The genesis and transmission of epidermal potentials in an amphibian embryo

The genesis and transmission of action potentials in epidermal cells of the newt (Cynops pyrrhogaster) embryo were investigated with special reference to cellular differentiation during development. Typical action potentials can be recorded from any of the epidermal cells at Stage 31. These potentials consist of a fast spike (18 msec) followed by a slow component (164 msec). The potential is graded with current intensity, and only the slow component initiates action potentials in adjacent cells and induces a transmission to other cells. The fast spike was found in all epidermal cells throughout the embryonic stages examined (Stages 26–47). The slow potential, however, appears at Stage 28, persists until Stage $\text{36}\text{37}$ just before hatching and then disappears at Stage $\text{38}\text{42}$. Electrical recordings from traumatic embryos (embryos without neural crest cells) or from cultured epidermal cell masses isolated from the pregastrula or the ventral region of the neurula, were compared with the intact embryo. No differences were observed in either the form of the action potential or its transmission. Thus these action potentials appear to be derived from epidermal cells, and are not of nervous origin. Evidence suggests that the transient establishment of excitable membranes in epidermal cells during differentiation is closely related to neural cell differentiation.     

Elevation of maternal glucocorticoid hormones alters neurotransmitter phenotypic expression in embryos

The tissue interaction between the notochord and the somites of the vertebrate embryo establishes the proper shape and constitution of the vertebral cartilage. Soon after somite formation, the somite differentiates into a cartilage-forming part, the sclerotome, and a muscle and skin-forming part, the dermamyotome. These components of the somite were dissected from $\text{3}\text{1}\text{2}\text{-}\text{day-old}$ chick embryos (stage $\text{18}\text{1}\text{2}\text{–19}$ and cultured in vitro in the presence or absence of notochord. It was found that the sclerotome cells respond to the notochord by an increased incidence of hyaline cartilage nodules, greater accumulation of sulfated glycosaminoglycans, synthesis of larger aggregates of proteoglycans, increased DNA accumulation, and accelerated DNA synthesis. The dermamyotome did not show these changes. These results indicate that the notochord enhances cartilage differentiation in the sclerotome. Under these conditions, the notochord did not elicit cartilage formation in the dermamyotome.     

Immunochemical localization of myosin heavy chain isoforms and paramyosin in developmentally and structurally diverse muscle cell types of the nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans contains two major groups of muscle cells that exhibit organized sarcomeres: the body wall and pharyngeal muscles. Several additional groups of muscle cells of more limited mass and spatial distribution include the vulval muscles of hermaphrodites, the male sex muscles, the anal-intestinal muscles, and the gonadal sheath of the hermaphrodite. These muscle groups do not exhibit sarcomeres and therefore may be considered smooth. Each muscle cell has been shown to have a specific origin in embryonic cell lineages and differentiation, either embryonically or postembryonically (Sulston, J. E., and H. R. Horvitz. 1977. Dev. Biol. 56:110-156; Sulston, J. E., E. Schierenberg, J. White, and J. N. Thomson. 1983. Dev. Biol. 100:64- 119). Each muscle type exhibits a unique combination of lineage and onset of differentiation at the cellular level. Biochemically characterized monoclonal antibodies to myosin heavy chains A, B, C, and D and to paramyosin have been used in immunochemical localization experiments. Paramyosin is detected by immunofluorescence in all muscle cells. Myosin heavy chains C and D are limited to the pharyngeal muscle cells, whereas myosin heavy chains A and B are localized not only within the sarcomeres of body wall muscle cells, as reported previously, but to the smooth muscle cells of the minor groups as well. Myosin heavy chains A and B and paramyosin proteins appear to be compatible with functionally and structurally distinct muscle cell types that arise by multiple developmental pathways.     

Myogenic defect in muscular dystrophy of the chicken.

Inherited muscular dystrophy of the chicken is thought to arise from abnormal development of trophic regulation of skeletal muscles by their innervating nerves. To determine whether expression of muscular dystrophy in the chicken is a property of the nerves or of the muscles, wing limb buds were transplanted between normal and dystrophic chick embryos at $\text{3}\text{1}\text{2}$ days of incubation (stage 19–20). Muscles of donor limbs innervated by nerves of the hosts were compared to contralateral unoperated host limb muscles in chicks from 6 to 25 weeks after hatching. Expression of normal or dystrophic phenotype was determined by examination of five different properties which are altered in dystrophic chick muscle: electromyographic evidence of myotonia; fiber diameter; acetylcholinesterase activity, localization, and isozymes; lactic dehydrogenase activity; and succinic dehydrogenase activity. Genetically normal muscle innervated by nerves of normal or dystrophic hosts was phenotypically normal while genetically dystrophic muscle innervated by normal nerves was phenotypically dystrophic. The results suggest that inherited muscular dystrophy of the chicken arises from a defect of muscle rather than from a lesion in the nerves themselves.     

In vitro development of early postimplantation rat embryos

Rat embryos explanted at $\text{7}\text{1}\text{2}$ or $\text{8}\text{1}\text{2}$post coitum were cultured throughout the major stages of organogenesis in a system of rotating bottles containing heat-inactivated, immediately centrifuged (I.C.) serum. About 80% of the $\text{8}\text{1}\text{2}\text{-}\text{day}$ explants and 50% of the $\text{7}\text{1}\text{2}\text{-}\text{day}$ explants developed a blood circulation in the yolk sac; in these embryos, organogenesis and growth rates were similar to those of embryos in vivo. In cultures continued for 4 or 5 days, many of the embryos developed 30–40 somites. There was little difference in the subsequent development of embryos cultured in maternal serum or male serum during the egg-cylinder stage except for a possible decrease in the frequency of normal axial rotation in embryos from the male serum. Development in rotator bottles was much better than in watchglass cultures.     

Divalent antibodies to mouse embryonal carcinoma cells inhibit compaction in the mouse embryo

Divalent antibodies from two antisera to embryonal carcinoma (EC) cells, F9 line, inhibited compaction in the preimplantation mouse embryo. One of these antisera, AF9-2, completely inhibited compaction at the 8-cell stage when the embryos were continuously incubated in a $\text{1}\text{100}$ dilution of the antiserum in culture medium from the 4-cell stage. Cell divisions continued and no cellular degeneration occurred. When cultured control embryos (preimmune and nonimmune sera) were normal blastocysts, many of the AF9-2-treated embryos were characterized by vacuolated cells and rounded rather than squamous, trophoectodermal cells. Anti-mouse spleen serum ($\text{1}\text{10}$, $\text{1}\text{100}$) had no effect on development. Purified divalent IgGs from AF9-2 (ammonium sulfate precipitation and DEAE chromatography) also were inhibitory at 30 μg/ml. Inhibition of compaction by AF9-2 was reversible. When uncompacted midmorula-stage embryos in AF9-2 ($\text{1}\text{10}$) were transferred to medium without serum, there was a threefold increase in the percentage (70%) of normal blastocysts at the end of culture. Fluorescence microscopy demonstrated that AF9-2 antibodies, unlike preimmune and nonimmune sera, were bound to the surface of 8-cell and early morula-stage embryos. Inhibition by AF9-2 does not occur merely by nonspecifically coating cell surfaces so that they no longer recognize each other, since antispleen antibodies show similar binding by immunofluorescence but no inhibition. This study provides strong evidence that AF9-2 has specific divalent antibodies which block morphogenesis. Since newly compacted embryos lost their compacted appearance in AF9-2, these divalent antibodies cause a loss of cell adhesion, do not extensively cross-link adjacent cell surfaces, and cannot cause the compacted phenotype by agglutination.     

Ultraviolet effects on presumptive primordial germ cells (pPGCs) in Xenopus laevis after the cleavage stage

The presumptive primordial germ cell (pPGC) number with development after the cleavage stage and the fate of pPGCs damaged by uv irradiation were studied in successive Epon sections (0.5 μm thick) with the light microscope in both uv-irradiated and unirradiated Xenopus embryos. taking survival rate and sterility into consideration. The pPGCs of the uv-irradiated embryos occupy nearly the same location in the embryos as those of the unirradiated embryos at stages 12, 17, 23, and 28 [see Ikenishi, K., and Kotani, M. (1975). Develop. Growth Different. 17, 101–110]. At stage $\text{33}\text{34}$ they are found in the central part of the endoderm cell mass in the uv-irradiated embryos, while they are situated in the lateral or dorsal part of the endoderm cell mass in the unirradiated. In the uv-irradiated embryos, a cavity which was never found in the unirradiated embryos was observed in the endoderm cell mass beneath the archenteron cavity and in the almost-median part of the posterior endoderm cell mass at stages 17 and 23, respectively, and some vacuoles in pPGCs as well as in somatic cells around those pPGCs were noticed at stages $\text{17–}\text{33}\text{34}$. The number of pPGCs of the unirradiated enbryos increases about three- or fourfold during stages 12–46, while the pPGCs of the uv-irradiated embryos slowly increase in number from stage 17 to stage 28, indicating that the division occurs in pPGCs, then decrease with development and finally disappear from the tadpole.     

Analysis of cartilage differentiation from skeletal muscle grown on bone matrix: II. Chondroitin sulfate synthesis and reaction to exogenous glycosaminoglycans

In the first paper in this series (Nathanson, M. A., and Hay, E. D. (1980). Develop. Biol. 78, 301–331), we described the ultrastructural alterations that take place when embryonic skeletal muscle is induced to form hyaline cartilage by demineralized bone matrix in vitro. In this paper, we analyze the pattern of appearance of chondroitin sulfates and dermatan sulfate in injured muscle in situ and in explants of muscle cultured either on bone matrix or on collagen gel. We also investigate the effects of exogenous glycosaminoglycans on the cultures to determine whether chondroitin sulfate (Ch-S) and hyaluronic acid (HA) can enhance or inhibit the biochemical differentiation of cartilage under these conditions. Our results indicate that during the first morphological phase, 1–3 days in vitro, there is an increased sulfate uptake, a shift in the relative abundance of Ch-S, and an increase in the ratio of chondroitin-4-sulfate (Ch-4-S) to chondroitin-6-sulfate (Ch-6-S); this change is correlated with the transformation of myoblasts to fibroblast-like cells in both types of cultures. A similar increase in the $\text{Ch-4-S}\text{Ch-6-S}$ ratio occurs in injured muscle in situ, suggesting that phase I is a regenerative response. Explants on bone matrix sustain Ch-4-S levels between 4 and 5 days (phase II) and show a large increase in Ch-4-S and sulfate incorporation when they form cartilage at 6–10 days (phase III). Explants on collagen gels regenerate muscle at 4–10 days with decreasing $\text{Ch-4-S}\text{Ch-6-S}$ ratios and decreasing sulfate incorporation. The data demonstrate that an environmental influence, such as trauma, is sufficient to alter the biosynthetic expression of skeletal muscle and that under appropriate conditions (such as the presence of bone matrix) this response may be augmented, leading to the synthesis of extracellular matrix components at ratios characteristic of cartilage. Exogenous Ch-S and HA did not significantly effect this overall pattern. These results are discussed in relation to the morphological observations presented in the preceding paper.     

A multifunctional cyclic nucleotide- and Ca2+-independent protein kinase from rabbit skeletal muscle

A cyclic nucleotide- and Ca²⁺-independent protein kinase, initially identified as a glycogen synthase kinase (Itarte, E. and Huang, K.-P. (1979) J. Biol. Chem. $\text{254}$, 4052–4057), was also found to phosphorylate phosphorylase kinase and troponin from skeletal muscle as well as myosin light chain and myosin light chain kinase from both smooth and skeletal muscles. With the exception of myosin light chain from skeletal muscle, all the above-mentioned proteins are also substrates for the multifunctional cAMP-dependent protein kinase. The results suggest that this cyclic nucleotide- and Ca²⁺-independent protein kinase, like cAMP-dependent protein kinase, may have multiple cellular functions.     

Evidence for dosage compensation of an X-linked gene in the 6-day embryo of the mouse.

The time at which dosage compensation of an X-linked gene in the mouse is established has been estimated by measuring the activity levels of two glycolytic enzymes, phosphoglycerate kinase (EC 2.7.2.3) and triosephosphate isomerase (EC 5.3.1.1), during early development of embryos from $\text{X}\text{X}$ and $\text{X}\text{O}$ mice. During preimplantation development the level of phosphoglycerate kinase in embryos from $\text{X}\text{X}$ mice was constant for the first 48 hr of development (2.55–2.71 nmoles/hr/embryo) and then dropped to one-half the earlier level (1.44 nmoles/hr/embryo) by 72 hr of development. The general developmental profile of phosphoglycerate kinase was similar in embryos from $\text{X}\text{O}$ mice; however, the absolute level of enzyme activity was reduced to approximately 1.4 nmoles/hr/embryo during the first 48 hr of development and to 0.9 nmoles/hr/embryo by 72 hr of development. These differences in phosphoglycerate kinase levels between embryos from $\text{X}\text{X}$ and $\text{X}\text{O}$ mice disappeared between the blastocyst and egg cylinder stages (150 hr postplug) during which time the activity levels had increased to 183 and 193 nmoles/hr/egg cylinder for embryos from $\text{X}\text{O}$ and $\text{X}\text{X}$ mice, respectively.The activity level for triosephosphate isomerase in embryos from $\text{X}\text{X}$ and $\text{X}\text{O}$ mothers did not differ at any stage of development; this suggests that the gene coding for triosephosphate isomerase is autosomal. In addition the level of activity remained constant during preimplantation development (approximately 3 nmoles/hr/embryo) and then, like phosphoglycerate kinase, increased 100-fold between the blastocyst and egg cylinder stages.The frequency distribution of the activity ratio (triosephosphate isomerase to phosphoglycerate kinase) in the egg cylinder of individual embryos from both $\text{X}\text{X}$ and $\text{X}\text{O}$ mothers was determined in order to detect differences among $\text{X}\text{X}\text{,}\text{X}\text{O}$ and $\text{X}\text{Y}$ embryos. These frequency distributions were unimodal, and hence these results coupled with the gene dosage differences observed during preimplantation development indicate that dosage compensation for an X-linked gene has been established in the 6-day mouse embryo.     

Effects of the Brachyury (T) mutation on morphogenetic movement in the mouse embryo

$\text{T}\text{T}$ embryos have been first distinguishable at 8 days post coitum by their gross morphological abnormalities. By quantitative morphometry of histological sections, anomalies in the homozygotes were expressed numerically. At 8 days p.c., morphologically identifiable T-homozygotes had an increased number of ectodermal and a reduced number of mesodermal cells compared to the wild type. At 7 days p.c., embryos with a low mesoderm/ectoderm ratio were found only in litters of $\text{T}\text{+}\text{×}\text{T}\text{+}$ matings at the expected frequency. At 6 days p.c., one-fourth of the embryos in $\text{T}\text{+}\text{×}\text{T}\text{+}$ litters showed a delay in the transition from cuboidal to squamous endoderm. No such embryos were found in the +/+ × +/+ matings. In 6-, 7-, and 8-day mutant embryos, cells proliferated at statistically normal rates. Therefore, it may be said that advanced morphological irregularities of 8-day homozygotes cannot be accounted for by anomalies in cell proliferation. When the total cell number was 5 × 10⁴/embryo (8 days), a sudden change was observed in the regional distribution of mesodermal and ectodermal cells along the anteroposterior axis of $\text{T}\text{T}$ embryos. Since no regional difference in the cell cycle time was observed, these abnormalities may best be explained by anomalies in cell migration. These results strongly suggest abnormal morphology of $\text{T}\text{T}$ mutants resulting from defects in morphogenetic movement.     

The effect of embryo quality at freezing on subsequent development of thawed cow embryos

Day 7 to 9 embryos were frozen by a rapid two-step method to ?38°C before being plunged into liquid nitrogen. Glycerol was used as the cryoprotectant and, following thawing, the embryos were cultured for 12 – 24 hours in PBS + 15% heat-treated steer serum. In Experiment 1, embryos were frozen in 2.0 ml glass ampoules or 0.5 ml Cassou straws. Two levels of glycerol (1.0M and 1.4M) gave comparable $\text{in vitro}$ survival rates ($\text{12}\text{20}$ and $\text{13}\text{25}$, respectively). A greater proportion of embryos developed in culture after freezing in straws. In Experiment 2, embryos were classified morphologically before and after freezing into 5 grades (1 = excellent; 2 = good; 3 = fair; 4 = poor; 5 = degenerate). Only embryos of grade 1, 2 and 3 were frozen. The post-thaw survival rates for embryos graded 1, 2 and 3 before freezing were 100% ($\text{11}\text{11}$), 86% ($\text{24}\text{28}$) and 83% ($\text{20}\text{24}$), respectively. Furthermore, the porportion of surviving embryos estimated to be of poor quality (grade 4) was greater for embryos graded 3 before freezing ($\text{13}\text{20}$) than for embryos graded 2 ($\text{6}\text{24}$) or 1 ($\text{1}\text{11}$). The percentage of embryos which developed normally after $\text{in vitro}$ culture for each of the pre-freezing grades 1, 2 and 3 was 91% ($\text{10}\text{11}$), 50% ($\text{14}\text{28}$) and 29% ($\text{7}\text{24}$), respectively. Of the total number of frozen-thawed embryos which developed in culture, $\text{5}\text{31}$ (16%) were of poor quality. The proportion of poor quality developing embryos was greater inembryos graded 3 before freezing ($\text{3}\text{7}$) than those graded 2 ($\text{2}\text{14}$). All of the embryos graded 1 before freezing and which developed in culture were of good quality. Results indicate that, if high post-thaw survival rates are to be obtained, stringent embryo selection processes will be required.     

DNA synthesis and the cell cycle in cultures of normal and mutant (mdg/mdg) embryonic mouse muscle cells.

The relationship between cell fusion, DNA synthesis and the cell cycle in cultured embryonic normal and dysgenic ($\text{mdg}\text{mdg}$) mouse muscle cells has been determined by autoradiography. The experimental evidence shows that the homozygous mutant myotubes form by a process of cell fusion and that nuclei within the myotubes do not synthesize DNA or undergo mitotic or amitotic division. The duration of the total cell cycle and its component phases was statistically the same in 2-day normal and mutant ($\text{mdg}\text{mdg}$) myogenic cultures with the approximate values: T, 21.5 hr; G₁, 10.5 hr; S, 7.5 hr; and G₂, 2.5 hr. In both kinds of cultures, labeled nuclei appeared in myotubes 15–16 hr after mononucleated cells were exposed to [³H]thymidine, and the rate of incorporation of labeled nuclei into multinucleated muscle cells was comparable in control and dysgenic cultures. Thus, homozygous $\text{mdg}\text{mdg}$ muscle cells in culture are similar to control cells with respect to their mechanism of myotube formation and the coordinate regulation of DNA synthesis and the cell cycle during myogenesis.     

Immunohistochemistry of chaetognath body wall muscles

Abstract. A light and electron immunohistochemical study was carried out on the body wall muscles of the chaetognath Sagitta friderici for the presence of a variety of contractile proteins (myosin, paramyosin, actin), regulatory proteins (tropomyosin, troponin), and structural proteins (α‐actinin, desmin, vimentin). The primary muscle (～80% of body wall volume) showed the characteristic structure of transversely striated muscles, and was comparable to that of insect asynchronous flight muscles. In addition, the body wall had a secondary muscle with a peculiar structure, displaying two sarcomere types (S1 and S2), which alternated along the myofibrils. S1 sarcomeres were similar to those in the slow striated fibers of many invertebrates. In contrast, S2 sarcomeres did not show a regular sarcomeric pattern, but instead exhibited parallel arrays of 2 filament types. The thickest filaments (～10–15 nm) were arranged to form lamellar structures, surrounded by the thinnest filaments (～6 nm). Immunoreactions to desmin and vimentin were negative in both muscle types. The primary muscle exhibited the classical distribution of muscle proteins: actin, tropomyosin, and troponin were detected along the thin filaments, whereas myosin and paramyosin were localized along the thick filaments; immunolabeling of α‐actinin was found at Z‐bands. Immunoreactions in the S1 sarcomeres of the secondary muscle were very similar to those found in the primary muscle. Interestingly, the S2 sarcomeres of this muscle were labeled with actin and tropomyosin antibodies, and presented no immunore‐actions to both myosin and paramyosin. α‐Actinin in the secondary muscle was only detected at the Z‐lines that separate S1 from S2. These findings suggest that S2 are not true sarcomeres. Although they contain actin and tropomyosin in their thinnest filaments, their thickest filaments do not show myosin or paramyosin, as the striated muscle thick myofilaments do. These peculiar S2 thick filaments might be an uncommon type of intermediate filament, which were labeled neither with desmin or vimentin antibodies.     

Retinoic acid induces neuronal differentiation of a cloned human embryonal carcinoma cell line in vitro

The human embryonal carcinoma cell lines $\text{NT2}\text{D1}$ and $\text{NT2}\text{B9}$, clonally derived from Tera-2, differentiate extensively in vitro when exposed to retinoic acid. This differentiation is marked by the appearance of several morphologically distinct cell types and by changes in cell surface phenotype, particularly by the disappearance of stage-specific embryonic antigen-3 (SSEA-3), which is characteristically expressed by human EC cells. Among the differentiated cells are neurons, which form clusters interconnected by extended networks of axon bundles, and which express tetanus toxin receptors and neurofilament proteins. These observations constitute the first instance of extensive somatic differentiation of a clonal human EC cell line in vitro.     

Genetic analysis of myosin assembly inCaenorhabditis elegans

The established observations and unresolved questions in the assembly of myosin are outlined in this article. Much of the background information has been obtained in classical experiments using the myosin and thick filaments from vertebrate skeletal muscle. Current research is concerned with problems of myosin assembly and structure in smooth muscle, a broad spectrum of invertebrate muscles, and eukaryotic cells in general. Many of the general questions concerning myosin assembly have been addressed by a combination of genetic, molecular, and structural approaches in the nematode Caenorhabditis elegans. Detailed analysis of multiple myosin isoforms has been a prominent aspect of the nematode work. The molecular cloning and determination of the complete sequences of the genes encoding the four isoforms of myosin heavy chain and of the myosin-associated protein paramyosin have been a major landmark. The sequences have permitted a theoretical analysis of myosin rod structure and the interactions of myosin in thick filaments. The development of specific monoclonal antibodies to the individual myosins has led to the delineation of the different locations of the myosins and to their special roles in thick filament structure and assembly. In nematode body-wall muscles, two isoforms, myosins A and B, are located in different regions of each thick filament. Myosin A is located in the central biopolar zones, whereas myosin B is restricted to the flanking polar regions. This specific localization directly implies differential behavior of the two myosins during assembly. Genetic and structural experiments demonstrate that paramyosin and the levels of expression of the two forms are required for the differential assembly. Additional genetic experiments indicate that several other gene products are involved in the assembly of myosin. Structural studies of mutants have uncovered two new structures. A core structure separate from myosin and paramyosin appears to be an integral part of thick filaments. Multifilament assemblages exhibit multiple nascent thick filament-like structures extending from central paramyosin regions. Dominant mutants of myosin that disrupt thick filament assembly are located in the ATP and actin binding sites of the heavy chain. A model for a cycle of reactions in the assembly of myosin into thick filaments is presented. Specific reactions of the two myosin isoforms, paramyosin, and core proteins with multifilament assemblages as possible intermediates in assembly are proposed.     

Triton X nonionic surfactants reversibly inhibit the EDTA/KC1 ATPase activity of myosin

The nonionic octylphenoxy polyethoxy series of surfactants, Triton X, reversibly inhibited the $\text{EDTA}\text{KCl}$ ATPase activity of purified rabbit skeletal muscle myosin at concentrations at or below their reported critical micelle concentrations. The maximum degree of enzyme inhibition increased with ethoxy content to 88% (with Triton X 102 average ethoxy content, 12.5 per molecule). The results suggest that binding of the surfactant to the myosin molecule occurs below the critical micelle concentrations and that the hydrophilic ethoxy chain forms a diffusion barrier against approach of ATP to the enzyme's active site. This model has implications for the organization of myosin in the plasma membrane of phagocytes.     

Early appearance in neural crest and crest-derived cells of an antigenic determinant present in avian neurons

A monoclonal antibody (“$\text{E}\text{C8}$”) against chicken dorsal root ganglion cells has been produced. The epitope (antigenic determinant) to which this antibody binds appears in neuronal cells—of both the peripheral and central nervous systems—and in a limited number of nonneuronal cell types in avian embryos. The epitope is intracellular and is probably part of a protein as judged by its susceptibility to proteases. This epitope appears very early in neuronal development. It may be detected in brain, spinal cord, and ventral root nerve fibers of Hamburger-Hamilton stage 16 chicken embryos (51–56 hr of incubation). At this same age, $\text{E}\text{C8}\text{-}\text{immunoreactive}$ cells can be found in the neural crest migratory space between the neural tube and the somite about a day before dorsal root ganglia begin to coalesce. Since some cultured neural crest cells (but not somitic mesenchymal cells) also express this epitope, we propose that the $\text{E}\text{C8}$ monoclonal antibody identifies an early differentiating subpopulation of neural crest cells which express this putative neuronal trait soon after the time of cessation of migration in vivo.     

β-Filagenin, a Newly Identified Protein Coassembling with Myosin and Paramyosin in Caenorhabditis elegans

Muscle thick filaments are stable assemblies of myosin and associated proteins whose dimensions are precisely regulated. The mechanisms underlying the stability and regulation of the assembly are not understood. As an approach to these problems, we have studied the core proteins that, together with paramyosin, form the core structure of the thick filament backbone in the nematode Caenorhabditis elegans. We obtained partial peptide sequences from one of the core proteins, β-filagenin, and then identified a gene that encodes a novel protein of 201–amino acid residues from databases using these sequences. β-Filagenin has a calculated isoelectric point at 10.61 and a high percentage of aromatic amino acids. Secondary structure algorithms predict that it consists of four β-strands but no α-helices. Western blotting using an affinity-purified antibody showed that β-filagenin was associated with the cores. β-Filagenin was localized by immunofluorescence microscopy to the A bands of body–wall muscles, but not the pharynx. β-filagenin assembled with the myosin homologue paramyosin into the tubular cores of wild-type nematodes at a periodicity matching the 72-nm repeats of paramyosin, as revealed by immunoelectron microscopy. In CB1214 mutants where paramyosin is absent, β-filagenin assembled with myosin to form abnormal tubular filaments with a periodicity identical to wild type. These results verify that β-filagenin is a core protein that coassembles with either myosin or paramyosin in C. elegans to form tubular filaments.     

An aortic spontaneously active phosphatase dephosphorylates myosin and inhibits actin-myosin interaction

Actin-myosin interaction in aortic actomyosin reportedly requires phosphorylation of the 20,000 dalton myosin light chains. A spontaneously active phosphatase which dephosphorylates phosphorylase $\text{a}$ and isolated phosphorylated cardiac myosin light chains was extracted from bovine aortic smooth muscle. This enzyme, when added to aortic native actomyosin (a) significantly suppressed phosphorylation of the light chains of the native hexameric smooth muscle myosin, (b) accelerated the rate and increased the magnitude of myosin light chain dephosphorylation in actomyosin that had been prephosphorylated, and (c) markedly attenuated the rate of actin-myosin interaction. These results support the hypothesis that myosin phosphorylation and subsequent actin-myosin interactions (contractility) in vascular smooth muscle may be modulated by spontaneously active aortic phosphatase.