scholarly journals Microfibril-associated Glycoprotein-2 (MAGP-2) Is Specifically Associated with Fibrillin-containing Microfibrils but Exhibits More Restricted Patterns of Tissue Localization and Developmental Expression Than Its Structural Relative MAGP-1

1998 ◽  
Vol 46 (8) ◽  
pp. 871-885 ◽  
Author(s):  
Mark A. Gibson ◽  
Merran L. Finnis ◽  
Jaliya S. Kumaratilake ◽  
Edward G. Cleary
Keyword(s):  
Skeletal Muscle ◽  
Expression Patterns ◽  
Fetal Lung ◽  
Northern Blotting ◽  
Developmental Expression ◽  
Mrna Levels ◽  
Western Blots ◽  
Fibrillin 1 ◽  
Significant Expression ◽  
Nuchal Ligament

SUMMARY We developed an affinity-purified anti-MAGP-2 peptide antibody that specifically identified MAGP-2 on Western blots of purified matrix proteins and extracts of nuchal ligament. Immunolocalization studies on tissues from a 210-day-old fetus and a mature bovine showed that MAGP-2 was located in similar regions to MAGP-1 and fibrillin-1 but that the distribution of MAGP-2 was more restricted. In fetal nuchal ligament, skeletal muscle, and spleen the distribution of MAGP-2 was indistinguishable from that of MAGP-1. In contrast to MAGP-1, MAGP-2 was not detected in the medial layer of fetal thoracic aorta and in much of the peritubular matrix of fetal and mature kidney and in the mature ocular zonule. Some differences in the immunolocalization patterns were also evident in fetal lung, cartilage, skin, and heart. Immunoelectron microscopy confirmed that MAGP-2 was specifically associated with fibrillin-containing microfibrils in nuchal ligament, dermis, adventitia of aorta, glomerular mesangium and perimysium. Northern blotting of RNA from tissues of a 210-day-old fetus indicated that steady-state MAGP-2 mRNA levels were highest in nuchal ligament. Significant expression was also detected in lung, heart, skeletal muscle, skin, and Achilles tendon. The tissue pattern of MAGP-2 expression differed significantly from that of MAGP-1. MAGP-2 expression appeared to be higher in nuchal ligament, heart, and skeletal muscle and lower in aorta and kidney. In nuchal ligament, MAGP-2 mRNA expression appeared to peak around 180 days of fetal development, which correlates with the period of onset of elastinogenesis in this tissue. Overall, the immunolocalization and expression patterns of MAGP-2 appeared to be distinct from those of other microfibrillar components. This is consistent with the view that MAGP-2 plays a unique role in the biology of the microfibrils, perhaps by mediating their interaction with cell surfaces at specific stages of development and differentiation.

scholarly journals Myostatin and insulin-like growth factor-I and -II expression in the muscle of rats exposed to the microgravity environment of the NeuroLab space shuttle flight

2000 ◽  
Vol 167 (3) ◽  
pp. 417-428 ◽  
Author(s):  
R Lalani ◽  
S Bhasin ◽  
F Byhower ◽  
R Tarnuzzer ◽  
M Grant ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Mass ◽  
Skeletal Muscle Mass ◽  
Recovery Period ◽  
Microgravity Environment ◽  
Mrna Levels ◽  
Adult Rats ◽  
Male Adult ◽  
Western Blots ◽  
Igf I

The mechanism of the loss of skeletal muscle mass that occurs during spaceflight is not well understood. Myostatin has been proposed as a negative modulator of muscle mass, and IGF-I and IGF-II are known positive regulators of muscle differentiation and growth. We investigated whether muscle loss associated with spaceflight is accompanied by increased levels of myostatin and a reduction in IGF-I and -II levels in the muscle, and whether these changes correlate with an increase in muscle proteolysis and apoptosis. Twelve male adult rats sent on the 17-day NASA STS-90 NeuroLab space flight were divided upon return to earth into two groups, and killed either 1 day later (R1) or after 13 days of acclimatization (R13). Ground-based control rats were maintained for the same periods in either vivarium (R3 and R15, respectively), or flight-simulated cages (R5 and R17, respectively). RNA and protein were isolated from the tibialis anterior, biceps femoris, quadriceps, and gastrocnemius muscles. Myostatin, IGF-I, IGF-II and proteasome 2c mRNA concentrations were determined by reverse transcription/PCR; myostatin and ubiquitin mRNA were also measured by Northern blot analysis; myostatin protein was estimated by immunohistochemistry; the apoptotic index and the release of 3-methylhistidine were determined respectively by the TUNEL assay and by HPLC. Muscle weights were 19-24% lower in the R1 rats compared with the control R3 and R5 rats, but were not significantly different after the recovery period. The myostatin/beta-actin mRNA ratios (means+/-s.e.m. ) were higher in the muscles of the R1 rats compared with the control R5 rats: 5.0-fold in tibialis (5.35 +/- 1.85 vs 1.07 +/- 0.26), 3.0-fold in biceps (2.46+/-0.70 vs 0.81 +/- 0.04), 1.9-fold in quadriceps (7.84 +/- 1.73 vs 4.08 +/- 0.52), and 2.2-fold in gastrocnemius (0.99 +/- 0.35 vs 0.44 +/- 0.17). These values also normalized upon acclimatization. Our antibody against a myostatin peptide was validated by detection of the recombinant human myostatin protein on Western blots, which also showed that myostatin immunostaining was increased in muscle sections from R1 rats, compared with control R3 rats, and normalized upon acclimatization. In contrast, IGF-II mRNA concentrations in the muscles from R1 rats were 64-89% lower than those in R3 animals. With the exception of the gastrocnemius, IGF-II was also decreased in R5 animals maintained in flight-simulated cages, and normalized upon acclimatization. The intramuscular IGF-I mRNA levels were not significantly different between the spaceflight rats and the controls. No increase was found in the proteolysis markers 3-methyl histidine, ubiquitin mRNA, and proteasome 2C mRNA. In conclusion, the loss of skeletal muscle mass that occurs during spaceflight is associated with increased myostatin mRNA and protein levels in the skeletal muscle, and a decrease in IGF-II mRNA levels. These alterations are normalized upon restoration of normal gravity and caging conditions. These data suggest that reciprocal changes in the expression of myostatin and IGF-II may contribute to the multifactorial pathophysiology of muscle atrophy that occurs during spaceflight.

scholarly journals Opioid Receptor Protein Expression During Development of the Rat Brainstem

2021 ◽  
Author(s):  
◽  
Bronwyn Maree Kivell
Keyword(s):  
Confocal Microscopy ◽  
Opioid Receptor ◽  
Glial Cells ◽  
Opioid Receptors ◽  
Cultured Cells ◽  
Expression Patterns ◽  
Receptor Expression ◽  
Developmental Expression ◽  
Receptor Protein ◽  
Western Blots

<p>Few satisfactory protocols exist for primary culture of postnatal brainstem neurons, and commonly used procedures often give poor survival rates in older foetal (>E16) and early postnatal brainstem cultures. The present study describes the first reliable method for establishing stable in vitro cultures of foetal and postnatal brainstem neurons up to six days postnatal age in a defined, serum-free culture medium. This novel culture method was used to study opioid receptor expression and distribution in developing brainstem cells. Opioids play an important role in brainstem functions, being involved in respiratory and cardiovascular modulation and pain control (Olsen et al., 1995; Olson et al., 1997; Vaccarino et al., 1999; Vaccarino and Kastin, 2001). These brainstem functions are particularly important for survival at birth, and opioid receptor distribution patterns and sensitivities to opioid ligands change during development. Using cultured cells and frozen sections of brainstem tissue, mu (MOR) and delta (DOR) opioid receptor localisation in neuronal and glial cells at different stages of foetal and postnatal development in the rat were examined by immunocytochemistry and confocal microscopy. Bipolar and multipolar neurons showed similar immunoreactivities; whereas, glial cells were more lightly stained than neurons. Developmentally advanced stages were more intensely stained for MOR (P<0.006, Mann-Whitney test); whereas, DOR immunoreactivity did not change during development. These developmental expression patterns observed in culture for MOR were similar to those obtained from Western blots of electrophoreses brainstem lysates. DOR, however, decreased in expression in brainstem lysates with increased developmental age, even though there was no difference in DOR expression in cultured cells. MOR and DOR were colocalised in specific brainstem regions and in the cerebellum of foetal and postnatal animals, although the distribution of both opioid receptors in the foetal brain was more diffuse than in the older animals. The intracellular distributions of MOR and DOR were investigated by confocal microscopy. In addition to plasma membrane staining, a population of internalised cytoplasmic receptors was present in neurons. MOR was down-regulated after exposure of either cultured brainstem cells or transfected or non-transfected SH-SY5Y neuroblastoma cells to the MOR agonist DAMGO. From the above investigation, it was concluded that opioid receptors are developmentally regulated during maturation of the brainstem of the rat, and that primary cell culture, immunocytochemistry, and immunoblotting of cell lysates are suitable techniques for investigating opioid systems in the foetal, postnatal, and adult rat.</p>

scholarly journals Insulin regulation of a novel WD-40 repeat protein in adipocytes

2001 ◽  
Vol 168 (2) ◽  
pp. 325-332 ◽  
Author(s):  
BD Rodgers ◽  
MA Levine ◽  
M Bernier ◽  
C Montrose-Rafizadeh
Keyword(s):  
Skeletal Muscle ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Tissue Expression ◽  
Northern Blotting ◽  
Predicted Amino Acid Sequence ◽  
Mrna Levels ◽  
Degenerate Primers ◽  
Hek 293 ◽  
Reading Frame

A 400 bp PCR product generated with degenerate primers derived from the glucagon-like peptide-1 receptor was used to screen a rat skeletal muscle cDNA library. The predicted amino acid sequence of the 978 bp open reading frame has a predicted M(r) of 35 804, an estimated isoelectric point (pI) of 5.31 and contains seven WD-40 repeats, which are common to G-protein beta subunits (Gbeta). Although chemically and structurally similar to Gbeta subunits, the predicted amino acid sequence, when compared with the previously cloned Gbeta isoforms, was found to be only 31-41% similar and thus was named Gbeta-like (GbetaL, 'Gable'). Western blotting of whole-cell lysates and immunoprecipitates of membrane and cytosolic fractions of HEK 293 cells stably overexpressing a carboxy-terminal His-tagged GbetaL indicates that the protein is cytosolic and that it migrates at 42 kDa. A 4 kb transcript was detected in all tissues surveyed by northern blotting; however, an additional 2 kb transcript was detected in testis. Expression of GbetaL mRNA was highest in the brain and testis, followed by lung, heart, kidney, skeletal muscle, spleen and liver. In addition, reverse transcriptase/PCR showed that several other tissues and cell lines express GbetaL. The ubiquitous nature of the tissue expression pattern of GbetaL is similar to that of the insulin receptor, which suggests that insulin may influence GbetaL expression. Indeed, GbetaL protein and mRNA levels, in fully differentiated 3T3-L1 adipocytes, were upregulated by insulin in a concentration-dependent fashion. These changes were highly sensitive to insulin stimulation, being minimally affected by doses as low as 0.1 nM and maximally elevated by 1 nM doses. These data suggest that insulin regulates GbetaL production and imply that some of the actions of insulin may be mediated, in part, by this novel intracellular protein.

scholarly journals Opioid Receptor Protein Expression During Development of the Rat Brainstem

2021 ◽  
Author(s):  
◽  
Bronwyn Maree Kivell
Keyword(s):  
Confocal Microscopy ◽  
Opioid Receptor ◽  
Glial Cells ◽  
Opioid Receptors ◽  
Cultured Cells ◽  
Expression Patterns ◽  
Receptor Expression ◽  
Developmental Expression ◽  
Receptor Protein ◽  
Western Blots

<p>Few satisfactory protocols exist for primary culture of postnatal brainstem neurons, and commonly used procedures often give poor survival rates in older foetal (>E16) and early postnatal brainstem cultures. The present study describes the first reliable method for establishing stable in vitro cultures of foetal and postnatal brainstem neurons up to six days postnatal age in a defined, serum-free culture medium. This novel culture method was used to study opioid receptor expression and distribution in developing brainstem cells. Opioids play an important role in brainstem functions, being involved in respiratory and cardiovascular modulation and pain control (Olsen et al., 1995; Olson et al., 1997; Vaccarino et al., 1999; Vaccarino and Kastin, 2001). These brainstem functions are particularly important for survival at birth, and opioid receptor distribution patterns and sensitivities to opioid ligands change during development. Using cultured cells and frozen sections of brainstem tissue, mu (MOR) and delta (DOR) opioid receptor localisation in neuronal and glial cells at different stages of foetal and postnatal development in the rat were examined by immunocytochemistry and confocal microscopy. Bipolar and multipolar neurons showed similar immunoreactivities; whereas, glial cells were more lightly stained than neurons. Developmentally advanced stages were more intensely stained for MOR (P<0.006, Mann-Whitney test); whereas, DOR immunoreactivity did not change during development. These developmental expression patterns observed in culture for MOR were similar to those obtained from Western blots of electrophoreses brainstem lysates. DOR, however, decreased in expression in brainstem lysates with increased developmental age, even though there was no difference in DOR expression in cultured cells. MOR and DOR were colocalised in specific brainstem regions and in the cerebellum of foetal and postnatal animals, although the distribution of both opioid receptors in the foetal brain was more diffuse than in the older animals. The intracellular distributions of MOR and DOR were investigated by confocal microscopy. In addition to plasma membrane staining, a population of internalised cytoplasmic receptors was present in neurons. MOR was down-regulated after exposure of either cultured brainstem cells or transfected or non-transfected SH-SY5Y neuroblastoma cells to the MOR agonist DAMGO. From the above investigation, it was concluded that opioid receptors are developmentally regulated during maturation of the brainstem of the rat, and that primary cell culture, immunocytochemistry, and immunoblotting of cell lysates are suitable techniques for investigating opioid systems in the foetal, postnatal, and adult rat.</p>

Identification of myosin light chains in Rana pipiens skeletal muscle and their expression patterns along single fibres

2001 ◽  
Vol 204 (24) ◽  
pp. 4237-4248 ◽  
Author(s):  
Gordon J. Lutz ◽  
Shannon N. Bremner ◽  
Michael J. Bade ◽  
Richard L. Lieber
Keyword(s):  
Skeletal Muscle ◽  
Rana Pipiens ◽  
Allelic Variation ◽  
Expression Patterns ◽  
Skeletal Muscle Fibre ◽  
Fibre Types ◽  
Muscle Fibre Types ◽  
Western Blots

SUMMARY Isoforms of myosin heavy chain (MHC) and myosin light chain (MLC) influence contractile kinetics of skeletal muscle. We previously showed that the four major skeletal muscle fibre types in Rana pipiens (type 1, type 2, type 3 and tonic; amphibian nomenclature) contain four unique MHC isoforms. In the present study we defined the MLCs expressed in each of these R. pipiens fibre types. The MLC composition of single MHC-typed fibres was determined from western blots using a panel of monoclonal MLC antibodies. A total of seven MLCs were identified, including four types of MLC1, two of MLC2 and a single MLC3. Twitch fibre types (types 1, 2 and 3) expressed MLC1f and MLC2f, while tonic fibres contained a unique set of isoforms, MLC1Ta, MLC1Tb and MLC2T. MLC3 was expressed primarily in type 1, type 1-2 and type 2 fibres. Surprisingly, some frogs displayed a striking pattern of MLC expression where a unique isoform of MLC1 (MLC1x) was coexpressed along with the normal MLC1 isoform(s) in all fibre types. MLC1x was either expressed in all fibres of a given frog or was completely absent. The intraspecific polymorphism in MLC1 expression is likely to have a genetic basis, but is unlikely to be caused by allelic variation. The ratio of MLC3/MLC1 increased in direct proportion to the percentage of type 1 MHC, but was only weakly correlated. The variability in MLC3/MLC1 within a fibre type was extremely large. Both the MHC isoform and MLC3/MLC1 ratio varied significantly between 1 mm segments along the length of fibres. For all segments combined, MLC3/MLC1 increased with the percentage of type 1 MHC, but the correlation between segments was weaker than between fibres.

Lung epithelial Na channel subunits are differentially regulated during development and by steroids

1995 ◽  
Vol 269 (3) ◽  
pp. C805-C812 ◽  
Author(s):  
S. Tchepichev ◽  
J. Ueda ◽  
C. Canessa ◽  
B. C. Rossier ◽  
H. O'Brodovich
Keyword(s):  
Fetal Lung ◽  
Developmental Expression ◽  
Na Channel ◽  
Mrna Levels ◽  
Adult Rats ◽  
Pregnant Rats ◽  
Lung Epithelial ◽  
Normal Rat ◽  
Epithelial Na Channel

Because the alpha-subunit of the rat lung epithelial Na channel (rENaC) is not expressed until late fetal gestation, the developmental immaturity of alpha-rENaC may be involved in the premature fetal lung's inability to mount a Na-absorptive response to appropriate agonists. As previous work has shown that the beta- and gamma-rENaC subunits of the Na channel are required for maximal alpha-rENaC activity, we determined their developmental expression in the fetal lung. In addition, because thyroid and corticosteroid therapy can mature the in vivo fetal lamb lung's ability to transport Na, we wished to determine whether such treatment increased the expression of alpha-, beta-, and gamma-rENaC. Lungs were harvested from normal rat fetuses of 17 through 22 days gestation (term = 22 days), normal rat pups during the first week of life, and adult rats. Initial expression of alpha-rENaC was detected at 19 days gestation and progressively increased in utero. beta- and gamma-rENaC mRNA were not detected until 21 and 22 days gestation, and then only at very low levels. During the first week after birth, the levels of alpha-rENaC declined, whereas beta- and gamma-rENaC mRNA levels increased. This pre- and postnatal pattern of alpha-rENaC expression correlates with the endogenous glucocorticosteroid levels in the fetus and the rat pup's early postnatal corticosteroid resistance. Combined or separate treatment of pregnant rats (16 through 22 days gestational age) with thyroid-releasing hormone (TRH) and/or dexamethasone (Dex) for 48 h showed that Dex, but not TRH, could increase fetal lung alpha-rENaC mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Developmental expression of the G protein-coupled receptor 54 and three GnRH mRNAs in the teleost fish cobia

2007 ◽  
Vol 38 (2) ◽  
pp. 235-244 ◽  
Author(s):  
J Shaik Mohamed ◽  
Abby D Benninghoff ◽  
G Joan Holt ◽  
Izhar A Khan
Keyword(s):  
G Protein ◽  
Teleost Fish ◽  
Expression Patterns ◽  
Developmental Expression ◽  
Mrna Levels ◽  
G Protein Coupled Receptor ◽  
Early Puberty ◽  
Cdna Sequences ◽  
Gnrh Release ◽  
G Protein Coupled

The cDNAs of the G protein-coupled receptor 54 (GPR54) and three prepro-gonadotropin-releasing hormones, GnRH-I (seabream GnRH), GnRH-II (chicken GnRH-II), and GnRH-III (salmon GnRH) were isolated and cloned from the brain of the teleost fish cobia, Rachycentron canadum. The cobia GPR54 cDNA was 95 and 51–56% identical to those of tilapia and mammalian models respectively. The GnRH cDNA sequences of cobia showed strong identities to those of tilapia, Atlantic croaker, red drum, and the seabass and seabream species. The real-time quantitative RT-PCR methods allowed detection of all three GnRH mRNAs on the first day after hatching (DAH). The GnRH-I mRNA levels, which were the lowest among the three GnRHs, increased gradually with two distinct peaks in larvae at 3 and 4 DAH. On the other hand, GnRH-II and GnRH-III mRNAs were significantly higher in larvae at 2 and 6 DAH compared with those on the preceding days. In addition, significant peaks of all the three GnRH mRNAs were observed in the brains of 26-day-old fish. The finding of higher GnRH-I and GnRH-II mRNAs in males than females at 153 DAH may be related to early puberty observed during the first year in laboratory-reared male cobia. Moreover, this study demonstrates for the first time the expression of GPR54 mRNA during larval development in a vertebrate species. The concomitant expression patterns of GPR54 and GnRH mRNAs during different stages of larval and juvenile developments, and during early puberty in male cobia suggest a potential relationship between GPR54 and multiple GnRHs during these stages of development consistent with the role of GPR54 in controlling GnRH release in mammals. The increase in GPR54 and GnRH mRNAs observed during early puberty in cobia is consistent with a similar change reported in pubertal rats. This finding together with the localization of GPR54 mRNAs on GnRH neurons in fish and mammals suggests that the GPR54–GnRH interactions may be conserved in different vertebrate groups.

scholarly journals The zebrafish as a model system for analyzing mammalian and native α-crystallin promoter function

2017 ◽  
Author(s):  
Mason Posner ◽  
Kelly Murray ◽  
Hayden Eighinger ◽  
Amy Drossman ◽  
Zachary Haley ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Promoter Activity ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Zebrafish Embryos ◽  
Mrna Levels ◽  
Gfp Expression ◽  
Promoter Function ◽  
Lens Crystallin ◽  
Αb Crystallin

Previous studies have used the zebrafish to investigate the biology of lens crystallin proteins and their roles in development and disease. However, little is known about zebrafish α-crystallin promoter function, how it compares to that of mammals, or whether mammalian α-crystallin promoter activity can be assessed using zebrafish embryos. We injected a variety of α-crystallin promoter fragments from each species combined with the coding sequence for green fluorescent protein (GFP) into zebrafish zygotes to determine the resulting spatiotemporal expression patterns in the developing embryo. We also measured mRNA levels and protein abundance for all three zebrafish α-crystallins. Our data showed that mouse and zebrafish αA-crystallin promoters generated similar GFP expression in the lens, but with earlier onset when using mouse promoters. Expression was also found in notochord and skeletal muscle in a small percentage of embryos. Mouse αB-crystallin promoter fragments drove GFP expression primarily in zebrafish skeletal muscle, with less common expression in notochord, lens, heart and in extraocular regions of the eye. A short fragment containing only a lens-specific enhancer region produced no GFP expression, suggesting that these lens responsive elements in the mouse are not used in the zebrafish. The two paralogous zebrafish αB-crystallin promoters produced subtly different expression profiles, with the αBa promoter driving expression equally in notochord and skeletal muscle while the αBb promoter resulted primarily in skeletal muscle expression. Messenger RNA for zebrafish αa, aBa and αBb were all detected by 1 day post fertilization (dpf). Parallel reaction monitoring (PRM) mass spectrometry was used to detect αA, αBa, and αBb peptides in digests of zebrafish embryos. In whole embryos, αA-crystallin was first detected by 2 dpf, peaked in abundance by 4-5 dpf, and was localized to the eye. αBa was also detected in whole embryo at nearly constant levels from 1-6 dpf, was also localized primarily to the eye, and its abundance in extraocular tissues decreased from 4-7 dpf. In contrast, due to its low abundance, no αBb protein could be detected in whole embryo, or dissected eye and extraocular tissues. Our results show that mammalian α-crystallin promoters can be efficiently screened in zebrafish embryos and that their controlling regions are well conserved, although their use in each species may reflect evolutionary changes in developmental roles for α-crystallins. An ontogenetic shift in zebrafish αBa-crystallin promoter activity provides an interesting system for examining the evolution and control of tissue specificity. Future studies that combine these promoter based approaches with the expanding ability to engineer the zebrafish genome via techniques such as CRISPR/Cas9 will allow the manipulation of protein expression to test hypotheses about lens crystallin function and its relation to lens biology and disease.

Abstract 632: Aortic Dilatation in Marfan Syndrome: A Result of Amplification of Molecular Mechanisms of Aging?

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Michael A Hagler ◽  
Grace Casaclang-Verzosa ◽  
Bin Zhang ◽  
Carolyn Roos ◽  
Nassir Thalji ◽  
...  
Keyword(s):  
Gene Expression ◽  
Marfan Syndrome ◽  
Aortic Root ◽  
Expression Patterns ◽  
Mutant Mice ◽  
Aortic Dilatation ◽  
Mrna Levels ◽  
Wild Type ◽  
Fibrillin 1 ◽  
Tgf Β1

Marfan syndrome (MFS) is a genetic disease with a mutation for the microfibrillar constituent protein fibrillin-1 being the most prevalent. MFS is often associated with progressive aortic root dilation, ultimately progressing to aortic aneurysm and dissection. While recent work has shown that increased angiotensin-II receptor type-1 and transforming growth factor beta (TGF-β) signaling contributes to aneurysm formation in aorta, efficacious therapeutic targets remain elusive. Given previous reports of progeriod phenotypes in a subset of Marfan patients, we sought to determine whether there are molecular changes that are consistent with accelerated aging in a mouse model of Marfan syndrome. In mice carrying a loss-of-function mutation in fibrillin-1, we assessed aortic root dimensions by echocardiography and gene expression levels of TGF-β1-3, runt related transcription factor 2 (RUNX2), and the cellular senescent marker CDKN2A by quantitative real time PCR at 3 and 14 months of age. As expected, aortic sinus dimensions did not change significantly with aging in wild type mice, but increased dramatically in fibrillin-1 mutant mice compared to wild-type littermate controls and with age (p < 0.05 for both). TGF-β1 ligand expression paralleled age and disease-dependent changes in aortic dimensions, however TGF-β2 and TGF-β3 mRNA levels did not. Aortic dilatation was associated with increased gene expression of RUNX2 with aging and in marfanoid mice. Interestingly, fibrillin-1 mutant mice demonstrated marked increases in expression of the anti-proliferative cell-cycle checkpoint protein CDKN2A at both time points, and correlated with changes in TGF-β1 (R 2 =0.54) and RUNX2 (R 2 =0.69) mRNA. CDNK2A gene expression patterns, however, demonstrated a poor correlation with expression of TGF-β2 and TGF-β3 (R 2 =0.04 and 0.06. respectively). Collectively, these data lend insight into novel mechanisms that may regulate development of aortic root dilation in patients MFS and are the first to implicate increased senescent cell burden in Marfan syndrome. Furthermore, we propose that clearance of senescent cells could be a viable therapeutic intervention to slow progression aortic root-dilation and aneurysm in patients with Marfan syndrome.

scholarly journals The zebrafish as a model system for analyzing mammalian and native α-crystallin promoter function

2017 ◽  
Author(s):  
Mason Posner ◽  
Kelly Murray ◽  
Hayden Eighinger ◽  
Amy Drossman ◽  
Zachary Haley ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Promoter Activity ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Zebrafish Embryos ◽  
Mrna Levels ◽  
Gfp Expression ◽  
Promoter Function ◽  
Lens Crystallin ◽  
Αb Crystallin

Previous studies have used the zebrafish to investigate the biology of lens crystallin proteins and their roles in development and disease. However, little is known about zebrafish α-crystallin promoter function, how it compares to that of mammals, or whether mammalian α-crystallin promoter activity can be assessed using zebrafish embryos. We injected a variety of α-crystallin promoter fragments from each species combined with the coding sequence for green fluorescent protein (GFP) into zebrafish zygotes to determine the resulting spatiotemporal expression patterns in the developing embryo. We also measured mRNA levels and protein abundance for all three zebrafish α-crystallins. Our data showed that mouse and zebrafish αA-crystallin promoters generated similar GFP expression in the lens, but with earlier onset when using mouse promoters. Expression was also found in notochord and skeletal muscle in a small percentage of embryos. Mouse αB-crystallin promoter fragments drove GFP expression primarily in zebrafish skeletal muscle, with less common expression in notochord, lens, heart and in extraocular regions of the eye. A short fragment containing only a lens-specific enhancer region produced no GFP expression, suggesting that these lens responsive elements in the mouse are not used in the zebrafish. The two paralogous zebrafish αB-crystallin promoters produced subtly different expression profiles, with the αBa promoter driving expression equally in notochord and skeletal muscle while the αBb promoter resulted primarily in skeletal muscle expression. Messenger RNA for zebrafish αa, aBa and αBb were all detected by 1 day post fertilization (dpf). Parallel reaction monitoring (PRM) mass spectrometry was used to detect αA, αBa, and αBb peptides in digests of zebrafish embryos. In whole embryos, αA-crystallin was first detected by 2 dpf, peaked in abundance by 4-5 dpf, and was localized to the eye. αBa was also detected in whole embryo at nearly constant levels from 1-6 dpf, was also localized primarily to the eye, and its abundance in extraocular tissues decreased from 4-7 dpf. In contrast, due to its low abundance, no αBb protein could be detected in whole embryo, or dissected eye and extraocular tissues. Our results show that mammalian α-crystallin promoters can be efficiently screened in zebrafish embryos and that their controlling regions are well conserved, although their use in each species may reflect evolutionary changes in developmental roles for α-crystallins. An ontogenetic shift in zebrafish αBa-crystallin promoter activity provides an interesting system for examining the evolution and control of tissue specificity. Future studies that combine these promoter based approaches with the expanding ability to engineer the zebrafish genome via techniques such as CRISPR/Cas9 will allow the manipulation of protein expression to test hypotheses about lens crystallin function and its relation to lens biology and disease.

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