Patterning of fast and slow fibers within embryonic muscles is established independently of signals from the surrounding mesenchyme.

During embryonic development, and before functional innervation, a highly stereotypic pattern of slow- and fast-contracting primary muscle fibers is established within individual muscles of the limbs, from distinct populations of myoblasts. A difference between the fiber-type pattern found within chicken and quail pectoral muscles was exploited to investigate the contributions of somite-derived myogenic precursors and lateral plate-derived mesenchymal stroma to the establishment of muscle fiber-type patterns. Chimeric chicken/quail embryos were constructed by reciprocal transplantation of somites or lateral plate mesoderm at stages prior to muscle formation. Muscle fibers derived from quail myogenic precursors that had migrated into chicken stroma showed a quail pattern of mixed fast- and slow-contracting muscle fibers. Conversely, chicken myogenic precursors that had migrated into quail stroma showed a chicken pattern of nearly exclusive fast muscle fiber formation. These results demonstrate in vivo an intrinsic commitment to fiber-type on the part of the myoblast, independent of extrinsic signals it receives from the mesenchymal stroma in which it differentiates.

Adhesive events in retinal development and function: the role of integrin receptors.

Cells in the developing retina contact a vast array of molecular cues in their microenvironment that are thought to guide their development. Many of these cues are embedded in the surface of neighboring cells or deposited within the extracellular matrix (ECM). Evidence has accumulated that cell-cell and cell-ECM interactions are essential in many phases of neural development, including neuroblast migration, determination of cell fate, axon outgrowth and synapse formation. In this chapter, we examine the developmental and functional roles fulfilled by integrins, a family of receptors for ECM molecules and cell adhesion molecules (CAMs). We have approached this problem by addressing a series of three questions: (1) which integrins are expressed in developing retina? (2) when and where are they expressed? and, (3) what functions do they carry out? Integrins have previously been implicated in axon extension, but new evidence suggests that they are also involved in earlier developmental events in preaxonal neuroblasts. High levels of expression of at least eight integrin subunits have been documented in these young retinal cells, and integrins containing the beta 1 subunit have been implicated in migration of adolescent retinal ganglion cells. Integrin expression persists through adulthood, both in the retina and in the neighboring layer of the retinal pigment epithelium (RPE). The integrin alpha v beta 5 has been shown to reside on the apical surface of the RPE and has been implicated in the phagocytosis of shed photoreceptor outer segments.

SF/HGF is a mediator between limb patterning and muscle development.

Scatter factor/hepatocyte growth factor (SF/HGF) is known to be involved in the detachment of myogenic precursor cells from the lateral dermomyotomes and their subsequent migration into the newly formed limb buds. As yet, however, nothing has been known about the role of the persistent expression of SF/HGF in the limb bud mesenchyme during later stages of limb bud development. To test for a potential role of SF/HGF in early limb muscle patterning, we examined the regulation of SF/HGF expression in the limb bud as well as the influence of SF/HGF on direction control of myogenic precursor cells in limb bud mesenchyme. We demonstrate that SF/HGF expression is controlled by signals involved in limb bud patterning. In the absence of an apical ectodermal ridge (AER), no expression of SF/HGF in the limb bud is observed. However, FGF-2 application can rescue SF/HGF expression. Excision of the zone of polarizing activity (ZPA) results in ectopic and enhanced SF/HGF expression in the posterior limb bud mesenchyme. We could identify BMP-2 as a potential inhibitor of SF/HGF expression in the posterior limb bud mesenchyme. We further demonstrate that ZPA excision results in a shift of Pax-3-positive cells towards the posterior limb bud mesenchyme, indicating a role of the ZPA in positioning of the premuscle masses. Moreover, we present evidence that, in the limb bud mesenchyme, SF/HGF increases the motility of myogenic precursor cells and has a role in maintaining their undifferentiated state during migration. We present a model for a crucial role of SF/HGF during migration and early patterning of muscle precursor cells in the vertebrate limb.

Sonic hedgehog enhances somite cell viability and formation of primary slow muscle fibers in avian segmented mesoderm.

Primary skeletal muscle fibers first form in the segmented portions of paraxial mesoderm called somites. Although the neural tube and notochord are recognized as crucial in patterning myogenic cell lineages during avian and mammalian somitic myogenesis, the source, identities, and actions of the signals governing this process remain controversial. It has been shown that signals emanating from the ventral neural tube and/or notochord alone or Shh alone serve to activate MyoD expression in somites. However, beyond a role in initiating MyoD expression, little is known about the effects of Shh on primary muscle fiber formation in somites of higher vertebrates. The studies reported here investigate how the ventral neural tube promotes myogenesis and compare the effects of the ventral neural tube with those of purified Shh protein on fiber formation in somites. We show that purified Shh protein mimics actions of the ventral neural tube on somites including initiation of muscle fiber formation, enhancement of numbers of primary muscle fibers, and particularly, the formation of primary fibers that express slow myosin. There is a marked increase in slow myosin expression in fibers in response to Shh as somites mature. The effects of ventral neural tube on fiber formation can be blocked by disrupting the Shh signaling pathway by increasing the activity of somitic cyclic AMP-dependent protein kinase A. Furthermore, it was demonstrated that apoptosis is a dominant fate of somite cells, but not somitic muscle fibers, when cultured in the absence of the neural tube, and that application of Shh protein to somites reduced apoptosis. The block to apoptosis by Shh is a manifestation of the maturity of the somite with a progressive increase in the block as somites are displaced rostrally from somite III forward. We conclude that purified Shh protein in mimicking the effects of the ventral neural tube on segmented mesoderm can exert pleiotropic effects during primary myogenesis, including: control of the proliferative expansion of myogenic progenitor cells, antagonism of cell death pathways within the precursors to muscle fibers, and during the crucial process of primary myogenesis, can exert an effect on diversification of muscle fiber types.

The alpha4 subunit of integrin is important for neural crest cell migration.

We identify the alpha4 subunit of integrin as a predominant integrin expressed by neural crest cells in both avian and murine embryos. Using degenerate primers, we obtained a PCR fragment of the chick integrin alpha4 subunit that was subsequently used to clone the full-length subunit with a predicted amino acid sequence 60% identical to human and mouse alpha4 subunits. In situ hybridization demonstrates that chick integrin alpha4 mRNA is expressed at high levels by migrating neural crest cells and neural crest-derived ganglia at both cranial and trunk levels. An antibody against the murine alpha4 subunit revealed similar distribution patterns in mouse to chick. In addition to neural crest cells, the integrin alpha4 subunit was later observed on the muscle masses of the limb, the apical ectodermal ridge, and the developing liver. To examine the functional role of the integrin alpha4 subunit in neural crest cell migration, we used an explant preparation that allows visualization of neural crest cells in their normal environment with or without perturbing reagents. In the presence of a blocking antibody against the mouse integrin alpha4 subunit, there was a profound abrogation of neural crest cell migration at trunk and hindbrain levels. Both the numbers of migrating neural crest cells and the total distance traversed were markedly reduced. Similarly, avian embryos injected with synthetic peptides that contain the integrin alpha4 binding site in fibronectin displayed abnormal neural crest cell migration. Our results suggest that the integrin alpha4 subunit is important for normal neural crest cell migration and may be one of the primary alpha subunits used for neural crest cell migration in vivo. Furthermore, the integrin alpha4 subunit represents a useful neural crest marker in the mouse.

Widespread expression of beta1 integrins in the developing chick retina: evidence for a role in migration of retinal ganglion cells.

During extension of axons, critical neuronal interactions with extracellular matrix (ECM) and other cells are thought to be mediated in part by heterodimeric beta1 integrin receptors. In this report, we examine the expression and function of beta1 integrins in the developing chick retina. Expression of the beta1 subunit, assayed by in situ hybridization and antibody staining of dissociated cells, was widespread in undifferentiated neuroepithelial cells, before the initiation of axons. Expression persisted in most retinal cell layers throughout embryonic development, during and after axon extension. The repertoire of beta1-associated alpha subunits was examined using reverse transcription-polymerase chain reaction. In addition to the alpha6 and alpha8 subunits previously reported, chick homologues of the alpha2 and alpha4 subunits were detected. Developmental Northern blots revealed varying patterns of integrin subunit expression and showed that expression of beta1 and the mRNAs of its associated alpha subunits are not always coregulated during retinal development. The timing and distribution of expression suggested that beta1 integrins may be involved in other developmental events in addition to axon extension. To address functions carried out by beta1 integrins in the early retina, explanted eye cups were incubated in the presence of function blocking anti-beta1 antibody and migration of newly born retinal ganglion cells (RGCs) was assessed. RGC migration from the ventricular zone to the vitreal border was significantly inhibited, suggesting that beta1 integrins play a role in neuroblast migration in the retina.

Temporal and spatial regulation of integrin vitronectin receptor mRNAs in the embryonic chick retina.

PURPOSE: To identify integrin vitronectin receptor subunit mRNAs in the developing avian retina and to track their expression. METHODS: Reverse transcription-polymerase chain reaction was used to identify integrin vitronectin receptor subunit mRNAs expressed in embryonic chick retina. cDNA clones encoding the beta 5 subunit were isolated and sequenced. Expression patterns of mRNAs encoding alpha v, beta 3, and beta 5 subunits were analyzed using northern analysis and in situ hybridization. RESULTS: Integrin beta 1, beta 3, and beta 5 subunit mRNA were identified in embryonic day 6 chick retina. The sequence of chicken beta 5 was 77% identical to that of human beta 5, and sequences with known signaling functions were highly conserved. Integrin alpha v, beta 3, and beta 5 mRNAs were expressed throughout the development of the embryonic retina, with the highest levels per retina observed around the embryonic day 9. In situ hybridization showed that both beta 3 and beta 5 were expressed throughout the developing retina, particularly in undifferentiated neuroepithelial precursor cells. At later times, beta 3 was expressed uniformly throughout the retina, whereas beta 5 expression was highest in a band throughout the central retina. CONCLUSIONS: The strong conservation of sequences with known signaling functions in chicken beta 5 suggests that it functions in a manner similar to human beta 5. Spatial expression patterns of vitronectin receptor subunits at early times of development point to a range of possible functions beyond axon outgrowth, including retinoblast proliferation, adhesion, and migration.

Integrin alpha 2 beta 1 mediates interactions between developing embryonic retinal cells and collagen.

In the developing nervous system, the extracellular matrix provides a source of extrinsic cues to guide determination of cell fate, neuroblast migration, axon outgrowth and synapse formation. In the neural retina, undifferentiated neuroepithelial precursor cells contact extracellular matrix that contains multiple collagen types. Collagens have been shown to support retinal cell adhesion and neurite outgrowth, but the integrin receptors mediating neuronal responses are not understood. Here we provide evidence that integrin alpha 2 beta 1 acts as a collagen receptor in the developing avian retina and examine its expression pattern. Using a recently described monoclonal antibody, MEP-17, alpha 2 protein was detected in the developing retina by immunofluorescence in tissue sections and dissociated cells, and by immunoprecipitation. At embryonic day 4 (E4), when the majority of retinal cells are undifferentiated neuroepithelial cells, alpha 2 immunoreactivity in sections was widespread and about half of cells dissociated in culture were alpha 2 positive. At E6, after the retinal ganglion cell layer had differentiated, immunoreactivity in sections decreased in the central, more developed portion of the retina and 25% of dissociated cells were alpha 2 positive. E6 retinal ganglion cells, identified by neurofilament immunoreactivity, did not express detectable alpha 2 immunoreactivity. Immunoprecipitation experiments using E6 extracts demonstrated that the alpha 2 subunit was paired with the beta 1 integrin subunit. By E12, alpha 2 immunoreactivity in sections was confined to the extreme peripheral retina, although the antigen may be masked since expression levels comparable to or slightly higher than E6 could be detected in dissociated cells and extracts. By employing function blocking antibodies, it was shown that alpha 2 beta 1 integrin is necessary for cell adhesion and process outgrowth by embryonic retinal cells on collagens I and IV. Although alpha 2 expression continued through E12, alpha 2 activity was down regulated with increasing embryonic age, since alpha 2-dependent adhesion and outgrowth declined. These data suggest a role for alpha 2 beta 1 in neuroepithelial cell interactions with collagen rather than for axon extension by retinal ganglion cells.

What happens to patients following open access gastroscopy? An outcome study from general practice.

BACKGROUND: Open access gastroscopy allows general practitioners to request a gastroscopy without prior referral to a specialist. The effect of open access gastroscopy upon patient management is poorly explored. Most studies have been hospital based and have focused on diagnostic yields and on means of tightening requests to reduce inefficient use. A user evaluation can only be made by measuring outcomes in primary care. AIM: A study was undertaken to determine the impact of open access gastroscopy in general practice and in particular, the value of a normal result. METHOD: All general practices in South Tees District Health Authority were asked to participate. Any of their patients who had had open access gastroscopy in the year prior to July 1990 were identified from the hospital computer and their general practitioner notes examined. Patient management during the year prior to the open access gastroscopy was compared with the year after. The main outcome measures were: detection rate and grade of lesion, change in graded score of prescribed drugs, consultation rate for dyspepsia and non-dyspepsia problems, and further hospital referral and investigations. Outcomes among those with normal and abnormal gastroscopy results were compared. RESULTS: The study sample comprised 715 patients, 36% of whom had a normal gastroscopy result, 34% a major abnormality and 26% a minor abnormality (4% of patients had miscellaneous diagnoses). It was found that 39% of all patients, and 60% of those with normal findings on open access gastroscopy had their drug treatment stopped or reduced in grade after the investigation. Of those with a major endoscopic abnormality 58% increased their treatment score. Consultations for dyspepsia in the year before and after gastroscopy fell by 57% overall among those with a normal gastroscopy result, by 37% among those with a minor finding and by 33% in those with a major finding. There was a 21% fall in consultations for all reasons among those with a normal gastroscopy result but those with a minor abnormality had a 23% increase in non-dyspepsia consultations. Of all patients 19% were referred to hospital subsequently. CONCLUSION: Open access gastroscopy has a major effect upon patient management in general practice, and a normal endoscopy result has an important an impact as an abnormal one. Open access gastroscopy is associated with a rationalization of drug therapy, reduced consultations and a low hospital referral rate.

A promoter element that exerts positive and negative control of the interleukin 2-responsive J-chain gene.

In a primary immune response a signal from interleukin 2 (IL-2) induces B lymphocytes to express the gene for the IgM joining component, the J chain. The signaling mechanism was pursued in this study by examining the J-chain gene 5' flanking region for regulatory sequences and interacting nuclear factors. The analyses identified a major control region located between -75 and -45 that encodes two adjacent elements: a T-rich sequence (JA) containing a single positive regulatory motif and an A+G-rich sequence (JB) containing overlapping positive and negative regulatory motifs. Dissection of the two elements indicated that the bifunctional JB sequence is the likely target of the IL-2 signal. The evidence was based on findings that (i) JB activity correlated with J-chain gene transcription--i.e., JB acts as a repressor in J-chain-silent B cells and as an activator in J-chain-expressing cells, and (ii) JB activator function is mediated by a B-cell-specific nuclear protein, NF-JB, that exhibits an IL-2-responsive binding pattern.

Immunoglobulin J chain gene from the mouse.

During a primary immune response, murine B lymphocytes are induced to express the gene for the immunoglobulin J chain. As a first step in determining the mechanism of induction, genomic DNA clones encoding the murine J chain were obtained from cell lines representative of B lymphocytes before and after J chain expression. Analysis of the coding regions showed that the J chain gene has a different structure from the other immunoglobulin genes. It consists of four exons organized in a simple 7.3-kilobase transcription unit that does not require DNA rearrangement or alternative processing for expression. These structural properties indicate that transcription of the J chain gene is initiated by changes in chromatin conformation, probably involving a J chain-specific DNA-binding factor. Analysis of the 5' flanking sequences of the J chain gene, on the other hand, showed that the promoter region contains two conserved elements that have been implicated in the lymphocyte-specific expression of the light chain genes. The sharing of these elements suggests that, once the J chain gene is activated, its transcription is regulated by mechanisms similar to those controlling the light chain genes.

Primary structure of the immunoglobulin J chain from the mouse.

The primary structure of the murine J chain was investigated by sequence analysis of the J chain cDNA inserts from two independently cloned chimeric plasmids. The sequence data showed that (i) the two cDNA inserts accounted for all but approximately 100 5' nucleotides of the J chain mRNA and (ii) the J chain mRNA encodes a prepeptide of at least 23 amino acids, a mature protein of 137 residues, and an untranslated 3' region of 707 nucleotides exclusive of the 3' poly(A) tract. The amino acid sequence deduced for the mature mouse J chain was found to be 74% identical with that previously determined for the human J chain. By analyzing the conserved features of the sequence, a two-domain structure was generated for the J chain which correlates well with its functions in the polymerization of IgM and IgA. Moreover, by comparing the homologies of the J and heavy chains in mouse and man, evidence was obtained that the structures involved in polymerization are the most conserved elements of immunoglobulin molecules.

Sequence-specific fragmentation of macronuclear DNA in a holotrichous ciliate.

Macronuclear DNA from the protozoan G. chattoni, a holotrichous ciliate, was analyzed. Most, if not all, of the macronuclear DNA is subchromosomal, ranging in size from above 100 kb down to 2.1 kb, with molecules in the lower molecular weight range being resolvable by gel electrophoresis into reproducible, specific, discrete size classes. A prominent class of linear 9.3 kb molecules consists of single free rRNA genes. Upon denaturation of total macronuclear DNA was found as single-stranded circles. Sequence analysis of showed that a minimum of 38 tandem repeats of the sequence CCCCAA is present in inverted orientation at each end of most or all Glaucoma macronuclear DNA molecules, including the rDNA. This sequence must therefore be recognized during site-specific fragmentation of chromosomes in macronuclear development.