Eye Regeneration Research Articles

Map3k1 suppresses terminal differentiation of migratory eye progenitors in planarian regeneration.

Proper stem cell targeting and differentiation is necessary for regeneration to succeed. In organisms capable of whole body regeneration, considerable progress has been made identifying wound signals initiating this process, but the mechanisms that control the differentiation of progenitors into mature organs are not fully understood. Using the planarian as a model system, we identify a novel function for map3k1, a MAP3K family member possessing both kinase and ubiquitin ligase domains, to negatively regulate terminal differentiation of stem cells during eye regeneration. Inhibition of map3k1 caused the formation of multiple ectopic eyes within the head, but without controlling overall head, brain, or body patterning. By contrast, other known regulators of planarian eye patterning like WntA and notum also regulate head regionalization, suggesting map3k1 acts distinctly. Eye resection and regeneration experiments suggest that unlike Wnt signaling perturbation, map3k1 inhibition did not shift the target destination of eye formation in the animal. Instead, map3k1(RNAi) ectopic eyes emerge in the regions normally occupied by migratory eye progenitors, and the onset of ectopic eyes after map3k1 inhibition coincides with a reduction to eye progenitor numbers. Furthermore, RNAi dosing experiments indicate that progenitors closer to their normal target are relatively more sensitive to the effects of map3k1, implicating this factors in controlling the site of terminal differentiation. Eye phenotypes were also observed after inhibition of map2k4, map2k7, jnk, and p38, identifying a putative pathway through which map3k1 prevents differentiation. Together, these results suggest that map3k1 regulates a novel control point in the eye regeneration pathway which suppresses the terminal differentiation of progenitors during their migration to target destinations.

Temporal Transcriptomic Profiling of the Developing Xenopus laevis Eye.

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.

Morphometric analysis and functional insights into the serotonergic system of Girardia tigrina (Tricladida, Platyhelminthes).

Using immunocytochemistry, serotonergic nerve elements were documented in the nervous system of the planarian Girardia tigrina. Serotonin-immunopositive components were observed in the brain, ventral, dorsal and longitudinal nerve cords, transverse nerve commissures connecting the nerve cords, and in the nerve plexus. Whole-mount preparations of G. tigrina were analyzed by fluorescent and confocal laser scanning microscopy. An essential quantitative morphometric measurement of serotonin-immunopositive structures was conducted in three body regions (anterior, middle, and posterior) of the planarian. The number of serotonin neurons was maximal in the head region. The ventral nerve cords gradually decreased in thickness from anterior to posterior body ends. Physiological action of exogenously applied serotonin was studied in G. tigrina for the first time. It was found that serotonin (0.1 and 1 µmol L-1) accelerated eye regeneration. The transcriptome sequencing performed for the first time for the planarian G. tigrina revealed the transcripts of the tryptophan hydroxylase (trph), amino acid decarboxylase (aadc) and serotonin transporter (sert) genes. The data obtained indicate the presence of the components of serotonin pathway in G. tigrina. The identified transcripts can take part in serotonin turnover and participate in the realization of biological effects of serotonin in planarians, associated with eyes regeneration and differentiation.

Temporal Transcriptomic Profiling of the Developing Xenopus laevis Eye.

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 included 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.

New characterization and safety evaluation of human limbal stem cells used in clinical application: fidelity of mitotic process and mitotic spindle morphologies

BackgroundLimbal stem cells (LSCs) are crucial for the regeneration of the corneal epithelium in patients with limbal stem cell deficiency (LSCD). Thus, LSCs during cultivation in vitro should be in highly homogeneous amounts, while potency and expression of stemness without tumorigenesis would be desirable. Therefore, further characterization and safety evaluation of engineered limbal grafts is required to provide safe and high-quality therapeutic applications.MethodsAfter in vitro expansion, LSCs undergo laboratory characterization in a single-cell suspension, cell culture, and in limbal grafts before transplantation. Using a clinically applicable protocol, the data collected on LSCs at passage 1 were summarized, including: identity (cell size, morphology); potency (yield, viability, population doubling time, colony-forming efficiency); expression of putative stem cell markers through flow cytometry, immunofluorescence, and immunohistochemistry. Then, mitotic chromosome stability and normal mitotic outcomes were explored by using live-cell imaging. Finally, impurities, bacterial endotoxins and sterility were determined.ResultsExpression of the stemness marker p63 in single-cell suspension and in cell culture showed high values by different methods. Limbal grafts showed p63-positive cells (78.7 ± 9.4%), Ki67 proliferation (41.7 ± 15.9%), while CK3 was negative. Impurity with 3T3 feeder cells and endotoxins was minimized. We presented mitotic spindles with a length of 11.40 ± 0.54 m and a spindle width of 8.05 ± 0.55 m as new characterization in LSC culture. Additionally, live-cell imaging of LSCs (n = 873) was performed, and only a small fraction < 2.5% of aberrant interphase cells was observed; 2.12 ± 2.10% of mitotic spindles exhibited a multipolar phenotype during metaphase, and 3.84 ± 3.77% of anaphase cells had a DNA signal present within the spindle midzone, indicating a chromosome bridge or lagging chromosome phenotype.ConclusionThis manuscript provides, for the first time, detailed characterization of the parameters of fidelity of the mitotic process and mitotic spindle morphologies of LSCs used in a direct clinical application. Our data show that p63-positive CK3-negative LSCs grown in vitro for clinical purposes undergo mitotic processes with extremely high fidelity, suggesting high karyotype stability. This finding confirms LSCs as a high-quality and safe therapy for eye regeneration in humans.

A Simple Method for Quantifying Blastema Growth in Regenerating Planarians.

Due to their strong regenerative capabilities, freshwater planarians are a well-suited model system for studying the effects of chemicals on stem cell biology and regeneration. After amputation, a planarian will regenerate the missing body parts within 1 to 2 weeks. Because planarians have a distinct head morphology that can be easily identified, head and eye regeneration has been a popular qualitative measure of toxicity. However, qualitative measures can only detect strong defects. Here, we present protocols for quantifying the rate of blastema growth to measure regeneration defects for assessment of chemical toxicity. Following amputation, a regenerative blastema forms at the wound site. Over the course of several days, the blastema grows and subsequently re-forms the missing anatomical structures. This growth can be measured by imaging the regenerating planarian. As the blastema tissue is unpigmented, it can be easily distinguished from the remaining pigmented body using standard image analysis techniques. Basic Protocol 1 provides a step-by-step guide for imaging regenerating planarians over several days of regeneration. Basic Protocol 2 describes the necessary steps for the quantification of blastema size using freeware. It is accompanied by video tutorials to facilitate adaptation. Basic Protocol 3 shows how to calculate the growth rate using linear curve fitting in a spreadsheet. The ease of implementation and low cost make this procedure suitable for an undergraduate laboratory teaching setting, in addition to typical research settings. Although we focus on head regeneration in Dugesia japonica, these protocols are adaptable to other wound sites and planarian species. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Imaging planarians during regeneration Basic Protocol 2: Quantitative analysis of blastema size with ImageJ Basic Protocol 3: Quantification of blastema growth rate.

Djsnon, a downstream gene of Djfoxk1, is required for the regeneration of the planarian central nervous system

Regulators of adult neurogenesis are crucial targets for neuronal repair. Freshwater planarians are ideal model systems for studying neuronal regeneration as they can regenerate their entire central nervous system (CNS) using pluripotent adult stem cells. Here, we identified Djfoxk1 in planarian Dugesia japonica to be required for planarian CNS regeneration. Knockdown of Djfoxk1 inhibits the regeneration of the cephalic ganglia, resulting in the failure of eye regeneration. By RNAi screening of Djfoxk1 downstream genes, we identified Djsnon as another regulator of planarian neuronal regeneration. Inhibition of Djsnon with RNA interference (RNAi) results in similar phenotypes caused by Djfoxk1 RNAi without affecting cell proliferation and wound healing. Our findings show that Djsnon as a downstream gene of Djfoxk1 regulates the regeneration of the planarian CNS.

Src acts with WNT/FGFRL signaling to pattern the planarian anteroposterior axis.

Tissue identity determination is crucial for regeneration, and the planarian anteroposterior (AP) axis uses positional control genes expressed from body wall muscle to determine body regionalization. Canonical Wnt signaling establishes anterior versus posterior pole identities through notum and wnt1 signaling, and two Wnt/FGFRL signaling pathways control head and trunk domains, but their downstream signaling mechanisms are not fully understood. Here, we identify a planarian Src homolog that restricts head and trunk identities to anterior positions. src-1(RNAi) animals formed enlarged brains and ectopic eyes and also duplicated trunk tissue, similar to a combination of Wnt/FGFRL RNAi phenotypes. src-1 was required for establishing territories of positional control gene expression in Schmidtea mediterranea, indicating that it acts at an upstream step in patterning the AP axis. Double RNAi experiments and eye regeneration assays suggest src-1 can act in parallel to at least some Wnt and FGFRL factors. Co-inhibition of src-1 with other posterior-promoting factors led to dramatic patterning changes and a reprogramming of Wnt/FGFRLs into controlling new positional outputs. These results identify src-1 as a factor that promotes robustness of the AP positional system that instructs appropriate regeneration.

MiR-8b is involved in brain and eye regeneration of Dugesia japonica in head regeneration.

ABSTRACTMicroRNAs (miRNAs) are a class of evolutionarily conserved small non-coding RNAs that regulate gene expression at the translation level in cell growth, proliferation and differentiation. In addition, some types of miRNAs have been proven to be key modulators of both CNS development and plasticity, such as let-7, miR-9 and miR-124. In this research, we found miR-8b acts as an important regulator involved in brain and eyespot regeneration in Dugesia japonica. miR-8b was highly conserved among species and was abundantly expressed in central nervous system. Here, we detected the expression dynamics of miR-8b by qPCR during the head regeneration of D. japonica. Knockdown miR-8b by anti-MIRs method caused severe defects of eyes and CNS. Our study revealed the evolutionary conserved role of miR-8b in the planarian regeneration process, and further provided more research ideas and available information for planarian miRNAs.

Neurological and regenerative defects in fresh water planarian exposed to lead

Lead (Pb) is a major heavy metal that augments environmental pollution and is a health risk to living organisms. This study was performed to investigate the effect of lead on characteristics of planarian, Dugesia japonica. Briefly, planarians were cultivated in water containing different concentrations (0–400 mg/L) of Pb, and incubated for varying durations (1, 3, and 5 hr). After termination of the incubation time, motility and seizure-like behaviors (c-like, head-bop, snake-like, and screw-like) of the planarians were counted in fresh water. Results showed that increasing Pb concentration and time resulted in decreased motility of the planarians (p<0.05). Increasing concentrations of Pb also resulted in significant increase in the seizure-like behavioral patterns, in particular the c-like and head-bop behaviors. In order to examine eye regeneration, the head region was cut, and rest of the body was incubated in water in the absence or presence of Pb, until emergence of the eye extrusion. Formation of eye spots was initiated in amputated fragments of the control group (without Pb) on day 4 after incubation, whereas appearance of the eye spots was delayed in planarian exposed to 200 mg/L Pb. Moreover, immunohistochemistry revealed that formation of the optic nerve was delayed in planarians exposed to Pb. Thus, our studies determined that planarians exposed to high concentrations of Pb resulted in decreasing motility and induction of seizure-like behaviors, as well as delayed eye regeneration. Results of the current study therefore validate that exposure to lead has a negative effect on the lifespan of aquatic organisms and can cause disturbance of the nervous system in animals, thereby implying the possibility of threatening health.

Effects of cadmium on the behavior and regeneration of Dugesia japonica

This study was undertaken to evaluate the toxic effect of cadmium sulfate (Cds) on planarians (Dugesia japonica), and the suitability of planarians as an alternative animal model for toxicity studies. Planarians were exposed to varying concentrations of experimental solutions containing 0.1–25 mg/L Cds, and incubated for 1, 3, and 5 hrs. Motility, seizure-like behaviors (c-like, head-bop, snake-like, and screw-like) and regeneration ability of the amputated fragment were subsequently evaluated. Results showed decreased motility with increasing concentrations of Cds (p&lt;0.05), with the lowest motility being observed at the highest concentration of 25 mg/L Cds. Results also indicate that seizure-like behavior patterns were significantly affected by increments in the Cds concentrations, especially the c-like and head-bop behaviors were notably increased. Compared to the control, the regeneration ability of the planarians was decreased in the experimental solutions containing Cds. Planarians exposed to Cds showed either delayed eye formation or no eye regeneration during incubation. Moreover, increased concentrations of Cds resulted in failure to regenerate and death of the planarians. In conclusion, this study confirm that the heavy metal Cds exerts a toxic effect on planarians. Furthermore, the performances of the planarians in the experimental period exhibit their suitability as an alternative animal model for toxicity studies.

Effects of cadmium on the behavior and regeneration of Dugesia japonica

This study was undertaken to evaluate the toxic effect of cadmium sulfate (Cds) on planarians (Dugesia japonica), and the suitability of planarians as an alternative animal model for toxicity studies. Planarians were exposed to varying concentrations of experimental solutions containing 0.1–25 mg/L Cds, and incubated for 1, 3, and 5 hrs. Motility, seizure-like behaviors (c-like, head-bop, snake-like, and screw-like) and regeneration ability of the amputated fragment were subsequently evaluated. Results showed decreased motility with increasing concentrations of Cds (p&lt;0.05), with the lowest motility being observed at the highest concentration of 25 mg/L Cds. Results also indicate that seizure-like behavior patterns were significantly affected by increments in the Cds concentrations, especially the c-like and head-bop behaviors were notably increased. Compared to the control, the regeneration ability of the planarians was decreased in the experimental solutions containing Cds. Planarians exposed to Cds showed either delayed eye formation or no eye regeneration during incubation. Moreover, increased concentrations of Cds resulted in failure to regenerate and death of the planarians. In conclusion, this study confirm that the heavy metal Cds exerts a toxic effect on planarians. Furthermore, the performances of the planarians in the experimental period exhibit their suitability as an alternative animal model for toxicity studies.

Serotonin is essential for eye regeneration in planaria Schmidteamediterranea.

Planaria is an ideal system to study factors involved in regeneration and tissue homeostasis. Little is known about the role of metabolites and small molecules in stem cell maintenance and lineage specification in planarians. Using liquid chromatography and mass spectrometry (LC-MS)-based quantitative metabolomics, we determined the relative levels of metabolites in stem cells, progenitors, and differentiated cells of the planarian Schmidteamediterranea. Tryptophan and its metabolic product serotonin are significantly enriched in stem cells and progenitor population. Serotonin biosynthesis in these cells is brought about by a noncanonical enzyme, phenylalanine hydroxylase. Knockdown of Smed-pah leads to complete disappearance of eyes in regenerating planaria, while exogenous supply of serotonin and its precursor rescues the eyeless phenotype. Our results demonstrate a key role for serotonin in eye regeneration.

INFLUENCE OF SEROTONIN ON PLANARIAN PHOTORECEPTORS’ REGENERATION

The paper presents data on the effect of biogenic amine, serotonin, on morphogenetic processes in planarians Schmidtea mediterranea and Girardia tigrina(Turbellaria, Platyhelminthes). For the study, cut-off median and tail fragments of the planarian body were used, in which eye regeneration was observed. Photoreceptor recovery occurred from the 3rd to the 6th day of regeneration. In experimental specimens exposed to serotonin at a concentration of 0.1–1 μM, acceleration of the photoreceptor regeneration process was observed. The stimulating effect was observed at 4–5 days after surgery. Planaria (Turbellaria, Platyhelminthes) are free-living flatworms related to parasitic trematodes, cestodes and monogenges. In addition to the nervous, muscular, digestive, excretory and reproductive systems, they have a pair of simple eyes (photoreceptors), so they can distinguish the intensity and direction of the light flux and are oriented in space [1]. Planarium eyes are located on the dorsal surface of the head part of the body and consist of photoreceptor and pigment cells that form an "eye cup". Planaria can regenerate the whole organism from a small fragment. In the process of regeneration, the planarium can completely restore the head ganglion (brain), as well as other organs, including the eyes.Our task was to study the dynamics of photoreceptor differentiation in planarians Schmidtea mediterranea, and to investigate the possible morphogenetic function of serotonin in S. mediterranea and G. tigrina

Eye Regeneration and Healing with Amniotic Membrane

If a person has suffered some tumor, ulcers, epithelial defects of the cornea and the conjunctiva, the treatment with amniotic membrane (MA) provides very satisfactory results to achieve the healing and reconstruction of the ocular surface in the short and long term.

Eye Regeneration and Healing

Eye Regeneration and Healing

The planarian Schmidtea mediterranea as a model system for the discovery and characterization of cell-penetrating peptides and bioportides.

The general utility of the planarian Schmidtea mediterranea, an organism with remarkable regenerative capacity, was investigated as a convenient three-dimensional model to analyse the import of cell-penetrating peptides (CPPs) and bioportides (bioactive CPPs) into complex tissues. The unpigmented planarian blastema, 3days post head amputation, is a robust platform to assess the penetration of red-fluorescent CPPs into epithelial cells and deeper tissues. Three planarian proteins, Ovo, ZicA and Djeya, which collectively control head remodelling and eye regeneration following decapitation, are a convenient source of novel cationic CPP vectors. One example, Djeya1 (RKLAFRYRRIKELYNSYR), is a particularly efficient and seemingly inert CPP vector that could be further developed to assist the delivery of bioactive payloads across the plasma membrane of eukaryotic cells. Eye regeneration, following head amputation, was utilized in an effort to identify bioportides capable of influencing stem cell-dependent morphogenesis. These investigations identified the tetradecapeptide mastoparan (INLKALAALAKKIL) as a bioportide able to influence the gross morphology of head development. We conclude that, compared with cellular monolayers, the S.mediterranea system provides many advantages and will support the identification of bioportides able to selectively modify the biology of totipotent neoblasts and, presumably, other mammalian stem cell types.

Smed-egfr-4 is required for planarian eye regeneration.

Planarians are remarkable organisms that can regenerate their entire body from a tiny portion thereof. This capability is made possible by the persistence throughout the lifespan of these animals of a population of pluripotent stem cells known as neoblasts. Planarian neoblasts include both pluripotent stem cells and specialized lineage-committed progenitors that give rise to all mature cell types during regeneration and homeostatic cell turnover. However, little is known about the mechanisms that regulate neoblast differentiation. A recent study demonstrated that Smed-egfr-1, a homologue of the epidermal growth factor receptor (EGFR) family, is required for final differentiation, but not specification, of gut progenitor cells into mature cells. Given the expression by planarians of several EGFR homologues, it has been proposed that these homologues may have diverged functionally to regulate the differentiation of distinct cell types in these animals. In this study, we investigated the role of Smed-egfr-4 in eye regeneration. Compared with controls, animals in which this gene was silenced by RNA interference (RNAi) regenerated smaller eyes. Moreover, the numbers of both mature eye cell types, photoreceptor neurons and cells of the pigment cup, were significantly reduced in Smed-egfr-4(RNAi) animals. By contrast, these animals exhibited an increase in the numbers of eye progenitor cells expressing the specific markers Smed-ovo and Smed-sp6-9. These results suggest that Smed-egfr-4 is required not for the specification of eye progenitor cells but for their final differentiation, and support the view that in planarians the EGFR pathway might play a general role in regulating the differentiation of lineage-committed progenitors.

Comparative transcriptomic analyses and single-cell RNA sequencing of the freshwater planarian Schmidtea mediterranea identify major cell types and pathway conservation

BackgroundIn the Lophotrochozoa/Spiralia superphylum, few organisms have as high a capacity for rapid testing of gene function and single-cell transcriptomics as the freshwater planaria. The species Schmidtea mediterranea in particular has become a powerful model to use in studying adult stem cell biology and mechanisms of regeneration. Despite this, systematic attempts to define gene complements and their annotations are lacking, restricting comparative analyses that detail the conservation of biochemical pathways and identify lineage-specific innovations.ResultsIn this study we compare several transcriptomes and define a robust set of 35,232 transcripts. From this, we perform systematic functional annotations and undertake a genome-scale metabolic reconstruction for S. mediterranea. Cross-species comparisons of gene content identify conserved, lineage-specific, and expanded gene families, which may contribute to the regenerative properties of planarians. In particular, we find that the TRAF gene family has been greatly expanded in planarians. We further provide a single-cell RNA sequencing analysis of 2000 cells, revealing both known and novel cell types defined by unique signatures of gene expression. Among these are a novel mesenchymal cell population as well as a cell type involved in eye regeneration. Integration of our metabolic reconstruction further reveals the extent to which given cell types have adapted energy and nucleotide biosynthetic pathways to support their specialized roles.ConclusionsIn general, S. mediterranea displays a high level of gene and pathway conservation compared with other model systems, rendering it a viable model to study the roles of these pathways in stem cell biology and regeneration.

A recipe for regeneration

Regeneration Unlike humans, planarian flatworms can regenerate certain tissues. During regeneration, existing tissues remodel, and undifferentiated and progenitor cells convert into specialized cell types at specified locations. Atabay et al. examined planarian eye regeneration (see the Perspective by Tanaka). Surgical and transplantation experiments revealed three properties governing regenerative progenitor behavior: cell self-organization, an extrinsic migratory target for progenitors, and a broad progenitor-specification zone. Predictions from this model enabled generation of animals with multiple stable eyes. Science , this issue p. [404][1]; see also p. [374][2] [1]: /lookup/doi/10.1126/science.aap8179 [2]: /lookup/doi/10.1126/science.aat4588