because of its total dependent on T3 and the dramatic changes affecting essentially all organs/tissues that can be easily manipulated. Earlier studies have shown that TRs are both necessary and sufficient for mediating the metamorphic effects of T3. Many candidate TR target genes have been identified during Xenopus tropicalis intestinal metamorphosis, a process that involves apoptotic degeneration of most of the larval epithelial cells and de novo development of adult epithelial stem cells. Among these putative TR target genes is mitochondrial fission process 1 (Mtfp1), a nuclear-encoded mitochondrial gene. Here, we report that Mtfp1gene expression peaks in the intestine during both natural and T3-induced metamorphosis when adult epithelial stem cell development and proliferation take place. Furthermore, we show that Mtfp1 contains a T3-response element within the first intron that is bound by TR to mediate T3-induced local histone H3K79 methylation and RNA polymerase recruitment in the intestine during metamorphosis. Additionally, we demonstrate that the Mtfp1 promoter can be activated by T3 in a reconstituted frog oocyte system in vivo and that this activation is dependent on the intronic TRE. These findings suggest that T3 activates Mtfp1 gene directly via the intronic TRE and that Mtfp1 in turn facilitate adult intestinal stem cell development/proliferation by affecting mitochondrial fission process.