TGF-beta signaling is required for multiple processes during Xenopus tail regeneration

Xenopus tadpoles can fully regenerate all major tissue types following tail amputation. TGF-β signaling plays essential roles in growth, repair, specification, and differentiation of tissues throughout development and adulthood. We examined the localization of key components of the TGF-β signaling pathway during regeneration and characterized the effects of loss of TGF-β signaling on multiple regenerative events. Phosphorylated Smad2 (p-Smad2) is initially restricted to the p63+ basal layer of the regenerative epithelium shortly after amputation, and is later found in multiple tissue types in the regeneration bud. TGF-β ligands are also upregulated throughout regeneration. Treatment of amputated tails with SB-431542, a specific and reversible inhibitor of TGF-β signaling, blocks tail regeneration at multiple points. Inhibition of TGF-β signaling immediately following tail amputation reversibly prevents formation of a wound epithelium over the future regeneration bud. Even brief inhibition immediately following amputation is sufficient, however, to irreversibly block the establishment of structures and cell types that characterize regenerating tissue and to prevent the proper activation of BMP and ERK signaling pathways. Inhibition of TGF-β signaling after regeneration has already commenced blocks cell proliferation in the regeneration bud. These data reveal several spatially and temporally distinct roles for TGF-β signaling during regeneration: (1) wound epithelium formation, (2) establishment of regeneration bud structures and signaling cascades, and (3) regulation of cell proliferation.     

MAPK/ERK signalling is required for zebrafish cardiac regeneration

Objectives

To better understand the molecular mechanisms of regeneration and explore the potential signalling pathways as therapeutic targets for heart attacks.

Results

After treatment with the MEK inhibitor AZD6244 upon cardiac injury, the core members in MAPK/ERK signalling—mek and erk—demonstrate elevated expression, and these proteins are deposited at the injury site in zebrafish. pERK is also induced in non-cardiomyocytes near the injury site. Furthermore, the induced expression of a dominant-negative form of MEK1 inhibits zebrafish cardiac regeneration, characterized by increased cardiac fibrosis (a hallmark of regenerative failure), reduced or delayed production of regenerative myocardium, and migration of FLI1⁺ endothelial cells, without direct inhibition of cardiomyocyte proliferation.

Conclusion

Appropriate activation of MAPK/ERK signalling is essential for zebrafish cardiac regeneration.

     

Laccase2 is required for cuticular pigmentation in stinkbugs

During the maturation of insect cuticle, protein-protein and protein-chitin crosslinkages are formed by the action of diphenoloxidases. Two types of diphenoloxidases, laccases and tyrosinases, are present in the insect cuticle. In coleopteran and hymenopteran insects, laccase2 gene has been identified as encoding an enzyme principally responsible for cuticular pigmentation and hardening, whereas biological roles of laccase genes in hemimetabolous insects remain to be established. Here we identified laccase2 genes from three hemipteran stinkbugs, Riptortus pedestris (Alydidae), Nysius plebeius (Lygaeidae) and Megacopta punctatissima (Plataspidae). In R. pedestris, laccase2 gene was highly expressed in epidermal tissues prior to molting. When the gene expression was suppressed by an RNA interference technique, cuticular pigmentation after molting were blocked depending on the dose of injected double-stranded RNA targeting the laccase2 gene. Similar results were obtained for N. plebeius and M. punctatissima. In all the stinkbug species, injecting 20 ng of double-stranded RNA was sufficient to prevent the cuticular maturation. These results indicate that laccase2 gene is generally required for cuticular pigmentation in different stinkbug families, highlighting its conserved biological function across diverse insect taxa.     

Canonical WNT/beta-catenin signaling is required for ureteric branching

WNT/beta-catenin signaling has an established role in nephron formation during kidney development. Yet, the role of beta-catenin during ureteric morphogenesis in vivo is undefined. We generated a murine genetic model of beta-catenin deficiency targeted to the ureteric bud cell lineage. Newborn mutant mice demonstrated bilateral renal aplasia or renal dysplasia. Analysis of the embryologic events leading to this phenotype revealed that abnormal ureteric branching at E12.5 precedes histologic abnormalities at E13.5. Microarray analysis of E12.5 kidney tissue identified decreased Emx2 and Lim1 expression among a small subset of renal patterning genes disrupted at the stage of abnormal branching. These alterations are followed by decreased expression of genes downstream of Emx2, including Lim1, Pax2, and the ureteric tip markers, c-ret and Wnt 11. Together, these data demonstrate that beta-catenin performs essential functions during renal branching morphogenesis via control of a hierarchy of genes that control ureteric branching.     

Shh is required for Tabby hair follicle development

In embryonic Eda mutant (“Tabby”) mice, the development of one of the two major types of hair, “primary” hair fails, but other “secondary” hairs develop in normal numbers, though shorter and slightly aberrant. In Tabby mice, Shh is undetectable in skin early on, but is activated during secondary hair formation. We inferred that Shh may be involved in primary hair formation, activated normally by Eda, and also possibly in secondary hair formation, activated by an Eda-independent pathway. Varying the dosage of Shh now supports these inferences. In Shh knockout mice, mice were totally hairless: primary and secondary hair follicle germs were formed, but further progression failed. Consistent with these findings, when Shh loss was restricted to the skin, secondary hair follicle germs were initiated on time in Tabby mice, but their subsequent development (down-growth) failed. An Shh transgene expressed in Tabby skin could not restore induction of primary hair follicles, but restored normal length to the somewhat aberrant secondary hair that was formed and prolonged the anagen phase of hair cycling. Thus, Shh is required for primary and secondary hair downgrowth and full secondary hair length, but is not itself sufficient to replace Eda or make fully normal secondary hair.Key words: Eda, Shh, Wnt, hair follicle subtypes, Tabby     

Shh is required for Tabby hair follicle development

In embryonic Eda mutant ("Tabby") mice, the development of one of the two major types of hair, "primary" hair fails, but other "secondary" hairs develop in normal numbers, though shorter and slightly aberrant. In Tabby mice, Shh is undetectable in skin early on, but is activated during secondary hair formation. We inferred that Shh may be involved in primary hair formation, activated normally by Eda, and also possibly in secondary hair formation, activated by an Eda-independent pathway. Varying the dosage of Shh now supports these inferences. In Shh knockout mice, mice were totally hairless: primary and secondary hair follicle germs were formed, but further progression failed. Consistent with these findings, when Shh loss was restricted to the skin, secondary hair follicle germs were initiated on time in Tabby mice, but their subsequent development (down-growth) failed. An Shh transgene expressed in Tabby skin could not restore induction of primary hair follicles, but restored normal length to the somewhat aberrant secondary hair that was formed and prolonged the anagen phase of hair cycling. Thus, Shh is required for primary and secondary hair down-growth and full secondary hair length, but is not itself sufficient to replace Eda or make fully normal secondary hair.     

TINP1 homolog is required for planarian regeneration

The planarian flatworm is an ideal system for the study of regeneration in vivo. In this study, we focus on TINP1, which is one of the most conserved proteins in eukaryotic organisms. We found that TINP1 was expressed in parenchymal region through whole body as well as central nervous system (CNS) during the course of regeneration. RNA interference targeting DjTINP1 caused lysis defects in regenerating tissues and a decreased in cell division and expression levels of DjpiwiA and Djpcna. Furthermore, the expression levels of DjTINP1 were decreased when we inhibited the TGF-β signal by knockdown of smad4, which is the sole co-smad and has been proved to control the blastema patterning and central nervous system (CNS) regeneration in planarians. These findings suggest that DjTINP1 participate in the maintenance of neoblasts and be required for proper cell proliferation in planarians as a downstream gene of the TGF-β signal pathway.     

Autophagy is required for zebrafish caudal fin regeneration

Regeneration is the ability of multicellular organisms to replace damaged tissues and regrow lost body parts. This process relies on cell fate transformation that involves changes in gene expression as well as in the composition of the cytoplasmic compartment, and exhibits a characteristic age-related decline. Here, we present evidence that genetic and pharmacological inhibition of autophagy – a lysosome-mediated self-degradation process of eukaryotic cells, which has been implicated in extensive cellular remodelling and aging – impairs the regeneration of amputated caudal fins in the zebrafish (Danio rerio). Thus, autophagy is required for injury-induced tissue renewal. We further show that upregulation of autophagy in the regeneration zone occurs downstream of mitogen-activated protein kinase/extracellular signal-regulated kinase signalling to protect cells from undergoing apoptosis and enable cytosolic restructuring underlying terminal cell fate determination. This novel cellular function of the autophagic process in regeneration implies that the role of cellular self-digestion in differentiation and tissue patterning is more fundamental than previously thought.     

Effect of thyroxin upon the thyroid gland and the regeneration and pigmentation of hair in Peromyscus

Development Genes and Evolution -     

FADD is required for multiple signaling events downstream of the receptor Fas.

To identify essential components of the Fas-induced apoptotic signaling pathway, Jurkat T lymphocytes were chemically mutagenized and selected for clones that were resistant to Fas-induced apoptosis. We obtained five cell lines that contain mutations in the adaptor FADD. All five cell lines did not express FADD by immunoblot analysis and were completely resistant to Fas-induced death. Complementation of the FADD mutant cell lines with wild-type FADD restored Fas-mediated apoptosis. Fas activation of caspase-2, caspase-3, caspase-7, and caspase-8 and the proteolytic cleavage of substrates such as BID, protein kinase Cdelta, and poly(ADP-ribose) polymerase were completely defective in the FADD mutant cell lines. In addition, Fas activation of the stress kinases p38 and c-Jun NH2 kinase and the generation of ceramide in response to Fas ligation were blocked in the FADD mutant cell lines. These data indicate that FADD is essential for multiple signaling events downstream of Fas.     

EGFR signaling is required for re-establishing the proximodistal axis during distal leg regeneration in the cricket Gryllus bimaculatus nymph

Nymphs of hemimetabolous insects, such as cockroaches and crickets, possess functional legs with a remarkable capacity for epimorphic regeneration. In this study, we have focused on the role of epidermal growth factor receptor (EGFR) signaling in regeneration of a nymphal leg in the cricket Gryllus bimaculatus. We performed loss-of-function analyses with a Gryllus Egfr homolog (Gb'Egfr) and nymphal RNA interference (RNAi). After injection of double-stranded RNA for Gb'Egfr in the body cavity of the third instar cricket nymph, amputation of the leg at the distal tibia resulted in defects of normal distal regeneration. The regenerated leg lacked the distal tarsus and pretarsus. This result indicates that EGFR signaling is required for distal leg patterning in regeneration during the nymphal stage of the cricket. Furthermore, we demonstrated that EGFR signaling acts downstream of the canonical Wnt/Wg signaling and regulates appendage proximodistal (PD) patterning genes aristaless and dachshund during regeneration. Our results suggest that EGFR signaling influences positional information along the PD axis in distal leg patterning of insects, regardless of the leg formation mode.     

DARPP-32 is required for MAPK/ERK signaling in thyroid cells

Modulation of MAPK signaling duration by cAMP defines its physiological output by driving cells toward proliferation or differentiation. Understanding how the kinetics of MAPK signaling are integrated with other cellular signals is a key issue in development and cancer. Here we show that dopamine and cAMP-regulated neuronal phosphoprotein, 32 kDa (DARPP-32), a protein required for thyroid cell differentiation, determines whether MAPK/ERK activation is sustained or transient. Serum, a stimulus that activates MAPK signaling and does not independently increase DARPP-32 levels results in transient activation of the MAPK pathway. By contrast, TSH + (IGF-I) activate MAPK signaling but also independently increase DARPP-32 levels. Our results are consistent with a model in which maintenance of DARPP-32 expression by TSH + IGF-I leads to sustained MAPK signaling. Moreover, the sensitivity of MAPK/ERK signaling in thyroid cells is lost when de novo DARPP-32 expression is blocked by small interfering RNA. Because both DARPP-32 levels and function as inhibitor of protein phosphatase 1, a key inhibitor of MAPK kinase activity, are governed by cAMP/protein kinase A, the results may explain why in thyroid cells cAMP signaling downstream from TSH controls the duration of MAPK pathway activity. Thus, fine-tuning of DARPP-32 levels leads to changes in the kinetics or sensitivity of MAPK/ERK signaling. Given the implications of MAPK signaling in thyroid cancer and the loss of DARPP-32 in tumor and transformed thyroid cells, DARPP-32 may represent a key therapeutic target.     

BMP signaling is required for development of the ciliary body

The ciliary body in the eye secretes aqueous humor and glycoproteins of the vitreous body and maintains the intraocular pressure. The ciliary muscle controls the shape of the lens through the ciliary zonules to focus the image onto the retina. During embryonic development, the ciliary epithelium is derived from the optic vesicle, but the molecular signals that control morphogenesis of the ciliary body are unknown. We report that lens-specific expression of a transgenic protein, Noggin, can block BMP signaling in the mouse eye and result in failure in formation of the ciliary processes. Co-expression of transgenic BMP7 restores normal development of the ciliary epithelium. Ectopic expression of Noggin also promotes differentiation of retinal ganglion cells. These results indicate that BMP signaling is required for development of the ciliary body and may also play a role in regulation of neuronal differentiation in the developing eye.