bluuebunny/crossref_metadata_2025 huggingface dataset:
92‐kDa type IV collagenase and TIMP‐3, but not 72‐kDa type IV collagenase or TIMP‐1 or TIMP‐2, are highly expressed during mouse embryo implantation
Authors: Reponen Paula , Leivo Ilmo , Sahlberg Carin , Apte Suneel S. , Thesleff Irma , Olsen Björn R. , Tryggvason Karl
Published: February 2005

Expression of 72 kDa and 92 kDa type IV collagenases and the metalloproteinase inhibitors TIMPs 1, 2, and 3 was studied by in situ hybridization in implanting mouse embryos of days 5.5 to 7.5. The 92 kDa type IV collagenase was strongly expressed in invading trophoblasts, signals above background not being observed in the embryonic proper or placental tissue. In contrast, signals above background were not seen for the 72 kDa enzyme in any cells of the implantation region, including trophoblasts and stromal cells of the decidual tissue. Only cells in the mucosal stroma outside the decidual region displayed some expression. TIMP‐3 was intensily expressed in maternal cells in the area surrounding the invading embryonic tissue. No expression was observed for TIMP‐1 or TIMP‐2 in the embryo proper, trophoblasts, or the area of the uterine decidual reactin. Weak signals appeared for TIMP‐1 only in the circular layer of myometrial smooth muscle and in some uterine stroma cells distant from the site of embryo implantation. The results suggest a central role for 92 kDa type IV collagenase and TIMP‐3 in the extracellular proteolysis associated with implantation of the early embryo. © 1995 Wiley‐Liss, Inc.
Differential expression of the chicken Pax‐1 and Pax‐9 Gene: In situ hybridization and immunohistochemical analysis
Authors: Peters Heiko , Doll Uwe , Niessing Jürgen
Published: February 2005

We report the cloning, partial sequence analysis, and spatiotemporal expression of the chicken Pax‐1 (chPax‐1) and Pax‐9 (chPax‐9) gene, two closely related members of the paired box‐containing (PAX) gene family. The chPax‐1 gene encodes RNAs of 2.0 and 4.3 kb and a 42 kD protein while the gene products of chPax‐9 are represented by 1.9 and 3.1 kb transcripts and a 39 kD protein. In situ hybridization and immunohistochemical analyses reveal chPax‐1 expression in the developing pectoral girdle, in cells of the ventral part of sclerotomes, in sclerotome cells of the perichordal tube, and, later in development, in sclerotome‐derived cells of the intervertebral disks. Other chPax‐1 expression domains detected in the mesenchyme surrounding the atlas and axis and in chondrocytes of immature vertebral bodies, so far unreported for mouse Pax‐1, correlate with as yet unexplained malformations in the mouse Pax‐1 mutant undulated and Undulated‐short tail. Overlapping expression of chPax‐1 and chPax‐9 is detected in epithelial cells of the embryonic and adult thymus and in cells of the developing intervertebral disks. Unlike chPax‐1, however, chPax‐9 is not expressed in those perichordal sclerotome cells which are thought to give rise to vertebral bodies. Furthermore, chPax‐9 gene products are detected in circumscribed areas of mesenchyme in the metatarsus and in entodermal derivatives, i.e., in the lining epithelium of the developing pharynx and of the embryonic and adult esophagus. ©1995 Wiley‐Liss, Inc.
Pattern of muscle fiber type formation in the pig
Authors: Lefaucheur L. , Edom F. , Ecolan P. , Butler‐Browne G. S.
Published: February 2005

The aim of this study was to analyze the temporal sequence of expression of the myosin isoforms in the populations of muscle fibers in the pig and to bring more information on the origin of the strikingly different pattern of fiber composition and distribution between the deep medial red (oxido‐glycolytic) and superficial white (glycolytic) portions of semitendinosus (ST) muscle. Muscle samples were taken from 49‐, 55‐, 75‐, 90‐, 103‐, and 113‐ (birth) day‐old fetuses, from 6‐, 11‐, 21‐, 35‐, 50‐, and 80‐day‐old piglets, and from a 3‐year‐old pig. Our results confirm the sequential formation of primary and secondary generation fibers. The use of immunohistochemistry and heterologous monoclonal antibodies (mAb) directed against specific myosin heavy chain (MHC) isoforms revealed a different pattern of gene expression between the two portions of the ST muscle for both generations of fibers. By 75 days of gestation (dg), primary myotubes from the deep medial portion stained positively for the anti‐slow MHC mAb and negatively for the adult anti‐fast MHC, whereas the opposite was observed in the superficial portion. Secondary fibers never expressed slow MHC until late gestation. Instead, they expressed an adult fast MHC isoform as soon as they formed in the deep medial portion and later on in the superficial portion. From late gestation to the first 3 postnatal weeks, slow MHC began to be expressed in a subpopulation of secondary fibers. These fibers were in the direct vicinity of primary myotubes in the deep medial portion, whereas their location could not be established in the superficial portion. The remaining secondary fibers matured to type IIA in the direct vicinity of these type I fibers and to type IIB at the periphery of the islets. In both portions of the muscle, a subpopulation of secondary fibers, the first ones to express slow MHC, also transitorily expressed a MHC that was identical or closely related to the αcardiac MHC during the early postnatal period. A third generation of small diameter fibers was observed shortly after birth and reacted with the anti‐fetal MHC mAb; their destiny remains to be established. The present work reveals a remarkable pattern of MHC gene expression in the pig and raises many questions on the real nature of these isoforms. In order to answer these questions, we have undertaken to make a cDNA library of pig skeletal muscle and to screen this library with the same mAbs used in the present study. ©1995 Wiley‐Liss, Inc.
Developmentally regulated neural protein EAP‐300 is expressed by myocardium and cardiac neural crest during chick embryogenesis
Authors: McCabe Craig F. , Gourdie Robert G. , Thompson Robert P. , Cole Gregory J.
Published: February 2005

The spatiotemporal distribution of EAP‐300 (embryonic avian polypeptide of 300 kDa) was analyzed in embryonic chick heart using immunohistochemistry and confocal microscopy. EAP‐300 is a developmentally regulated protein initially characterized in neural cells from chick retina. Myocardial cells all along the early tubular heart were ubiquitously immunolabeled for EAP‐300 by embryonic day 2 (E2, Stage 13). At E5 (Stage 24), myocardial EAP‐300 expression levels remained significant in both atrial and ventricular myocardium. At E6 (Stage 28), distinct populations of EAP‐300 immunolabeled cells were also observed external to the heart, in septal mesenchymal tissue and neural ganglia adjacent to the outflow tract; these cell populations were confirmed as neural crest‐derived by co‐localization of EAP‐300 and HNK‐1. At E13 (Stage 39), myocardial immunolabeling for EAP‐300 was no longer ubiquitous, but increasingly restricted to conduction tissues, including the atrioventricular bundle and subendocardial Purkinje cells. This restriction of immunolabeling could be demonstrated definitively at E15 (stage 41), by which stage subendocardial and periarterial Purkinje fibers were clearly immunoreactive for EAP‐300 and several known markers of chick conduction tissue, including specific myosin heavy chain isoforms and connexin42, a gap junctional protein preferentially expressed by Purkinje fibers. Just prior to hatching at E21 (Stage 46), immunolabeling of conduction tissues was reduced, although still above that of non‐conductile myocardium. This spatiotemporal map of cardiac EAP‐300 expression indicates that it is independently and transiently expressed in early myocardium, cardiac conduction tissue, and neural crest derivatives during development. ©1995 Wiley‐Liss, Inc.
Expression of runt in the mouse embryo
Authors: Simeone Antonio , Daga Antonio , Calabi Franco
Published: February 2005

The Drosophila runt locus controls early events in embryogenesis. A human homologue (CBFA2) was criginally identified because of its involvement in the t(8;21) associated with a subtype of acute myeloid leukaemia. The phylogenetically conserved region (runt box) was reported to correspond to a DNA binding domain. In order to investigate whether runt also plays a role in mammalian development, we have conducted a preliminary survey of its expression in the mouse embryo. Expression in embryonic tissues was detected starting from day 9.2 post coitum. From day 10.5 post coitum, highest levels are found in the neural tube, sensory ganglia, specialised sensory epithelial structures (olfactory and gustatory mucosa, follicles of the vibrissae), all chondrogenic centres (both of neural crest and of mesodermal origin), and the genital system (the gonad, the paramesonephros, and the genital tubercle). Unambiguous expression in the haemo‐poietic system could be established for the thymus. The data suggest a pleiotropic role for mammalian runt in embryogenesis. ©1995 Wiley‐Liss, Inc.
Distribution of vitronectin mRNA during murine development
Authors: Seiffert Dietmar , Iruela‐Arispe Maria Luisa , Sage E. Helene , Loskutoff David J.
Published: February 2005

Vitronectin (Vn) is not only a major adhesive glycoprotein in plasma but also regulates cell‐mediated proteolytic enzyme cascades, including the complement, coagulation, and fibrinolytic systems. This broad functional activity suggests that Vn may also play a critical role in development. To begin to investigate this possibility, we studied Vn gene expression during murine embryogenesis. In situ hybridization analysis of embryonic tissues revealed Vn mRNA primarily in the liver and the central nervous system (CNS). In the liver, Vn mRNA was detected by day 10, the level increasing at later developmental stages. In the CNS, Vn mRNA was also detected as early as day 10 and was confined to the floor plate. However, as development proceeded, high levels of Vn transcripts became prominent in the meninges of the cortex and spinal cord, and in close proximity to brain capillaries. The perikarya of most neurons lacked Vn mRNA. Unexpectedly, high levels of Vn mRNA were associated with capillaries of the CNS, but not with blood vessels of peripheral organs. These results indicate that Vn is expressed in a spatially and temporally distinct pattern during murine embryogenesis, and suggest that the Vn transcript may be a CNS‐specific vascular marker. ©1995 Wiley‐Liss, Inc.
Inhibitory effects of ouabain on early heart development and cardiomyogenesis in the chick embryo
Authors: Linask Kersti K. , Gui Yong‐Hao
Published: February 2005

Pericardial cavity formation and epithelialization of the cardiac precursor cell population constitute a critical developmental period that precedes stable cardiac cell commitment and differentiation. These events delineate the myocardial and endocardial precursor population in the embryo. Restriction of Na/K‐ATPase (the sodium pump) expression to the pre‐cardiomyocyte lateral membranes coincides with these events. Na/K‐ATPase has been implicated developmentally in cavitation and in maintaining membrane potential. Experiments were undertaken to determine if the effects of perturbing sodium pump activity will affect pericardial cavity formation and, in turn, whether heart formation and/or cardiac cell commitment will be affected. We incubated whole chick embryos in vitro between stages 5 to 8 in the presence of the highly specific Na/K‐ATPase inhibitor ouabain. Exposure of whole embryos to 10 μM ouabain (10−5 M) demonstrated that heart development and precardiomyocyte differentiation are inhibited principally between stage 5 through stage 7. In each stage the degree of inhibition follows a rostrocaudal gradient as development proceeds along the anterior to posterior axis. After stage 8 ouabain no longer affects heart development or cardiomyogenesis. The inhibition is concentration‐ and developmental stage‐dependent. The inhibition is reversible by elevating the outside potassium ion concentration [Ko] in the culture medium or by transferring the embryos into normal medium minus ouabain even after 20 hr of ouabain exposure. The results also suggest that the regulation of the formation of the three‐dimensional organ is independent from regulation of myogenesis. ©1995 Wiley‐Liss, Inc.
Steel and c‐kit in the development of avian melanocytes: A study of normally pigmented birds and of the hyperpigmented mutant silky fowl
Authors: Lecoin Laure , Lahav Ronit , Martin Francis H. , Teillet Marie‐Aimée , Le Douarin Nicole M.
Published: February 2005

We describe here the expression of c‐kit and Steel (Sl) genes during the development of melanocytes in normally pigmented strains of chick and quail compared to unpigmented (White Leghorn) and hyperpigmented (Silky Fowl) strains of chickens. By using the quail/chick chimera system, we found that the neural crest cells, which migrate dorso‐laterally in the subectodermal mesenchyme to give rise to the melanocytes, express c‐kit as early as E4, that is about 2 days after they have left the neural primordium. The Sl gene is expressed from E4 onward in the epidermis but not at all in the dermis at any developmental stage. As feather buds develop, Sl mRNA becomes restricted to the apical region of the feather filaments. During formation of the barbs and barbules of the down feather, production of the Steel factor is restricted to the external epidermal cells of the barbules. The cell bodies of the c‐kit‐positive melanocytes are then located in the internal border of the epidermal ridges and extend their processes toward the source of the Steel factor. We propose that the spatial restriction of Sl gene activity at that stage accounts for the morphology of the melanocytes and their vectorial secretion of melanin to the external barbule cells. As a whole, these results show that during skin development c‐kit positive cells are present in the Steel factor‐producing areas at the time when melanoblasts proliferate and differentiate. Interestingly, in the mouse, previous studies showed that the Sl gene is activated in the dermis where melanoblasts undergo most of their expansion (Nishikawa et al. [1991] EMBO J. 10:2111‐2118). In the unpigmented and hyperpigmented mutants that we studied, expression of the Sl message, as judged quantitatively in Northern blots (for the SF embryos) or spatially by in situ hybridization, is similar to that observed in normal birds. In SF embryos the c‐kit expressing melanoblasts migrate initially in the dorso‐lateral migration pathway as in normal birds. However their number increases considerably in the dermis from E5 onward. From E7, they invade mesodermally derived organs that do not express the Sl gene. This suggests that another, still unknown, factor(s) is responsible for the survival, the proliferation, and the extensive spreading of melanocytic cells within the mesoderm of this mutant. ©1995 Wiley‐Liss, Inc.
Molecular cloning of tyrosine kinases in the early Xenopus embryo: Identification of eck‐related genes expressed in cranial neural crest cells of the second (Hyoid) Arch
Authors: Brändli A. W. , Kirschner M. W.
Published: February 2005

Growth factors and their receptors play an important role in controlling cellular proliferation, migration, and differentiation during vertebrate embryogenesis. We have used the reverse transcription‐polymerase chain reaction to survey the repertoire of receptor tyrosine kinases (TK) expressed during early embryogenesis of Xenopus laevis. Twelve distinct Xenopus TK cDNA classes were identified among a total of 352 cDNAs screened. A single TK cDNA class has been described previously and encodes the fibroblast growth factor receptor FGFR‐A1. The remaining 11 TK cDNA classes appear to encode novel genes of the FGFR, platelet‐derived growth factor receptor (PDGFR), Eph, Csk, Tyk2, and Klg subfamilies. By RNase protection assays, Xenopus TK mRNAs are rare transcripts (≤107 mRNA mol‐ecules/embryo), and are usually found to be expressed also maternally in the embryo. Most Xenopus TK genes examined by whole‐mount in situ hybridization were expressed widely in tissues derived from multiple germ layers. Two Eck‐related genes, however, were found to be restricted in their expression to neural crest of the second (hyoid) arch. Our findings are consistent with the proposed function of TKs in the regulation of specification and differentiation of embryonic tissues. ©1995 Wiley‐Liss, Inc.
Syndecan‐3, tenascin‐C, and the development of cartilaginous skeletal elements and joints in chick limbs
Authors: Koyama Eiki , Leatherman Judith L. , Shimazu Atsushi , Nah Hyun‐Duck , Pacifici Maurizio
Published: February 2005

The mechanisms by which the early limb cell condensations and interzone mesenchyme give rise to skeletal elements and joints are poorly understood. Previous work from this laboratory has shown that the extracellular matrix protein tenascin‐C is associated with articular cartilage and joint tissue development; others have shown that tenascin‐C may exert its biological activities via interactions with cell surface receptors, such as syndecans. To further analyze the roles of tenascin‐C and its putative receptors in skeletal development, we carried out a detailed in situ hybridization analysis of tenascin‐C and syndecan‐3 gene expression during development of chick limb skeletal elements and joints. We found that as the early mesenchymal condensations chondrify around day 5 (E5) of development, they become surrounded by a thick syndecan‐3 rich perichondrium while tenascin‐C transcripts are much fewer and restricted to diaphyseal perichondrium and developing interzones. Similar patterns were observed as distal carpal and digit condensations formed in older embryos. As the cartilaginous long bone models elongated proximo‐distally and joint formation proceeded with age, we observed that syndecan‐3 transcripts decrease significantly along the diaphysis and remain very abundant along the metaphysis and in the epiphyseal articular cap and interzone. Conversely, tenascin‐C RNAs remain abundant along the diaphysis and begin to increase at the epiphysis and in interzone‐derived tissues, such as menisci and joint capsule. By E10, the skeletal elements have well‐defined morphologies, endochondral ossification has initiated in their diaphysis, and diaphyseal perichondrium has become periosteum. These developmental changes were accompanied by equally marked changes in gene expression; these included a marked increase in tenascin‐C gene expression in articular cap, fragmentation of tenascin‐C gene expression along the periosteum, reinitiation of syndecan‐3 gene expression in periosteum, and differential gene expression in osteoprogenitor cells. The sheer complexity of the gene expression patterns documented in this study attests to the complexity of processes that bring about normal skelatogenesis. Clearly, tenascin‐C and syndecan‐3 appear to be closely associated with several of these processes, particularly in establishing tissue boundaries (perichondrium and periosteum) between condensations and surrounding mesenchymal cells, in regulating perichondral cell differentiation and incorporation into the growing skeletal elements, and in the genesis of epiphyseal chondrocytes and associated joint tissues. ©1995 Wiley‐Liss, Inc.
Expression of the helix‐loop‐helix gene Id3 during murine embryonic development
Authors: Ellmeier Wilfried , Weith Andreas
Published: February 2005

The family of dominant‐negative helix‐loop‐helix (dnHLH) transcriptional modulators consists of four mammalian genes known to date: Id1, Id2, Id3 (Id3 was formerly designated HLH462 or HEIR1 or HLH1R21), and Id4. We have performed a detailed study of Id3 expression in mouse embryos in order to gain insight into the possible developmental control functions of this gene and to compare its expression to that of other mammalian dnHLH genes. Employing RNA in situ hybridization on sectioned mouse embryos of gestational stages E9.5–17.5 and neonatal head, we generally detected a high abundance of transcripts at early stages that gradually declined at most sites toward E15.5. Expression of Id3 in embryos was detected in brain, spinal cord, olfactory system, branchial arches, limbs, sclerotome, endocardiac cushions, the outer lining of the gut, lung, retina, the collecting system of the kidney, and in tooth anlagen. Although the abundance of mRNA decreased toward later stages in most tissues, it remained high in teeth and kidney. This expression pattern suggests that Id3 functions both in undifferentiated tissues and in organs which are in the process of differentiation. When compared to the expression of other dnHLH genes, it becomes obvious that the pattern of Id3 mainly coincides with that of Id1. This may reflect a partial redundancy in gene function. Furthermore, previous experiments suggested a mutually exclusive expression of the proto‐oncogene N‐myc an Id3. The results of the present study confirm these data. ©1995 Wiley‐Liss, Inc.
Expression of genes encoding bone morphogenetic proteins and sonic hedgehog in talpid (ta3) limb buds: Their relationships in the signalling cascade involved in limb patterning
Authors: Francis‐West P. H. , Robertson K. E. , Ede D. A. , Rodriguez C. , Izpisúa‐Belmonte J. C. , Houston B. , Burt D. W. , Gribbin C. , Brickell P. M. , Tickle C.
Published: February 2005

The chicken mutant talpid3 (ta3) has polydactylous limbs with up to 7–8 morphologically similar digits. This lack of antero–posterior polarity in digit pattern is correlated with symmetrical expression of genes of the HoxD complex. We determined the distribution of polarizing activity in limb buds of the chick mutant ta3 by assessing the ability of mesenchyme from various positions along the antero‐posterior axis to induce digit duplications when grafted anteriorly into a normal limb. Cells with highest polarizing activity were found at the posterior margin of the wing as in the polarizing region of normal limb buds. However, in contrast to normal limb buds, ta3 anterior mesenchyme also had low polarizing activity. Application of retinoic acid or a polarizing region graft to the anterior of ta3 limb buds changed digit morphology but did not induce digit duplications or digits with any characteristic a–p pattern. To determine which genes are associated with polarizing activity and which are associated with patterning of the digits, we examined expression of the genes Sonic hedgehog (shh), Bmp‐2, and Bmp‐7, whose expression is normally confined to the posterior margin of the early wing bud and is associated with the polarizing region. In addition, we determined the distribution of Fgf‐4 transcripts which in normal limb buds are restricted to the posterior part of the apical ectodermal ridge. In ta3 limb buds, shh expression is restricted to the posterior limb mesenchyme, which has high polarizing activity, but is not expressed in regions which have low polarizing activity. In contrast, Bmp‐2 and Bmp‐7 are expressed uniformly along the a–p axis. Fgf‐4 transcripts are present throughout the apical ectodermal ridge in ta3 limb buds. In the ta3 mutant, there is both an abnormal distribution of signalling activity and response to polarizing signals. In addition, the dissociation between the expression of shh and Bmps suggests distinct roles for the encoded molecules in signalling and response in a–p patterning of limb buds. ©1995 Wiley‐Liss, Inc.
Tissue‐Specific and differential expression of alternatively spliced α1(II) collagen mRNAs in early human embryos
Authors: Lui Vincent C. H. , Ng Ling Jim , Nicholls John , Tam Patrick P. L. , Cheah Kathryn S. E.
Published: February 2005

Expression of the α1(II) procollagen gene is not confined to chondrogenic tissues during vertebrate development. Transcripts of the human gene (COL2A1) are alternatively spliced to give mRNAs which either exclude (type IIB mRNA) or include (type IIA mRNA) an exon encoding a cysteine‐rich domain in the amino‐propeptide. The distribution of COL2A1 mRNAs in 27‐ to 44‐day human embryos and 8‐ to 24‐week fetuses was studied by in situ hybridization and RNase protection analyses. Type IIA mRNAs were expressed in prechondrogenic cells and were also preferentially expressed in chondrogenic tissues at regions of chondrocyte commitment and cartilage growth. During maturation of chondrocytes, there is a switch to expression of type IIB mRNAs. In non‐chondrogenic tissues of early embryos, type IIA mRNA expression was associated with active tissue remodeling, epithelial organization, and sites of tissue interaction. Type IIA mRNAs were also expressed in some non‐chondrogenic tissues where expression had previously been undetected, such as the tooth bud, liver, adrenal cortex, apical ectodermal ridge, and indifferent gonad. In older fetuses type IIA mRNAs were the sole or major transcript in most non‐chondrogenic tissues except the choroid plexus and tendon. In the meninges there was a unique switch from type IIB to type IIA expression. The expression pattern of COL2A1 transcripts suggests that, in addition to contributing to the structural integrity of the cartilage extracellular matrix, type II procollagen may serve a morphogenetic role in embryonic development. Our findings clearly show that the pattern of expression of type II procollagen mRNAs is largely conserved between man and mouse. However, some differences exist, and these should be taken into consideration when animal models are used to study human diseases associated with COL2A1. ©1995 Wiley‐Liss, Inc.
New gene, nel, encoding a Mr 93 K protein with EGF‐like repeats is strongly expressed in neural tissues of early stage chick embryos
Authors: Matshuhashi Sachiko , Noji Sumihare , Koyama Eiki , Myokai Fumio , Ohuchi Hideyo , Taniguchi Shigehiko , Hori Katsuji
Published: February 2005

A new gene, nel, was isolated from a 9‐day‐old chick embryonic cDNA library. The gene encoes a protein of 835 amino acids (93,407 Mr) consisting of two hydrophobic domains presumed to be the signal and transmembrane sequences, a histidine rich domain, two repeats of a cysteine rich structure similar to the C‐terminal domain of von Willebrand factor, five EGF‐like repeats, and again two repeats of the cysteine rich sequence similar to the C‐terminal domain of von Willebrand factor in the presumed cytoplasmic domain. The expression of the nel gene was studied by Northern blot and in situ hybridization analyses of chick embryos. The mRNA of the gene was found in all tissues of 10‐ and 17‐day‐old embryos by Northern blot hybridization. Among the tissues examined, the level in the brain was highest and increased with age. After hatching, gene expression was retained in the brain at about the same level found in old embryos, increased in the retina, and disappeared from the other tissues. In situ hybridization with a nel gene probe revealed that the gene was strongly expressed in neural tissues such as brain, spinal cord, and dorsal root ganglia of early embryos. Gene expression was observed in the mantle layer of the neurepithelium of the brain and of the spinal cord. Gene expression in early embryos was not restricted to the neural tissues, but was also detected in the cells around cartilage, myocardium, lung mesenchymal cells, and in the liver, etc. One band of about 4.5 Kb mRNA was detected in all tissues and stages by Northern blot hybridization analysis. The possible function of the gene is discussed. ©1995 Wiley‐Liss, Inc.
Pattern of keratinocyte growth factor and keratinocyte growth factor receptor expression during mouse fetal development suggests a role in mediating morphogenetic mesenchymal‐epithelial interactions
Authors: Finch Paul W. , Cunha Gerald R. , Rubin Jeffrey S. , Wong Jane , Ron Dina
Published: February 2005

Mesenchymal cells are required for the induction of epithelial development during mammalian organogenesis. Keratinocyte growth factor (KGF) is a mesenchymally derived mitogen with specific activity for epithelial cells, suggesting that it may play a role in mediating these interactions. To further evaluate this hypothesis, in situ hybridization was used to examine the spatial distribution of KGF and KGF receptor (KGFR) transcripts during organogenesis and limb formation in mouse embryos (days 14.5 through 16.5). To facilitate this aim, mouse KGF cDNA clones were isolated. There was extensive identity between the deduced mouse KGF protein sequence and that of its human and rat cognates, indicating that this gene has been highly conserved during mammalian evolution. In addition, mouse KGF protein was purified from fibroblasts and demonstrated to be structurally and functionally similar to human KGF protein. For organs within the integumental, respiratory, gastrointestinal, and urogenital systems, whose development is dependent upon mesenchymal‐epithelial interactions, KGF mRNA was detected in mesenchymal cells, while epithelial cells expressed transcripts for the KGFR. KGF and KGFR mRNA was also expressed in certain other tissues such as perichondrium, cartilage of developing bones, developing skeletal muscle, and visceral smooth muscle whose development is not regulated by mesenchymal‐epithelial interactions. KGF expression was also detected in tissues isolated from human embryos, suggesting similar functions for KGF in human development. Taken together, our results suggest that KGF plays an important role in mediating mesenchymal‐epithelial interactions during organogenesis, but may also have other developmental functions in tissues not governed by such interactions. ©1995 Wiley‐Liss, Inc.
Drosophila glutamate receptor RNA expression in embryonic and larval muscle fibers
Authors: Currie Douglas A. , Truman James W. , Burden Steven J.
Published: February 2005

Glutamate is the excitatory transmitter at neuromuscular synapses in Drosophila, and electrophysiological studies indicate that the receptors for glutamate are concentrated in muscle fibers at synaptic sites. Acetylcholine is the excitatory transmitter at vertebrate neuromuscular synapses, and previous studies have shown that accumulation of acetylcholine receptors (AChRs) at synaptic sites is controlled both by transcriptional and post‐translational mechanisms. The transcriptional pathway culminates in selective expression of AChR subunit genes in nuclei near the synaptic site, causing AChR mRNA to accumulate in the synaptic region of the muscle fiber. We used a cDNA encoding a subunit of the Drosophila muscle glutamate receptor (DGluR‐II) to determine the temporal and spatial expression pattern of the DGluR‐II gene during embryogenesis and in larval muscle. We show that DGluR‐II mRNA is first expressed at stage 12 of embryogenesis and that expression is detected in developing dorsal, lateral, and ventral somatic muscles within the next 2 hr. By stage 16 DGluR‐II mRNA is expressed in all somatic muscles and in pharyngeal muscles. In third instar larvae DGluR‐II mRNA is expressed in all body‐wall muscle fibers. DGluR‐II mRNA, however, is expressed throughout the larval muscle fibers and is not concentrated within muscle fibers at neuromuscular synapses. These results indicate that although the DGluR‐II gene is expressed in somatic muscle cells it is not selectively expressed in nuclei near the synaptic site. ©1995 Wiley‐Liss, Inc.
Expression of transforming growth factor β in the embryonic avian lens coincides with the presence of mitochondria
Authors: Potts Jay D. , Bassnett Steven , Beebe David C.
Published: February 2005

During their maturation, lens cells lose all membrane bound organelles, including mitochondira. In chicken embryos this process begins in the central lens fibers beginning around embryonic day 12 (E12). Transforming growth factor β (TGFβ) is a multipotent growth modulator thought to play a role in numerous developmental processes. TGFβ1 has been localized to mitochondria in rat liver cells and muscle cells. In the present study, we examined the expression of TGFβ isoform mRNAs and proteins during chicken embryonic lens development. PCR analysis demonstrated TGFβ2 and TGFβ3 trasncripts in the lens epithelium and fibers throughout pre‐ and post‐hatching development. TGFβ isoforms were detected throughout the lens epithelium and fibers early in development (E6). However by E19, the distribution of TGFβ2 and TGFβ3 transcripts and proteins coincided with regions of the lens that contained mitochondria. In addition, intense TGFβ staining was observed in the basal portions of the equatorial epithelial cells, a region with abundant mitochondria. Transcripts for TGFβ1 and TGFβ4 were not detected in any tissue or time frame examined. Similarly, no immunostaining for TGFβ1 was observed. ©1995 Wiley‐Liss, Inc.
Migration of myogenic cells from the somites to the fore‐limb buds of developing mouse embryos
Authors: Sze Lung‐Yum , Kenneth Lee K. H. , Webb Sarah E. , Li Zhenlin , Paulin Denise
Published: February 2005

In this study, we have isolated newly formed somites from the caudal regions of 8.5 day mouse embryos and transplanted them orthotopically into correspondingly staged hosts at the level of the prospective limb‐forming region. The experimental embryos were then cultured intact for 32‐36 hr. The donor somites used were pre‐labelled with DiI, a fluorescent lipophilic dye, or were obtained from transgenic embryos that carried a 1 kb 5′ regulatory sequence of the desmin gene linked to the gene encoding Escherichia coli β‐galactosidase. The transgene is specifically expressed in skeletal muscles (Li et al. [1993] Development 117:947‐959). The aim of these experiments was to show definitively that the musculature of the mammalian limb is derived from the somites. The results demonstrated that DiI‐labelled cells from the implanted somites were able to invade the proximal region of the fore‐limb bud during the course of development. The use of transgenic somites as grafts confirmed that some of the somitic cells found in the limbs were myogenic cells. To determine whether the displacement of somitic cells is an active or passive process, somatopleure obtained from the prospective limb‐forming regions of day 8.5 day embryos was implanted into 8.5 day hosts. We did not detect the presence of DiI‐labelled somatopleural cells in the fore‐limb after 32‐36 hr of culture. This suggests that somitic cells reached the limb bud via active locaomotion rather than as a result of being passively dragged there, as the limb elongates during development. In addition, we injected latex beads into the somites, as probes, to determine whether extracellular matrix‐driven translocation plays a role in driving the somitic cells to the limb bud. In a majority of the specimens examined, we could not detect the presence of these beads in the limb bud. However, in the trunk of these embryos, the beads were found dispersed throughout the ventral neural crest pathway.
Expression of a type II collagen gene in the zebrafish embryonic axis
Authors: Yan Yi‐Lin , Hatta Kohei , Riggleman Bob , Postlethwait John H.
Published: February 2005

To understand the hierarchy of developmental controls underlying axis specification in vertebrate embryos, it is helpful to identify relationships between regulatory molecules and the genes that given axial cells their differentiated phenotypes. This work reports the cloning and expression pattern of one of these differentiation genes, a type II collagen (col2a1) gene from the zebrafish Danio rerio. Along th8e embryonic axis, col2a1 is expressed dynamically in three rows that are each a single cell wide: the notochord and the rows of cells immediately dorsal and ventral to it—the floor plate of the central nervous system, and the hypochord. In addition, col2a1 is expressed in the pharyngeal arches, the epithelium of the otic capsule, and in the mesenchyme of the neurocranium. Experiments probed the expression pattern of col2a1 relative to that of known or potential regulators of axis development, including axial, sonic hedgehog, twist, and cyclops. The results showed that the spatial and temporal pattern of col2a1 expression in axial mesoderm follows the expression of twist closer than other genes tested. In cyclops embryos, which lack an intact floor plate, col2a1 expression was usually low, but not missing in cells in the ventral spinal cord. Because col2a1 expression reveals abnormalities in the notochord of cyclopsb16 embryos, and less col2a1‐expressing mesenchyme accumulates rostral to the notochord in cyclops embryos, the effects of the cyclopsb16 mutation are not confined to the central nervous system. ©1995 Wiley‐Liss, Inc.
Novel FGF receptor (Z‐FGFR4) is dynamically expressed in mesoderm and neurectoderm during early zebrafish embryogenesis
Authors: Thisse Bernard , Thisse Christine , Weston James A.
Published: February 2005

We have identified a novel FGF receptor, Z‐FGFR4, in zebrafish embryos. Z‐FGFR4 is closely related to both chicken FREK (Marcelle et al. [1994] Development 120:683–694) and the Pleurodeles cDNA clone Pw‐FGFR4 (also named PFR4). The Z‐FGFR4 cDNA clones contain consensus sequences for two groups of two Ig‐like domains, separated by eight acidic residues referred to as the “acid box”. Z‐FGFR4, therefore, is the first FGFR molecule yet described in vertebrates that contains four Ig domains in its aminoterminal region.

Whole‐mount in situ hybridization of staged zebrafish embryos, using probes prepared from a variety of domains of the Z‐FGFR4 cDNA, reveal complex temporal and spatial expression patterns. Expression of Z‐FGFR4 mRNA is first detected in embryos prior to gastrulation and then appears in prechordal plate mesendoderm. At this time, Z‐FGFR mRNA is expressed in the epiblast in two distinct stripes which ultimately contribute to the brain. Eventually Z‐FGFR4 transcripts are observed in forebrain, anterior hindbrain (rhombomeres 1, 3), and caudal hindbrain (rhombomere 7), as well as in the dorsal‐most portion of the rostral spinal cord. Expression in axial mesendoderm appears transiently in notochord and segmental plate mesoderm. Eventually, Z‐FGFR4 mRNA becomes restricted to the posterior somites and is absent in differentiated notochord. These detailed expression studies provide the basis for understanding FGFR function through an analysis, currently in progress, of the developmental consequences of Z‐FGFR4 misexpression. ©1995 Wiley‐Liss, Inc.
Mouse half embryos: Viability and allocation of cells in the blastocyst
Authors: Papaioannou V. E. , Ebert K. M.
Published: February 2005

We have investigted the developmental capacity of mouse embryos in which one blastomere was destroyed by lysis at the 2‐cell stage. The allocation of cells to the trophectoderm and inner cell mass (ICM) was documented by differential cell counts on single embryos after 2 days under different culture conditions. Viability and further developmental potential were tested by embryo transfer to foster mothers. The conditions used were: (1) in vitro culture in modified BMOC‐2 medium, (2) in vivo oviduct transfer to immature (prepuberal) females, and (3) in vivo oviduct transfer to pseudopregnant females. Half embryos almost always fared less well for all parameters of development than control embryos developing under the same conditions. Lower total cell numbers in half embryos were accounted for by decreases in both ICM and trophectoderm with a disproportionate decrease in ICM in smaller embryos. In both half and control embryos, the growth conditions affected the rate of morphological development, the total cell number, and embryo viability. Unlike the effect of halving embryos, the growth condition effects on total cell number can be accounted for primarily by differences in ICM cell number, with trophectoderm cell number remaining constant. These results provide new information on the ability of the mouse embryo to differentially regulate ICM and trophectoderm cell number under different conditions, and confirm our previous work showing the advantage of short‐term development in vivo over short‐term in vitro culture (Papaioannou and Ebert [1986] J. Reprod. Fertil. 76:603–608). They also clearly document that lower total cell number and a lower proportion of ICM is correlated with compromised development.
Modulation of cell migration and vessel formation by vascular endothelial growth factor and basic fibroblast growth factor in cultured embryonic heart
Authors: Ratajska Anna , Torry Ronald J. , Kitten Gregory T. , Kolker Sandra J. , Tomanek Robert J.
Published: February 2005

Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) stimulate endothelial cell proliferation, migration, and vascular tube formation. We tested the hypotheses that these growth factors stimulate (1) cell migration and (2) assembly into cord‐like structures in embryonic rat heart explants cultured on collagen gels. Atrial and ventricular explants from rat embryos at 12 (E12, avascular) and 14 (E14, early vascularization stage) days of gestation were cultured on a collagen substrate. Western blot analysis of the explants indicated that endogenous VEGF was present in both atria and ventricles during incubation. Addition of bFGF to E12 explants markedly increased cell migration, whereas VEGF had no significant effect. In E14 explants neither growth factor influenced cell migration. Cotreatment with VEGF and bFGF did not have a synergistic effect on the migration distance of cells from either E12 or E14 embryonic hearts. However, VEGF stimulated the appearance of cord‐like structures in E14, but not E12, explants. Transmission electron microscopy analysis showed that these cord‐like structures consist of elongated cells, some of which aggregate into clusters, or form tube‐like structures, similar to capillaries. Serial sections of monolayers revealed that tube formation occurs beneath the surface of collagen gel. We conclude that in this model system VEGF and bFGF play distinct roles, at specific time points, in coronary vascular tube formation in the developing heart. ©1995 Wiley‐Liss, Inc.
Characterization of α1, β1, and γ1 laminin subunits during rabbit fetal lung development
Authors: Durham Paul L. , Snyder Jeanne M.
Published: February 2005

Laminin‐1 is an extracellular matrix protein composed of three polypeptide chains that are designated α1, βT1, and γ1. We investigated the expression of laminin α1, β1, and γ1 subunit chains during several stages of rabbit fetal lung development. Utilizing polyclonal antibodies directed against human placental laminin and immunoblot analysis, we found that the highest levels of laminin α1, β1, and γ1 subunit chains in the fetal lung were present on day 26 of gestation (term = 31 days), coincident with the initiation of alveolar epithelial cell differentiation. Levels of the laminin chains were approximately five times higher in fetal lung at day 26 of gestation than in adult lung tissue. Different temporal patterns of laminin α1, β1, and γ1 subunit chain expression were observed, data suggestive that the chains are independently regulated during lung development. Laminin was localized to the basement membranes of bronchi, bronchioles, preal‐veolar ducts, and blood vessels in fetal lung tissue, as shown by immunostaining with polyclonal laminin antibodies. A similar staining pattern was observed in adult lung tissue, but the alveolar wall was also stained. Laminin was also observed surrounding a few mesenchymal cells in fetal lung on day 19 of gestation; the number of positive mesenchymal cells increased with lung development. Laminin α1 subunit chains, detected using a monoclonal antibody, were present in the basement membranes of bronchi, bronchioles, preal‐veolar ducts, and blood vessels in fetal lung tissue. No laminin α1 chain staining was observed in the mesenchyme of early fetal lung tissue. Using a monoclonal antibody, laminin β1 subunit chains were immunolocalized in the basement membranes of bronchi, bronchioles, in prealveolar ducts, and surrounding some mesenchymal cells in fetal lung tissue. Laminin α1 and β1 subunit chains in adult lung tissue were present in basement membranes of airways, blood vessels, and alveoli. Thus, changes in the localization and accumulation of laminin near the time of alveolar type I and type II epithelial cell differentiation suggest that laminin may play a role in mediating the differentiation of these cell types during rabbit fetal lung development. ©1995 Wiley‐Liss, Inc.
Number of adrenergic and Islet‐1 immunoreactive cells is increased in avian trunk neural crest cultures in the presence of human recombinant osteogenic protein‐1
Authors: Varley Joel E. , Wehby Richard G. , Rueger David C. , Maxwell Gerald D.
Published: February 2005

OP‐1, also known as BMP‐7, is a member of the TGF‐β superfamily of proteins and was originally identified on the basis of its ability to induce new bone formation in vivo. OP‐1 mRNA is found in the developing kidney and adrenal gland as well as in some brain regions (Özkaynak et al. [1991] Biochem. Biophys. Res. Commun. 179:116–123). We have tested the effect of recombinant human OP‐1 on quail trunk neural crest cultures. The number of catechol‐amine‐positive cells which developed after 7 days in vitro in the presence of OP‐1 was increased in a dose‐dependent manner, with a greater than 100‐fold maximal stimulation observed. The increase in the number of catecholamine‐positive cells in the presence of OP‐1 was paralleled by an increase in the number of tyrosine hydroxylase (TH)‐positive cells. In contrast, total and melanocyte cell number were unaffected by the presence of OP‐1. The number of Islet‐1‐immunoreactive cells was also increased by OP‐1, but to only about half the value seen for TH. Double label experiments revealed these Islet‐1‐positive cells were a subset of the TH‐positive cells. Inhibitors of DNA synthesis prevented the OP‐1‐mediated increase in adrenergic cell number, indicating that OP‐1 does not act on a postmitotic cell population. However, labeling studies with bromodeoxyuridine indicated that OP‐1 did not increase the proportion of the cell population engaged in DNA synthesis. Thus, the OP‐1‐mediated increase in adrenergic cell number most likely occurs as a result of the enhanced survival of a subpopulation of adrenergic precursors or an increase in their probability of adrenergic differentiation, but not by increasing the mitotic rate of adrenergic precursors or adrenergic cells themselves. In contrast to OP‐1, TGF‐β1 decreased adrenergic cell number. When OP‐1 and TGF‐β1 were added simultaneously, TGF‐β1 antagonized the OP‐1‐mediated increase in adrenergic cell number in a dose‐dependent manner. �1995 Wiley‐Liss, Inc.
Transient expression of glutamate decarboxylase and γ‐amino butyric acid in embryonic lens fibers of the rat
Authors: Li Xuan , Ma Wu , Barker Jeffery L. , Piatigorsky Joram
Published: February 2005

We have determined the localization and developmental expression of glutamate decarboxylase (GAD67) in the rat lens. Immunofluorescence experiments showed that GAD67 was transiently expressed in the nuclear fiber cells of the lens between embryonic days (E) 15 and 20, with maximal immunostaining occurring on E17 and E18. γ‐amino butyric acid (GABA) co‐localized with GAD67 in the embryonic nuclear fiber cells. Reverse transcription‐polymerase chain reaction (RT‐PCR) tests showed that at least three alternatively spliced forms of GAD67 mRNA, including mRNAs with and without the I80 and the I86 insert, were transiently co‐expressed with GAD67 in the embryonic lens. The major GAD67 protein in the lens was 67 kDa. We conclude that enzymatically active GAD67 is transiently expressed in the lens nuclear fiber cells of the embryonic rat. The transient expression is regulated by transcriptional and/or posttranscriptional processes. We speculate on the basis of possible common gene regulatory elements for glutamate and ornithine decarboxylases and the involvement of these enzymes with polyamine synthesis, that the transient expression of GAD67 may be connected to nuclear and/or DNA breakdown during lens fiber cell differentiation. ©1995 Wiley‐Liss, Inc.