Age-Associated Differences in Recovery from Exercise-Induced Muscle Damage.

Understanding the intricate mechanisms governing the cellular response to resistance exercise is paramount for promoting healthy aging. This narrative review explored the age-related alterations in recovery from resistance exercise, focusing on the nuanced aspects of exercise-induced muscle damage in older adults. Due to the limited number of studies in older adults that attempt to delineate age differences in muscle discovery, we delve into the multifaceted cellular influences of chronic low-grade inflammation, modifications in the extracellular matrix, and the role of lipid mediators in shaping the recovery landscape in aging skeletal muscle. From our literature search, it is evident that aged muscle displays delayed, prolonged, and inefficient recovery. These changes can be attributed to anabolic resistance, the stiffening of the extracellular matrix, mitochondrial dysfunction, and unresolved inflammation as well as alterations in satellite cell function. Collectively, these age-related impairments may impact subsequent adaptations to resistance exercise. Insights gleaned from this exploration may inform targeted interventions aimed at enhancing the efficacy of resistance training programs tailored to the specific needs of older adults, ultimately fostering healthy aging and preserving functional independence.

Lectin-mediated, time-efficient, and high-yield sorting of different morphologically intact nephron segments.

The kidney is a highly complex organ equipped with a multitude of miniscule filter-tubule units called nephrons. Each nephron can be subdivided into multiple segments, each with its own morphology and physiological function. To date, conventional manual approaches to isolate specific nephron segments are very laborious, time-consuming, often limited to only a specific segment, and typically have low yield. Here, we describe a novel, unconventional method that is superior in many aspects to previous protocols by combining low-cost fluorophore-conjugated lectins or agglutinins (Flaggs) with flow sorting. This allows the simultaneous separation of different nephron segments with preserved 3D morphology from mouse or human samples in under 3 h. Using a 200-µm nozzle and 5 psi, glomeruli, proximal, or distal convoluted tubules are sorted with Cy3-labeled Sambucus Nigra agglutinin (SNA-Cy3), Fluorescein-labeled Lotus Tetragonolobus lectin (LTL-FITC), or Pacific Blue-labeled soybean agglutinin (SBA-PB), respectively. Connecting tubules and collecting ducts are sorted by double-positive SBA-PB and SNA-Cy3 signals, while thick ascending limb segments are characterized by the absence of any Flaggs labeling. From two mouse kidneys, this yields 37-521 ng protein/s or 0.71-16.71 ng RNA/s, depending on the specific nephron segment. The purity of sorted segments, as assessed by mRNA expression level profiling of 15 genes, is very high with a 96.1-fold median enrichment across all genes and sorted segments. In summary, our method represents a simple, straightforward, cost-effective, and widely applicable tool yielding high amounts of pure and morphologically largely intact renal tubule materials with the potential to propel nephron segment-specific research.

Effects of an Omega-3 Supplemented, High-Protein Diet in Combination with Vibration and Resistance Exercise on Muscle Power and Inflammation in Old Adults: A Pilot Randomized Controlled Trial.

BACKGROUND: Inflammaging is considered to drive loss of muscle function. Omega-3 fatty acids exhibit anti-inflammatory properties. Therefore, we examined the effects of eight weeks of vibration and home-based resistance exercise combined with a whey-enriched, omega-3-supplemented diet on muscle power, inflammation and muscle biomarkers in community-dwelling old adults. METHODS: Participants were randomized to either exercise (3x/week, n = 20), exercise + high-protein diet (1.2-1.5 g/kg, n = 20), or exercise + high-protein and omega-3-enriched diet (2.2 g/day, n = 21). Muscle power (watt/m2) and chair rise test (CRT) time (s) were assessed via CRT measured with mechanography. Furthermore, leg strength (kg/m2) and fasting concentrations of inflammatory (interleukin (IL-) 6, IL-10, high-mobility group box-1 (HMGB-1)) and muscle biomarkers (insulin-like growth factor (IGF-) 1, IGF-binding protein-3, myostatin) were assessed. RESULTS: Sixty-one participants (70.6 ± 4.7 years; 47% men) completed the study. According to generalized linear mixed models, a high-protein diet improved leg strength and CRT time. Only IGF-1 increased with additional omega-3. Sex-specific analyses revealed that muscle power, IL-6, IL-6/IL-10 ratio, and HMGB-1 improved significantly in the male high-protein, omega-3-enriched group only. CONCLUSION: Vibration and home-based resistance exercise combined with a high-protein, omega-3-enriched diet increased muscle power and reduced inflammation in old men, but not in old women. While muscle biomarkers remained unchanged, a high-protein diet combined with exercise improved leg strength and CRT time.

Tissue chaperoning-the expanded functions of fetuin-A beyond inhibition of systemic calcification.

Traditionally, fetuin-A embodies the prototype anti-calcification protein in the blood, preventing cardiovascular calcification. Low serum fetuin-A is generally associated with mineralization dysbalance and enhanced mortality in end stage renal disease. Recent evidence indicates that fetuin-A is a crucial factor moderating tissue inflammation and fibrosis, as well as a systemic indicator of acute inflammatory disease. Here, the expanded function of fetuin-A is discussed in the context of mineralization and inflammation biology. Unbalanced depletion of fetuin-A in this context may be the critical event, triggering a vicious cycle of progressive calcification, inflammation, and tissue injury. Hence, we designate fetuin-A as tissue chaperone and propose the potential use of exogenous fetuin-A as prophylactic agent or emergency treatment in conditions that are associated with acute depletion of endogenous protein.

Dichotomous Responses to Chronic Fetal Hypoxia Lead to a Predetermined Aging Phenotype.

Hypoxia-induced intrauterine growth restriction increases the risk for cardiovascular, renal, and other chronic diseases in adults, representing thus a major public health problem. Still, not much is known about the fetal mechanisms that predispose these individuals to disease. Using a previously validated mouse model of fetal hypoxia and bottom-up proteomics, we characterize the response of the fetal kidney to chronic hypoxic stress. Fetal kidneys exhibit a dichotomous response to chronic hypoxia, comprising on the one hand cellular adaptations that promote survival (glycolysis, autophagy, and reduced DNA and protein synthesis), but on the other processes that induce a senescence-like phenotype (infiltration of inflammatory cells, DNA damage, and reduced proliferation). Importantly, chronic hypoxia also reduces the expression of the antiaging proteins klotho and Sirt6, a mechanism that is evolutionary conserved between mice and humans. Taken together, we uncover that predetermined aging during fetal development is a key event in chronic hypoxia, establishing a solid foundation for Barker's hypothesis of fetal programming of adult diseases. This phenotype is associated with a characteristic biomarker profile in tissue and serum samples, exploitable for detecting and targeting accelerated aging in chronic hypoxic human diseases.

Fetuin-A is a HIF target that safeguards tissue integrity during hypoxic stress.

Intrauterine growth restriction (IUGR) is associated with reduced kidney size at birth, accelerated renal function decline, and increased risk for chronic kidney and cardiovascular diseases in adults. Precise mechanisms underlying fetal programming of adult diseases remain largely elusive and warrant extensive investigation. Setting up a mouse model of hypoxia-induced IUGR, fetal adaptations at mRNA, protein and cellular levels, and their long-term functional consequences are characterized, using the kidney as a readout. Here, we identify fetuin-A as an evolutionary conserved HIF target gene, and further investigate its role using fetuin-A KO animals and an adult model of ischemia-reperfusion injury. Beyond its role as systemic calcification inhibitor, fetuin-A emerges as a multifaceted protective factor that locally counteracts calcification, modulates macrophage polarization, and attenuates inflammation and fibrosis, thus preserving kidney function. Our study paves the way to therapeutic approaches mitigating mineral stress-induced inflammation and damage, principally applicable to all soft tissues.

Nephrotoxicity of Calcineurin Inhibitors in Kidney Epithelial Cells is Independent of NFAT Signaling.

Background: Calcineurin inhibitors (CNIs) such as cyclosporine A and tacrolimus are commonly used after renal transplantation to suppress the immune system. In lymphoid cells, cyclosporine A acts via the calcineurin/nuclear factor of activated T-cell (NFAT) axis. In non-lymphoid cells, such as kidney epithelial cells, cyclosporine A induces calcineurin inhibitor toxicity. It is unknown via which off-targets cyclosporine A induces calcineurin inhibitor toxicity in kidney epithelial cells. Methods: To measure a compound's potential to induce nephrotoxicity, the expression of the surrogate marker Fn14 was measured by flow cytometry. Compounds were tested for their potential to induce Fn14 either chemically or plasmid-mediated. Mice were injected with various compounds, and changes in nephrotoxic gene expression levels of the kidney epithelial cells were then analyzed. Results: Fn14 is specifically upregulated due to calcineurin inhibitor toxicity inducing agents. Inhibition of the NFAT axis showed no increase of the Fn14 expression on the surface of kidney cells. However, inhibition of p38 MAPK, phosphoinositide-3-kinase (PI3K)/Akt, protein kinase C (PKC), and inhibitor of nuclear factor-κB (IκB) kinase (IKK) showed clear induction of Fn14 and increased expressions of nephrotoxic, inflammatory, and fibrotic genes in vitro and in vivo. Conclusions: These findings show that cyclosporine A acts independently of NFAT on kidney epithelial cells. Moreover, inhibition of serine/threonine protein kinases mimics cyclosporine A's activity on kidney epithelial cells. This mimicking effect indicates that these protein kinases are off-targets of cyclosporine A and damage structural renal cells when inhibited and therefore contributes likely to the development and progression of calcineurin inhibitor toxicity.

Physiological and Molecular Responses to Altered Sodium Intake in Rat Pregnancy.

Background In pregnancy, a high plasma volume maintains uteroplacental perfusion and prevents placental ischemia, a condition linked to elevated maternal blood pressure ( BP ). Reducing BP by increasing Na+ intake via plasma volume expansion appears contra-intuitive. We hypothesize that an appropriate Na+ intake in pregnancy reduces maternal BP and adapts the renin-angiotensin system in a pregnancy-specific manner. Methods and Results BP was measured by implanted telemetry in Sprague-Dawley rats before and throughout pregnancy. Pregnant and nonpregnant animals received either a normal-salt (0.4%; NS ), high-salt (8%; HS ), or low-salt (0.01%; LS ) diet, or HS (days 1-14) followed by LS (days 14-20) diet ( HS / LS ). Before delivery (day 20), animals were euthanized and organs collected. Food, water, and Na+ intake were monitored in metabolic cages, and urinary creatinine and Na+ were analyzed. Na+ intake and retention increased in pregnancy ( NS , LS ), leading to a positive Na+ balance ( NS , LS ). BP was stable during LS , but reduced in HS conditions in pregnancy. The renin-angiotensin system was adapted as expected. Activating cleavage of α- and γ-subunits of the renal epithelial Na+ channel and expression of-full length medullary ß-subunits, accentuated further in all LS conditions, were upregulated in pregnancy. Conclusions Pregnancy led to Na+ retention adapted to dietary changes. HS exposure paradoxically reduced BP . Na+ uptake while only modestly linked to the renin-angiotensin system is enhanced in the presence of posttranslational renal epithelial Na+ channel modifications. This suggests (1) storage of Na+ in pregnancy upon HS exposure, bridging periods of LS availability; and (2) that potentially non-renin-angiotensin-related mechanisms participate in EN aC activation and consecutive Na+ retention.

The TWEAK/Fn14 pathway is required for calcineurin inhibitor toxicity of the kidneys.

Calcineurin inhibitor toxicity (CNT) is a frequent occurrence in transplanted renal grafts and autochthone kidneys from patients undergoing long-term treatment with calcineurin inhibitors, notably cyclosporin A (CsA) and tacrolimus. Here, we show an indispensable role of the tumor necrosis factor superfamily (TNFS) molecule TNF-related weak inducer of apoptosis (TWEAK) (TNFSF12) in the pathogenesis of acute CNT lesions in mice. A deficiency in TWEAK resulted in limited tubulotoxicity after CsA exposure, which correlated with diminished expression of inflammatory cytokines and reduced intraparenchymal infiltration with immune cells. We further identified tubular epithelial cells of the kidney as major targets of CsA activity and found that Fn14 (tumor necrosis factor receptor superfamily 12A), the receptor for TWEAK, is a highly CsA-inducible gene in these cells. Correlating with this, CsA pretreatment sensitized tubular epithelial cells specifically to the pro-inflammatory activities of recombinant TWEAK in vitro. Moreover, injection of rTWEAK alone into mice induced moderate disease similar to CsA, and rTWEAK combined with CsA resulted in synergistic nephrotoxicity. These findings support the importance of tubular epithelial cells as cellular targets of CsA toxicity and introduce TWEAK as a critical contributor to CNT pathogenesis.

Reduced ß-catenin expression affects patterning of bone primordia, but not bone maturation.

Wnt/ß-catenin signaling is involved in patterning of bone primordia, but also plays an important role in the differentiation of chondrocytes and osteoblasts. During these processes the level of ß-catenin must be tightly regulated. Excess ß-catenin leads to conditions with increased bone mass, whereas loss of ß-catenin is associated with osteoporosis or, in extreme cases, the absence of limbs. In this study, we examined skeletogenesis in mice, which retain only 25% of ß-catenin. These embryos showed severe morphological abnormalities of which the lack of hindlimbs and misshaped front paws were the most striking. Surprisingly however, calcification of bone primordia occurred normally. Moreover, the Wnt-dependent regulatory network of transcription factors driving the differentiation of cartilage and bone, as well as the expression of extracellular matrix components, were preserved. These findings show that 25% ß-catenin is insufficient for the correct patterning of bone primordia, but sufficient for their mineralization. Our approach helps to identify bone morphogenetic processes that can proceed normally even at low ß-catenin levels, in contrast to those that require high ß-catenin dosages. This information could be exploited to improve the treatment of bone diseases by fine-tuning the individual ß-catenin dosage requirements.

Bidirectional signalling between EphA2 and ephrinA1 increases tubular cell attachment, laminin secretion and modulates erythropoietin expression after renal hypoxic injury.

Acute kidney injury (AKI) is common in hospitalized patients and has a poor prognosis, the severity of AKI being linked to progression to chronic kidney disease. This stresses the need to search for protective mechanisms during the acute phase. We investigated kidney repair after hypoxic injury using a rat model of renal artery branch ligation, which led to an oxygen gradient vertical to the corticomedullary axis. Three distinct zones were observed: tubular necrosis, infarction border zone and preserved normal tissue. EphA2 is a receptor tyrosine kinase with pivotal roles in cell architecture, migration and survival, upon juxtacrine contact with its membrane-bound ligand EphrinA1. Following hypoxia, EphA2 was up-regulated in cortical and medullary tubular cells, while EphrinA1 was up-regulated in interstitial cells adjacent to peritubular capillaries. Moreover, erythropoietin (EPO) messenger RNA (mRNA) was strongly expressed in the border zone of infarcted kidney within the first 6 h. To gain more insight into the biological impact of EphA2 and EphrinA1 up-regulation, we activated the signalling pathways in vitro using recombinant EphrinA1/Fc or EphA2/Fc proteins. Stimulation of EphA2 forward signalling in the proximal tubular cell line HK2 increased cell attachment and laminin secretion at the baso-lateral side. Conversely, activation of reverse signalling through EphrinA1 expressed by Hep3B cells promoted EPO production at both the transcriptional and protein level. Strikingly, in co-culture experiments, juxtacrine contact between EphA2 expressing MDCK and EphrinA1 expressing Hep3B was sufficient to induce a significant up-regulation of EPO mRNA production in the latter cells, even in the absence of hypoxic conditions. The synergistic effects of EphA2 and hypoxia led to a 15-20-fold increase of EPO expression. Collectively, our results suggest an important role of EphA2/EphrinA1 signalling in kidney repair after hypoxic injury through stimulation of (i) tubular cell attachment, (ii) secretion of basal membrane proteins and (iii) EPO production. These findings could thus pave the way to new therapeutic approaches.

Beta-catenin is vital for the integrity of mouse embryonic stem cells.

ß-Catenin mediated Wnt-signaling is assumed to play a major function in embryonic stem cells in maintaining their stem cell character and the exit from this unique trait. The complexity of ß-catenin action and conflicting results on the role of ß-catenin in maintaining the pluripotent state have made it difficult to understand its precise cellular and molecular functions. To attempt this issue we have generated new genetically modified mouse embryonic stem cell lines allowing for the deletion of ß-catenin in a controlled manner by taking advantage of the Cre-ER-T2 system and analyzed the effects in a narrow time window shortly after ablation. By using this approach, rather then taking long term cultured ß-catenin null cell lines we demonstrate that ß-catenin is dispensable for the maintenance of pluripotency associated genes. In addition we observed that the removal of ß-catenin leads to a strong increase of cell death, the appearance of multiple clustered functional centrosomes most likely due to a mis-regulation of the polo-like-kinase 2 and furthermore, alterations in chromosome segregation. Our study demonstrates the importance of ß-catenin in maintaining correct cellular functions and helps to understand its role in embryonic stem cells.

A thymosin beta15-like peptide promotes intersegmental myotome extension in the chicken embryo.

Beta-thymosins constitute a group of small actin-sequestering peptides. These highly conserved peptides are involved in cytoskeleton dynamics and can influence different cell properties such as motility, substrate adhesion, shape and chemotaxis. As a marker for tumour metastasis, the mammalian thymosin beta15 is believed to have an important diagnostic relevance in cancer prognosis, although little is known about its physiological function. In order to study the role of thymosin beta15(avian) in embryogenesis, we cloned the chicken and quail orthologues of thymosin beta15 and used the chicken as a model for vertebrate development. Avian thymosin beta15, the first known non-mammalian thymosin beta15-like gene, encodes a peptide that possesses a cysteine at position one after the methionine which is a significant difference compared to its mammalian counterparts. Thymosin beta15(avian) expression starts at an early stage of development. The expression pattern changes rapidly with development and differs from that of the related thymosin beta4 gene. The most prominent expression domain is seen in developing muscles of limbs and trunk. Gain-of-function experiments revealed that thymosin beta15(avian) has a function in normal myotome development. Ectopic over-expression of thymosin beta15(avian) leads to premature elongation of myotome cells trespassing segment borders. We conclude that thymosin beta15(avian) has a still undescribed function in promoting myocyte elongation.

E-cadherin is required for the proper activation of the Lifr/Gp130 signaling pathway in mouse embryonic stem cells.

The leukemia inhibitory factor (Lif) signaling pathway is a crucial determinant for mouse embryonic stem (mES) cell self-renewal and pluripotency. One of the hallmarks of mES cells, their compact growth morphology, results from tight cell adhesion mediated through E-cadherin, ß-catenin (Ctnnb1) and α-catenin with the actin cytoskeleton. ß-catenin is also involved in canonical Wnt signaling, which has also been suggested to control mES cell stemness. Here, we analyze Ctnnb1(-/-) mES cells in which cell adhesion is preserved by an E-cadherin-α-catenin (Eα) fusion protein (Ctnnb1(-/-)Eα mES cells), and show that mimicking only the adhesive function of ß-catenin is necessary and sufficient to maintain the mES cell state, making ß-catenin/Wnt signaling obsolete in this process. Furthermore, we propose a role for E-cadherin in promoting the Lif signaling cascade, showing an association of E-cadherin with the Lifr-Gp130 receptor complex, which is most likely facilitated by the extracellular domain of E-cadherin. Without Eα, and thus without maintained cell adhesion, Ctnnb1(-/-) mES cells downregulate components of the Lif signaling pathway, such as Lifr, Gp130 and activated Stat3, as well as pluripotency-associated markers. From these observations, we hypothesize that the changes in gene expression accompanying the loss of pluripotency are a direct consequence of dysfunctional cell adhesion. Supporting this view, we find that the requirement for intact adhesion can be circumvented by the forced expression of constitutively active Stat3. In summary, we put forward a model in which mES cells can be propagated in culture in the absence of Ctnnb1, as long as E-cadherin-mediated cell adhesion is preserved.

Differential requirements for ß-catenin during mouse development.

Embryogenesis relies on the precise interplay of signaling cascades to activate tissue-specific differentiation programs. An important player in these morphogenetic processes is ß-catenin, which is a central component of adherens junctions and canonical Wnt signaling. Lack of ß-catenin is lethal before gastrulation, but mice heterozygous for ß-catenin (Ctnnb1) develop as wild type. Here, we confine ß-catenin amounts below the heterozygous expression level to study the functional consequences for development. We generate embryonic stem (ES) cells and embryos expressing ß-catenin only from the ubiquitously active ROSA26 promoter and thereby limit ß-catenin expression to ~12.5% (ROSA26(ß/+)) or ~25% (ROSA26(ß/ß)) of wild-type levels. ROSA26(ß/+) is sufficient to maintain ES cell morphology and pluripotent characteristics, but is insufficient to activate canonical target genes upon Wnt stimulation. This Wnt signaling deficiency is incompletely restored in ROSA26(ß/ß) ES cells. We conclude that even very low ß-catenin levels are able to sustain cell adhesion, but not Wnt signaling. During development, ROSA26(ß/ß) as well as ROSA26(ß/+) partially rescues the knockout phenotype, yet proper gastrulation is absent. These embryos differentiate according to the neural default hypothesis, indicating that gastrulation depends on high ß-catenin levels. Strikingly, if ROSA26(ß/+) or ROSA26(ß/ß) is first activated after gastrulation, subsequent development correlates with the dosage of ß-catenin. Moreover, molecular evidence indicates that the amount of ß-catenin controls the induction of specific Wnt target genes. In conclusion, by restricting its expression we determine the level of ß-catenin required for adhesion or pluripotency and during different morphogenetic events.

Wnt/ß-catenin signaling regulates telomerase in stem cells and cancer cells.

Telomerase activity controls telomere length and plays a pivotal role in stem cells, aging, and cancer. Here, we report a molecular link between Wnt/ß-catenin signaling and the expression of the telomerase subunit Tert. ß-Catenin-deficient mouse embryonic stem (ES) cells have short telomeres; conversely, ES cell expressing an activated form of ß-catenin (ß-cat(ΔEx3/+)) have long telomeres. We show that ß-catenin regulates Tert expression through the interaction with Klf4, a core component of the pluripotency transcriptional network. ß-Catenin binds to the Tert promoter in a mouse intestinal tumor model and in human carcinoma cells. We uncover a previously unknown link between the stem cell and oncogenic potential whereby ß-catenin regulates Tert expression, and thereby telomere length, which could be critical in human regenerative therapy and cancer.

Avian somitogenesis: translating time and space into pattern.

Vertebrates have a metameric bodyplan that is based on the presence of paired somites. Somites develop from the segmental plate in a cranio-caudal sequence. At the same time, new material is added from Hensen's node, the primitive streak and the tailbud. In this way, the material residing in the segmental plate remains constant and comprises 12 prospective somites on each side. Prospective segment borders are not yet determined in the caudal segmental plate. Prior to segmentation, the cranial segmental plate undergoes epithelialization, which is controlled by signals from the neural tube and ectoderm. The bHLH transcription factor Paraxis is critically involved in this process. Formation of a new somite from the cranial end of the segmental plate is a highly controlled process involving complex cell movements in relation to each other. Hox genes specify regional identity of the somites and their derivatives. In the chicken a transposition of thoracic into cervical vertebrae has occurred as compared to the mouse. Transcription factors of the bHLH and homeodomain type also specify the cranio-caudal polarity and that of particular cell groups within the somites. According to segmentation models, somitogenesis is under the control of a "segmentation clock" in combination with a morphogen gradient. This hypothesis has recently found support from molecular data, especially the cycling expression of genes such as cHairy1 and Lunatic Fringe, which depend on the Notch/Delta pathway of signal transduction. FGF8 has been described to be distributed along a cranio-caudal gradient. The first oscillating gene described shown to be independent of Notch is Axin2, encoding a negative regulator of the canonical Wnt pathway and a target of Wnt3a. Wnt3a and Axin2 show a similar distribution as FGF8 with high levels in the tailbud. The chick embryo has recently become accessible to molecular approaches such as overexpression by electroporation and RNA interference which can be expected to help elucidating some of the still open questions concerning somitogenesis.

From segment to somite: segmentation to epithelialization analyzed within quantitative frameworks.

One of the most visually striking patterns in the early developing embryo is somite segmentation. Somites form as repeated, periodic structures in pairs along nearly the entire caudal vertebrate axis. The morphological process involves short- and long-range signals that drive cell rearrangements and cell shaping to create discrete, epithelialized segments. Key to developing novel strategies to prevent somite birth defects that involve axial bone and skeletal muscle development is understanding how the molecular choreography is coordinated across multiple spatial scales and in a repeating temporal manner. Mathematical models have emerged as useful tools to integrate spatiotemporal data and simulate model mechanisms to provide unique insights into somite pattern formation. In this short review, we present two quantitative frameworks that address the morphogenesis from segment to somite and discuss recent data of segmentation and epithelialization.

Twist is an integrator of SHH, FGF, and BMP signaling.

Development of vertebrate embryos is regulated by a number of different signaling pathways. These pathways are frequently not independent of each other but are connected by crosstalk between cells and tissues. Furthermore, different signaling pathways have been found to interact at the cellular level. Development of cranial and limb structures is an example, in which FGF, BMP, and SHH signaling interact. Mutations in the different signaling pathways may therefore result in complex but similar phenotypes. This indicates the existence of integrator molecules, which depend in their expression or activity on the combination of different signaling pathways. Here we show that expression of the bHLH transcription factor Twist in the paraxial mesoderm requires an induction from the notochord. This induction can only be substituted by a combination of FGF and SHH signaling, but not by individual application of FGF8 or SHH alone. Furthermore, the expression of Twist can be modified by BMP2 in a complex, age-dependent manner. We propose that Twist is one of the integrating parts of the three signaling pathways and mediates some of the common effects.