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Growth of long bones and vertebrae is maintained postnatally by a long-lasting pool of progenitor cells. Little is known about the molecular mechanisms that regulate the output and maintenance of the cells that give rise to mature cartilage. Here we demonstrate that postnatal chondrocyte-specific deletion of a transcription factor Stat3 results in severely reduced proliferation coupled with increased hypertrophy, growth plate fusion, stunting and signs of progressive dysfunction of the articular cartilage. This effect is dimorphic, with females more strongly affected than males. Chondrocyte-specific deletion of the IL-6 family cytokine receptor gp130, which activates Stat3, phenocopied Stat3-deletion; deletion of Lifr, one of many co-receptors that signals through gp130, resulted in a milder phenotype. These data define a molecular circuit that regulates chondrogenic cell maintenance and output and reveals a pivotal positive function of IL-6 family cytokines in the skeletal system with direct implications for skeletal development and regeneration. Liu et al. demonstrate the role of gp130/STAT3 signalling in the development and homeostasis of chondrocytes in the growth plate and articular cartilage. The authors report that tamoxifen-induced deletion of STAT3 or gp130 in chondrocytes after birth results in defective chondrocyte proliferation, growth plate fusion and stunting, and signs of progressive dysfunction of the articular cartilage, with female mice more strongly affected than males.

The type I TGFβ receptor TGFβRI (encoded by Tgfbr1) was ablated in cartilage. The resulting Tgfbr1Col2 mice exhibited lethal chondrodysplasia. Similar defects were not seen in mice lacking the type II TGFβ receptor or SMADs 2 and 3, the intracellular mediators of canonical TGFβ signaling. However, we detected elevated BMP activity in Tgfbr1Col2 mice. As previous studies showed that TGFβRI can physically interact with ACVRL1, a type I BMP receptor, we generated cartilage-specific Acvrl1 (Acvrl1Col2 ) and Acvrl1/Tgfbr1 (Acvrl1/Tgfbr1Col2 ) knockouts. Loss of ACVRL1 alone had no effect, but Acvrl1/Tgfbr1Col2 mice exhibited a striking reversal of the chondrodysplasia seen in Tgfbr1Col2 mice. Loss of TGFβRI led to a redistribution of the type II receptor ACTRIIB into ACVRL1/ACTRIIB complexes, which have high affinity for BMP9. Although BMP9 is not produced in cartilage, we detected BMP9 in the growth plate, most likely derived from the circulation. These findings demonstrate that the major function of TGFβRI in cartilage is not to transduce TGFβ signaling, but rather to antagonize BMP signaling mediated by ACVRL1.

Neurodegenerative proteinopathies such as Alzheimer’s Disease are characterized by abnormal protein aggregation and neurodegeneration. Neuroresilience or regenerative strategies to prevent neurodegeneration, preserve function, or restore lost neurons may have the potential to combat human proteinopathies; however, the adult human brain possesses a limited capacity to replace lost neurons. In contrast, axolotls (Ambystoma mexicanum) show robust brain regeneration. To determine whether axolotls may help identify potential neuroresilience or regenerative strategies in humans, we first interrogated whether axolotls express putative proteins homologous to human proteins associated with neurodegenerative diseases. We compared the homology between human and axolotl proteins implicated in human proteinopathies and found that axolotls encode proteins highly similar to human microtubule-binding protein tau (tau), amyloid precursor protein (APP), and β-secretase 1 (BACE1), which are critically involved in human proteinopathies like Alzheimer’s Disease. We then tested monoclonal Tau and BACE1 antibodies previously used in human and rodent neurodegenerative disease studies using immunohistochemistry and western blotting to validate the homology for these proteins. These studies suggest that axolotls may prove useful in studying the role of these proteins in disease within the context of neuroresilience and repair.

Diabetes is a condition characterized by a loss of pancreatic β-cell function which results in the dysregulation of insulin homeostasis. Using a partial pancreatectomy model in axolotl, we aimed to observe the pancreatic response to injury. Here we show a comprehensive histological assessment of pancreatic islets in axolotl. Following pancreatic injury, no apparent blastemal structure was observed. We found a significant, organ-wide increase in cellular proliferation post-resection in the pancreas compared to sham-operated controls. This proliferative response was most robust at the site of injury. We found that β-cells actively contributed to the increased rates of proliferation upon injury. β-cell proliferation manifested in increased β-cell mass in injured tissue at two weeks post injury. At four weeks post injury, we found organ-wide proliferation to be extinguished while proliferation at the injury site persisted, corresponding to pancreatic tissue recovery. Similarly, total β-cell mass was comparable to sham after four weeks. Our findings suggest a non-blastema-mediated regeneration process takes place in the pancreas, by which pancreatic resection induces whole-organ β-cell proliferation without the formation of a blastemal structure. This process is analogous to other models of compensatory growth in axolotl, including liver regeneration.

Previous studies have reported that amputation invokes body-wide responses in regenerative organisms, but most have not examined the implications of these changes beyond the region of tissue regrowth. Specifically, long-range epidermal responses to amputation are largely uncharacterized, with research on amputation-induced epidermal responses in regenerative organisms traditionally being restricted to the wound site. Here, we investigate the effect of amputation on long-range epidermal permeability in two evolutionarily distant, regenerative organisms: axolotls and planarians. We find that amputation triggers a long-range increase in epidermal permeability in axolotls, accompanied by a long-range epidermal downregulation in MAPK signaling. Additionally, we provide functional evidence that pharmacologically inhibiting MAPK signaling in regenerating planarians increases long-range epidermal permeability. These findings advance our knowledge of body-wide changes due to amputation in regenerative organisms and warrant further study on whether epidermal permeability dysregulation in the context of amputation may lead to pathology in both regenerative and non-regenerative organisms.

CCN5/WISP2 is a matricellular protein, the expression of which is under the regulation of Wnt signaling and IGF-1. Our initial characterization supports the notion that CCN5 promotes the proliferation and survival of pancreatic β-cells leading to metabolic benefits [1]. Recently, the effects of CCN5 gene deficiency and ectopic, transgenic overexpression of CCN5 have been established. A systemic deficiency of CCN5 gene expression caused adipocyte hypertrophy, increased adipogenesis, and lipid accumulation, resulting in insulin resistance and glucose intolerance which are further exacerbated upon high-fat diet (HFD) feeding [2]. On the other hand, an adipocyte-specific and systemic overexpression of CCN5 caused an increase in lean body mass, improved insulin sensitivity, hyperplasia of cardiomyocytes and increased heart mass but decreased fasting glucose levels [3]. CCN5 is clearly a regulator of adipocyte proliferation and maturation, affecting lean/fat mass ratio and insulin sensitivity. In order to consolidate those findings and further establish the metabolic roles played by endogenous CCN5, we characterized CCN5 knockout mice fed in chow diet or 60% HFD. Unlike being reported [2], however, CCN5 knockout mice in our hands, both male and female, exhibited no significant change in lean/fat mass, insulin sensitivity, nor glucose tolerance when fed a chow diet, despite 2-5-fold elevations in serum insulin concentration. Upon HFD feeding, CCN5 knockout mice gained similar amounts of weight as their wild-type counterparts (male 2.0 vs. 1.9-fold; female 2.7 vs. 2.4-fold) but demonstrated sexual dimorphic changes in insulin sensitivity. Interestingly, male knockout mice displayed significant improvements in insulin sensitivity and glucose tolerance, despite being equally obese as their wild-type controls. The smaller improvements in female knockout mice were not as significant. At the end of the 27 weeks of HFD, the increased levels of serum insulin in response to obesity, were 30-60% lower in knockout mice (both male and female) than in wild-type counterparts, also supporting improved insulin sensitivity. Although our previous work has demonstrated that CCN5 stimulates pancreatic β-cell proliferation and survival, our assessment of the CCN5 knockout mice seems to indicate that normal, endogenous expression of CCN5/WISP2 gene is rather detrimental to metabolic compensations against diet-induced obesity, especially in male mice. These observations are not only unexpected but also contradicts to a previous report [2]. We are further characterizing potential changes in pancreatic islet morphometry and molecular markers of beta-cell function, in knockout vs. wild-type mice after HFD feeding. References 1. Chowdhury, S.; et al. Endocrinology 2014, 155, 1629-1642. 2. Kim, J.; et al. PLoS One 2018, 13, e0207228. 3. Grunberg, J.R.; et al. Sci Rep 2017, 7, 43515. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Many species regenerate lost body parts following amputation. Most limb regeneration research has focused on the immediate injury site. Meanwhile, body-wide injury responses remain largely unexplored but may be critical for regeneration. Here, we discovered a role for the sympathetic nervous system in stimulating a body-wide stem cell activation response to amputation that drives enhanced limb regeneration in axolotls. This response is mediated by adrenergic signaling, which coordinates distant cellular activation responses via the α -adrenergic receptor, and local regeneration responses via β-adrenergic receptors. Both α - and β-adrenergic signaling act upstream of mTOR signaling. Notably, systemically-activated axolotls regenerate limbs faster than naïve animals, suggesting a potential selective advantage in environments where injury from cannibalism or predation is common. This work challenges the predominant view that cellular responses underlying regeneration are confined to the injury site and argues instead for body-wide cellular priming as a foundational step that enables localized tissue regrowth.

Salamanders are capable of regenerating whole limbs throughout life, a feat that is unmatched within tetrapods. Limb regeneration is dependent upon the formation of a blastema, which contains undifferentiated cells capable of giving rise to most cells of the regenerated limb. Innervation is required for regeneration, along with many signaling pathways, including FGF, BMP and Wnt, but the role of VEGF signaling during salamander limb regeneration is not well understood. Here we show that VEGF signaling is essential for limb regeneration and that blastema cells and limb fibroblasts display impaired proliferation in the absence of VEGF signaling. By performing analogous experiments in planaria, which lack vasculature, we show a potential evolutionarily conserved role for VEGF in the expansion of blastema tissues that is separable from angiogenesis. Moreover, loss of VEGF signaling reduces induction of EMT-like processes, suggesting VEGF signaling functions upstream of the expression of EMT transcription factors, including Snai2. These findings highlight potential roles for VEGF signaling during regeneration which may extend beyond typical findings related to angiogenesis.

Growth of long bones and vertebrae is maintained postnatally by a long-lasting pool of progenitor cells. Little is known about the molecular mechanisms that regulate the output and maintenance of the cells that give rise to mature cartilage. Here we demonstrate that postnatal chondrocyte-specific deletion of a transcription factor Stat3 results in severely reduced proliferation coupled with increased hypertrophy, growth plate fusion, stunting and signs of progressive dysfunction of the articular cartilage. This effect is dimorphic, with females more strongly affected than males. Chondrocyte-specific deletion of the IL-6 family cytokine receptor gp130, which activates Stat3, phenocopied Stat3-deletion; deletion of Lifr, one of many co-receptors that signals through gp130, resulted in a milder phenotype. These data define a new molecular circuit that regulates chondrogenic cell maintenance and output and reveals a novel, hitherto unrecognized function of IL-6 cytokines in the skeletal system with direct implications for skeletal development and regeneration. Competing Interest Statement The authors have declared no competing interest.