Effects of hyperthyroidism in the development of the appendicular skeleton and muscles of zebrafish, with notes on evolutionary developmental pathology (Evo-Devo-Path).

The hypothalamus-pituitary-thyroid (HPT) axis plays a crucial role in the metabolism, homeostasis, somatic growth and development of teleostean fishes. Thyroid hormones regulate essential biological functions such as growth and development, regulation of stress, energy expenditure, tissue compound, and psychological processes. Teleost thyroid follicles produce the same thyroid hormones as in other vertebrates: thyroxin (T4) and triiodothyronine (T3), making the zebrafish a very useful model to study hypo- and hyperthyroidism in other vertebrate taxa, including humans. Here we investigate morphological changes in T3 hyperthyroid cases in the zebrafish to better understand malformations provoked by alterations of T3 levels. In particular, we describe musculoskeletal abnormalities during the development of the zebrafish appendicular skeleton and muscles, compare our observations with those recently done by us on the normal developmental of the zebrafish, and discuss these comparisons within the context of evolutionary developmental pathology (Evo-Devo-Path), including human pathologies.

The role of folate metabolism in orofacial development and clefting.

Folate deficiency has been associated with numerous diseases and birth defects including orofacial defects. However, whether folate has a role in the face during early orofacial development has been unclear. The present study reveals that pharmacological and antisense oligonucleotide mediated inhibition of DHFR, an integral enzyme in the folate pathway, results in specific changes in the size and shape of the midface and embryonic mouth. Such defects are accompanied by a severe reduction in the muscle and cartilage jaw elements without significant change in neural crest pattern or global levels of methylation. We propose that the orofacial defects associated with DHFR deficient function are the result of decreased cell proliferation and increased cell death via DNA damage. In particular, localized apoptosis may also be depleting the cells of the face that express crucial genes for the differentiation of the jaw structures. Folate supplementation is widely known to reduce human risk for orofacial clefts. In the present study, we show that activating folate metabolism can reduce median oral clefts in the primary palate by increasing cell survival. Moreover, we demonstrate that a minor decrease in DHFR function exacerbates median facial clefts caused by RAR inhibition. This work suggests that folate deficiencies could be a major contributing factor to multifactorial orofacial defects.

Klhl31 attenuates ß-catenin dependent Wnt signaling and regulates embryo myogenesis.

Klhl31 is a member of the Kelch-like family in vertebrates, which are characterized by an amino-terminal broad complex tram-track, bric-a-brac/poxvirus and zinc finger (BTB/POZ) domain, carboxy-terminal Kelch repeats and a central linker region (Back domain). In developing somites Klhl31 is highly expressed in the myotome downstream of myogenic regulators (MRF), and it remains expressed in differentiated skeletal muscle. In vivo gain- and loss-of-function approaches in chick embryos reveal a role of Klhl31 in skeletal myogenesis. Targeted mis-expression of Klhl31 led to a reduced size of dermomyotome and myotome as indicated by detection of relevant myogenic markers, Pax3, Myf5, myogenin and myosin heavy chain (MF20). The knock-down of Klhl31 in developing somites, using antisense morpholinos (MO), led to an expansion of Pax3, Myf5, MyoD and myogenin expression domains and an increase in the number of mitotic cells in the dermomyotome and myotome. The mechanism underlying this phenotype was examined using complementary approaches, which show that Klhl31 interferes with ß-catenin dependent Wnt signaling. Klhl31 reduced the Wnt-mediated activation of a luciferase reporter in cultured cells. Furthermore, Klhl31 attenuated secondary axis formation in Xenopus embryos in response to Wnt1 or ß-catenin. Klhl31 mis-expression in the developing neural tube affected its dorso-ventral patterning and led to reduced dermomyotome and myotome size. Co-transfection of a Wnt3a expression vector with Klhl31 in somites or in the neural tube rescued the phenotype and restored the size of dermomyotome and myotome. Thus, Klhl31 is a novel modulator of canonical Wnt signaling, important for vertebrate myogenesis. We propose that Klhl31 acts in the myotome to support cell cycle withdrawal and differentiation.

Identification of compounds with anti-convulsant properties in a zebrafish model of epileptic seizures.

The availability of animal models of epileptic seizures provides opportunities to identify novel anticonvulsants for the treatment of people with epilepsy. We found that exposure of 2-day-old zebrafish embryos to the convulsant agent pentylenetetrazole (PTZ) rapidly induces the expression of synaptic-activity-regulated genes in the CNS, and elicited vigorous episodes of calcium (Ca(2+)) flux in muscle cells as well as intense locomotor activity. We then screened a library of ∼2000 known bioactive small molecules and identified 46 compounds that suppressed PTZ-inducedtranscription of the synaptic-activity-regulated gene fos in 2-day-old (2 dpf) zebrafish embryos. Further analysis of a subset of these compounds, which included compounds with known and newly identified anticonvulsant properties, revealed that they exhibited concentration-dependent inhibition of both locomotor activity and PTZ-induced fos transcription, confirming their anticonvulsant characteristics. We conclude that this in situ hybridisation assay for fos transcription in the zebrafish embryonic CNS is a robust, high-throughput in vivo indicator of the neural response to convulsant treatment and lends itself well to chemical screening applications. Moreover, our results demonstrate that suppression of PTZ-induced fos expression provides a sensitive means of identifying compounds with anticonvulsant activities.

Maternal dietary chromium restriction programs muscle development and function in the rat offspring.

Intrauterine growth retardation programs the fetus to manipulated metabolic changes that lead to adult diseases. Considering that chromium (Cr) supplements influence lean body mass (LBM) in both humans and experimental animals, we have studied the effect of maternal Cr restriction on muscle development and function in the rat offspring. Female weanling Wistar/NIN rats received, for 12 weeks, a control or 65% Cr-restricted diet ad libitum and mated with control males. While control mothers/offspring received control diet throughout (CrC), some restricted mothers were switched to control diet from conception (CrRC) and parturition (CrRP) and their offspring were weaned on to control diet. Half of the remaining restricted pups were weaned on to control diet (CrRW) and the other half continued on restricted diet throughout (CrR). Maternal CrR significantly decreased the percent of LBM (LBM %) and fat-free mass (FFM %) in the offspring and this was associated with decreased expression of the myogenic genes: MyoD, Myf5 and MyoG. Surprisingly, expression of the muscle atrophy genes, Atrogin and MuRF 1, was also decreased in CrR offspring. Although basal glucose uptake by muscle was higher in CrR than in CrC offspring, the stimulation with insulin was comparable, implying no change in its insulin sensitivity. Rehabilitation partly corrected myogenic and atrophic gene expression but had no effect on LBM % or FFM % or glucose uptake by muscle. The results show that maternal Cr restriction in rats may irreversibly impair muscle development and glucose uptake by muscle. Modulation of muscle atrophy appears to be an adaptive mechanism to preserve muscle mass in CrR offspring.

Maternal vitamin D in pregnancy may influence not only offspring bone mass but other aspects of musculoskeletal health and adiposity.

Osteoporotic fractures, falls and obesity are major health problems in developed nations. Evidence suggests that there are antenatal factors predisposing to these conditions. Data are emerging from Australia and elsewhere to suggest that maternal vitamin D status in pregnancy affects intrauterine skeletal mineralisation and skeletal growth together with muscle development and adiposity. Given that low levels of vitamin D have been documented in many urbanised populations, including those in countries with abundant sunlight, an important issue for public health is whether maternal vitamin D insufficiency during pregnancy has adverse effects on offspring health. The developing fetus may be exposed to low levels of vitamin D during critical phases of development as a result of maternal hypovitaminosis D. We hypothesise that this may have adverse effects on offspring musculoskeletal health and other aspects of body composition. Further research focused on the implications of poor gestational vitamin D nutrition is warranted as these developmental effects are likely to have a sustained influence on health during childhood and in adult life. We suggest that there is a clear rationale for randomised clinical trials to assess the potential benefits and harmful effects of vitamin D supplementation during pregnancy.

Loss of selenoprotein N function causes disruption of muscle architecture in the zebrafish embryo.

Mutations in the gene coding for selenoprotein N (SelN), a selenium containing protein of unknown function, cause different forms of congenital muscular dystrophy in humans. These muscular diseases are characterized by early onset of hypotonia which predominantly affect in axial muscles. We used zebrafish as a model system to understand the function of SelN in muscle formation during embryogenesis. Zebrafish SelN is highly homologous to its human counterpart and amino acids corresponding to the mutated positions in human muscle diseases are conserved in the zebrafish protein. The sepn1 gene is highly expressed in the somites and notochord during early development. Inhibition of the sepn1 gene by injection of antisense morpholinos does not alter the fate of the muscular tissue, but causes muscle architecture disorganization and greatly reduced motility. Ultrastructural analysis of the myotomes reveals defects in muscle sarcomeric organization and in myofibers attachment, as well as altered myoseptum integrity. These studies demonstrate the important role of SelN for muscle organization during early development. Moreover, alteration of myofibrils architecture and tendon-like structure in embryo deficient for SelN function provide new insights into the pathological mechanism of SelN-related myopathy.

Developmental and evolutionary aspects of the basic helix-loop-helix transcription factors Atonal-like 1 and Achaete-scute homolog 2 in the jellyfish.

The close functional link of nerve and muscle cells in neuromuscular units has led to the hypothesis of a common evolutionary origin of both cell types. Jellyfish are well suited to evaluate this theory since they represent the most basal extant organisms featuring both striated muscle and a nervous system. Here we describe the structure and expression of two novel genes for basic helix-loop-helix (bHLH) transcription factors, the Achaete-scute B family member Ash2 and the Atonal-like gene Atl1, in the hydrozoan jellyfish Podocoryne carnea. Ash2 is expressed exclusively in larval and adult endoderm cells and may be involved in differentiation of secretory cells. Atl1 expression is more widespread and includes the developing striated muscle as well as mechanosensory and nerve cell precursors in the medusa tentacles. Moreover, Atl1 expression is upregulated in proliferating nerve cell precursors arising from adult striated muscle cells by transdifferentiation in vitro. Likewise, the neuronal marker gene NP coding for the RFamide neuropeptide is expressed not only in mature nerve cells but also transiently in the developing muscle. The molecular evidence is concurrent to the hypothesis that muscle and nerve cells are closely linked in evolution and derive from a common myoepithelial precursor.

Erbin is a protein concentrated at postsynaptic membranes that interacts with PSD-95.

Neuregulin is a factor essential for synapse-specific transcription of acetylcholine receptor genes at the neuromuscular junction. Its receptors, ErbB receptor tyrosine kinases, are localized at the postjunctional membrane presumably to ensure localized signaling. However, the molecular mechanisms underlying synaptic localization of ErbBs are unknown. Our recent studies indicate that ErbB4 interacts with postsynaptic density (PSD)-95 (SAP90), a PDZ domain-containing protein that does not interact with ErbB2 or ErbB3. Using as bait the ErbB2 C terminus, we identified Erbin, another PDZ domain-containing protein that interacts specifically with ErbB2. Erbin is concentrated in postsynaptic membranes at the neuromuscular junction and in the central nervous system, where ErbB2 is concentrated. Expression of Erbin increases the amount of ErbB2 labeled by biotin in transfected cells, suggesting that Erbin is able to increase ErbB2 surface expression. Furthermore, we provide evidence that Erbin interacts with PSD-95 in both transfected cells and synaptosomes. Thus ErbB proteins can interact with a network of PDZ domain-containing proteins. This interaction may play an important role in regulation of neuregulin signaling and/or subcellular localization of ErbB proteins.

The maternal transcript for truncated voltage-dependent Ca2+ channels in the ascidian embryo: a potential suppressive role in Ca2+ channel expression.

Ca2+ entry during electrical activity plays several critical roles in development. However, the mechanisms that regulate Ca2+ influx during early embryogenesis remain unknown. In ascidians, a primitive chordate, development is rapid and blastomeres of the muscle and neuronal lineages are easily identified, providing a simple model for studying the expression of voltage-dependent Ca2) channels (VDCCs) in cell differentiation. Here we isolate an ascidian cDNA, TuCa1, a homologue of the alpha(1)-subunit of L-type class Ca2+ channels. We unexpectedly found another form of Ca2+ channel cDNA (3-domain-type) potentially encoding a truncated type which lacked the first domain and a part of the second domain. An analysis of genomic sequence suggested that 3-domain-type RNA and the full-length type have alternative transcriptional start sites. The temporal pattern of the amount of 3-domain-type RNA was the reverse of that of the full-length type; the 3-domain type was provided maternally and persisted during early embryogenesis, whereas the full-length type was expressed zygotically in neuronal and muscular lineage cells. Switching of the two forms occurred at a critical stage when VDCC currents appeared in neuronal or muscular blastomeres. To examine the functional roles of the 3-domain type, it was coexpressed with the full-length type in Xenopus oocyte. The 3-domain type did not produce a functional VDCC current, whereas it had a remarkable inhibitory effect on the functional expression of the full-length form. In addition, overexpression of the 3-domain type under the control of the muscle-specific actin promoter in ascidian muscle blastomeres led to a significant decrease in endogenous VDCC currents. These findings raise the possibility that the 3-domain type has some regulatory role in tuning current amplitudes of VDCCs during early development.

Identification and characterization of newt rad (ras associated with diabetes), a gene specifically expressed in regenerating limb muscle.

Formation of the blastema is a key event for limb regeneration in urodele amphibians, and skeletal muscle has been thought to be a major origin of the multipotent blastemal mesenchyme. In the present study, we used differential display to identify the genes expressed differentially in the muscle at the amputation site. We have isolated a cDNA clone that was upregulated during limb regeneration of the Japanese newt, Cynops pyrrhogaster. Deduced amino acid sequence revealed that the cloned cDNA was a newt homolog of rad (ras associated with diabetes), a gene overexpressed in skeletal muscle of Type II diabetic patients. Expression of newt rad (nrad) was not observed in unamputated normal limb muscle, increased within 4 hr after amputation, and then decreased to the level of normal muscle between 11 and 21 days after amputation. In situ hybridization showed that the transcripts of nrad were localized around most of the nuclei of skeletal muscle near the amputation site, indicating the expression of nrad in the multinucleate myotubes. This expression gradually decreased along the distal to proximal axis. No signals were observed in apical epidermal cap or blastemal mesenchyme. However, reverse transcription-PCR analysis detected a very low level of nrad expression in blastema, suggesting the carry-over of nrad expression in blastema from muscle. Administration of retinoic acid, which has been shown to cause an enhanced dedifferentiation in the regenerating limbs, increased nrad expression in more proximally located limb muscle tissues and prolonged the expression period. Thus, it was strongly suggested that the nrad expression is correlated with the dedifferentiation of myotubes of regenerating limbs. We also analyzed the expression of nrad during development. Transcripts were observed in immature oocytes, seen faintly or not seen thereafter until stage 57 when its expression increased again. These results indicated that nrad may play a role(s) in the developmental process as well as limb regeneration.

The dynamics of myogenin site-specific demethylation is strongly correlated with its expression and with muscle differentiation.

The molecular mechanisms underlying the activation of tissue-specific genes have not yet been fully clarified. We analyzed the methylation status of specific CCGG sites in the 5'-flanking region and exon 1 of myogenin gene, a very important myogenic differentiation factor. We demonstrated a loss of methylation, at the onset of C2C12 muscle cell line differentiation, limited to the CCGG site of myogenin 5'-flanking region, which was strongly correlated with the transcriptional activation of this gene and with myogenic differentiation. The same CCGG site was also found to be hypomethylated, in vivo, in embryonic mouse muscle (a myogenin-expressing tissue), as opposed to nonmuscle (nonexpressing) tissues that had a fully methylated site. In a C2C12-derived clone with enhanced myogenic ability, demethylation occurred within 2 h of induction of differentiation, suggesting the involvement of some active demethylation mechanism(s) that occur in the absence of DNA replication. Exposure to drugs that inhibit DNA methylation by acting on the S-adenosylmethionine metabolism produced a further reduction, to a few minutes, in the duration of the demethylation dynamics. These effects suggest that the final site-specific DNA methylation pattern of tissue-specific genes is defined through a continuous, relatively fast interplay between active DNA demethylation and re-methylation mechanisms.

Activated Raf inhibits avian myogenesis through a MAPK-dependent mechanism.

Chronic overexpression of the oncogenic form of Ras is a potent inhibitor of skeletal myogenesis. However, the intracellular signaling pathways that mediate the repressive actions of Ras on myogenic differentiation have yet to be identified. We examined the role of Raf-mediated signaling as a modulator of avian myogenesis. Raf overexpression elicited pronounced effects on both myoblasts and mature myocytes. Most notably, the embryonic chick myoblasts overexpressing a constitutively active form of Raf (RCAS-Raf CAAX or RCAS-Raf BXB) fail to form the large multinucleated myofibers characteristic of myogenic cultures. While residual myofibers were apparent in the RCAS-Raf BXB and RCAS-Raf CAAX infected cultures, these fibers had an atrophic phenotype. The altered morphology is not a result of reinitiation of the myonuclei cell cycle nor is it due to apoptosis. Furthermore, the mononucleated myoblasts misexpressing Raf BXB are differentiation-defective due to overt MAPK activity. Supplementation of the culture media with the MAPK kinase (MEK) inhibitor, PD98059, caused a reversal of the phenotype and allowed the formation of multinucleated myofibers at levels comparable to controls. Our results indicate that the Raf/MEK/MAPK axis is intact in chick myoblasts and that persistent activation of this signaling cascade is inhibitory to myogenesis.

Effects of prepartum supplementary fat and muscle hypertrophy genotype on cold tolerance in newborn calves.

Effects of feeding pregnant dams supplemental dietary fat during the last 55 d of gestation on cold tolerance of newborn crossbred calves with (Piedmontese cross, P, n = 15) or without (Hereford cross, H, n = 16) the muscle hypertrophy allele was determined. Primiparous F1 dams gestating F2 calves of the respective breeds were assigned randomly within breed to receive gestation diets containing either 2.2 (Low Fat; LF) or 5.1% fat (High Fat; HF). Safflower (Carthamus tinctorius L.) seeds containing 37% oil with 79% linoleic acid were the supplemental fat source in diets formulated to be isocaloric-isonitrogenous. At parturition, calves were separated from their dams, fed 38 degrees C pooled dairy cow colostrum (30 mL/kg BW), muzzled to prevent suckling, and returned to their dams in a heated (22 degrees C) room for 3.5 h. At 4 h of age (birth = 0 h), a catheter was inserted into the jugular vein. At 5 h of age, calves were placed in a 0 degrees C room for 140 min, and rectal temperatures and blood samples were obtained at 10- and 20-min intervals. Blood was assayed for cortisol and glucose. Rectal temperature was affected by diet (P<.05), time, diet x time, and breed x time (P<.01 for time and the interactions). Cortisol and glucose concentrations were not affected by diet, breed, or the diet x breed interaction, but they were affected by time, breed x time (both P<.01), and diet x time (P = .06). Calves from HF dams had higher rectal temperatures than calves from LF dams, and the HF calves maintained higher rectal temperatures throughout cold exposure. Cortisol concentrations were lower (P = .06) in calves from HF dams, and these calves had more (P = .06) glucose available for metabolic heat production than calves from LF dams. Piedmontese-cross calves maintained higher (P<.01) rectal temperatures and had higher cortisol and glucose (both P<.01) concentrations than did H-cross calves. We conclude that feeding dams supplemental fat during late gestation increased heat production in newborn calves and potentially could increase calf survival; calves with muscle hypertrophy may have a different ratio of shivering vs nonshivering thermogenesis due to differences in body composition or relationships among uncoupling proteins.

A role of midkine in the development of the neuromuscular junction.

Midkine (MK) is a member of a family of developmentally regulated neurotrophic and heparin-binding growth factors. It is expressed during the midgestation period in a retinoid-acid dependent manner during embryogenesis in the mouse. In vitro, it promotes neurite outgrowth from spinal cord neurons and cell migration. It expression is strongest in the central nervous system, thus suggesting a function for this protein in neural development. In this study, the role of MK in synaptogenesis was examined in the Xenopus system. A Xenopus MK cDNA was cloned from an embryonic library encompassing neurulation and synaptogenesis stages. By Northern blot analysis, MK mRNA was detected from the onset of neurulation and throughout the stages of synaptogenesis in the Xenopus embryo. This suggests that MK is also an important growth regulator in Xenopus embryogenesis. To study the function of MK in the development of the neuromuscular junction (NMJ), fusion proteins were made and their ability to induce the formation of acetylcholine receptor (AChR) clusters in cultured muscle cells was studied. Beads coated with MK strongly induce AChR clustering. When nerve-muscle cocultures were labeled with antibodies made against the MK fusion protein, MK immunoreactivity was detected at the NMJ. Unlike heparin-binding growth-associated molecule (HB-GAM), another member of this growth factor family, MK expression cannot be detected in the muscle but is present in spinal cord neurites. Consistent with these in vitro data is the observation that MK mRNA is only localized in the central nervous system but the protein is deposited at the intersomitic junction where the NMJ is located in vivo. Exogenously applied MK does bind to the heparan sulfate proteoglycan on the surface of Xenopus muscle cells. Agrin, a heparan-sulfate proteoglycan that induces the formation of AChR clusters in cultured muscle cells, binds strongly to MK. Bath application of MK in conjunction with agrin results in a change in the pattern of AChR clustering induced by agrin alone. These data suggest that MK is a neuron-derived factor that participates in the signal transduction process during NMJ development.

Two muscle-specific LIM proteins in Drosophila.

The LIM domain defines a zinc-binding motif found in a growing number of eukaryotic proteins that regulate cell growth and differentiation during development. Members of the cysteine-rich protein (CRP) family of LIM proteins have been implicated in muscle differentiation in vertebrates. Here we report the identification and characterization of cDNA clones encoding two members of the CRP family in Drosophila, referred to as muscle LIM proteins (Mlp). Mlp60A encodes a protein with a single LIM domain linked to a glycine-rich region. Mlp84B encodes a protein with five tandem LIM-glycine modules. In the embryo, Mlp gene expression is spatially restricted to somatic, visceral, and pharyngeal muscles. Within the somatic musculature, Mlp84B transcripts are enriched at the terminal ends of muscle fibers, whereas Mlp60A transcripts are found throughout the muscle fibers. The distributions of the Mlp60A and Mlp84B proteins mirror their respective mRNA localizations, with Mlp84B enrichment occurring at sites of muscle attachment. Northern blot analysis revealed that Mlp gene expression is developmentally regulated, showing a biphasic pattern over the course of the Drosophila life cycle. Peaks of expression occur late in embryogenesis and during metamorphosis, when the musculature is differentiating. Drosophila Mlp60A and Mlp84B, like vertebrate members of the CRP family, have the ability to associate with the actin cytoskeleton when expressed in rat fibroblast cells. The temporal expression and spatial distribution of muscle LIM proteins in Drosophila are consistent with a role for Mlps in myogenesis, late in the differentiation pathway.

Expression of the met receptor tyrosine kinase in muscle progenitor cells in somites and limbs is absent in Splotch mice.

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates proliferation, dissociation, migration and morphogenesis of cells in culture. To investigate a possible role for HGF/SF and its receptor, the Met tyrosine kinase, in embryonic development, we have analyzed their expression in mouse embryos from day 7.5 of gestation by whole-mount in situ hybridization. Met expression is first detected in the ventral portion of somites at day 9.25 of gestation (22 somite embryo) at the level of fore limb buds. As somites mature, met expression is detected in caudal somites, and is confined to the lateral and media] tips of the dermomyotome and dermomyotome/myotome respectively. In contrast, HGF/SF is expressed exclusively in the mesodermal core of the limb bud. As the dermomyotome elongates ventrolaterally, the met-expressing cells at the lateral tip appear to detach from the somite, invade the limb bud and localize at the dorsal and ventral limb sides in close proximity to HGF/SF-expressing cells. At later stages, both met- and HGF/SF-expressing cells appear to migrate distally and localize to the digit forming area of the developing hand plate. Met expression in the lateral dermomyotome and limb bud coincides with expression of Pax-3, a marker for migrating muscle precursor cells in the somite and limb. Splotch-2H and Splotch-delayed mice, which harbor mutations in Pax-3, show major disruptions in early limb muscle development. Significantly, no met-expressing cells were observed in the limbs of homozygous Splotch-2H and Splotch-delayed animals, whereas HGF/SF expression was not affected. The restricted expression of met to a sub-population of Pax-3-expressing cells in the lateral tip of the dermomyotome, demonstrates that met represents a unique molecular marker for this migratory cell population. From these observations, together with the biological activities of HGF/SF, we propose that in homozygous Splotch embryos the failure of muscle precursors to migrate into and populate the limb bud results from a loss of met expression in the cells at the ventrolateral edge of the somitic dermomyotome.

Effects of Prudhoe Bay crude oil on hatching success and associated changes in pipping muscles in embryos of domestic chickens (Gallus gallus).

Fertile white leghorn chicken eggs were exposed to 0, 1, 2, 4, 6, 8 or 16 microliters of Prudhoe Bay Crude oil (PBCO) on day 9 of incubation. The effects of oil on pipping and hatching success, body weight gain after hatching, serum creatine kinase levels, and pathological changes in organ systems were assessed in embryos that had survived acute toxic effects and were alive on day 18 of incubation. Exposure to oil greatly reduced pipping and hatching success. Severe edema and hemorrhage in the pipping muscle, multifocal subcapsular hepatic necrosis, marked depletion of lymphocytes in the bursa of Fabricius with infiltration by heterophils, and occasional dorso-caudal subcutaneous edema were observed in treated embryos. Pipping muscles were heavier in oil-exposed embryos. Embryos exposed to 4 microliters of PBCO had significantly reduced gain in body weight post-hatching. Serum creatine kinase levels were significantly elevated in the oil-exposed embryos only at the time of hatching. There was no evidence that exposure to oil caused degenerative changes in pipping muscle cells.

Injection of a K+ channel (Kv1.3) cRNA in fertilized eggs leads to functional expression in cultured myotomal muscle cells from Xenopus embryos.

The synthetic cRNA encoding for the major T lymphocyte K+ channel (Kv1.3) was injected into Xenopus fertilized eggs. Somites from embryos of stage 20-22 (about 40 h post-fertilization at 19 degrees C) were dissociated and myotomal muscle cells were cultured in vitro for 2 days. The whole cell configuration of the tight seal patch-clamp technique was used to record K+ channel activity in cultured myocytes. These myocytes have two endogenous delayed-rectifiers (sustained and transient) and an inward-rectifier K+ currents, all of which are insensitive to the scorpion toxin charybdotoxin. Cultured myocytes dissociated from embryos injected with the Kv1.3 cRNA expressed the exogenous Kv1.3 channel. The Kv1.3 channel was identified by its physiological (a very low recovery from inactivation) and its pharmacological properties (a high sensitivity to charybdotoxin). This work demonstrates that Xenopus cultured myotomal muscle cells represent a very efficient and practical assay system for the functional expression of cloned ion channels.

Bifunctional transcriptional properties of YY1 in regulating muscle actin and c-myc gene expression during myogenesis.

Oncogene expression is generally incompatible with terminal cell differentiation as in myogenesis. We present evidence that this incompatibility can be caused in part by the dual activity of a Kruppel-related zinc finger, YY1 (formerly F-ACT1), in differentially regulating oncogene and muscle-specific gene expression. The c-myc and skeletal alpha-actin gene promoters contain YY1 binding sites thought to act either as positive or negative cis-acting elements. Through manipulating the intracellular level of YY1 by treating primary myoblasts with bromodeoxyuridine (BrdU), which inhibited myogenesis and increased the YY1 protein content, or by transfecting YY1 cDNA expression vector, we show that it can simultaneously inhibit and activate expression of the skeletal alpha-actin and c-myc genes, respectively. The transrepression activity of YY1 solely depends on its C-terminal zinc finger region (amino acids 297-407) while its transactivation function requires an additional N-terminal domain (amino acids 1-90) normally masked in the full-length protein. We propose that the high level of YY1 in proliferating myoblasts might serve to maintain c-myc expression and suppress muscle actin expression, which can then be gradually reversed by downregulating YY1 activity toward myogenesis.