bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of contractile protein gene expression in fetal murine crural muscles: Emergence of muscle diversity
Authors: Ontell Martin P. , Sopper Maggie M. , Lyons Gary , Buckingham Margaret , Ontell Marcia
Published: February 2005

The modulation of contractile protein gene expression in mouse crural muscles (i.e., muscles located in the region between the knee and ankle) during the fetal period (defined as 15 days gestation to birth), resulting in diversity among and within these muscles, has been evaluated with in situ hybridization and correlated with morphogenetic events in the extensor digitorum longus and soleus muscles. During the fetal period extensive secondary myotube formation occurs in the crural muscles, and the myotubes become innervated (Ontell and Kozeka [1984a, b] Am. J. Anat. 171:133–148, 149–161; Ontell et al. [1988a, b] Am. J. Anat. 181:267–278, 181:278–288). At 15 days gestation, hybridization with 35S‐labeled antisense cRNA probes demonstrates the accumulation of transcripts forα‐cardiac andα‐skeletal actin; MLC1A, MLC1F, and MLC3F; and MHCemb, MHCpn, and MHCβ/slow. At 16 days gestation, accumulation of MHCemb transcripts is reduced (as compared with earlier developmental stages); intensity of signal following hybridization with the probe forα‐skeletal actin is, for the first time, equal to that for the cardiac isoform; and MLC1V mRNA accumulation is discernible. At this stage, variation in transcript accumulation for some mRNAs among and within crural muscles becomes evident. Two factors may play a role in the selective distribution of these transcripts: (1) the stage of muscle maturation; and (2) the future myofiber type. At 16 days gestation anterior crural muscles (which mature ˜ 2 days before posterior crural muscles; Ontell and Kozeka [1984a, b], ibid., Ontell et al. [1988a, b], ibid.) exhibit a greater accumulation of transcripts forα‐skeletal actin and for MLC3F than is found in posterior crural muscles. In muscles that in the neonate are composed, in large part, of slow myofibers, MHCβ/slow and MLC1V mRNAs accumulate in greater amounts, whereas MHCpn transcripts are less abundant in the soleus muscle than in other crural muscles. By 19 days gestation regionalization of transcript accumulation is more pronounced. The soleus muscle, a predominantly slow twitch muscle in the newborn mouse (Wirtz et al. [1983] J. Anat. 137:109–126) exhibits strong signal after hybridization with probes specific for MHCβ/slow and MLC1V. While the level of transcript accumulation for the developmetal isoforms, MHCemb, MLC1A, andα‐cardiac actin, is greatly reduced in most crural muscles at 19 days gestation, these transcripts persist in the soleus muscle at levels equal to or exceeding their amount in limb muscles of 13 day gestation mouse embryos. By 19 days gestation both MyoD and myogenin are “down‐regulated” (as compared with their expression at earlier developmental stages) in all muscle masses. Alterations in contractile protein gene expression are correlated with changes in the myogenic regulatory factors present in fetal hindlimbs during development. © 1993 Wiley‐Liss, Inc.
Expression of tumor necrosis factor‐α (TNFα)‐cross‐reactive proteins during early chick embryo development
Authors: Wride M. A. , Sanders E. J.
Published: February 2005

We have investigated the expression of tumor necrosis factor‐α (TNFα)‐cross‐reactive proteins during the early development of the chick embryo from day 1 to day 6 (H‐H stages 5–29) using a polyclonal antibody and two monoclonal antibodies to recombinant mouse TNFα. We have confirmed the cross‐reactivity of the antibodies with chicken tissue in Western blotting studies. Proteins of 50 kDa and 70 kDa, showing anti‐TNFα cross‐reactivity, have been identified during early chick development. In addition, both monoclonal antibodies recognize a 120 kDa protein. These molecules probably represent cytosolic or transmembrane TNF‐α‐like proteins, similar to those previously identified on the surface of cytotoxic T‐lymphocytes. We show by ultrastructural cytochemistry that immunoreactivity can be detected at the surfaces of some cells, suggesting that at least some of the antigen is membrane‐associated. The proteins are shown to have a widespread tissue distribution during this period of development. Immunoreactivity is first detected in the gastrulating embryo, in the mesoderm and the endoderm. By day 2, expression is confined to the ectoderm and the endoderm, while at day 3 expression appears in the myotome, the notochord, and in nervous tissue. At day 4 the distribution of reactivity is more extensive and includes the notochord, the sclerotome, and the myotome, while the cranial and spinal nerves also become intensely immunoreactive. Also at this stage, neural tube reactivity becomes localized to the marginal neuroepithelial zone, and the lens fibers become positive. This distribution of staining then persists until 6 days of development. We hypothesize that the expression of TNFα‐cross‐reactive proteins in early development could be indicative of a role for them in programmed cell death (apoptosis) during differentiation of the notochord, the lens, and the nervous system, and in tissue remodeling. © 1993 Wiley‐Liss, Inc.
Retinoic acid receptor isoform β2 is an early marker for alimentary tract and central nervous system positional specification in the chicken
Authors: Smith Susan M.
Published: February 2005

In this study I describe the distribution of one variant of retinoic acid receptor‐β (RAR‐β), the RAR‐β2 isoform, during the stages before organogenesis of the chick embryo. Unlike the situation in older embryos, at these stages its distribution does not differ qualitatively from that of all RAR‐β transcripts. During the presomite headfold stage, RAR‐β2 transcripts are simultaneously upregulated in two different locations. These locations define positional identities within the anlage of the chick alimentary tract and within the central nervous system (CNS). As development proceeds the transcript expression maintains its spatial restriction within those two regions. At presomite stages RAR‐β2 transcripts are enriched within the proamnion, which contains the presumptive foregut and precardiac cells; somewhat later it is present within the foregut endoderm at the site where foregut and the lateral amniocardiac vesicles fuse to form the coelom and cardiac tube. As the foregut continues its caudal extension, RAR‐β2 expression defines an anteroposterior boundary at the level of the pharynx within the alimentary tract. The second expression site of RAR‐β2 mRNA first appears within the posterior neural plate at the level where Hensen's node commences its caudal regression. This boundary lies at the border between the future rhombomeres 5 and 6 within the hindbrain. Expression of RAR‐β2 transcripts is also spatially restricted within some migrating cranial neural crest cells. The expression of RAR‐β2 in cranial neural crest cells is consistent with what is known about the mechanisms by which cranial neural crest cell fate is determined. These data support the hypothesis that retinoids may contribute to positional specification of anteroposterior body axis, and perhaps also to the formation and identity of the developing alimentary tract and heart tube. © 1994 Wiley‐Liss, Inc.
Male‐sterile phenotype of the neurological mouse mutant weaver
Authors: Harrison Susan M. W. , Roffler‐Tarlov Suzanne
Published: February 2005

The autosomal recessive murine mutation weaver (wv) affects postnatal differentiation in at least three neuronal populations in the brain: dopamine‐containing neurons in the midbrain, and granule and Purkinje cells in the cerebellum. Neuronal populations vulnerable to the actions of weaver die in the midst of development. In addition, homozygous weaver males are sterile. We show by a histological analysis of epididymides and testes that the cause of male sterility in weaver is lack of sperm. The epididymides of the adult weaver mice examined were devoid of sperm, and few seminiferous tubules in the adult weaver's testes contained elongated spermatids. Most tubules were marked by moderate to severe degeneration of the seminiferous epithelium. A developmental study showed that the mutant phenotype emerged after the third postnatal week. By postnatal day 28, the development of weaver sperm lagged behind that of the wild‐type and some seminiferous tubules contained degenerating germ cells. By postnatal day 35, terminal differentiation of spermatids appeared to be arrested in many tubules and degeneration of the seminiferous epithelium was widespread. The heterozygotes were unaffected at all ages sampled. We conclude that the normal allele at the weaver locus is necessary for spermiogenesis and the maintenance of spermatogenesis. © 1994 Wiley‐Liss, Inc.
Effect of in ovo retinoic acid exposure on forebrain neural crest: In vitro analysis reveals up‐regulation of N‐CAM and loss of mesenchymal phenotype
Authors: Shankar Kavita R. , Chuong Cheng‐Ming , Jaskoll Tina , Melnick Michael
Published: February 2005

In a prior study of in ovo exogenous retinoic acid (RA) exposure, we observed a prolonged expression of cell surface N‐CAM in cranial neural crest (NC) cells exhibiting migratory failure. In the present studies, we employed an experimental strategy in which embryos were first exposed to exogenous RA in ovo and incubated for 45–60 hr; this was followed by extirpation and in vitro culturing of these same RA‐exposed cranial neural tubes. NC cell outgrowth from the explant was assayed, as was the immunohistochemical localization of HNK‐1 and N‐CAM antigens. In RA‐exposed explants, the size of the NC cell outgrowths were comparable to controls. However, almost all NC cells lost their mesenchymal phenotype and were arranged in an “epithelioid” pattern of tightly packed polygonal cells that expressed N‐CAM at adjacent cell boundaries. By contrast, control NC cells were flattened and multipolar in shape and expressed HNK‐1, rarely co‐expressing N‐CAM. These observations indicate that RA modulates NC cell N‐CAM expression and microanatomical phenotype, a finding consistent with prior in ovo studies of RA‐exposure. Several possible explanations are considered. © 1994 Wiley‐Liss, Inc.
Precardiac mesoderm is specified during gastrulation in quail
Authors: Antin Parker B. , Taylor Richard G. , Yatskievych Tatiana
Published: February 2005

The establishment of precardiac mesoderm and the role of anterolateral endoderm and ectoderm in regulating heart muscle cell development have been studied in quail using explant cultures. Mesoderm from precardiac regions of stage 4+–6 embryos was explanted alone or in combination with adjacent endoderm or ectoderm, cultured for 12 to 72 hr in several types of culture media, and then assayed by morphological and immunocytochemical criteria for the presence of differnetiated cardiac myocytes. Results show that mesoderm from heart forming regions is capable of differentiating into beating cardiac myocytes in a defined medium lacking potential signaling molecules by stage 4+, the earliest time at which we could isolate mesoderm from adjacent cell layers. Although an interaction with anterolateral endoderm from stage 4+onward is therefore not required for the specification of precardiac mesoderm in quail, explants consisting of mesoderm plus endoderm show an enhanced rate of myocyte differentiation and a shortened delay between expression of myosin heavy chain and the onset of beating. Endoderm also plays a central role in early heart morphogenesis since beating heart tubes from only in explants that contain both mesoderm and endoderm. In contrast, ectoderm from stage 4+–5+embryos does not support development of precardiac mesoderm. These results suggeest that early heart muscle cell development involves an initial specification step that occurs prior to or during gastrulation and which leads to the appearance of myocardial precursor cells, and a subsequent differentiation step during which endoderm plays a central role in enhancing the rate of myocyte differentiation and the degree of heart tube morphogenesis. © 1994 Wiley‐Liss, Inc.
Murine PGK‐1 promoter drives widespread but not uniform expression in transgenic mice
Authors: McBurney Michael W. , Staines William A. , Boekelheide Kim , Parry David , Jardine Karen , Pickavance Lucy
Published: February 2005

Pgk‐1 is an X‐linked gene encoding 3‐phosphoglycerate kinase, an enzyme necessary in every cell for glycolysis. The regulatory sequences of the Pgk‐1 gene were used to drive the E. coli lacZ reporter gene and 2 strains of transgenic animals created with this Pgk‐lacZ transgene carried on autosomes. The levels of expression of Pgk‐1 varied from one adult tissue to another and the transgene was similarly regulated. However, in situ staining of the β‐galactosidase encoded by the transgene indicated extensive cell‐to‐cell variability in its level of expression. A reproducible subset of cells stained darkly for the transgene product. Some of these β‐galactosidase positive cells were rapidly proliferating while others appeared to be metabolically very active, suggesting that the Pgk‐1 promoter is regulated so as to be more active in cells requiring high levels of glycolysis. Although Pgk‐1 is X‐linked and subject to X chromosome inactivation, the transgenes were not inactivated in either female somatic or male germ cells. Thus, the Pgk‐1 promoter drives transgene expression in all tissues but the levels of expression are not uniform in each cell. © 1994 Wiley‐Liss, Inc.
Heart specification in the Mexican axolotl (Ambystoma mexicanum)
Authors: Easton Heather S. , Armstrong John B. , Smith Steven C.
Published: February 2005

The concept of the morphogenetic field has been used extensively in developmental biology. However, little is known about the mechanisms that partition these broad areas of tissue into the smaller areas which actually form the corresponding structures, and the remaining tissue. In the Mexican axolotl, the heart field forms as the anterior lateral plate mesoderm migrates over the underlying pharyngeal endoderm between stages 14 and 28. We have previously shown that both the mid‐ventral and lateral walls of the pharyngeal cavity have considerable inductive capacity at stage 14. If this inductive capability, and the competence of the mesoderm to respond, is retained between stages 14 and 28, a much broader area of mesoderm would be induced than actually participates in heart development. In this paper, we use explant cultures to establish that pharyngeal endoderm retains its inductive activity, and that both pre‐cardiac mesoderm and lateral plate mesoderm caudal to the pharyngeal cavity remain competent to respond to the induction throughout this period. We also map the specified region of the antero‐lateral mesoderm between stages 14 and 28 by placing carefully measured areas of mesoderm in culture without inductive endoderm. We found that the region capable of initiating a spontaneous beat approximately doubles in size during this period. Since the specified region is larger than the actual heart primordium, some mechanism must exist to partition “induced” mesoderm into heart‐forming and non‐heart‐forming areas. One possibility is a reaction‐diffusion mechanism involving local activation of the first mesodermal cells to contact the inductive endoderm, accompanied by production of a diffusible inhibitor that limits the extent of the heart‐forming region. © 1994 Wiley‐Liss, Inc.
Retarded chondrogenesis in transgenic mice with a type II collagen defect results in fracture healing abnormalities
Authors: Hiltunen Ari , Metsäranta Marjo , Virolainen Petri , Aro Hannu T. , Vuorio Eero
Published: February 2005

We have examined the biological and biomechanical consequences of defective type II collagen production for fracture repair employing a genetically engineered mouse line Del1 which was generated by microinjection of a 39‐kb mouse proα1(II) collagen gene construct containing a deletion of exon 7 and intron 7 (Metsäranta et al. [1992] J. Cell Biol. 118:203–212). Standardized tibial fractures were produced in transgenic Del1 mice and their nontransgenic littermates were used as controls. The fracture callus tissues were analyzed at days 7, 9, 14, 28, and 42 using radiography, histomorphometry, biomechanical testing, and Northern analysis of mRNAs for several tissue‐specific matrix components. Deficient production of cartilage in Del1 mice resulted in reduced radiographic callus size, smaller cross‐sectional area, and impaired biomechanical properties when compared with fractures of nontransgenic control mice. The differences were most evident in 14‐day fracture calluses. Consequently mRNAs for cartilage‐specific type IX and X collagens and aggrecan were also reduced in Del1 calluses. Levels of type II collagen mRNAs were unaffected since the mutated transgene produced additional type II collagen mRNA molecules. Further abnormalities in the fracture repair process of Del1 mice were observed in callus remodeling. In the control animals a typical feature of external callus remodeling was reduction of callus size during endochondral ossification between days 14 and 28. Such reduction was not observed in the transgenic mice. Histological examination of fracture calluses suggested also a reduction in trabecular surface area, which was found to be even more pronounced in metaphyseal bone of Del1 mice. Despite these differences the biomechanical properties of the calluses in the two groups became similar by day 28 of fracture healing. The results thus suggest that reduced chondrogenesis due to the presence of mutated transgenes in Del1 mice not only causes a temporary impairment in biomechanical properties of healing fractures but also affects later stages of callus remodeling. © 1994 Wiley‐Liss, Inc.
Time‐lapse total internal reflection fluorescence video of acetylcholine receptor cluster formation on myotubes
Authors: Wang Michelle Dong , Axelrod Daniel
Published: February 2005

To study when and where acetylcholine receptor (AChR) clusters appear on developing rat myotubes in primary culture, we have made time‐lapse movies of total internal reflection fluorescence (TIRF) overlaid with schlieren transmitted light images. The receptors, including the ones newly incorporated into the membrane, were labeled with rhodamine α‐bungarotoxin (R‐BT) continuously present in the medium. Since TIRF illuminates only cell‐substrate contact regions where almost all of the AChR clusters are located, background fluorescence from fluorophores either in the bulk solution or inside the cells can be suppressed. Also, because TIRF minimizes the exposure of the cell interior to light, the healthy survival of the cell culture during imaging procedures is much enhanced relative to standard epi‐ (or trans‐) illumination. During the experiment, cells were kept alive on the microscope stage at 37°C in an atmosphere of 10% CO2· Two digital images were recorded by a CCD camera every 20 min: the schlieren image of the cells and the TIRF image of the clusters. After background subtraction, the cluster image was displayed in pseudocolors, overlaid onto the cell images, and recorded as 3 frames on a videotape. The final movies are thus able to summarize a week‐long experiment in less than a minute. These movies and images show that clusters form often shortly after the myoblast fusion but sometimes much later, and the formation takes place very rapidly (a few hours). The clusters have an average lifetime of around a day, much shorter than the lifetime of a typical myotube. The brightest and largest clusters tend to be the longest‐lived. The cluster formation seems to be associated with the contacts of myotubes at the glass substrate, but not with cell‐cell contacts or myoblast fusion into myotubes. New AChR continuously appear in preexisting clusters: after photobleaching, the fluorescence of some clusters recovers within an hour. © 1994 Wiley‐Liss, Inc.
Enhancement of skeletal muscle regeneration
Authors: Bischoff Richard , Heintz Catherine
Published: February 2005

We have studied the effect of adding extra satellite cells or soluble factors from crushed muscle on regeneration of minced fragments from rat tibialis muscle. The muscle mince was wrapped in an artificial epimysium to prevent adhesions and cell immigration from adjacent muscles. Regeneration was quantitatively assessed by electrophoretic determination of the muscle‐specific form of creatine kinase. Control minces exhibited three periods of change in creatine kinase activity during a 7‐week regeneration period. Activity fell rapidly during the first week, then rose gradually from 1–3 weeks and increased more rapidly from 3–7 weeks. To augment the original complement of myogenic cells, satellite cells were isolated from the contralateral muscle, purified by density gradient centrifugation, and expanded in culture for 3 days before adding to the muscle mince. The added cells resulted in a 3‐fold enhancement of creatine kinase activity throughout the regeneration period. Soluble muscle extract incorporated into a collagen matrix also stimulated regeneration when added to muscle mince. The extract accelerated the rate of creatine kinase increase during the 1–3 week period beyond that observed in the control or cell augmented mince, suggesting that factors in the extract may facilitate revascularization or reinnervation. The specific activity of creatine kinase was increased in regenerates augmented with both cells and extract, indicating that the effects enhance primarily myogenic processes. © 1994 Wiley‐Liss, Inc.
Immunohistochemical studies indicate multiple enteroendocrine cell differentiation pathways in the mouse proximal small intestine
Authors: Aiken Kimberly D. , Kisslinger Jeffrey A. , Roth Kevin A.
Published: February 2005

The enteroendocrine cell system of the mammalian gastrointestinal tract is comprised of at least 16 different subpopulations. Each subpopulation shows a characteristic distribution along both the crypt‐villus and cephalocaudal axes. In both the small intestine and colon of adult mice, multilabel immunohistochemistry has demonstrated that two or more neuroendocrine products can be coexpressed in various combinations in single cells along the crypt‐villus axis, suggesting that enteroendocrine phenotypes may be actively regulated. Using bromodeoxyuridine (BrdU) incorporation and multilabel immunohistochemistry, we have previously demonstrated an enteroendocrine cell differentiation pathway consisting of two subpopulations of cells in the mouse proximal small intestine—one involving the sequential expression of substance P, serotonin, and secretin in cells migrating out of the crypts into the villi, and a second involving the expression of substance P and serotonin in cells which remain in the crypts. In this report, we use double label immunohistochemistry and BrdU incorporation to define the temporal and spatial interrelationships between gastrin, cholecystokinin (CCK), glucagon‐like peptide‐1 (GLP‐1), and gastric inhibitory peptide (GIP) immunoreactive cells in the mouse proximal small intestine. The expression of these products was compared with that of substance P, serotonin, and secretin. Minimal overlap of expression was found in cells immunoreactive for substance P or serotonin with gastrin, CCK, GLP‐1, or GIP; however, secretin was found colocalized in villus‐associated gastrin, CCK, and GLP‐1 containing cells. We demonstrate that, similar to the bidirectionally migrating substance P and serotonin expressing cells, gastrin, CCK, GLP‐1, and secretin are expressed in upwardly migrating cells, and gastrin, CCK, and GLP‐1 are expressed in downwardly migrating cells that fail to express secretin. GIP containing cells only rarely coexpressed any of the products examined, but were found both in the villi and the crypts, suggesting both upwardly and downwardly migrating populations. These findings demonstrate several novel enteroendocrine cell differentiation pathways. In addition, the expression of secretin in the villi, but not in the crypts, by two otherwise distinct differentiation pathways, and the lack of secretin expression by villus‐associated GIP expressing cells, suggests that local factors present in the crypts and/or on the villi are necessary, but not sufficient, for secretin expression. © 1994 Wiley‐Liss, Inc.
Caenorhabditis elegans sqt‐3 mutants have mutations in the col‐1 collagen gene
Authors: van der Keyl Harjeet , Kim Hwaok , Espey Richard , Oke C. Valerie , Edwards M. Kaye
Published: February 2005

sqt‐3 mutants of Caenorhabditis elegans form dumpy larvae and adults and display allele‐specific defects in locomotion, fertility, and viability. We have determined that the sqt‐3 locus encodes COL‐1 collagen. We physically mapped the col‐1 gene to a cosmid on chromosome V whose position is consistent with the location of the sqt‐3 gene. We also observed morphological defects in sqt‐3 mutants at stages that correlate with the mRNA expression patterns of col‐1. Sequence analysis of the col‐1 gene in the three temperature‐sensitive mutants revealed that each allele of sqt‐3 has a unique missense mutation causing arginine or glutamic acid to replace glycine in a Gly‐X‐Y triple helical domain. These glycine substitutions may result in longer non‐collagenous domains, which may decrease the thermal stability or impart additional flexibility to mutant trimers. In addition, we describe four corrections to the published sequence of col‐1, including one fifteen nucleotide addition that completes a conserved domain in the amino terminal coding region. © 1994 Wiley‐Liss, Inc.
Differentiation potentialities of distinct myogenic cell precursors in avain embryos
Authors: Auda‐Boucher G. , Fontaine‐Pérus J.
Published: February 2005

Interspecific grafting experiments between chick and quail embryos were carried out to investigate the differentiation capacities of myoblasts from different development stages. Grafts consisted of 3.5‐day‐old embryonic quail dermomyotomes isolated from the cranial level, 7‐ to 10‐day‐old and 16‐day‐old embryonic quail pectoralis muscles, 15‐day‐old postnatal quail pectoralis muscle, and 3‐ to 10‐day‐old embyonic quail cardiac and gut muscles. Grafts were implanted into 2‐day‐old chick embryos in place of the dorsal halves of somites from the prospective wing level. After implantation of dermomyotome fragments, we observed that quail cells participated in trunk and limb musculature. After implantation of 7‐ to 10‐day‐old embryonic muscle, quail cells were rarely found in the limb but systematically took part in the formation of trunk muscles. All these capacities were totally lost in 16‐day‐old embryonic and 15‐day‐old postnatal muscles. After implantation of nonsomitic derivatives such as embryonic cardiac and gut muscles, implanted cells never participated either in wing or trunk musculature. After dermomyotome, embryonic muscle, and gut implantation, quail cells were capable of invading the dermis and aggregating into feather germs. Our results extend those previously reported and indicate that somitic myogenic derivatives which do not migrate in the normal course of embryogenesis have migratory potentialities and are able to give rise to axial muscles. All these potentialities are lost as myogenesis proceeds in embryos. © 1994 Wiley‐Liss, Inc.
Tumor necrosis factor‐α and embryonic mouse lung morphogenesis
Authors: Jaskoll Tina , Boyer Paul D. , Melnick Michael
Published: February 2005

The ontogeny of the embryonic and fetal lung involves complex interactions between epithelial and mesenchymal primordia which require a specific program of gene regulation and signal transduction. Past studies in our laboratory using congenic mouse strains indicate that one or more genes which map to the H‐2 region of chromosome 17 regulate the rate of lung morphogenesis, defined in this context as differentiative heterochrony among strains. Since hormones and growth factors are the messengers of morphogenesis, it was logical to propose that tumor necrosis factor‐α (TNF‐α), a well‐characterized cytokine whose gene maps to the D‐region of the H‐2 complex, is a putative mediator of lung morphogenesis. We investigated this proposition using immunochemical methods and a serumless, chemically defined in vitro model system. Our results demonstrate that: (1) TNF‐α has a specific spatiotemporal localization, in vivo and in vitro; (2) TNF‐α receptor, in vivo and in vitro, is localized throughout the embryonic lung; (3) TNF‐α supplementation in vitro of embryonic lung primordia has a marked dose‐dependent, stimulatory effect on branching morphogenesis and surfactant‐associated protein (SP‐A) expression; (4) multiple immunoreactive proteins, including 17, 26, and 68 kDa species, are expressed during development in vivo, and a subset of these are expressed in vitro; and (5) both time‐ and glucocorticoid‐dependent changes occur in the in vivo expression pattern of TNF‐α immunoreactive proteins after 4 and 7 days in vitro, including the up‐regulation of a novel 40 kDa protein. Given that glucocorticoids (CORT) regulate TNF‐α expression and TNF‐α's ability to stimulate pulmonary morphodifferentiation and histodifferentiation, we conclude that TNF‐α is an autocrine/paracrine pulmonary cytokine, probably a component of the lung morphogenesis pathway regulated by CORT. © 1994 Wiley‐Liss, Inc.
Expression patterns of the bone morphogenetic protein genes Bmp‐4 and Bmp‐2 in the developing chick face suggest a role in outgrowth of the primordia
Authors: Francis‐West Philippa H. , Tatla Taranjit , Brickell Paul M.
Published: February 2005

Bone morphogenetic proteins BMP‐4 and BMP‐2 are closely‐related members of the transforming growth factor‐β superfamily that have been implicated in signalling in a number of developmental systems. To determine whether they could be involved in the epithelial‐mesenchymal interactions that control face development, we mapped the distribution of Bmp‐4 and Bmp‐2 gene transcripts in the developing chick facial primordia. At stages when primordia were becoming established, Bmp‐4 transcripts were present in specific regions of epithelium in all facial primordia, but were undetectable in the mesenchyme. Bmp‐4 transcripts appeared subsequently in specific regions of mesenchyme at the distal tips of the primordia. This mesenchymal expression first appeared in the frontonasal mass and then, in turn, in the lateral nasal processes, the maxillary primordia and the mandibular primordia. There was a complex relationship between domains of epithelial and mesenchymal Bmp‐4 expression, and at many sites there was an inverse correlation between epithelial and mesenchymal Bmp‐4 expression. Bmp‐2 transcripts were found in the epithelium and mesenchyme of the maxillary and mandibular primordia at early stages in facial development. Bmp‐2 transcripts appeared in the frontonasal mass and lateral nasal processes at later stages, with epithelial expression preceding mesenchymal expression. In general, mesenchymal Bmp‐2 expression was associated with overlying epithelial Bmp‐2 expression. The domains of Bmp‐4 expression overlapped with those of Bmp‐2, but detailed examination showed that there was no precise correlation between the expression patterns of the two genes. Indeed, in some places the Bmp‐4 and Bmp‐2 expression domains were complementary. The expression of the Bmp‐4 and Bmp‐2 genes in the epithelium and distal mesenchyme of the facial primordia suggests that BMP‐4 and BMP‐2 may be involved in the epithelial‐mesenchymal interactions that control outgrowth of these primordia. © 1994 Wiley‐Liss, Inc.
Differential expression during embryogenesis of three genes clustered in the Ras1 region of Drosophila melanogaster
Authors: Ezer Sini T. , Sahar Dvora , Salzberg Adi , Lev Zeev
Published: February 2005

Transcription mapping and nucleotide sequence analysis reveal that the genomic region of the Drosophila Ras1 gene contains a cluster of three closely localized genes. A gene termed Rlb1 is located nearby and upstream of Ras1, and is oriented in the opposite polarity relative to Ras1. In addition, a third gene termed Rlc1, is found at a very close proximity downstream to Rlb1. Ras1, the homologue of the human transforming ras genes, has been shown to be active in the posterior termini of the Drosophila embryo and in the eye imaginal disc in pathways of cell fate determination. We demonstrate that during embryogenesis Ras1 transcripts are restricted mainly to the embryonic central nervous system, suggesting that the gene product also may have a role in these nerve cells. Rlb1 encodes for a novel, lysine‐rich basic protein. It is expressed mainly in the developing midgut and in the somatic mesoderm. Rlc1 also encodes for a novel, basic protein. The expression of Rlc1 during embryogenesis is similar, but not identical, to the expression pattern detected for Ras1. The vertebrate p21Ras proteins are bound to the inner face of the cell membrane. Ras1, the Drosophila homologue of p21, and the Rlb1 protein, are also non‐cytoplasmic, membranous proteins. Rlb1 is found in the cell membrane of larval midgut epithelial cells. In addition, Rlb1 is detected in the nuclei of these cells, and in the nuclei of the midgut imaginal cells. © 1994 Wiley‐Liss, Inc.
Developmental relations between sixth nerve motor neurons and their targets in the chick embryo
Authors: Wahl Christina M. , Noden Drew M. , Baker Robert
Published: February 2005

The developmental relations between abducens (VI) nerves and their targets, the lateral rectus, quadratus, and pyramidalis muscles, have been examined in the chick embryo from early neural tube stages through 10 days of incubation. Sites of myoblast origins were determined by microinjection of replication‐incompetent retroviruses containing the LacZ reporter into paraxial mesoderm corresponding to somitomeres 3‐5. Motor neurons and axons were identified by Bodian staining, immunocytochemistry, and application of DiI and DiO to dissected peripheral nerves. Anlage of the dorsal oblique originate in somitomere 3, close to the ventrolateral margin of the mid‐to‐caudal mesencephalon. Precursors of the lateral rectus arise deep within somitomere 4, beside the future metencephalon (rhombomere “A”). Quadratus and pyramidalis precursors are located between and partially segregated from these other two anlage. VIth nerve axons exit rhombomeres 5 and 6 via multiple median roots, fasciculate, and by stage 17 have elongated rostrally beneath the hindbrain. Immediately caudal to a mesenchymal pre‐muscle condensation located deep to rhombomere 2, the VIth nerve separates into two branches. One branch enters the rostral portion of the condensation, from which quadratus and pyramidalis muscles will segregate. This branch projects exclusively from rhombomere 5 and is the accessory abducens nerve. The other branch enters the caudal, presumptive lateral rectus, region of the condensation. This is the abducens nerve, and it projects from cells located in both rhombomeres 5 and 6. These findings indicate that specific matching of motor nerves with their presumptive targets begins prior to the differentiation and segregation of myogenic populations, and that spatial organization of developing eye muscles is initiated well before they interact with connective tissue precursors derived from the neural crest. © 1994 Wiley‐Liss, Inc.
Mapping the origin of the avian enteric nervous system with a retroviral marker
Authors: Epstein M. L. , Mikawa T. , Brown A. M. C. , McFarlin D. R.
Published: February 2005

The enteric nervous system is largely formed from the vagal neural crest which arises from the neuroaxis between somites 1—7. In order to evaluate the contribution of different regions of the vagal crest to the enteric nervous system, we marked crest cells by injecting somites 1—10 with a replication‐defective spleen necrosis virus vector which contains the marker gene lacZ. After incubation in X‐gal, lacZ‐positive blue cells were found in the wall of the gut in three locations. Most were found at the peripheral edge of the developing circular muscle and within the developing submucosa, sites characteristic of developing ganglia. LacZ‐positive cells in these ganglionic sites were always surrounded by HNK‐1 immunostained cells, confirming their neural crest origin. LacZ‐positive cells were also seen in a third location, the circular muscle layer of the esophagus and crop, and were separated from the HNK‐1 positive ganglionic elements. These cells in the circular muscle are probably muscle cells derived from labeled mesodermal cells of the somite. Injection of somites 3, 4, 5, and 6 resulted in the largest percentage of preparations with lacZ‐positive crest‐derived cells and in the largest number of positive cells in the gut. After injection of these somites, lacZ‐positive crest‐derived cells were found in all regions of the gut from the proventriculus to the rectum. Very few positive crest‐derived cells were found in the esophagus. Injection of somites 1, 2, and 7 resulted in a smaller percentage of preparations with positive crest‐derived cells and in a smaller number of positive crest‐derived cells, which were confined to the fore and midgut. The gizzard was the gut region most frequently containing labeled cells and the rectum was the region least frequently containing such cells. This suggests that the number of crest cells available for colonization of the gut decreases as the distance from the gizzard increases. We conclude that the region of the neuroaxis between somites 3—6 is the major source of crest cells to the gut and that crest cells from different segments of the neuroaxis do not appear to be segregated to different regions of the gut. © 1994 Wiley‐Liss, Inc.
Stage‐ and region‐dependent responses of chick wing‐bud mesenchymal cells to retinoic acid in serum‐free microcultures
Authors: Paulsen Douglas F. , Chen Wei‐Dang , Okello David , Johnson Benita
Published: February 2005

Retinoic acid (RA) has been shown to affect skeletal patterning in vivo in both developing and regenerating limbs. Regional differences in RA concentrations alone cannot account for the region‐specific cell behaviors involved in limb‐skeletal morphogenesis. The present study explores a role for regional differences in signal interpretation in RA's effects along the anteroposterior and proximodistal axes of stage 21–22 and 23–24 chick wing‐buds. Mesenchymal cells isolated from specific limb regions were grown in chemically defined medium and exposed to 5 or 50 ng/ml of RA for 4 days in high‐density microtiter cultures. Previous studies showed that RA's effects on chondrogenesis and growth in such cultures differed depending on the position along the limb's proximodistal axis from which the cells were isolated. The present study is the first to show that such differences in RA‐responsiveness also exist along the limb's anteroposterior axis, especially in the distal subridge mesenchyme. The region‐dependent relationships between RA's effects on growth and chondrogenesis suggest that RA affects these two behaviors through different mechanisms. The regional differences in the responsiveness of these cells to exogenous RA are discussed with respect to their correspondence to the in vivo patterns of expression of RA‐binding proteins, RA‐receptors, and other patterning‐related genes. © 1994 Wiley‐Liss, Inc.
Characterization of cDNAs encoding the chick retinoic acid receptor γ2 and preferential distribution of retinoic acid receptor γ transcripts during chick skin development
Authors: Michaille Jean‐Jacques , Blanchet Sandrine , Kanzler Benoit , Garnier Jean‐Marie , Dhouailly Danielle
Published: February 2005

Retinoic acid receptors α, β and γ (RARα, β and γ) are ligand‐inductible transcriptional activators which belong to the steroid/thyroid hormone receptor superfamily. At least two major isoforms (1 and 2) of each RAR arise by differential use of two promoters and alternative splicing. In mouse, the three RAR genes are expressed in stage‐ and tissue‐specific patterns during embryonic development. In order to understand the role of the different RARs in chick, RARγ2 cDNAs were isolated from an 8.5‐day (stage 35 of Hamburger and Hamilton) chick embryo skin library. The deduced chick RARγ2 amino acid sequence displays uncommon features such as 21 specific amino acid replacements, 12 of them being clustered in the amino‐terminal region (domains A2 and B), and a truncated acidic carboxy‐terminal region (F domain). However, the pattern of RARγ expression in chick embryo resembles that reported in mouse, particularly in skin where RARγ expression occurs in both the dermal and epidermal layers at the beginning of feather formation, and is subsequently restricted to the differentiating epidermal cells. Northern blot analysis suggests that different RARγ isoforms could be successively required during chick development. © 1994 Wiley‐Liss, Inc.
Quantitative switch in integrin expression accompanies differentiation of F9 cells treated with retinoic acid
Authors: Burdsal Carol A. , Lotz Margaret M. , Miller Jeffrey , McClay David R.
Published: February 2005

F9 embryonal carcinoma cells resemble epithelial cells when in monolayer culture. After treatment with retinoic acid these cells differentiate into fibroblastic‐like cells in a sequence that has been modeled as the mammalian equivalent of the differentiation from stem cells of the inner cell mass to parietal endoderm. This study examined the changes in integrin subtypes that accompany retinoic acid‐induced differentiation of F9 cells. Although several integrins were found to be present on the surface of F9 cells and retinoic acid‐induced (RA) cells, the two dominant integrins were α3β1 and α5β1. Differentiation of F9 cells resulted in about 10‐ to 25‐fold increase in the amount of α3β1 integrin protein as measured by immunoprecipitation of cell surface labeled material. There was a corresponding several‐fold reduction of α5β1 protein. The concentration of α3 mRNA was about the same in F9 and RA cells while the concentration of α5 mRNA dropped several‐fold after retinoic acid treatment. Thus α3 regulation appeared to be largely posttranscriptional while the drop in α5 protein may have been a result of transcriptional down‐regulation. Quantitative measurement of adhesion suggested that most of the F9 and RA cell‐substrate adhesion to fibronectin or laminin is mediated by these integrins. They are the dominant integrins present, and antibodies to either these integrins or to the substrate blocked the adhesion.

Despite the large switch in integrin subtype protein expression there was little difference between the two cell types in initial cell interactions when adhesive affinities were measured quantitatively. Also there was no difference between the two phenotypes in rate of initial adhesive strengthening. The phenotypic difference was first observed with later events in the attachment and spreading of the RA‐treated cells to the substrate. These results show that retinoic acid treatment alters the amounts of α5 and α3 integrin subunits during the F9 to RA phenotypic switch. The data show that these integrins are important in the cell‐substrate adhesion to fibronectin and laminin. They show, however, that the phenotypic changes observed with differentiation are not associated with the initial preferential adhesions to the substrate, but rather with consequences that alter the cytoskeletal architecture of the cell. © 1994 Wiley‐Liss, Inc.
Protooncogene c‐ski is expressed in both proliferating and postmitotic neuronal populations
Authors: Lyons G. E. , Micales B. K. , Herr M. J. , Horrigan S. K. , Namciu S. , Shardy D. , Stavnezer E.
Published: February 2005

The cellular protooncogene, c‐ski, is expressed in all cells of the developing mouse at low but detectable levels. In situ hybridization and Northern blot analyses reveal that some cells and tissues express this gene at higher levels at certain stages of embryonic and postnatal development. RT‐PCR results indicate that alternative splicing of exon 2, known to occur in chickens (Sutrave and Hughes [1989] Mol. Cell. Biol. 9:4046–4051; Grimes et al. [1993] Oncogene 8:2863–2868) does not occur in adult mouse tissues. In the embryo, neural crest cells express the c‐ski gene during migration at 8.5 to 9.5 days post coitum (p.c.). Neural crest derivatives such as dorsal root ganglia and melanocytes stain positively with an antibody to the ski protein. At 9 days p.c., the entire neural tube has high levels of c‐ski gene expression. By 12–13.5 days only the ependymal layer expresses c‐ski above background levels. At 14–16 days p.c., c‐ski mRNAs are detected at high levels in the cortical layers of the brain and in the olfactory bulb. In 2 week and 6 week postnatal brains, c‐ski gene transcripts are also detected in the hippocampus and in the granule cell layer of the cerebellum. The allantois and placenta exhibit high levels of c‐ski mRNAs. Neonatal lung tissue increases c‐ski gene expression approximately two‐fold compared to prenatal levels. These results suggest that ski plays a role in both the proliferation and differentiation of specific cell populations of the central and peripheral nervous systems and of other tissues. © 1994 Wiley‐Liss, Inc.
Differential expression of myogenic regulatory genes and Msx‐1 during dedifferentiation and redifferentiation of regenerating amphibian limbs
Authors: Simon Hans‐George , Nelson Craig , Goff Debbie , Laufer Ed , Morgan Bruce A. , Tabin Cliff
Published: February 2005

An amputated limb of an adult urodele amphibian is capable of undergoing regeneration. The new structures form from an undifferentiated mass of cells called the regenerative blastema. The cells of the blastema are believed to derive from differentiated tissues of the adult limb. However, the exact source of these cells and the process by which they undergo dedifferentiation are poorly understood. In order to elucidate the molecular and cellular basis for dedifferentiation we isolated a number of genes which are potential regulators of the process. These include Msx‐1, which is believed to support the undifferentiated and proliferative state of cells in the embryonic limb bud; and two members of the myogenic regulatory gene family, MRF‐4 and Myf‐5, which are expressed in differentiated muscle and regulate muscle‐specific gene activity. As anticipated, we find that Msx‐1 is strongly up‐regulated during the initiation of regeneration. It remains expressed throughout regeneration but is not found in the fully regenerated limb. The myogenic gene MRF‐4 has the reverse expression pattern. It is expressed in adult limb muscle, is rapidly shut off in early regenerative blastemas, and is only reexpressed at the completion of regeneration. These kinetics are paralleled by those of a musclespecific Myosin gene. In contrast Myf‐5, a second member of the myogenic gene family, continues to be expressed throughout the regenerative process. Thus, MRF‐4 and Myf‐5 are likely to play distinct roles during regeneration. MRF‐4 may directly regulate muscle phenotype and as such its repression may be a key event in dedifferentiation. Myf‐5 may play a role in maintaining a distinct myogenic lineage during regeneration. © 1995 Wiley‐Liss, Inc.