bluuebunny/crossref_metadata_2025 huggingface dataset:
Renal anatomy of the manatee, Trichechus manatus, linnaeus
Authors: Maluf N. S. R.
Published: February 2005

The manatee kidney is com posed of several closely apposed lobes. These are formed by cortical folds (plicae corticales) that completely isolate the medullae, except where the medullae of adjacent lobes are partially fused. The cortex is continuous; its folds usually are separated, but only partially, by interlobar septa ex tending from the renal capsule. The cortex makes up ∼57% of renal mass in adults and 68% in the calf. There are about 3 million glomeruli per kidney. The average is somewhat less than that expected of an adult eutherian of equal mass. The glomeruli, however, are large; they form 7.38%±1.33 of cortical mass, which is above that for at least ten unrelated adult eutherians. The number of glomeruli per gram of cortex is considerably greater in the calf than in the adult.

The medullae are about 43% of renal mass. The cortico‐medullary thickness ratio is 0.08 to 0.24. All terminal collecting ducts open at a crater (cra‐tera cribrosa) of varying depth. Hair‐pin loops occur at all levels of medulla, and apparently all loops bend at their thick segment. Cortical loops occur in the medullary rays. Vascular bundles were evident at the cortico‐medullary border and thin tubules extended into the medulla from the central ends of the medullary rays (cortical) in seven out of the nine kidneys. The renal pelvis is separated from the central ends of the cortical folds by delicate fascia through which pass the interlobar vessels. There are no fornices and no infundibula. The collagenous tissue of the pelvic wall extends across most of the pelvic surface of the outer medulla.
Differences in the solubility and susceptibility to proteolytic degradation of basement‐membrane components in adult and embryonic mouse tissues
Authors: Dziadek Marie , Mitrangas Katherine
Published: February 2005

We have studied susceptibility of basement membranes in a variety of tissues to solubility in guanidine hydrochloride and to proteolytic degradation by trypsin and thermolysin. Unfixed sections from embryonic and adult mouse tissues and the EHS tumor were subjected to sol vent buffers or digested with enzymes. The retention or disappearance of the basement‐membrane components nidogen, laminin, collagen IV, and heparan sulfate proteoglycan was subsequently assayed by immunofluorescence. Our data show ed that in all tissues nidogen was the most readily solubilized component and the most susceptible to proteolytic degradation. With few exceptions, nidogen in embryonic tissues was more susceptible to degradation than that in adult tissues, and this correlated well with the susceptibility of the other basement‐membrane components to be degraded. We conclude that basement membranes differ quite markedly in their solubility and their susceptibility to proteolytic degradation and that these properties reflect differences in their molecular structure.
Development of the stratified squamous epithelium of the human tympanic membrane and external canal: The origin of auditory epithelial migration
Authors: Michaels Leslie , Soucek Sava
Published: February 2005

The development of the stratified squamous epithelium of the tympanic membrane and external canal was studied in 167 embryonic, fetal, and postnatal human ears. It originates as a tube derived from the epithelium of the fundus of the primary external canal (zone 1). The tube is composed of a thin, flat epithelium on the medial side (zone 2), continuous with a thicker one (zone 3) on the lateral side; zone 3 thereafter merges with the external epithelium of the primary external canal (zone 4). Proliferative activity, as indicated by a thickened epithelium, with rete ridges in later fetal life, is present mainly in zones 1 and 3.

Cornification at 18 weeks gestation is followed by clearing of keratinous debris to the exterior. Subsequently the canal widens, zone 1 now covering the pars flaccida region, a tongue‐shaped area passing inferiorly from it and a part of the postero‐superior deep canal adjacent to it; zone 2 covers the pars tensa and zone 3 most of the deep external canal.

On the basis of the original embryonic growth, migratory epithelial movement throughout life is postulated to be generated in zone 1 by mitotic interposition and then to pass to zone 2. It then moves en masse through to zone 3, where unilateral progression by mitotic means takes the epithelium up to the cartilaginous canal. Such a path way is approximately that seen in the marked, living eardrum and canal.
Ultrastructural morphometric study of efferent nerve terminals on murine bone marrow stromal cells, and the recognition of a novel anatomical unit: The “neuro‐reticular complex”
Authors: Yamazaki Kazuto , Allen Terence D.
Published: February 2005

In order to extend our understanding of the role of nerve fibers in the structure and function of bone marrow stroma, we have examined nerve terminals, arterioles, and capillaries in femoral bone marrow tissues of 50 C57BL strain mice, using electron microscopy and mor‐phometric methods.

Within the adventitia of arterioles, a particular type of cell, termed periarterial adventitial (PAA) cell, is characterized by a thin veil‐like cytoplasm which concentrically surrounds both nerves and arterioles. Nerve fibers containing both unmyelinated and myelinated axons are distributed mainly between the layers of PAA cells, but are found rarely on the sinus walls or within the hematopoietic parenchyma. Quantitatively, the efferent nerve terminals with many synaptic vesicles are distributed mainly beside arterial smooth muscle cells (Type I: 58.8%) or between the layers of PAA cells (Type III: 33.2%), and rarely in hematopoietic parenchyma (Type II: 5.3%) or on sinus walls (Type IV: 2.7%).

In the case of Type II–IV nerve terminals, efferent (autonomic) nerves and bone marrow stromal cells which are connected by gap junctions (sinus adventitial reticular cells, intersinusoidal reticular cells, and PAA cells) appear to constitute a potential functional unit for signal conduction. We would like to propose a new term for this anatomical unit in marrow, the “neuro‐reticular complex.”
Origin of the proximal coronary artery stems and a review of ventricular vascularization in the chick embryo
Authors: Waldo Karen L. , Willner Will , Kirby Margaret L.
Published: February 2005

The objective of this study was to determine how the coronary artery stems develop in the chick embryo. The hearts of 51 inkinjected and cleared chick embryos, aged embryonic days 6, 6.5, 7, 7.5, 9, and 10, were dissected, examined, and selectively photographed. Two representative hearts from each group were paraffin embedded, serially sectioned at 10 ¨︁m, and examined for aortic endothelial budding. We found that the proximal coronary artery did not appear to grow outward from the aorta as commonly described in the literature. It appeared to originate from a capillary ring which encircled the aortic and pulmonary outflow tracts. On embryonic day 7.5, one to three channels arising from this ring penetrated each aortic sinus, in an area of darker textured endothelium. Histologically and grossly, multiple channels were still apparent on day 9, particularly in the left coronary artery. One of these channels always became dominant to form the stem. Each stem, which varied in length from embryo to embryo, always ended in a plexus of sinusoidal endothelial tubes. By day 10, the coronary artery stems were longer, with many major branches. Histologically, evidence of multiple channels still was visible. It is significant that channels from the bulbar vascular ring penetrated the aorta at very specific points in the aortic sinuses and did not penetrate the pulmonary trunk or other aortic sites. We believe this fact indicates that the penetration of the aortic sinuses by channels from the bulbar vascular ring represents a controlled invasion of the aorta.
Further serial transmission electron microscopy studies of auditory hair cell innervation in lizards and in a snake
Authors: Miller Malcolm R. , Beck Janet
Published: February 2005

Auditory hair cells of three lizard and one snake species were studied by serial transmission electron microscopy (TEM) sections of two unidirectinal hair cells (UHC) and two bidirectional hair cells (BHC) and by nonserial section montages of each entire papilla cut at 2‐μm intervals across the papillar width. The unidirectional hair cell region of the agamid lizard, Acanthosaura crucigera, lacked efferent innervation. Another agamid lizard, Agama agama, studied by nonserial section only, lacked efferent innervation to the UHC. Afferent innervation to both the UHC and BHC of Acanthosaura was primarily exclusive (each nerve fiber innervates only one hair cell), although an occasional nerve fiber innervated two hair cells. Both the UHC and the BHC of the anguid, Celestus costatus, were exclusively innervated. Both hair cell types of the varanid, Varanus exanthematicus, were nonexclusively innervated (all afferent nerve fibers innervate two or more hair cells). The auditory papilla of the colubrid snake, Elaphe obsoleta, has only one type of hair cell and each is nonexclusively innervated. The numbers of afferent and efferent nerve fibers and of afferent synapses are presented in tabular form.
Characterization of different microenvironments at the surface of the frog's taste organ
Authors: Sbarbati A. , Zancanaro C. , Franceschini F. , Balercia G. , Morroni M. , Osculati F.
Published: February 2005

We used a panel of histochemical techniques to identify and characterize the cell‐associated extracellular material at the surface of the frog's taste organ. We employed morphological and histochemical techniques using both the light microscope and the electron microscope. Results show that the apical, external aspect of cells reaching the surface of the taste organ is in close contact with a layer of amorphous material. The histochemical characteristics of this material vary according to the cell type with which it is in contact. Three different microenvironments can be identified at the surface of the frog's taste organ: type 1 microenvironment is associated with the superficial layer of mucus (secretory) cells; type 2 microenvironment characterizes the surface of the so‐called wing cells, which reach the surface of the taste organ as thin laminae running among mucus cells; and type 3 microenvironment shrouds the free endings of putative taste cells and is rich in calcium and lipids. Type 2 and type 3 microenvironments fix peroxidase (a sapid macromolecule) with increasing affinity. We conclude that highly differentiated microenvironments exist at the surface of the frog's taste organ, and these could play a role in the chain of biological events leading to the taste sensation. Furthermore, characterization of the cellassociated, specific microenvironments can help clarify the role of the different cell types in the frog's taste organ.
Neural organization of spindles in three hindlimb muscles of the rat
Authors: Kucera Jan , Walro Jon M. , Reichler Judith
Published: February 2005

The neuroanatomical organization of the dynamic (bag1) and static (bag2 and chain) intrafusal systems was compared by light and electron microscopy of serial sections among 71 poles of muscle spindle in soleus (SOL), extensor digitorum longus (EDL), and lumbrical (LUM) muscles in the rat. Eighty‐four percent of 195 fusi‐motor (γ) axons to the spindles innervated either the dynamic bag1 fiber or the static bag2 and/or chain fibers. Sixteen percent of the γ axons coin‐nervated the dynamic and static intrafusal fibers. Some of these nonselective axons were branches of efferents that also gave rise to axons selective to either the dynamic or static types of intrafusal fibers in one or more spindles. Thus activation of individual stem γ efferents might not have a purely dynamic or purely static effect on the integrated afferent outflow from spindles of a hind‐limb muscle in the rat. In addition, primary afferents in all muscles had terminations that cross‐innervated the dynamic bag1 and static bag2 and/or chain intrafusal fibers in individual spindles, an arrangement that may enhance the mixed dynamic/static behavior of afferents when different intrafusal fibers are activated concurrently. Spindles of the slow SOL and fast EDL muscles had similar features, whereas differences were observed in the organization of the proximal (SOL and EDL) and distal (LUM) muscles. Spindles in LUM muscles had fewer static intrafusal fibers, a higher ratio of dynamic to static γ axons, and a higher incidence of skeletofusimotor (β) innervation to intrafusal fibers than spindles in the SOL or EDL muscles. Thus, the relative contribution of dynamic and static systems to muscle afferent outflow may differ among spindles located in different segments of the rat hindlimb. However, the dynamic and static intrafusal systems of spindle were less sharply demarcated in each of the three hindlimb rat muscles than in the cat tenuissimus muscle.
Inflammatory macrophages in the dog contain high amounts of intravesicular ferritin and are associated with pouches of connective tissue fibers
Authors: Águas Artur P. , Grande Nuno Rodrigues , Carvalho Emanuel
Published: February 2005

We have studied the subcellular distribution of ferritin in inflammatory macrophages present in regional lymph nodes from dogs subjected to a pulmonary inflammatory reaction. The inflammatory reaction was induced by intrabronchial instillation of calcium tungstate (CaWO4), a water‐insoluble powder. Ferritin was identified by electron microscopy, and its electron density was enhanced by the use of a modified Perls method. From day 14 on after the CaWO4deposition, tungsten‐positive lymph node macrophages showed a massive accumulation of ferritin. Most of the ferritin was stored in membrane‐bounded vesicles that showed heterogeneous concentrations of the protein. A significant complement of ferritin was also detected in the cytoplasmic ground substance of phagocytes. The cell surface of the ferritin‐rich, tungsten‐positive macrophages showed deep infoldings that encompassed small pockets of connective tissue fibers. These features were not observed in control samples or in lymph nodes from dogs subjected to CaWO4‐induced inflammation for periods shorter than 1 week. Our data indicate that inflammatory macrophages greatly increase their content of ferritin and that ferritin is stored predominantly by a membrane‐bounded vesicular compartment. This is in contrast with suggestions that the inflammation‐induced increase in macrophage iron is restricted to the labile pool of iron and it does not involve the iron bound to ferritin molecules. Our observation of nodules of connective‐tissue fibers in intimate topographical association with ferritin‐rich macrophages may indicate that the increase in intracellular ferritin in the macrophage is in some way related to the secretion of factors by the phagocyte that will stimulate fibrillogenesis by neighboring fibroblasts.
Developing nephrons in adolescent dogfish, Scyliorhinus caniculus (L.), with reference to ultrastructure of early stages, histogenesis of the renal countercurrent system, and nephron segmentation in marine elasmobranchs
Authors: Hentschel Hartmut
Published: February 2005

Light and electron microscopy of the excretory kidney of adolescent dogfish, Scyliorhinus caniculus (L.), revealed immature and mature nephrons as well as four developmental stages of nephrons. At stage I the nephron was characterized by a condensed mass of mesenchymal cells in the center of several concentric layers of connective tissue. At stage II of the nephron, the S‐shaped body was an elongate cyst with a high prismatic epithelium that was connected by a developing collecting tubule with the collecting duct system. At stage III, the developing nephrons already possess the essential features of the mature nephron but lack complete differentiation. Developing renal corpuscles had one afferent arteriole and two efferent vessels. Developing tubules ran four times between the lateral bundle zone and the mesial tissue zone before they joined the collecting duct system. A continuous sheath of flat cells, encompassing the collecting duct system, extended around the developing lateral bundle. A rudimentary central vessel ran from the developing lateral bundle to the venous sinusoid capillaries between the mesial convolutions. Developmental stage IV was similar to the mature nephron, however, renal corpuscles and tubular segments were smaller than those of mature nephrons.

Conclusive evidence for morphological homology of elasmobranch nephron segments and collecting tubule‐collecting duct system with those of other vertebrates is provided. The origin and nature of the central vessel and the bundle sheath is clarified. These specific structures of marine elasmobranch kidney supposedly are of great functional relevance for the renal countercurrent system that in turn is essential for ion‐ and osmoregulation.
Regional differences in the morphology of the goat epididymis: A light microscopic and ultrastructural study
Authors: Goyal Hari Om , Williams Carol S.
Published: February 2005

The goat epididymis, based on morphological differences, was divided into five regions; regions I and II, and the proximal part of region III constituted the head; the distal part of region III and region IV, the body; and region V, the tail. The epithelium of all regions contained principal and basal epithelial cells and intraepithelial lymphocytes and macrophages. In addition, regions II to IV also contained a few apical cells. Clear cells were absent.

The epithelium varied in height from the tallest in region I (88 ± 33 μm) to the shortest in region V (38 ± 5 μm). Conversely, the luminal diameter, thickness of smooth muscle wall, and luminal sperm concentration were highest in region V. The irregular epithelial height of regions I and IV accounted for a stellate lumen in contrast to the oval lumen of the other regions. Whereas the lumen of region I contained only a few sperm, those of regions II, III, and IV were filled with sperm.

Principal cells were the only cell type that showed striking cytological differences between regions. While they contained absorptive features (canaliculi, pinocytotic and coated vesicles, and subapical vacuoles) in all regions, the principal cells of region II were filled with large, heterogeneous vacuoles (up to 5 μm in diameter), suggesting that they may be preferentially involved in transporting and digesting particulate material. Besides absorptive features, principal cells of all regions contained morphological correlates of protein synthesis such as highly developed Golgi complexes in the supranuclear area and numerous cisternae of RER near the Golgi body and in the infranuclear cytoplasm. The cisternae of RER were more developed in region IV, and in some instances, they were distended with flocculent material resembling newly synthesized protein. Unlike the protein synthesizing organelles, principal cells of all regions lacked morphological correlates of steroid hormone synthesis.

These results are compared with previously published data on the regional differences in the epididymis of other species, especially with those of the rat and the bull, in an effort to understand the significance of the epididymis in sperm maturation.
Connexin 43 expression in the mouse embryo: Localization of transcripts within developmentally significant domains
Authors: Ruangvoravat Christine P. , Lo Cecilia W.
Published: February 2005

The expression of the gap junction gene, Cx43, during mouse embryogenesis was characterized by an in situ hybridization analysis of mouse embryos from gestation days 4.5 to 12.5. This analysis revealed that Cx43 transcripts are differentially expressed as a function of development beginning at the blastocyst stage. In many regions of the embryo, Cx43 transcripts were found in discrete spatially restricted domains. This was observed in conjunction with the development of the brain, neural tube, prevertebra, limb, and various aspects of organogenesis. In some cases, the differential localization of Cx43 transcripts is associated with developmental processes mediated by inductive interactions, such as that of the eye, otic vesicle, kidney, and the branchial arches. In addition, in the 10.5 day embryo, Cx43 transcripts appear to be distributed as a gradient in regions spanning the midbrain/hindbrain junction, in the telencephalon, and in the limb mesenchyme. Surprisingly, our results also suggest that neural crest and sclerotomal cells, i.e., cells that are presumably migratory, express high levels of Cx43 transcripts. Overall, these results suggest that gap junctions encoded by Cx43 may play a role in various aspects of mouse development, possibly including relaying second messengers emanating from signal transduction pathways that mediate inductive interactions. © 1992 Wiley‐Liss, Inc.
Regulation of anterior cell‐specific mec‐3 expression during asymmetric cell division in C. elegans
Authors: Way Jeffrey C. , Run Jin‐Quan , Wang Alice Y.
Published: February 2005

The homeobox‐containing mec‐3 gene of C. elegans is expressed in 10 mechanosensory neurons and is necessary for these cells to acquire their fate. All the mec‐3‐expressing cells are anterior daughters from an asymmetric cell division. In this paper, we examine the expression of a mec‐3–lacZ fusion in the presence of mutations that may disrupt asymmetric cell division or anterior‐posterior positional information, as well as mutations that may specifically alter mec‐3 expression. A mutation in lin‐17 causes production of additional mec‐3‐expressing cells and can have its effect on the cell division that produces a mec‐3‐expressing cell. In a lin‐5 mutant, in which postembryonic blast cells do not complete cell division and become polyploid, blast cells that would give rise to mec‐3‐expressing daughters instead express mec‐3 themselves. In a lin‐12 glp‐1 double mutant, which is disrupted for many cell interactions in which two cells compete for the same fate, mec‐3 expression is unaffected. These results are consistent with a model for asymmetric cell division in which the mec‐3‐expressing cell and its sister are different immediately upon cell division, rather than acquiring differences through later interaction with each other or their surroundings. lin‐17 mutant animals also show defects in the position of the PVM cell and the PLM axons. Animals mutant in unc‐73 and unc‐40, known to have axon outgrowth defects, also show errors in PVM position and a low frequency of additional mec‐3‐expressing cells, as well as occasional secondary vulval protrusions, a common phenotype of lin‐17 animals. Many other mutations have either no effect on mec‐3 expression or an effect that can be largely predicted from previously known phenotypes: these include mab‐5, mig‐1, unc‐53, egl‐5, lin‐32, and egl‐27. unc‐11 shows an unexpected and specific defect in mec‐3 expression in the PVD neurons, but not in the other mec‐3‐expressing cells. Two mutations that suppress the egg‐laying defect of unc‐86 have no effect on the mec‐3 expression defect in an unc‐86 mutant. © 1992 Wiley‐Liss, Inc.
Expression of collagenase and IL‐1α in developing rat hearts
Authors: Nakagawa Masao , Terracio Louis , Carver Wayne , Birkedal‐Hansen H. , Borg Thomas K.
Published: February 2005

During development, extracellular matrix (ECM) molecules are thought to play a major role in regulating the formation of the heart. The change in the heart from a simple tube to a complex, four‐chambered organ requires the modification of both the cellular components as well as the surrounding ECM. Matrix metalloproteinases (MMP), which include collagenases, are enzymes present in the ECM that have the potential to modify the existing ECM during the development of the heart. Using both monoclonal and polyclonal antisera against collagenase, specific temporal and spatial patterns have been documented during critial periods of heart development. The cytokine interleukin 1α (IL‐1α), a potent inducer of the MMP expression, was also shown to have a similar staining pattern in the developing heart.

The monoclonal anti‐rat collagenase (Mab) intensely stained the surfaces of the myocytes in the trabeculae and the ventricular and atrial walls of the 11.5 or 12.5 embryonic day (ED) rat hearts. In contrast, the polyclonal anti‐human collagenase (Pab) stained not only the cardiomyocytes but also the hypertrophic endocardial cells. Pab appeared to stain the leading edge of the mesenchymal cells that migrate into the cardiac jelly of the 11.5 or 12.5 ED hearts. Immunohistochemical staining showed IL‐1α on the endocardial endothelium and the surface of cardiomyocytes near the cardiac jelly just before or coincident with the appearance of migrating cells.

IL‐1α was detected on the endocardial endothelium, cardiomyocytes in the trabeculae, and the ventricular and atrial walls, as well as in the myocardial basement membrane of the truncal or atrioventricular region. However, no staining could be detected on the migrating cells in the cardiac cushions. These results indicate the presence of collagenase and IL‐1α on the surface of cardiomyocytes and mesenchymal cells at times when the heart is undergoing acute remodeling during septation and trabeculation. These data suggest a role for collagenase/cytokine interaction in tissue remodeling during critical stages of cardiac embryogenesis where modification of the ECM is essential to cardiac morphogenesis. © 1992 Wiley Liss, Inc.
Fibrillin delineates the primary axis of the early avian embryo
Authors: Gallagher Betty C. , Sakai Lynn Y. , Little Charles D.
Published: February 2005

Chicken embryos were immunolabeled with antibodies to fibrillin, an extracelular matrix molecule implicated in the Marfan syndrome. The pattern of labeling, established using both whole‐mounted and sectioned embryos, showed a striking localization at the primary axis of the early embryo, including Hensen's node. After stage 8, the labeling of Hensen's node diminished while other structures in the embryo exhibited increased immunolabeling. These structures include the region of the presumptive dorsal aorta, the ventral surface of the notochord, and the mesocardium. Injections of the antibody between the vitelline membrane and the blastoderm in vivo failed to perturb the development of embryos, thus precluding any conclusions regarding the function of fibrillin. © 1993 wiley‐Liss, Inc.
gli, a zinc finger transcription factor and oncogene, is expressed during normal mouse development
Authors: Walterhouse David , Ahmed Maqbool , Slusarski Diane , Kalamaras Julie , Boucher Diane , Holmgren Robert , Iannaccone Philip
Published: February 2005

The oncogene GLI is amplified and expressed in some cases of human malignant glioma and undifferentiated childhood sarcoma a and is the prototype for a gene family characterized by a highly conserved set of five tandem zinc fingers and a consensus cysteine‐histidine link. This zinc finger motif has been shown to bind DNA with sequence specificity and may mediate transcriptional regulation. Since GLI is expressed in embryonal carcinoma cell lines but not in most normal adult tissues and shows significant sequence similarity within its zinc finger domain to cubitus interruptus dominant (ciD), a Drosophila segmentation gene known to be important in the morphogenesis of the posterior portion of each larval segment, we established the temporal and tissue expression patterns of the mouse homologue of human GLI in day 10 through 18 mouse embryos with Northern blotting, reverse transcriptase coupled PCR, and in situ hybridization. gli transcripts were demonstrated on days 10 through 18 of mouse embryonic development as well as in normal adult uterus, brain, testis, and limb. Tissue expression of gli during gestation was demonstrated in Meckel's precartilage mesenchyme, the basis occipitus, rib mesenchymal condensations, primordial vertebral bodies, digital mesenchymal condensations in forefoot and hindfoot plates, the ependymal layer of the spnal cord, and the mesoderm of the gastrointestinal tract. Expression persisted throughout gestation in developing bone and cartilage of the extremities, the ribs, and the vertebral bodies, as well as the gastrointestinal tract mesoderm. These findings support a role for gli family genes in normal craniofacial and digital development in mammals first suggested by the demonstration of translocation breakpoints within the GLI3 gene in families with the Greig cephalopolysyndactyly syndrome and subsequently by reduced gli3 expression in the mouse mutant extra toes. It is surprising that a single gene would be expressed in such a wide range of mesenchymal structures. © 1993 wiley‐Liss, Inc.
Analysis of LacZ reporter genes in transgenic embryos suggests the presence of several cis‐acting regulatory elements in the murine Hoxb‐6 gene
Authors: Eid Roland , Koseki Haruhiko , Schughart Klaus
Published: February 2005

Homeobox genes are expressed in stage‐, region‐, and tissue‐specific patterns during embryonic development of the mouse. In order to understand the underlying regulatory mechanisms the murine homeobox gene Hoxb‐6 was analyzed for the presence of cis‐acting regulatory elements. Transgenic mouse embryos and lines were generated which contained the LacZ reporter gene under the control of different fragments from the Hoxb‐6 gene. In total, 13.2 kb of genomic DNA covering the entire Hoxb‐6 region were analyzed. Our results suggested the presence of three regulatory regions in the Hoxb‐6 gene: a limb/LPM element which directed gene expression into restricted regions of the developing limb buds and the ventro‐lateral and visceral mesenchyme, a regulatory element required for gene expression into ventral regions of the developing spinal cord, and a third element necessary for directing gene expression into developing mesonephric tubules and mesonephric ducts. We demonstrated that the limb/LPM element functions as an enhancer in a promoter‐ and orientiation‐independent manner. The comparison of the expression patterns of the reporter gene constructs and the endogenous Hoxb‐6 gene revealed that the regulatory regions were able to reproduce part of the Hoxb‐6 pattern. However, not all control elements necessary to completely mimic the endogenous Hoxb‐6 expression pattern could be detected, although the entire Hoxb‐6 genomic region has been analyzed. These observations suggest that for some aspects of Hoxb‐6 gene regulation the integrity of large genomic regions of the Hoxb cluster is required. © 1993 wiley‐Liss, Inc.
N‐cadherin mediates sertoli cell‐spermatogenic cell adhesion
Authors: Newton Sean C. , Blaschuk Orest W. , Millette Clarke F.
Published: February 2005

The complex topological association of Sertoli cells and spermatogenic cells in the testis suggests the existence of cell surface adhesion molecules that regulate cellular interactions within the seminiferous epithelium. The recent report of N‐cadherin mRNA expression in the mouse testis implies the involvement of this known adhesion molecule in testicular cell binding. Accordingly, here we report that (1) N‐cadherin is found on the surface membranes of rat spermatogenic cells and on Sertoli cells, and (2) that N‐cadherin is a partial mediator of Sertoli cell‐germ cell adhesion as tested in an in vitro cell‐cell binding assay. Antiserum directed against the N‐cadherin cell adhesion recognition sequence was used for Western blot analysis of purified plasma membranes from Sertoli cells and from spermatogenic cells. Both membrane preparations exhibited reactivity at an appropriate Mr of about 130 kDa. In addition, immunofluorescence assays demonstrated that both germ cells and Sertoli cells were labeled by anti‐N‐cadherin. Finally, the antiserum was included in a cytometer‐assisted cell‐cell binding test to determine its inhibitory ability. The antiserum consistently reduced specific testicular cell‐cell adhesion by 30%–50%. This is the first demonstration that antibodies directed against the cadherin cell adhesion recognition sequence are capable of inhibiting cell‐cell interactions. Pre‐incubation of either rat Sertoli cells or spermatogenic cells alone was sufficient to achieve statistically significant inhibition of intercellular adhesion. We conclude, therefore, that N‐cadherin is expressed by both Sertoli cells and spermatogenic cells and that N‐cadherin is one of a number of regulatory molecules mediating local cellular associations in the mammalian seminiferous tubule. © 1993 Wiley‐Liss, Inc.
Differential expression of thrombospondin 1, 2, and 3 during murine development
Authors: Iruela‐Arispe M. Luisa , Liska DeAnn J. , Sage E. Helene , Bornstein Paul
Published: February 2005

Thrombospondin 1 is a secreted, trimeric glycoprotein that mediates interactions between cells and extracellular matrix and exhibits cell‐specific effects on migration and proliferation. Recently, two additional thrombospondin genes (thrombospondin 2 and 3) have been identified. To study the functions of these proteins, we have used in situ hybridization and RNAse protection assays to compare the expression of the genes encoding thrombospondin 1, 2, and 3 during murine embryogenesis. Thrombospondin mRNAs were associated with ossification, neuronal organogenesis, and lung development, although transcripts were differentially expressed. Thrombospondin 1 was predominant from days 10 to 13. During this period, high but transient levels of expression were observed in the neural tube, head mesenchyme, and cardiac cushions. In contrast, a more constant level of thrombospondin 1 mRNA was apparent in resident megakaryocytes of the liver, as well as in circulating megakaryocytes; neither thrombospondin 2 nor 3 was detected in these cells. Thrombospondin 1 was also produced by cells of the developing kidney and gut. The expression of thrombospondin 2 was confined principally to organized connective tissue that included pericardium, pleura, perichondrium, periosteum, meninges, ligaments, and reticular dermis. Thrombospondin 2 was also produced by differentiating skeletal myoblasts and by cells of the kidney and gut. Moreover, high levels of expression were detected in blood vessels. Thrombospondin 3 mRNA was restricted to brain, cartilage, and lung. Although thrombospondin 1, 2, and 3 belong to a family of structurally related genes, the differences observed in the spatiotemporal distribution of the corresponding mRNAs indicate unique functions for these secreted proteins. © 1993 Wiley‐Liss, Inc.
Developmental analysis of the external granular layer in the Meander tail mutant mouse: Do cerebellar microneurons have independent progenitors?
Authors: Napieralski Julie A. , Eisenman Leonard M.
Published: February 2005

The cerebellum of the meander tail mutant mouse (mea/mea) is characterized by an apparently normal cytoarchitecture posteriorly with an abrupt transition to an abnormal anterior region. Anteriorly, there is abnormal folition, a drastic reduction in the granule cells (GC) population, disorganization of the Purkinje cells (PC), and a virtual absence of Bergmann glial processes. In this paper we analyze the prenatal and postnatal development of the cerebellum in the mea/mea and attempt to determine the phenotypic onset of the mutation in the anterior region. Hematoxylin and eosin stained sections reveal a morphological difference in the cerebellum of the mea/mea as early as embryonic day 16 characterized by a reduction in the external granule cell layer (EGL). The reduction in the EGL becomes increasingly apparent as development proceeds. This deficit in the EGL most probably results in the absence of GC, but it is unclear at this point whether reduced migration, proliferation, and/or increased cell death is the major factor. Interestingly, immunohistochemical staining with a monoclonal antibody against parvalbumin reveals that the basket and stellate cells, which are also thought to arise from the EGL, are present in the anterior region of the mea/mea cerebellum. These results suggest that the lack of GC in the meander tail is due to an early expressed abnormality of the EGL. However, the presence of the basket and/or stellate cells raises some interesting questions concerning the lineage of the cerebellar microneurons. © 1993 Wiley‐Liss, Inc.
Cellular origin of the dermal‐epidermal basement membrane
Authors: Marinkovich M. Peter , Keene Douglas R. , Rimberg Clytie S. , Burgeson Robert E.
Published: February 2005

The basement membrane underlying epithelium of skin is generally believed to be of epithelial origin, but a mesenchymal contribution to the basement membrane has not been directly examined. The purpose of this study was to directly evaluate both epithelial and mesenchymal contributions to the basement membrane. Fetal bovine keratinocytes cultured on the surface of collagen gels in the absence of fibroblasts did not produce an ultrastructurally recognizable basement membrane; however, when these cells were cultured in the presence of dermal fibroblasts a basement membrane at the keratinocyte‐fibroblast interface was produced after 1 week which was very similar in biochemical composition and ultrastructural appearance to dermal‐epidermal basement membrane in human skin. When dual species cultures of bovine keratinocytes and human fibroblasts were analyzed by indirect immunofluorescent microscopy (IF)1 with human specific antibodies against basement membrane components, dermal fibroblasts were shown to synthesize and deposit type IV collagen, type VII collagen, and laminin in a linear manner into the basement membrane zone. Fetal bovine keratinocytes cultured in the presence or absence of fibroblasts synthesized and deposited type IV collagen, type VII collagen, laminin, K‐laminin, kalinin, and basement membrane associated heparan sulfate proteoglycan (HSPG) into the underlying basement membrane zone. In organ culture, a subpopulation of fibroblasts initially migrating from human foreskin explants was found to stain strongly for types VII and IV collagen and laminin by IF whereas after subculture all cells showed a uniform low staining. Based on these observations we propose that differentiated fibroblasts exist adjacent to epithelial tissues in vivo which produce basement membrane components and assist in basement membrane components and assist in basement membrane assembly. © 1993 Wiley‐Liss, Inc.
Effect of epidermal growth factor on expression of transforming growth factor‐β1 mRNA in stellate reticulum cells of rat mandibular molars
Authors: Lin Fan , Wise Gary E.
Published: February 2005

Cultured stellate reticulum cells isolated from rat mandibular molars respond to incubation in EGF by increasing their level of expression of TGF‐β1 mRNA. Northern blots showed that incubation in EGF for 6 hours stimulated over a two‐fold increase in TGF‐β1 mRNA in the cells. In contrast, indicating that TGF‐β1 does not have an autocrine effect on these cells. Immunocytochemistry showed that EGF receptor was present on the surface of many but not all of the cultured stellate reticulum cells. Because EGF does stimulate premature eruption of teeth, it is possible that its effect on the stellate reticulum region of the enamel organ would be to stimulate synthesis of TGF‐β1 mRNA which, in turn, could lead to increased synthesis of TGF‐β1 by these cells. The cells do contain the TGF‐β1 protein as revealed by immunocytostaining. The newly synthesized TGF‐β1 may exert its effect on the adjacent dental follicle to either initiate the onset of the cellular events of tooth eruption or to increase the secretion of extracellular matrix proteins by the follicle for formation of the periodontal ligament. © 1993 Wiley‐Liss, Inc.
Expression of Xenopus snail in mesoderm and prospective neural fold ectoderm
Authors: Essex Linda J. , Mayor Roberto , Sargent Michael G.
Published: February 2005

Expression of the Xzna gene during Xenopus laevis embryogenesis has been analysed by in situ hybridisation. Like its homologue snail in Drosophila, Xsna is expressed zygotically in all early mesoderm. Expression starts during stage 9 in the dorsal marginal zone and spreads to the ventral side by stage 10. During gastrulation, each cell begins to express as it involutes so that cells newly expressing Xsna are added to the forming mesoderm mantle in an anterior‐to‐posterior progression. Xsna expression is then down‐regulated in a tissue‐specific fashion that reveals the subdivision of the mesoderm before its derivatives are overtly differentiated; e.g., the appearance of the notochord, myotomes, and pronephroi are preceded by the disappearance of Xsna mRNA, while undifferentiated mesoderm remins labelled, even into tadpole stages. Xsna is expressed in the suprablastoporal endoderm during gastrultion and in its derivatives, the prechordal and sub‐notochordal endoderm, during neurulation. Relationships between Xbra, Xtwi and Xsna expression are examined.

Xsna is also expressed in the prospective neural fold ectoderm from stage 11 in a low arc above the dorsal marginal zone, precisely identifying a distinct band of cells that surrounds the prospective neural plate that we designate the neural plate border. The anterior transverse neural fold, which becomes forebrain, ceases Xsna expression during neurulation. In the longitudinal neural folds, the deep and superficial ectoderm compartments labelled by Xsna expression are the prospective neural crest and prospective roof of the neural tube, respectively. Xsna expression persists in the neural crest during migration and in some derivatives at least until metamorphosis but ceases in the roof of the neural tube soon after neurulation. © 1993 Wiley‐Liss, Inc.
Collagen type VI in neural crest development: Distribution in situ and interaction with cells in vitro
Authors: Perris Roberto , Kuo Heuy‐Ju , Glanville Robert W. , Bronner‐Fraser Marianne
Published: February 2005

We have examined the spatiotemporal distribution of collagen type VI (Col VI) during neural crest development in vivo and its ability to promote neural crest cell attachment and migration in vitro. An affinity purified antiserum and chain‐specific monoclonal antibodies against chicken Col VI were employed to immunolocalize the collagen in tissue sections and by immunoblotting. At stages of initial neural crest cell migration, the α1(VI) and α2(VI) chains were immunolocalized in apposition with basement membrances of the neural tube, somites, notochord and ectoderm, whereas no immunoreactivity was seen for the α3(VI) chain. Immunoblotting analysis confirmed the expression of α1(VI) and α2(VI) chains and the lack of detectable immunoreactivity for the α3(VI) chain at these early phases of neural crest development. Conversely, at advanced phases of migration and following gangliogenesis, expression of α3(VI) chain coincided with that of α1(VI) and α2(VI) chains in apposition with basement membrances, around the dorsal root ganglia, and in fibrillar arrangements within the developing dermis and ventral sclerotome. The ability of Col VI to promote neural crest cell attachment and migration was tested in vitro using quantitative assays for these processes. Both native microfilaments and isolated tetramers of Col VI strongly promoted neural crest cell attachment and migration. Optimal stimulation of neural crest cell adhesion and migration was dependent upon structural integrity of Col VI since unfolded and disassembled α chains only weakly promoted cell attachment and were virtually inactive in supporting cell movement. The importance of a native macromolecular organization of Col VI further was analyzed in experiments in which dissociated tetramers were reassociated by Ca2+‐ and temperature‐dependent self‐aggregation. In contrast to native microfilaments, these oligomeric complexes were less effective in promoting neural crest cell movement, but still retained the ability to stimulate maximal cell attachment. The results indicate that Col VI is a primary component of the extracellular matrix deposited along neural crest migratory pathways, where it may participate in the regulation of cell movement by functioning as a migratory substrate. The ability of Col VI to promote neural crest cell adhesion and motility is highly dependent upon maintainance of a native macromolecular arrangement. © 1993 Wiley‐Liss, Inc.
Expression of the dystrophin‐related protein (utrophin) gene during mouse embryogenesis
Authors: Schofield Julian , Houzelstein Denis , Davies Kay , Buckingham Margaret , Edwards Yvonne H.
Published: February 2005

The utrophin (UTRN) locus is the autosomal homologue of the DMD (Duchenne muscular dystrophy) gene and encodes a protein, utrophin which is thought to be upregulated in the absence of dystrophin. In this study the spatial and temporal expression of the UTRN gene has been examined during mouse embryogenesis and compared with that of the DMD gene. The patterns of expression of these two genes are very different. Whilst DMD is expressed largely in mesodermal derivatives such as cardiac and striated muscle, UTRN shows a more widespread distribution and is expressed in neural tube, tissues which originate from neural crest and a variety of other sites of non‐neural origin. In early embryos UTRN transcripts initially accumulate in the mid‐neural plate and thereafter in the caudal neural tube. UTRN mRNA then becomes abundant in a subset of neural crest cell derived tissues, in particular the spinal and facial ganglia and ossifying facial cartilages. UTRN is also expressed in a variety of other sites and organs such as the tendon primordia in the digits, the pituitary, thyroid and adrenal glands, cardiac muscle, kidney and lung, follicies of the vibrissae and the outflow tract of the heart. Several patterns of UTRN expression are apparent and we discuss the possibility that these can be ascribed to a family of mRNAs transcribed from the UTRN gene using alternative promoters. © 1993 Wiley‐Liss, Inc.
Pro‐opiomelanocortin gene is expressed in post‐implantation mouse embryos and enhances growth potential of myogenic cells
Authors: De Angelis L. , Cusella‐De Angelis M. G. , Monaco L. , Raschellá G. , Cossu G.
Published: February 2005

We previously showed that the adrenocorticotropin hormone (ACTH) is a mitogen for myoblasts and is present in post‐implantation mammalian embryos (Cossu et al. [1989] Dev. Biol. 131:331–336; De Angelis et al. [1992] Dev. Biol. 151:446–458). In this paper, we investigated the expression of the corresponding gene, pro‐opiomelanocortin (POMC), by in situ hybridization and polymerase chain reaction. In situ analysis revealed low level expression in the basal layer of 10.5 d.p.c. neural tube and in several discrete areas around the dorsal aorta. By more sensitive Reverse‐transcription polymerase chain reaction (RT‐PCR) analysis, expression was detected also in developing limb buds and in cultured myogenic cells, but not in fibroblasts.

To investigate the possible role of POMC gene expression in myogenesis, we induced its over‐expression in proliferating myoblasts. Upon sub‐optimal growth conditions, over‐expressing cells were found to give rise to clones larger than control cells. The differentiation potential of POMC over‐expressing myogenic cells was unchanged. © 1993 Wiley‐Liss, Inc.