Highly regenerative species-specific genes improve age-associated features in the adult Drosophila midgut.

BACKGROUND: The remarkable regenerative abilities observed in planarians and cnidarians are closely linked to the active proliferation of adult stem cells and the precise differentiation of their progeny, both of which typically deteriorate during aging in low regenerative animals. While regeneration-specific genes conserved in highly regenerative organisms may confer regenerative abilities and long-term maintenance of tissue homeostasis, it remains unclear whether introducing these regenerative genes into low regenerative animals can improve their regeneration and aging processes. RESULTS: Here, we ectopically express highly regenerative species-specific JmjC domain-encoding genes (HRJDs) in Drosophila, a widely used low regenerative model organism. Surprisingly, HRJD expression impedes tissue regeneration in the developing wing disc but extends organismal lifespan when expressed in the intestinal stem cell lineages of the adult midgut under non-regenerative conditions. Notably, HRJDs enhance the proliferative activity of intestinal stem cells while maintaining their differentiation fidelity, ameliorating age-related decline in gut barrier functions. CONCLUSIONS: These findings together suggest that the introduction of highly regenerative species-specific genes can improve stem cell functions and promote a healthy lifespan when expressed in aging animals.

Examining the liver-pancreas crosstalk reveals a role for the molybdenum cofactor in ß-cell regeneration.

Regeneration of insulin-producing ß-cells is an alternative avenue to manage diabetes, and it is crucial to unravel this process in vivo during physiological responses to the lack of ß-cells. Here, we aimed to characterize how hepatocytes can contribute to ß-cell regeneration, either directly or indirectly via secreted proteins or metabolites, in a zebrafish model of ß-cell loss. Using lineage tracing, we show that hepatocytes do not directly convert into ß-cells even under extreme ß-cell ablation conditions. A transcriptomic analysis of isolated hepatocytes after ß-cell ablation displayed altered lipid- and glucose-related processes. Based on the transcriptomics, we performed a genetic screen that uncovers a potential role of the molybdenum cofactor (Moco) biosynthetic pathway in ß-cell regeneration and glucose metabolism in zebrafish. Consistently, molybdenum cofactor synthesis 2 (Mocs2) haploinsufficiency in mice indicated dysregulated glucose metabolism and liver function. Together, our study sheds light on the liver-pancreas crosstalk and suggests that the molybdenum cofactor biosynthesis pathway should be further studied in relation to glucose metabolism and diabetes.

Mechanisms of regeneration: to what extent do they recapitulate development?

One of the enduring debates in regeneration biology is the degree to which regeneration mirrors development. Recent technical advances, such as single-cell transcriptomics and the broad applicability of CRISPR systems, coupled with new model organisms in research, have led to the exploration of this longstanding concept from a broader perspective. In this Review, I outline the historical parallels between development and regeneration before focusing on recent research that highlights how dissecting the divergence between these processes can uncover previously unreported biological mechanisms. Finally, I discuss how these advances position regeneration as a more dynamic and variable process with expanded possibilities for morphogenesis compared with development. Collectively, these insights into mechanisms that orchestrate morphogenesis may reshape our understanding of the evolution of regeneration, reveal hidden biology activated by injury, and offer non-developmental strategies for restoring lost or damaged organs and tissues.

A wound-induced differentiation trajectory for neurons.

Animals capable of whole-body regeneration can replace any missing cell type and regenerate fully functional new organs, including new brains, de novo. The regeneration of a new brain requires the formation of diverse neural cell types and their assembly into an organized structure with correctly wired circuits. Recent work in various regenerative animals has revealed transcriptional programs required for the differentiation of distinct neural subpopulations, however, how these transcriptional programs are initiated in response to injury remains unknown. Here, we focused on the highly regenerative acoel worm, Hofstenia miamia, to study wound-induced transcriptional regulatory events that lead to the production of neurons and subsequently a functional brain. Footprinting analysis using chromatin accessibility data on a chromosome-scale genome assembly revealed that binding sites for the Nuclear Factor Y (NFY) transcription factor complex were significantly bound during regeneration, showing a dynamic increase in binding within one hour upon amputation specifically in tail fragments, which will regenerate a new brain. Strikingly, NFY targets were highly enriched for genes with neuronal function. Single-cell transcriptome analysis combined with functional studies identified soxC+ stem cells as a putative progenitor population for multiple neural subtypes. Further, we found that wound-induced soxC expression is likely under direct transcriptional control by NFY, uncovering a mechanism for the initiation of a neural differentiation pathway by early wound-induced binding of a transcriptional regulator.

A cytidine deaminase regulates axon regeneration by modulating the functions of the Caenorhabditis elegans HGF/plasminogen family protein SVH-1.

The pathway for axon regeneration in Caenorhabditis elegans is activated by SVH-1, a growth factor belonging to the HGF/plasminogen family. SVH-1 is a dual-function factor that acts as an HGF-like growth factor to promote axon regeneration and as a protease to regulate early development. It is important to understand how SVH-1 is converted from a protease to a growth factor for axon regeneration. In this study, we demonstrate that cytidine deaminase (CDD) SVH-17/CDD-2 plays a role in the functional conversion of SVH-1. We find that the codon exchange of His-755 to Tyr in the Asp-His-Ser catalytic triad of SVH-1 can suppress the cdd-2 defect in axon regeneration. Furthermore, the stem hairpin structure around the His-755 site in svh-1 mRNA is required for the activation of axon regeneration by SVH-1. These results suggest that CDD-2 promotes axon regeneration by transforming the function of SVH-1 from a protease to a growth factor through modification of svh-1 mRNA.

Tert-expressing cells contribute to salivary gland homeostasis and tissue regeneration after radiation therapy.

Salivary gland homeostasis and regeneration after radiotherapy depend significantly on progenitor cells. However, the lineage of submandibular gland (SMG) progenitor cells remains less defined compared with other normal organs. Here, using a mouse strain expressing regulated CreERT2 recombinase from the endogenous Tert locus, we identify a distinct telomerase-expressing (TertHigh) cell population located in the ductal region of the adult SMG. These TertHigh cells contribute to ductal cell generation during SMG homeostasis and to both ductal and acinar cell renewal 1 year after radiotherapy. TertHigh cells maintain self-renewal capacity during in vitro culture, exhibit resistance to radiation damage, and demonstrate enhanced proliferative activity after radiation exposure. Similarly, primary human SMG cells with high Tert expression display enhanced cell survival after radiotherapy, and CRISPR-activated Tert in human SMG spheres increases proliferation after radiation. RNA sequencing reveals upregulation of "cell cycling" and "oxidative stress response" pathways in TertHigh cells following radiation. Mechanistically, Tert appears to modulate cell survival through ROS levels in SMG spheres following radiation damage. Our findings highlight the significance of TertHigh cells in salivary gland biology, providing insights into their response to radiotherapy and into their use as a potential target for enhancing salivary gland regeneration after radiotherapy.

Efficient In Vitro Regeneration System and Comparative Transcriptome Analysis Offer Insight into the Early Development Characteristics of Explants from Cotyledon with Partial Petiole in Small-Fruited Pepper (Capsicum annuum).

In our research, we utilized six small-fruited pepper germplasms as materials, selected cotyledons with the petiole and hypocotyls as explants, and conducted in vitro regeneration studies. Our outcomes specify that the most suitable explant is cotyledon with the petiole, and the suitable genotype is HNUCA341. The optimal medium for inducing and elongating adventitious buds for this genotype is Murashige and Skoog medium (MS) + 9.12 µM Zeatin (ZT) + 0.57 µM 3-Indoleacetic acid (IAA), with a bud induction rate of 44.4%. The best rooting induction medium is MS + 1.14 µM IAA, with a rooting rate of 86.7%. Research on the addition of exogenous hormones has revealed that the induction speed of buds in small-fruited pepper (HNUCA341) in the combination of ZT and IAA hormones (abbreviated as ZI) is quicker, and the induction effect is better. The histological observations indicate that ZI treatment accelerates the initiation of explant division and differentiation, causing a shorter duration of vascular-bundle tissue production. The plant hormone signaling pathway was significantly enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, including ARR9 (LOC107843874, LOC107843885), ARR4 (LOC107848380, LOC107862455), AHK4 (LOC107870540), AHP1 (LOC107839518), LAX2 (LOC107846008), SAUR36 (LOC107852624), IAA8 (LOC107841020), IAA16 (LOC107839415), PYL4 (LOC107843441), and PYL6 (LOC107871127); these significantly enriched genes may be associated with in vitro regeneration. In addition, the carbon metabolism pathway and plant mitogen-activated protein kinase (MAPK) signaling pathway are also significantly enriched in KEGG. The results of the Gene Ontology (GO) analysis revealed that differentially expressed genes related to carbon metabolism and fixation, photosynthesis and MAPK signaling pathways were upregulated under ZI treatment. It was found that they might be associated with enhanced regeneration in vitro. Furthermore, we also screened out differentially expressed transcription factors, primarily from the MYB, bHLH, AP2/ERF, and NAC families. Overall, our work accumulated important data for the in-depth analysis of the molecular mechanism of in vitro regeneration of pepper, and provides valuable germplasm for establishing an efficient stable pepper genetic-transformation system based on tissue culture.

Time-course study of genetic changes in periodontal ligament regeneration after tooth replantation in a mouse model.

This research focused on analyzing gene expression changes in the periodontal ligament (PDL) after tooth re-plantation to identify key genes and pathways involved in healing and regeneration. Utilizing a mouse model, mRNA was extracted from the PDL at various intervals post-replantation for RNA sequencing analysis, spanning from 3 to 56 days. The results revealed significant shifts in gene expression, particularly notable on day 28, supported by hierarchical clustering and principal component analysis. Gene ontology (GO) enrichment analysis highlighted an upregulation in olfactory receptor and G protein-coupled receptor signaling pathways at this time point. These findings were validated through reverse transcription-quantitative PCR (RT-qPCR), with immunochemical staining localizing olfactory receptor gene expression to the PDL and surrounding tissues. Moreover, a scratch assay indicated that olfactory receptor genes might facilitate wound healing in human PDL fibroblasts. These results underscore the importance of the 28-day post-transplant phase as a potential "tipping point" in PDL healing and regeneration. In conclusion, this research sheds light on the potential role of olfactory receptor genes in PDL regeneration, providing a foundation for developing new therapeutic approaches in tooth replantation and transplantation, with broader implications for regenerative medicine in oral health.

Direct Somatic Embryogenesis in Carica papaya L. Genotypes for Genetic Modification Purposes.

This chapter presents an efficient protocol for regenerating Carica papaya plants via somatic embryogenesis from immature zygotic embryos from economically important papaya genotypes. To achieve regenerated plants from somatic embryos, in the present protocol, four induction cycles are required, followed by one multiplication cycle and one regeneration cycle. With this optimized protocol, 80% of somatic embryos can be obtained in only 3.5 months. At this stage, calli containing more than 50% globular structures can be used for transformation (via agrobacterium, biobalistics, or any other transformation method). Once transformed, calli can be transferred to the following steps (multiplication, elongation, maturation, rooting, and ex vitro acclimatization) to regenerate a transformed somatic embryo-derived full plant.

The long noncoding RNA lncMPD2 inhibits myogenesis by targeting the miR-34a-5p/THBS1 axis.

Long noncoding RNAs (lncRNAs) participate in regulating skeletal muscle development. However, little is known about their role in regulating chicken myogenesis. In this study, we identified a novel lncRNA, lncMPD2, through transcriptome sequencing of chicken myoblasts at different developmental stages. Functionally, gain- and loss-of-function experiments showed that lncMPD2 inhibited myoblast proliferation and differentiation. Mechanistically, lncMPD2 directly bound to miR-34a-5p, and miR-34a-5p promoted myoblasts proliferation and differentiation and inhibited the mRNA and protein expression of its target gene THBS1. THBS1 inhibited myoblast proliferation and differentiation in vitro and delayed muscle regeneration in vivo. Furthermore, rescue experiments showed that lncMPD2 counteracted the inhibitory effects of miR-34a-5p on THBS1 and myogenesis-related gene mRNA and protein expression. In conclusion, lncMPD2 regulates the miR-34a-5p/THBS1 axis to inhibit the proliferation and differentiation of myoblasts and skeletal muscle regeneration. This study provides more insight into the molecular regulatory network of skeletal muscle development, identifying novel potential biomarkers for improving chicken quality and increasing chicken yield. In addition, this study provides a potential goal for breeding strategies that minimize muscle damage in chickens.

Planarian LDB and SSDP proteins scaffold transcriptional complexes for regeneration and patterning.

Sequence-specific transcription factors often function as components of large regulatory complexes. LIM-domain binding protein (LDB) and single-stranded DNA-binding protein (SSDP) function as core scaffolds of transcriptional complexes in animals and plants. Little is known about potential partners and functions for LDB/SSDP complexes in the context of tissue regeneration. In this work, we find that planarian LDB1 and SSDP2 promote tissue regeneration, with a particular function in anterior regeneration and mediolateral polarity reestablishment. We find that LDB1 and SSDP2 interact with one another and with characterized planarian LIM-HD proteins Arrowhead, Islet1, and Lhx1/5-1. We also show that SSDP2 and LDB1 function with islet1 in polarity reestablishment and with lhx1/5-1 in serotonergic neuron maturation. Finally, we find new roles for LDB1 and SSDP2 in regulating gene expression in the planarian intestine and parenchyma; these functions are likely LIM-HD-independent. Together, our work provides insight into LDB/SSDP complexes in a highly regenerative organism. Further, our work provides a strong starting point for identifying and characterizing potential binding partners of LDB1 and SSDP2 and for exploring roles for these proteins in diverse aspects of planarian physiology.

Progressive modifications during evolution involving epigenetic changes have determined loss of regeneration mainly in terrestrial animals: A hypothesis.

In order to address a biological explanation for the different regenerative abilities present among animals, a new evolutionary speculation is presented. It is hypothesized that epigenetic mechanisms have lowered or erased regeneration during the evolution of terrestrial invertebrates and vertebrates. The hypothesis indicates that a broad regeneration can only occur in marine or freshwater conditions, and that life on land does not allow for high regeneration. This is due to the physical, chemical and microbial conditions present in the terrestrial environment with respect to those of the aquatic environment. The present speculation provides examples of hypothetic evolutionary animal lineages that colonized the land, such as parasitic annelids, terrestrial mollusks, arthropods and amniotes. These are the animals where regeneration is limited or absent and their injuries are only repaired through limited healing or scarring. It is submitted that this loss derived from changes in the developmental gene pathways sustaining regeneration in the aquatic environment but that cannot be expressed on land. Once regeneration was erased in terrestrial species, re-adaptation to freshwater niches could not reactivate the previously altered gene pathways that determined regeneration. Therefore a broad regeneration was no longer possible or became limited and heteromorphic in the derived, extant animals. Only in few cases extensive healing abilities or regengrow, a healing process where regeneration overlaps with somatic growth, have evolved among arthropods and amniotes. The present paper is an extension of previous speculations trying to explain in biological terms the different regenerative abilities present among metazoans.

The brittle star genome illuminates the genetic basis of animal appendage regeneration.

Species within nearly all extant animal lineages are capable of regenerating body parts. However, it remains unclear whether the gene expression programme controlling regeneration is evolutionarily conserved. Brittle stars are a species-rich class of echinoderms with outstanding regenerative abilities, but investigations into the genetic bases of regeneration in this group have been hindered by the limited genomic resources. Here we report a chromosome-scale genome assembly for the brittle star Amphiura filiformis. We show that the brittle star genome is the most rearranged among echinoderms sequenced so far, featuring a reorganized Hox cluster reminiscent of the rearrangements observed in sea urchins. In addition, we performed an extensive profiling of gene expression during brittle star adult arm regeneration and identified sequential waves of gene expression governing wound healing, proliferation and differentiation. We conducted comparative transcriptomic analyses with other invertebrate and vertebrate models for appendage regeneration and uncovered hundreds of genes with conserved expression dynamics, particularly during the proliferative phase of regeneration. Our findings emphasize the crucial importance of echinoderms to detect long-range expression conservation between vertebrates and classical invertebrate regeneration model systems.

Single-cell genomic profiling to study regeneration.

Regenerative capacities and strategies vary dramatically across animals, as well as between cell types, organs, and with age. In recent years, high-throughput single-cell transcriptomics and other single-cell profiling technologies have been applied to many animal models to gain an understanding of the cellular and molecular mechanisms underlying regeneration. Here, we review recent single-cell studies of regeneration in diverse contexts and summarize key concepts that have emerged. The immense regenerative capacity of some invertebrates, exemplified by planarians, is driven mainly by the differentiation of abundant adult pluripotent stem cells, whereas in many other cases, regeneration involves the reactivation of embryonic or developmental gene-regulatory networks in differentiated cell types. However, regeneration also differs from development in many ways, including the use of regeneration-specific cell types and gene regulatory networks.

Sall4 regulates downstream patterning genes during limb regeneration.

Many salamanders can completely regenerate a fully functional limb. Limb regeneration is a carefully coordinated process involving several defined stages. One key event during the regeneration process is the patterning of the blastema to inform cells of what they must differentiate into. Although it is known that many genes involved in the initial development of the limb are re-used during regeneration, the exact molecular circuitry involved in this process is not fully understood. Several large-scale transcriptional profiling studies of axolotl limb regeneration have identified many transcription factors that are up-regulated after limb amputation. Sall4 is a transcription factor that has been identified to play essential roles in maintaining cells in an undifferentiated state during development and also plays a unique role in limb development. Inactivation of Sall4 during limb bud development results in defects in anterior-posterior patterning of the limb. Sall4 has been found to be up-regulated during limb regeneration in both Xenopus and salamanders, but to date it function has been untested. We confirmed that Sall4 is up-regulated during limb regeneration in the axolotl using qRT-PCR and identified that it is present in the skin cells and also in cells within the blastema. Using CRISPR technology we microinjected gRNAs specific for Sall4 complexed with cas9 protein into the blastema to specifically knockout Sall4 in blastema cells only. This resulted in limb regenerate defects, including missing digits, fusion of digit elements, and defects in the radius and ulna. This suggests that during regeneration Sall4 may play a similar role in regulating the specification of anterior-proximal skeletal elements.

The IRE1α/XBP1 signaling axis drives myoblast fusion in adult skeletal muscle.

Skeletal muscle regeneration involves a signaling network that regulates the proliferation, differentiation, and fusion of muscle precursor cells to injured myofibers. IRE1α, one of the arms of the unfolded protein response, regulates cellular proteostasis in response to ER stress. Here, we demonstrate that inducible deletion of IRE1α in satellite cells of mice impairs skeletal muscle regeneration through inhibiting myoblast fusion. Knockdown of IRE1α or its downstream target, X-box protein 1 (XBP1), also inhibits myoblast fusion during myogenesis. Transcriptome analysis revealed that knockdown of IRE1α or XBP1 dysregulates the gene expression of molecules involved in myoblast fusion. The IRE1α-XBP1 axis mediates the gene expression of multiple profusion molecules, including myomaker (Mymk). Spliced XBP1 (sXBP1) transcription factor binds to the promoter of Mymk gene during myogenesis. Overexpression of myomaker in IRE1α-knockdown cultures rescues fusion defects. Inducible deletion of IRE1α in satellite cells also inhibits myoblast fusion and myofiber hypertrophy in response to functional overload. Collectively, our study demonstrates that IRE1α promotes myoblast fusion through sXBP1-mediated up-regulation of the gene expression of multiple profusion molecules, including myomaker.

A large-scale CRISPR screen reveals context-specific genetic regulation of retinal ganglion cell regeneration.

Many genes are known to regulate retinal regeneration after widespread tissue damage. Conversely, genes controlling regeneration after limited cell loss, as per degenerative diseases, are undefined. As stem/progenitor cell responses scale to injury levels, understanding how the extent and specificity of cell loss impact regenerative processes is important. Here, transgenic zebrafish enabling selective retinal ganglion cell (RGC) ablation were used to identify genes that regulate RGC regeneration. A single cell multiomics-informed screen of 100 genes identified seven knockouts that inhibited and 11 that promoted RGC regeneration. Surprisingly, 35 out of 36 genes known and/or implicated as being required for regeneration after widespread retinal damage were not required for RGC regeneration. The loss of seven even enhanced regeneration kinetics, including the proneural factors neurog1, olig2 and ascl1a. Mechanistic analyses revealed that ascl1a disruption increased the propensity of progenitor cells to produce RGCs, i.e. increased 'fate bias'. These data demonstrate plasticity in the mechanism through which Müller glia convert to a stem-like state and context specificity in how genes function during regeneration. Increased understanding of how the regeneration of disease-relevant cell types is specifically controlled will support the development of disease-tailored regenerative therapeutics.

Mapping spatially resolved transcriptomes in human and mouse pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with poor prognosis and limited treatment options. Efforts to identify effective treatments are thwarted by limited understanding of IPF pathogenesis and poor translatability of available preclinical models. Here we generated spatially resolved transcriptome maps of human IPF (n = 4) and bleomycin-induced mouse pulmonary fibrosis (n = 6) to address these limitations. We uncovered distinct fibrotic niches in the IPF lung, characterized by aberrant alveolar epithelial cells in a microenvironment dominated by transforming growth factor beta signaling alongside predicted regulators, such as TP53 and APOE. We also identified a clear divergence between the arrested alveolar regeneration in the IPF fibrotic niches and the active tissue repair in the acutely fibrotic mouse lung. Our study offers in-depth insights into the IPF transcriptional landscape and proposes alveolar regeneration as a promising therapeutic strategy for IPF.

CircRNA8388 functions as the sponge for miR-2392 during intestinal regeneration in sea cucumber Apostichopus japonicus.

The sea cucumber Apostichopus japonicus can expel internal organs under stress and regenerate them subsequently. However, growth is delayed during regeneration, significantly impacting the industry. Circular RNAs (circRNAs) are single-stranded circular RNA molecules produced through alternative splicing of mRNA precursors. They play crucial roles in regulating gene expression via the ceRNA mechanism. In this study, circRNA profiles of control and regenerated intestines were constructed. A total of 15,874 circRNAs were identified, with a length of 300-350 nucleotides (nt) being the most abundant. Sanger sequencing confirmed the circular structure of circRNA398. Compared with the normal intestine, 50 and 83 differentially expressed circRNAs (DE-circRNAs) were identified in the regenerated intestine at 1 and 3 days post evisceration (dpe), respectively. Gene ontology (GO) terms for signal transduction and development regulation were most significantly enriched in 1dpeVScon and 3dpeVScon treatments, respectively. The dual-luciferase assay revealed that circRNA8388 functions as a sponge for miR-2392, participating in the remodeling of the extracellular matrix (ECM). In conclusion, these findings will contribute to the enhancement of the non-coding RNA database for echinoderms and lay the groundwork for future investigations into circRNA regulation during intestinal regeneration.

Single-cell transcriptomic atlas reveals increased regeneration in diseased human inner ear balance organs.

Mammalian inner ear hair cell loss leads to permanent hearing and balance dysfunction. In contrast to the cochlea, vestibular hair cells of the murine utricle have some regenerative capacity. Whether human utricular hair cells regenerate in vivo remains unknown. Here we procured live, mature utricles from organ donors and vestibular schwannoma patients, and present a validated single-cell transcriptomic atlas at unprecedented resolution. We describe markers of 13 sensory and non-sensory cell types, with partial overlap and correlation between transcriptomes of human and mouse hair cells and supporting cells. We further uncover transcriptomes unique to hair cell precursors, which are unexpectedly 14-fold more abundant in vestibular schwannoma utricles, demonstrating the existence of ongoing regeneration in humans. Lastly, supporting cell-to-hair cell trajectory analysis revealed 5 distinct patterns of dynamic gene expression and associated pathways, including Wnt and IGF-1 signaling. Our dataset constitutes a foundational resource, accessible via a web-based interface, serving to advance knowledge of the normal and diseased human inner ear.