Notch Signaling Pathway in Tooth Shape Variations throughout Evolution.

Evolutionary changes in vertebrates are linked to genetic alterations that often affect tooth crown shape, which is a criterion of speciation events. The Notch pathway is highly conserved between species and controls morphogenetic processes in most developing organs, including teeth. Epithelial loss of the Notch-ligand Jagged1 in developing mouse molars affects the location, size and interconnections of their cusps that lead to minor tooth crown shape modifications convergent to those observed along Muridae evolution. RNA sequencing analysis revealed that these alterations are due to the modulation of more than 2000 genes and that Notch signaling is a hub for significant morphogenetic networks, such as Wnts and Fibroblast Growth Factors. The modeling of these tooth crown changes in mutant mice, via a three-dimensional metamorphosis approach, allowed prediction of how Jagged1-associated mutations in humans could affect the morphology of their teeth. These results shed new light on Notch/Jagged1-mediated signaling as one of the crucial components for dental variations in evolution.

Fibroblast Growth Factors for Nonalcoholic Fatty Liver Disease: Opportunities and Challenges.

Nonalcoholic fatty liver disease (NAFLD), a chronic condition associated with metabolic dysfunction and obesity, has reached epidemic proportions worldwide. Although early NAFLD can be treated with lifestyle changes, the treatment of advanced liver pathology, such as nonalcoholic steatohepatitis (NASH), remains a challenge. There are currently no FDA-approved drugs for NAFLD. Fibroblast growth factors (FGFs) play essential roles in lipid and carbohydrate metabolism and have recently emerged as promising therapeutic agents for metabolic diseases. Among them, endocrine members (FGF19 and FGF21) and classical members (FGF1 and FGF4) are key regulators of energy metabolism. FGF-based therapies have shown therapeutic benefits in patients with NAFLD, and substantial progress has recently been made in clinical trials. These FGF analogs are effective in alleviating steatosis, liver inflammation, and fibrosis. In this review, we describe the biology of four metabolism-related FGFs (FGF19, FGF21, FGF1, and FGF4) and their basic action mechanisms, and then summarize recent advances in the biopharmaceutical development of FGF-based therapies for patients with NAFLD.

Focusing on Phosphorus Loads: From Healthy People to Chronic Kidney Disease.

Phosphorus is an essential micromineral with a key role in cellular metabolism and tissue structure. Serum phosphorus is maintained in a homeostatic range by the intestines, bones, and kidneys. This process is coordinated by the endocrine system through the highly integrated actions of several hormones, including FGF23, PTH, Klotho, and 1,25D. The excretion kinetics of the kidney after diet phosphorus load or the serum phosphorus kinetics during hemodialysis support that there is a "pool" for temporary phosphorus storage, leading to the maintenance of stable serum phosphorus levels. Phosphorus overload refers to a state where the phosphorus load is higher than is physiologically necessary. It can be caused by a persistently high-phosphorus diet, renal function decline, bone disease, insufficient dialysis, and inappropriate medications, and includes but is not limited to hyperphosphatemia. Serum phosphorus is still the most commonly used indicator of phosphorus overload. Trending phosphorus levels to see if they are chronically elevated is recommended instead of a single test when judging phosphorus overload. Future studies are needed to validate the prognostic role of a new marker or markers of phosphorus overload.

Short-term semaglutide treatment improves FGF21 responsiveness in primary hepatocytes isolated from high fat diet challenged mice.

Metabolic functions of GLP-1 and its analogues have been extensively investigated. In addition to acting as an incretin and reducing body weight, we and others have suggested the existence of GLP-1/fibroblast growth factor 21 (FGF21) axis in which liver mediates certain functions of GLP-1 receptor agonists. In a more recent study, we found with surprise that four-week treatment with liraglutide but not semaglutide stimulated hepatic FGF21 expression in HFD-challenged mice. We wondered whether semaglutide can also improve FGF21 sensitivity or responsiveness and hence triggers the feedback loop in attenuating its stimulation on hepatic FGF21 expression after a long-term treatment. Here, we assessed effect of daily semaglutide treatment in HFD-fed mice for 7 days. HFD challenge attenuated effect of FGF21 treatment on its downstream events in mouse primary hepatocytes, which can be restored by 7-day semaglutide treatment. In mouse liver, 7-day semaglutide treatment stimulated FGF21 as well as genes that encode its receptor (FGFR1) and the obligatory co-receptor (KLB), and a battery of genes that are involved in lipid homeostasis. In epididymal fat tissue, expressions of a battery genes including Klb affected by HFD challenge were reversed by 7-day semaglutide treatment. We suggest that semaglutide treatment improves FGF21 sensitivity which is attenuated by HFD challenge.

Protection from alcohol intoxication: Must be FGF21 to enter.

Fibroblast growth factor 21 (FGF21) is generally known as a stress-induced metabolic regulator with enormous therapeutic potential to treat metabolic diseases, but a more specific role of FGF21 concerns physiological handling of alcohol in mammals. In this issue of Cell Metabolism, Choi et al. demonstrate that FGF21 mediates the recovery from alcohol intoxication by directly activating noradrenergic neurons in mice, thus advancing our knowledge on FGF21 biology and further diversifying its therapeutic potential.

FGF21 counteracts alcohol intoxication by activating the noradrenergic nervous system.

Animals that consume fermenting fruit and nectar are at risk of exposure to ethanol and the detrimental effects of inebriation. In this report, we show that the hormone FGF21, which is strongly induced by ethanol in murine and human liver, stimulates arousal from intoxication without changing ethanol catabolism. Mice lacking FGF21 take longer than wild-type littermates to recover their righting reflex and balance following ethanol exposure. Conversely, pharmacologic FGF21 administration reduces the time needed for mice to recover from ethanol-induced unconsciousness and ataxia. FGF21 did not counteract sedation caused by ketamine, diazepam, or pentobarbital, indicating specificity for ethanol. FGF21 mediates its anti-intoxicant effects by directly activating noradrenergic neurons in the locus coeruleus region, which regulates arousal and alertness. These results suggest that this FGF21 liver-brain pathway evolved to protect against ethanol-induced intoxication and that it might be targeted pharmaceutically for treating acute alcohol poisoning.

FGF19-mediated ELF4 overexpression promotes colorectal cancer metastasis through transactivating FGFR4 and SRC.

Background: Metastasis accounts for the high lethality of colorectal cancer (CRC) patients. Unfortunately, the molecular mechanism manipulating metastasis in CRC is still elusive. Here, we investigated the function of E74-like factor 4 (ELF4), an ETS family member, in facilitating CRC progression. Methods: The expression of ELF4 in human CRC samples and CRC cell lines was determined by quantitative real-time PCR, immunohistochemistry and immunoblotting. The migratory and invasive phenotypes of CRC cells were evaluated by in vitro transwell assays and in vivo metastatic models. The RNA sequencing was used to explore the downstream targets of ELF4. The luciferase reporter assays and chromatin immunoprecipitation assays were used to ascertain the transcriptional regulation related to ELF4. Results: We found elevated ELF4 was positively correlated with distant metastasis, advanced AJCC stages, and dismal outcomes in CRC patients. ELF4 expression was also an independent predictor of poor prognosis. Overexpression of ELF4 boosted CRC metastasis via transactivating its downstream target genes, fibroblast growth factor receptor 4 (FGFR4) and SRC proto-oncogene, non-receptor tyrosine kinase, SRC. Fibroblast growth factor 19 (FGF19) upregulated ELF4 expression through the ERK1/2/SP1 axis. Clinically, ELF4 expression had a positive correlation with FGF19, FGFR4 and SRC, and CRC patients who positively coexpressed FGF19/ELF4, ELF4/FGFR4, or ELF4/SRC exhibited the worst clinical outcomes. Furthermore, the combination of the FGFR4 inhibitor BLU-554 and the SRC inhibitor KX2-391 dramatically suppressed ELF4-mediated CRC metastasis. Conclusions: We demonstrated the essentiality of ELF4 in the metastatic process of CRC, and targeting the ELF4-relevant positive feedback circuit might represent a novel therapeutic strategy.

Differential regulation of cardiac sodium channels by intracellular fibroblast growth factors.

Voltage-gated sodium (NaV) channels are responsible for the initiation and propagation of action potentials. In the heart, the predominant NaV1.5 α subunit is composed of four homologous repeats (I-IV) and forms a macromolecular complex with multiple accessory proteins, including intracellular fibroblast growth factors (iFGF). In spite of high homology, each of the iFGFs, iFGF11-iFGF14, as well as the individual iFGF splice variants, differentially regulates NaV channel gating, and the mechanisms underlying these differential effects remain elusive. Much of the work exploring iFGF regulation of NaV1.5 has been performed in mouse and rat ventricular myocytes in which iFGF13VY is the predominant iFGF expressed, whereas investigation into NaV1.5 regulation by the human heart-dominant iFGF12B is lacking. In this study, we used a mouse model with cardiac-specific Fgf13 deletion to study the consequences of iFGF13VY and iFGF12B expression. We observed distinct effects on the voltage-dependences of activation and inactivation of the sodium currents (INa), as well as on the kinetics of peak INa decay. Results in native myocytes were recapitulated with human NaV1.5 heterologously expressed in Xenopus oocytes, and additional experiments using voltage-clamp fluorometry (VCF) revealed iFGF-specific effects on the activation of the NaV1.5 voltage sensor domain in repeat IV (VSD-IV). iFGF chimeras further unveiled roles for all three iFGF domains (i.e., the N-terminus, core, and C-terminus) on the regulation of VSD-IV, and a slower time domain of inactivation. We present here a novel mechanism of iFGF regulation that is specific to individual iFGF isoforms and that leads to distinct functional effects on NaV channel/current kinetics.

HMGA1 induces FGF19 to drive pancreatic carcinogenesis and stroma formation.

High mobility group A1 (HMGA1) chromatin regulators are upregulated in diverse tumors where they portend adverse outcomes, although how they function in cancer remains unclear. Pancreatic ductal adenocarcinomas (PDACs) are highly lethal tumors characterized by dense desmoplastic stroma composed predominantly of cancer-associated fibroblasts and fibrotic tissue. Here, we uncover an epigenetic program whereby HMGA1 upregulates FGF19 during tumor progression and stroma formation. HMGA1 deficiency disrupts oncogenic properties in vitro while impairing tumor inception and progression in KPC mice and subcutaneous or orthotopic models of PDAC. RNA sequencing revealed HMGA1 transcriptional networks governing proliferation and tumor-stroma interactions, including the FGF19 gene. HMGA1 directly induces FGF19 expression and increases its protein secretion by recruiting active histone marks (H3K4me3, H3K27Ac). Surprisingly, disrupting FGF19 via gene silencing or the FGFR4 inhibitor BLU9931 recapitulates most phenotypes observed with HMGA1 deficiency, decreasing tumor growth and formation of a desmoplastic stroma in mouse models of PDAC. In human PDAC, overexpression of HMGA1 and FGF19 defines a subset of tumors with extremely poor outcomes. Our results reveal what we believe is a new paradigm whereby HMGA1 and FGF19 drive tumor progression and stroma formation, thus illuminating FGF19 as a rational therapeutic target for a molecularly defined PDAC subtype.

Direct and indirect effects of fibroblast growth factor 23 on the heart.

Fibroblast growth factor (FGF)23 is a bone-derived phosphotropic hormone that regulates phosphate and mineral homeostasis. Recent studies have provided evidence that a high plasma concentration of FGF23 is associated with cardiac disease, including left ventricular hypertrophy (LVH), heart failure, atrial fibrillation, and cardiac death. Experimental studies have shown that FGF23 activates fibroblast growth factor receptor 4 (FGFR4)/phospholipase Cγ/calcineurin/nuclear factor of activated T-cells signaling in cardiomyocytes and induces cardiac hypertrophy in rodents. Activation of FGFR4 by FGF23 normally requires the co-receptor α-klotho, and klotho-independent signaling occurs only under conditions characterized by extremely high FGF23 concentrations. Recent studies have demonstrated that FGF23 activates the renin-angiotensin-aldosterone system (RAAS) and induces LVH, at least in part as a result of lower vitamin D activation. Moreover, crosstalk between FGF23 and RAAS results in the induction of cardiac hypertrophy and fibrosis. In this review, we summarize the results of studies regarding the relationships between FGF23 and cardiac events, and describe the potential direct and indirect mechanisms whereby FGF23 induces LVH.

Fibroblast growth factor 21 inhibits vascular calcification by ameliorating oxidative stress of vascular smooth muscle cells.

Vascular calcification is very common in clinical. Severe vascular calcification is related to the occurrence of adverse events. Oxidative stress (OS) plays a pathophysiological role in the formation of vascular calcification. Previous studies have demonstrated that fibroblast growth factor 21(FGF21) could inhibit vascular calcification both in vivo and in vitro. FGF21 has also been proved to promote the recovery of superoxide dismutase (SOD) and thereby alleviate OS. Thus, our assumption was that FGF21 inhibit vascular calcification partly by restoring the level of antioxidant SOD and reducing OS. In this study, we established the vascular calcification by 5/6 nephrectomy plus high phosphate diet chronic kidney disease (CKD) model. The results showed the receptor of FGF21, fibroblast growth factor receptor 1 (FGFR1) and ßKlotho in the aorta increased in CKD group, and mainly located in the media of the artery. Ulteriorly, immunofluorescence (IF) and IHC staining showed that FGFR1 and ßKlotho mainly existed in arterial vascular smooth muscle cells (VSMCs). When FGF21 was knock out, the calcification was more severe in FGF21 KO + CKD mice, compared to wild type (WT)+ CKD mice. The transcriptional level of vascular calcification-related genes was significantly higher in FGF21 KO mice than control group. The dihydroethidium (DHE) staining reactive oxygen species (ROS) level in the CKD group was higher compared to the control group, but lower in FGF21 KO + CKD group, and the transcriptional level of SOD1 and SOD2 in FGF21 KO + CKD group was significantly higher than that in CKD group. In conclusion, FGF21 could inhibit vascular calcification, partly by restoring the level of antioxidant SOD and reducing vascular oxidative stress. This study provides further evidence for FGF21 as a candidate drug for cardiovascular protective agents.

A human laboratory study on the link between alcohol administration and circulating fibroblast growth factor 21 (FGF21) in individuals with alcohol use disorder.

Growing evidence indicates that the crosstalk between the central nervous system and the periphery plays an important role in the pathophysiology of neuropsychiatric conditions, including addictive disorders. Fibroblast growth factor 21 (FGF21) is part of the liver-brain axis and regulates energy homeostasis, metabolism, and macronutrient intake. In addition, FGF21 signaling modulates alcohol intake and preference, and changes in FGF21 levels are observed following alcohol consumption. To further elucidate the relationship between alcohol use and FGF21, we assessed serum FGF21 concentrations in 16 non-treatment seeking individuals with alcohol use disorder (AUD) in a naturalistic outpatient setting, as well as a controlled laboratory experiment that included alcohol cue-reactivity, alcohol priming, and alcohol self-administration in a bar-like setting. FGF21 levels were stable during the outpatient phase when participants received placebo and had no significant lifestyle changes. During the bar-like laboratory experiment, a robust increase in serum FGF21 concentrations was found after the 2-hr alcohol self-administration session (F3, 49 = 23.39, p < 0.001). Percent change in FGF21 levels positively correlated with the amount of alcohol self-administered but did not reach statistical significance. No significant changes in FGF21 levels were found after exposure to alcohol cues or consuming the priming drink. Given the bidirectional link between FGF21 and alcohol, targeting the FGF21 system may be further examined as a potential pharmacotherapy for AUD.

Heparin is essential for optimal cell signaling by FGF21 and for regulation of ßKlotho cellular stability.

While important insights were gained about how FGF21 and other endocrine fibroblast growth factors (FGFs) bind to Klotho proteins, the exact mechanism of Klotho/FGF receptor assembly that drives receptor dimerization and activation has not been elucidated. The prevailing dogma is that Klotho proteins substitute for the loss of heparan sulfate proteoglycan (HSPG) binding to endocrine FGFs by high-affinity binding of endocrine FGF molecules to Klotho receptors. To explore a potential role of HSPG in FGF21 signaling, we have analyzed the dynamic properties of FGF21-induced FGF21-ßKlotho-FGFR1c complexes on the surface of living wild-type (WT) or HSPG-deficient Chinese hamster ovary (CHO) cells by employing quantitative single-molecule fluorescence imaging analyses. Moreover, detailed analyses of FGF21 and FGF1 stimulation of cellular signaling pathways activated in WT or in HSPG-deficient CHO cells are also analyzed and compared. These experiments demonstrate that heparin is required for the formation of FGF21-ßKlotho-FGFR1c complexes on the cell membrane and that binding of heparin or HSPG to FGFR1c is essential for optimal FGF21 stimulation of FGFR1c activation, mitogen-activated protein kinase responses, and intracellular Ca2+ release. It is also shown that FGF1 binding stimulates assembly of ßKlotho and FGFR1c on cell membranes, resulting in endocytosis and degradation of ßKlotho. We conclude that heparin or HSPG is essential for FGF21 signaling and for regulation of ßKlotho cellular stability by acting as a coligand of FGFR1c.

Identification, Characterization and Functional Analysis of Fibroblast Growth Factors in Black Rockfish (Sebastes schlegelii).

Fibroblast growth factors (FGFs) are short polypeptides that play essential roles in various cellular biological processes, including cell migration, proliferation, and differentiation, as well as tissue regeneration, immune response, and organogenesis. However, studies focusing on the characterization and function of FGF genes in teleost fishes are still limited. In this study, we identified and characterized expression patterns of 24 FGF genes in various tissues of embryonic and adult specimens of the black rockfish (Sebates schlegelii). Nine FGF genes were found to play essential roles in myoblast differentiation, as well as muscle development and recovery in juvelines of S. schlegelii. Moreover, sex-biased expression pattern of multiple FGF genes was recorded in the species' gonads during its development. Among them, expression of the FGF1 gene was recorded in interstitial and sertoli cells of testes, promoting germ-cell proliferation and differentiation. In sum, the obtained results enabled systematic and functional characterization of FGF genes in S. schlegelii, laying a foundation for further studies on FGF genes in other large teleost fishes.

Postprandial triglycerides and fibroblast growth factor 19 as potential screening tools for paediatric non-alcoholic fatty liver disease.

BACKGROUND: Better screening tools for paediatric NAFLD are needed. We tested the hypothesis that the postprandial triglyceride (TG) and fibroblast growth factor 19 (FGF19) response to an abbreviated fat tolerance test (AFTT) could differentiate adolescents with NAFLD from peers with obesity and normal weight. METHODS: Fifteen controls with normal weight (NW), 13 controls with obesity (OB) and 9 patients with NAFLD completed an AFTT. Following an overnight fast, participants consumed a high-fat meal. TG and FGF19 were measured at baseline and 4 h post-meal. Liver steatosis and fibrosis were measured via Fibroscan. RESULTS: Fasting TG and FGF19 did not differ among groups; 4 h TG in the NAFLD and OB groups were greater (197 ± 69 mg/dL; 157 ± 72 mg/dL, respectively) than NW (105 ± 45 mg/dL; p < 0.05) and did not differ from one another. Within the entire cohort, 4 h TG were stratified by high and low steatosis. Adolescents with high steatosis had 98% greater 4 h TG than adolescents with low steatosis. 4 h FGF19, but not fasting FGF19, was higher in children with low steatosis compared with high steatosis (p < 0.05). Using area under the receiver operating curve (AUROC), the only biochemical outcome with diagnostic accuracy for NAFLD was 4 h TG (0.77 [95% CI: 0.60-0.94; p = 0.02]). CONCLUSIONS: The postprandial TG response is increased in adolescents with obesity with hepatic steatosis, with or without NAFLD. Our preliminary analysis demonstrates 4 h TG differentiate patients with NAFLD from those without, supporting a role for the AFTT as a screening tool for paediatric NAFLD.

Quantifying endodermal strains during heart tube formation in the developing chicken embryo.

In the early avian embryo, the developing heart forms when bilateral fields of cardiac progenitor cells, which reside in the lateral plate mesoderm, move toward the embryonic midline, and fuse above the anterior intestinal portal (AIP) to form a straight, muscle-wrapped tube. During this process, the precardiac mesoderm remains in close contact with the underlying endoderm. Previous work has shown that the endoderm around the AIP actively contracts to pull the cardiac progenitors toward the midline. The morphogenetic deformations associated with this endodermal convergence, however, remain unclear, as do the signaling pathways that might regulate this process. Here, we fluorescently labeled populations of endodermal cells in early chicken embryos and tracked their motion during heart tube formation to compute time-varying strains along the anterior endoderm. We then determined how the computed endodermal strain distributions are affected by the pharmacological inhibition of either myosin II or fibroblast growth factor (FGF) signaling. Our data indicate that a mediolateral gradient in endodermal shortening is present around the AIP, as well as substantial convergence and extension movements both anterior and lateral to the AIP. These active endodermal deformations are disrupted if either actomyosin contractility or FGF signaling are inhibited pharmacologically. Taken together, these results demonstrate how active deformations along the anterior endoderm contribute to heart tube formation within the developing embryo.

Fibroblast growth factor 19 secretion and function in perinatal development.

Limited work has focused on fibroblast growth factor-19 (FGF19) secretion and function in the perinatal period. FGF19 is a potent growth factor that coordinates development of the brain, eye, inner ear, and skeletal system in the embryo, but after birth, FGF19 transitions to be an endocrine regulator of the classic pathway of hepatic bile acid synthesis. FGF19 has emerged as a mediator of metabolism and bile acid synthesis in aged animals and adults in the context of liver disease and metabolic dysfunction. FGF19 has also been shown to have systemic insulin-sensitizing and skeletal muscle hypertrophic effects when induced or supplemented at supraphysiological levels in adult rodent models. These effects could be beneficial to improve growth and nutritional outcomes in preterm infants, which are metabolically resistant to the anabolic effects of enteral nutrition. Existing clinical data on FGF19 secretion and function in the perinatal period in term and preterm infants has been equivocal. Studies in pigs show that FGF19 expression and secretion are upregulated with gestational age and point to molecular and endocrine factors that may be involved. Work focused on FGF19 in pediatric diseases suggests that augmentation of FGF19 secretion by activation of gut FXR signaling is associated with benefits in diseases such as short bowel syndrome, parenteral nutrition-associated liver disease, and biliary atresia. Future work should focus on characterization of FGF19 secretion and the mechanism underpinning the transition of FGF19 function as an embryological growth factor to metabolic and bile acid regulator.

Exercise and chronic kidney disease: potential mechanisms underlying the physiological benefits.

Increasing evidence indicates that exercise has beneficial effects on chronic inflammation, cardiorespiratory function, muscle and bone strength and metabolic markers in adults with chronic kidney disease (CKD), kidney failure or kidney transplants. However, the mechanisms that underlie these benefits have received little attention, and the available clinical evidence is mainly from small, short-duration (<12 weeks) exercise intervention studies. The available data, mainly from patients with CKD or on dialysis, suggest that exercise-mediated shifts towards a less inflammatory immune cell profile, enhanced activity of the NRF2 pathway and reduced monocyte infiltration into adipose tissue may underlie improvements in inflammatory biomarkers. Exercise-mediated increases in nitric oxide release and bioavailability, reduced angiotensin II accumulation in the heart, left ventricular remodelling and reductions in myocardial fibrosis may contribute to improvements in left ventricular hypertrophy. Exercise stimulates an anabolic response in skeletal muscle in CKD, but increases in mitochondrial mass and satellite cell activation seem to be impaired in this population. Exercise-mediated activation of the canonical wnt pathway may lead to bone formation and improvements in the levels of the bone-derived hormones klotho and fibroblast growth factor 23 (FGF23). Longer duration studies with larger sample sizes are needed to confirm these mechanisms in CKD, kidney failure and kidney transplant populations and provide evidence for targeted exercise interventions.

Ascidian gastrulation and blebbing activity of isolated endoderm blastomeres.

Gastrulation is the first dynamic cell movement during embryogenesis. Endoderm and mesoderm cells are internalized into embryos during this process. Ascidian embryos provide a simple system for studying gastrulation in chordates. Gastrulation starts in spherical late 64-cell embryos with 10 endoderm blastomeres. The mechanisms of gastrulation in ascidians have been investigated, and a two-step model has been proposed. The first step involves apical constriction of endoderm cells, followed by apicobasal shortening in the second step. In this study, isolated ascidian endoderm progenitor cells displayed dynamic blebbing activity at the gastrula stage, although such a dynamic cell-shape change was not recognized in toto. Blebbing is often observed in migrating animal cells. In ascidians, endoderm cells displayed blebbing activity, while mesoderm and ectoderm cells did not. The timing of blebbing of isolated endoderm cells coincided with that of cell invagination. The constriction rate of apical surfaces correlated with the intensity of blebbing activity in each endoderm-lineage cell. Fibroblast growth factor (FGF) signaling was both necessary and sufficient for inducing blebbing activity, independent of cell fate specification. In contrast, the timing of initiation of blebbing and intensity of blebbing response to FGF signaling were controlled by intrinsic cellular factors. It is likely that the difference in intensity of blebbing activity between the anterior A-line and posterior B-line cells could account for the anteroposterior difference in the steepness of the archenteron wall. Inhibition of zygotic transcription, FGF signaling, and Rho kinase, all of which suppressed blebbing activity, resulted in incomplete apical constriction and failure of the eventual formation of cup-shaped gastrulae. Blebbing activity was involved in the progression and maintenance of apical constriction, but not in apicobasal shortening in whole embryos. Apical constriction is mediated by distinct blebbing-dependent and blebbing-independent mechanisms. Surface tension and consequent membrane contraction may not be the sole mechanical force for apical constriction and formation of cup-shaped gastrulae. The present study reveals the hidden cellular potential of endodermal cells during gastrulation and discusses the possible roles of blebbing in the invagination process.

Heating-mediated purification of active FGF21 and structure-based design of its variant with enhanced potency.

Fibroblast growth factor 21 (FGF21) has pharmaceutical potential against obesity-related metabolic disorders, including non-alcoholic fatty liver disease. Since thermal stability is a desirable factor for therapeutic proteins, we investigated the thermal behavior of human FGF21. FGF21 remained soluble after heating; thus, we examined its temperature-induced structural changes using circular dichroism (CD). FGF21 showed inter-convertible temperature-specific CD spectra. The CD spectrum at 100 °C returned to that at 20 °C when the heated FGF21 solution was cooled. Through loop swapping, the connecting loop between ß10 and ß12 in FGF21 was revealed to be associated with the unique thermal behavior of FGF21. According to surface plasmon resonance (SPR) experiments, in vitro cell-based assays, and model high-fat diet (HFD)-induced obesity studies, heated FGF21 maintained biological activities that were comparable to those of non-heated and commercial FGF21s. Based on sequence comparison and structural analysis, five point-mutations were introduced into FGF21. Compared with the wild type, the heated FGF21 variant displayed improved therapeutic potential in terms of body weight loss, the levels of hepatic triglycerides and lipids, and the degree of vacuolization of liver in HFD-fed mice.