Biomedical subjects
Publications and source records attributed to B Christ.
We report on a muscular variation in the human foot. The tendon of the tibialis posterior muscle was attached to the peroneus longus tendon by means of a trapezoid tendinous connection. This variation was observed only in the left foot.
Wilhelm Roux's doctoral thesis described the relationship between the angle and diameter of bifurcating blood vessels. We have re-read this work in the light of biophysics and developmental biology and found two remarkable aspects hidden among a multitude of observations, rules and exceptions to these rules. First, the author identified the major determinants involved in vascular development; genetics, cybernetics, and mechanics; moreover, he knew that he could not deal with the genetic and regulatory aspects, and could hardly treat the mechanical part adequately. Second, he was deeply convinced that the laws of physics determine the design of organisms, and that a necessity for optimality was inherent in development. We combined the analysis of diameter relationships with the requirement for optimality in a stochastic biophysical model, and concluded that a constant wall-stress condition could define a minimum wall-tissue optimum during arterial development. Hence, almost 120 years after Wilhelm Roux's pioneering work, our model indicates one possible way in which physical laws have determined the evolution of regulatory and structural properties in vessel wall development.
In cultured rat hepatocytes, the gluconeogenic key enzyme, phosphoenolpyruvate carboxykinase (PCK), is induced by glucagon via elevation of cyclic 3',5' adenosine monophosphate (cAMP). The proinflammatory cytokine, interleukin-6 (IL-6), which in the liver together with IL-1beta and tumor necrosis factor alpha triggers the acute-phase response, had been shown to attenuate the glucagon-induced increase in PCK gene transcription, messenger (mRNA) levels, and enzyme activity. The molecular mechanism of this inhibition was investigated in the present study. Glucagon increased cyclic cAMP and PCK mRNA levels to a transient maximum twofold and fivefold, respectively. The increases were attenuated by IL-6. Forskolin, which stimulates adenylate cyclase activity, increased cAMP and PCK mRNA levels 1.6-fold and fivefold, respectively. However, IL-6 attenuated the forskolin-stimulated increase in PCK mRNA but not the increase in cAMP. This showed that IL-6 inhibited PCK mRNA increase in part by the attenuation of cAMP increase, but also beyond cAMP formation. This was confirmed in experiments in which PCK mRNA levels were increased by the nonhydrolyzable cAMP-analogue, chlorophenylthio (CPT)-cAMP. The increase in PCK mRNA was again attenuated by IL-6. In pertussis toxin- and in isobutylmethylxanthine-treated hepatocytes, IL-6 still inhibited the glucagon-stimulated increase in cAMP, indicating that IL-6 did not activate an inhibitory G-protein or phosphodiesterase, which could cause the impairment of cAMP increase. To demonstrate whether the inhibition of PCK gene expression by IL-6 beyond cAMP might be caused by the inhibition of the activation of the PCK gene promoter by cAMP, cultured rat hepatocytes were transfected with a luciferase reporter gene construct under the control of a PCK gene promoter fragment (base -979 to base +32). Luciferase activity was determined after stimulation of the cells with CPT-cAMP in the absence or presence of IL-6. CPT-cAMP increased luciferase activity by 1.7-fold, which was inhibited in the presence of IL-6. It is concluded that IL-6 had a dual inhibitory effect on the stimulation of PCK gene expression by glucagon. It inhibited the increase in cAMP at a site before cAMP formation by adenylate cyclase and at a site after cAMP formation, the activation of the PCK gene promoter by cAMP.
The skeletal muscle progenitor cells of the vertebrate body originate in the dermomyotome epithelium of the embryonic somites. To precisely locate myotome precursor cells, fluorescent vital dyes were iontophoretically injected at specific sites in the dermomyotome in ovo and the fates of dye-labeled cells monitored by confocal microscopy. Dye-labeled myotome myofibers were generated from cells injected along the entire medial boundary and the medial portion of the cranial boundary of the dermomyotome, regions in close proximity to the dorsal region of the neural tube where myogenic-inducing factors are thought to be produced. Other injected regions of the dermomyotome did not give rise to myotome fibers. Analysis of nascent myotome fibers showed that they elongate along the embryonic axis in cranial and caudal directions, or in both directions simultaneously, until they reach the margins of the dermomyotome. Finally, deposition of myotome fibers and expansion of the dermomyotome epithelium occurs in a lateral-to-medial direction. This new model for early myotome formation has implications for myogenic specification and for growth of the epaxial domain during early embryonic development.
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The essential function of vascular endothelial growth factor (VEGF) in embryonic angiogenesis has clearly been documented in murine embryos with targeted deletions of either VEGF or its receptors. The effects of VEGF in the organogenetic phase of development have not been studied to date. Therefore, we applied 0.7 to 0.9 microgram of VEGF via methylcellulose carriers into the midbrain or onto the right forelimb of 4.5-day-old quail embryos. Another group of embryos was treated with 1 microgram of platelet-derived growth factor and controls were carried out using carriers without any growth factor. VEGF-induced cardiovascular malformations resulted in embryonic lethality. The venous area of the vasculature was dilated in almost all organs. The heart was most seriously affected and showed typical characteristics of insufficiency. VEGF strongly increased endocardial cell proliferation and obviously induced impairment of the growth rates of myocardium and endocardium. The myocardium of the ventricles was extremely thin, and septation defects were observed. As a result of the disturbed outflow, the atria were extremely dilated and thin-walled. The morphology of the hearts was reminiscent of that observed in congenital malformations such as Uhl's and Osler's syndromes. Our results show that expression of VEGF has to be tightly controlled during development.
The influence of the inflammatory mediators interleukin 1 beta (IL1 beta) and tumour necrosis factor alpha (TNF alpha) on the glucagon-induced expression of phosphoenolpyruvate carboxykinase (PCK) and on glucose formation via gluconeogenesis was investigated in cultured rat hepatocytes. Gene expression was monitored by determination of mRNA levels and of enzyme activity. Glucose formation was estimated with newly synthesized radioactive glucose derived from a radiolabelled lactate precursor. Glucagon (0.1 or 1 nM) induced PCK mRNA transiently to a maximum 2 h after its application. In the presence of recombinant human (rh) IL1 beta or rhTNF alpha the increase in PCK mRNA levels was totally inhibited at 0.1 nM glucagon, whereas at 1 nM glucagon the maximal increase was inhibited by only 25%. Glucagon (0.1 or 1 nM) induced PCK activity to a maximum after 4 h (4-fold and 6-fold over prestimulatory activity respectively). In the presence of rhIL1 beta or rhTNF alpha the maximal increase was inhibited by approx. 50%. Addition of rhIL1 beta or rhTNF alpha 2 h after glucagon, at the maximal glucagon-induced PCK mRNA levels, accelerated the decay of PCK mRNA. Glucagon (1 or 10 nM) [corrected] increased glucose formation from lactate by 1.3-fold and 1.7-fold respectively over unstimulated rates. In the presence of rhIL1 beta or rhTNF alpha this increase in glucose formation was inhibited by 60-90%. At 0.1 nM, glucagon doubled the intracellular cAMP concentration. This increase was prevented by rhIL1 beta or rhTNF alpha. At 1 nM, glucagon increased cAMP concentrations by 10-fold. In the presence of rhIL1 beta or rhTNF alpha this increase was inhibited by 70%. From the results it is suggested that rhIL1 beta and rhTNF alpha prevented glucagon-stimulated PCK gene expression and gluconeogenesis at least in part by inhibition of the glucagon-stimulated increase in cAMP concentrations.
The initiating event in the migration of myogenic cells to the limb buds is an epitheliomesenchymal transformation of cells located at the lateral edge of the dermomyotome. Recently, a targeted mutation of c-met in mice demonstrated an essential role of this tyrosine kinase receptor and its ligand, scatter factor/hepatocyte growth factor (SF/HGF), in the migration of myogenic cells to the limb buds. Here, we show that ectopic application of exogenous SF/HGF induces emigration of Pax-3-positive myogenic cells into the lateral plate mesoderm. During this process, the lateral portions of the dermomyotomes deepithelialize and the basement membrane disintegrates. Detaching myogenic cells do not lose N-cadherin from their surfaces. We conclude that an HGF/SF- and c-met-mediated signal detaches myogenic precursor cells from the somites and thus plays a necessary role in the initiation of myoblast migration.
We report on the normal and experimentally altered expression of two structurally related genes, Follistatin and Follistatin-like (Flik), in the somites of avian embryos. In normal chick embryos, Follistatin expression can first be seen in the cells of the dorsolateral somite quarter. During somite maturation, the cells of the dorsomedial quarter also express this gene. Within the dermomyotome it seems that only the muscle precursors are Follistatin-positive. The migrating precursors of limb and tongue muscle as well as the myotome cells show Follistatin expression. The manipulation experiments reveal that the expression of Follistatin in the somites can be inhibited by notochord signals. This effect can be mimicked by sonic hedgehog protein. Flik is expressed in the dorsomedial compartment of the somite and later on in the myotome. Unlike Follistatin, Flik expression requires signals emanating from the neural tube. Notochordal influences do not alter Flik expression. The expression of both genes does not depend on signals of intermediate or lateral mesoderm. Since the products of both genes are proposed to antagonize TGF-beta superfamily proteins during gastrulation and neuralization, we postulate that during myogenesis follistatin and flik counteract inhibiting effects of related molecules on muscle differentiation.
Pax1 and Pax9 represent a subfamily of paired-box-containing genes. In vertebrates, Pax1 and Pax9 transcripts have been found specifically in mesodermal tissues and the pharyngeal endoderm. Pax1 expression in the sclerotomes has been shown to be indispensable for proper formation of the axial skeleton, but expression of Pax1 in the endoderm has not been studied in detail. We have cloned the chick homologue of the murine Pax9 gene. Our results show that transcripts of Pax1 and Pax9 are first detectable in the prospective foregut endoderm of headfold-stage avian embryos. Endodermal expression correlates with the highly proliferative zones of the folding foregut and evaginating pharyngeal pouches. In later stages, Pax1 and Pax9 are expressed in overlapping but distinct patterns within the developing sclerotomes and limb buds. From grafting experiments we conclude that activation of pharyngeal Pax1 and Pax9 expression is an intrinsic property of the endoderm, not requiring midline structures or head mesoderm. In contrast, notochord is required to induce Pax1 in competent sclerotomes. Here we show that in vitro there is a cranio-caudal gradient of inductive capacity in the notochord. This coincides with the graded expression of Pax1 and Pax9 along the cranio-caudal axis in 2- to 3-day-old embryos. Furthermore, paraxial head mesoderm shows no competence to express Pax1. Finally, in vitro we find counteracting influences on notochord signaling by lateral tissues (lateral plate, intermediate mesoderm), leading to an inhibition of Sonic hedgehog (Shh) expression in notochord and floor plate, as well as Pax1 and Pax9 expression in sclerotomes. Taken together, our results demonstrate that different mechanisms regulate expression of Pax1 and Pax9 in foregut and sclerotome, but suggest a common function for both genes in the two tissues that is promoting proliferation and preventing fusion of neighboring blastemas.
Limb muscle formation involves invasion of the limb bud mesoderm by myogenic precursor cells from the dermomyotomes at limb bud level. Directed cell migration, homing, and differentiation of myogenic cells are controlled by the stationary cells of the limb bud mesoderm. At the level of the extracellular matrix, the molecular basis of migration control has been suggested to be exerted by the distribution of hyaluronan. Here, we demonstrate that N-cadherin-mediated interactions play a role at cell-membrane level in myoblast distribution and differentiation. N-cadherin is strongly expressed by myogenic cells in the chick limb bud and more moderately expressed by stationary mesodermal cells in the myogenic zones and progress zone. After in vivo injection of antibodies and Fab-fragments against the homophilic binding site of N-cadherin into the wing bud mesoderm, aggregates of myoblasts are found predominantly in the dorsal myogenic zone 36 hr after injection apparently due to immobilization. In the same position, areas of myf-5-positive cells are also observed. In injected limb buds, Pax-3-positive cells are less evenly distributed than in uninjected limbs. They are found to spread up to the epidermis and also form loosely arranged aggregates. After prolonged reincubation periods, injected limbs show ectopic myoblasts that are rich in desmin and areas of strongly desmin-expressing myoblasts within muscle blastemas. These effects were not observed after application of antibodies against other parts of the N-cadherin molecule. We conclude that N-cadherin is involved in myoblast migration in the limb buds via homophilic interactions and that it plays a role in signal transduction during myogenesis.
We have studied the effect of VEGF(121) homodimer and VEGF(121/165) heterodimer on the chorioallantoic membrane (CAM) of 13-day-old chick embryos. The factors were applied in doses of 2-4 micrograms and the effects were evaluated macroscopically after 2 and 3 days. Histological studies were performed on semi- and ultrathin sections. Proliferation was studied according to the BrdU-anti-BrdU method on whole mounts and sections. The labeling density was quantified in whole mounts. The fractal dimension, D, of the vascular tree was assessed as a value for vascular bifurcation density. Both forms of VEGF induce brush-like vessel formation in the precapillary region. New capillaries are found in the stroma of the CAM, which normally does not contain capillaries. Our results show that VEGF(121) is a specific endothelial cell mitogen. A fourfold increase of BrdU-labeled endothelial cells is found after VEGF(121) application. The fractal dimension of the vascular tree increases from 1.26 in the controls to 1.44 (VEGF(121)) and 1.41 (VEGF(121/165)). The endothelial cells of the newly formed capillaries possess many mitochondria and micropinocytotic vesicles, but no fenestrations. These capillaries are obviously formed by intussusceptive microvascular growth. Signs of sprouting are almost absent. An effect on the lymphatic vessels of the CAM is not detectable. Compared to VEGF(165) and VEGF(121/165), VEGF(121) diffuses over a slightly greater distance. Using in situ hybridization, VEGF receptor-2 (flk-1/Quek1) and the homologous flt-4 (Quek2) receptor were studied in the CAM of normal quail embryos and after VEGF(121) application on the CAM of 11-day-old quail embryos. During normal development, flk-1 expression becomes restricted to vascular endothelial cells of large vessels in the stroma of the CAM. VEGF(121) application induces expression of flk-1 in capillaries that normally do not express the receptor. In the normal development of the CAM, flt-4 becomes restricted to endothelial cells of vessels that appear to be lymphatic vessels. Application of VEGF(121) does not alter flt-4 expression.
In human liver, phosphoenolpyruvate carboxykinase (PCK; EC 4.1.1.32) is about equally distributed between cytosol and mitochondria in contrast with rat liver in which it is essentially a cytosolic enzyme. Recently, the isolation of the gene and cDNA of the human cytosolic enzyme has been reported [Ting, Burgess, Chamberlian, Keith, Falls and Meisler (1993) Genomics 16, 698-706; Stoffel, Xiang, Espinosa, Cox, Le Beau and Bell (1993) Hum. Mol. Genet. 2, 1-4]. It was the goal of this investigation to isolate the cDNA of the human mitochondrial form of hepatic PCK. A human liver cDNA library was screened with a rat cytosolic PCK cDNA probe comprising sequences from exons 2 to 9. A cDNA clone was isolated which had overall a 68% DNA sequence and a 70% deduced amino acid sequence identity with the human cytosolic PCK cDNA. Without the flanking 270 bases (=90 amino acids) each at the 5' and 3' end, the sequence identity was 73% on the DNA and 78% on the amino acid level. The isolated cDNA had an open reading frame of 1920 bp; it was 54 bp (equivalent to 18 amino acids) longer than that of human or rat cytosolic PCK cDNA. The isolated cDNA was cloned into the eukaryotic expression vector pcDNAI and transfected into human embryonal kidney cells HEK293; PCK activity was increased by 3-fold in the mitochondria, which normally contain 70% of total PCK activity, but not in the cytosol. The isolated cDNA was also transfected into cultured rat hepatocytes; again, PCK activity was enhanced by about 40-fold in the mitochondria, which normally possess only 10% of total PCK activity, but not in the cytosol. In the rat hepatocytes only the endogenous cytosolic PCK and not the transfected mitochondrial PCK was induced 3-fold with glucagon. Comparison of the amino acid sequences deduced from the isolated cDNA with human and rat cytosolic PCK showed that the additional 18 amino acids were located at the N-terminus of the protein and probably constitute a mitochondrial targeting signal. Northern-blot analyses revealed the human mitochondrial PCK mRNA to be 2.25 kb long, about 0.6 kb shorter than the mRNA of the cytosolic PCK. Primer extension experiments showed that the 5'-untranslated region of mitochondrial PCK mRNA was 134 nucleotides in length.
We studied the early pattern of neural tube (NT) vascularization in quail embryos and chick-quail chimeras. Angioblasts appeared first in the dorsal third at Hamburger and Hamilton (HH) stage 19 as single, migrating cells. Their distribution did not correspond to a segmental pattern. After this initial dorsal immigration, endothelial sprouts invaded the NT on either side of the floor plate (HH stage 21). These cells remained continuous with their arterial vascular sources, connected to the venous perineural vascular plexus at HH-stage 22, and formed the first perfused vessels of the NT at HH-stage 23. The same pattern of angiotrophic vascularization was observed in a craniocaudal sequence starting caudal to the rhombencephalic NT. Extremely long filopodia were observed on sprouting cells, extending toward the central canal and the mantle layer. The exclusively extraneuroectodermal origin of angioblastic cells was demonstrated with chick-quail chimeras. Following replacement of quail NT by chick NT graft, angioblast and sprout distribution in chimeras was the same as in controls. We conclude that the NT receives its first blood vessels by a combination of two different processes, dorsal immigration of isolated migrating angioblastic cells and ventral sprouting of endothelial cells, which derive from perfused vessels. The dorsal invasive angioblasts contribute to the developing intraneural vascular plexus after having traversed the neural tube. The initial distribution of blood vessels within the neuroepithelium corresponds to intrinsic random motility of angioblastic cells; a more regular pattern is seen later. The floor plate apparently prohibits connections between sprouts in both NT sides, whereas in the dorsal NT, such a separating effect on the migrating angioblasts does not exist.
Basic fibroblast growth factor (bFGF, FGF-2) mediates several biological functions during embryonic development. With regard to skeletal muscle formation, it has been suggested that FGF-2 is involved in the growth and differentiation of myogenic precursor cells. To identify the FGF-responsive cells we studied the expression of FGF receptor type I (FGFR-1) during early embryonic development of the chick. FGFR-1 immunoreactivity is present at all stages examined (embryonic day [E] 2-E5). Expression of FGFR-1 is found in the somite myotome, limb bud muscle cells, eye and tongue muscle cells, and myocardium. Transplantation of an additional notochord into the paraxial mesoderm, which prevents the formation of a myotome, reveals the absence of FGFR-1 immunoreactivity on the operated side. The distinct expression pattern of FGFR-1 in migrating and differentiating muscle cells indicates that in addition to the stimulation of proliferation of myoblasts, FGF-2 exerts other (nonmitogenic) effects on postmitotic myocytes.
In the midline of the embryo an invisible barrier exists that keeps endothelial cells from migrating to the contralateral side. Interspecific grafting experiments between chick and quail were carried out in order to investigate the role of the axial structures in maintaining this barrier. The quail endothelial cells of the graft were therefore stained with QH1 antibody. In all experimental series quail paraxial mesoderm was used as a source of endothelial cells. First, a quail somite was transplanted either ipsilaterally or contralaterally. The results not only show the existence of laterality in the distribution pattern, but also demonstrate that the laterality does not depend on the origin of the graft but on the environment of the host embryo. Laterality in the distribution pattern of endothelial cells means that the endothelial cells of the two body halves migrate independently and do not change from one side to the other. Single cells do not know whether they are cells from the right or from the left half of the body. In the next series of experiments axial structures were removed in order to modify the barrier. In addition, paraxial mesoderm was exchanged with the corresponding quail tissue in order to determine the migration behaviour of the grafted endothelial cells. The removal of the neural tube does not influence the barrier. After notochordectomy, however, the endothelial cells exhibited a balanced distribution pattern over both halves of the embryo. We concluded that the notochord forms a barrier for endothelial cells that presumably operates on the basis of chemical substances. It is conceivable that our results can explain the lateralization of illnesses of the vascular system, as the Klippel-Trénaunay syndrome or the Sturge-Weber syndrome.
In this paper we investigate the somitic origin of the individual muscles of the forearm and hand using quail-chick chimeras. Our results show that only somites 16-21 give rise to wing muscle, but they take part in muscle formation to different extents. Somite 21 does not always participate in the formation of muscle of the forearm and hand. The most cranial somite (16) takes part in the radial muscles and the most caudal somites (20, 21) in the ulnar muscles, reflecting their position with respect to the limb bud. The centrally located somites (17, 18, 19) are involved in all (18) or most (17, 19) muscle primordia. This pattern of distribution is clearest in the forearm, whereas the participation of somites in particular muscle groups is not so distinct in the hand. Hand muscles are mainly made up of cells from somites 18-20. All brachial somites participate in dorsal (extensor) as well as ventral (flexor) muscles of the forearm and hand. Each somite takes part in more than three muscle primordia in a reproducible fashion, and every muscle primordium is derived from at least three somites. Especially the M. ulnimetacarpalis ventralis takes origin from all somites involved in limb muscle formation (16-21). Apart from muscle cells, endothelial cells also and a few fibroblasts of quail origin are found in the limb bud after somite grafting.
Supply with nutrients is essential from early embryonic stages onwards. Therefore, circulatory organs form the first functioning organ system. With the exception of the heart, this system is at first formed by only one cell type, the endothelial cell. Emergence, behavior, and differentiation of endothelial cells are discussed in this review. At first, endothelial cells develop from angioblasts (primary angiogenesis/angioblastic development), later they develop from preexisting endothelial cells (secondary angiogenesis/angiotrophic growth). The composition of the extracellular matrix may promote or inhibit angiogenesis. Various growth factors which can be bound to the extracellular matrix may have been found, but only two of them (VEGF, P1GF) seem to influence endothelial cell behavior directly. Heterogeneity and organ-typical differentiation of endothelial cells seem to be dependent on cell-cell signaling within each organ.