bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of transcriptional isoforms of collagen types IX, II, and I in the developing avian cornea by competitive polymerase chain reaction
Authors: Fitch John M. , Gordon Marion K. , Gibney Eileen P. , Linsenmayer Thomas F.
Published: February 2005

The genes for the α1(IX), α1(IX), α1(II), and α2(I) collagen chains can give rise to different isoforms of mRNA, generated by alternative promoter usage [for α(IX) and α2(I)] or alternative splicing [for α1(II)]. In this study, we employed competitive reverse transcriptase PCR to quantitate the amounts of transcriptional isoforms for these genes in the embryonic avian cornea from its inception (about 3 1/2 days of development) to 11 days. In order to compare values at different time points, the results were normalized to those obtained for the “housekeeping” enzyme, glycerol‐3‐phosphate dehydrogenase (G3PDH). These values were compared to those obtained from other tissues (anterior optic cup and cartilage) that synthesize different combinations of the collagen isoforms. We found that, in the cornea, transcripts from the upstream promotor of α1(IX) collagen (termed “long IX”) were predominant at stage 18–20 (about 3 1/2 days), but then fell rapidly, and remained at a low level. By 5 days (just before stromal swelling) the major mRNA isoform of α1(IX) was from the downstream promotor (termed “short IX”). The relative amount of transcript for the short form of type IX collagen rose to a peak at about 6 days of development, and then declined. Throughout this period, the predominant transcriptional isoform of the collagen type II gene was IIA (i.e., containing the alternatively spliced exon 2). This indicates that the molecules of type II collagen that are assembled into heterotypic fibrils with type I collagen possess, at least transiently, an amino‐terminal globular domain similar to that found in collagen types I, III, and V. For type I, the “bone/tendon” mRNA isoform of the α2(I) collagen gene was predominant; transcripts from the downstream promotor were at basal levels. In other tissues expressing collagen types IX and II, long IX was expressed predominantly with the IIA form in the anterior optic cup at stage 22/23; in 14 1/2 day cartilage, long IX was expressed predominantly along with the IIB form of α1(II). The downstream transcript of the α2(I) gene (Icart) was found at high levels only in cartilage. © 1995 Wiley‐Liss, Inc.
Studies on insulin‐like growth factor‐I and insulin in chick limb morphogenesis
Authors: Dealy Caroline N. , Kosher Robert A.
Published: February 2005

The apical ectodermal ridge (AER) promotes the proliferation and directed outgrowth of the subridge mesodermal cells of the developing limb bud, while suppressing their differentiation. Insulin‐like growth factor‐I (IGF‐I) and its receptor are expressed by the subridge mesodermal cells of the chick limb bud growing out in response to the AER, and specific insulin receptors are present in the limb bud during its outgrowth. To study the possible roles of IGF‐I and insulin in limb outgrowth, we have examined their effects on the morphogenesis of posterior and anterior portions of the distal tip of stage 25 embryonic chick wing buds subjected to organ culture in serum‐free medium in the presence or absence of the AER and limb ectoderm. The distal mesoderm of control posterior explants lacking an AER or all limb ectoderm ceases expressing IGF‐I mRNA, exhibits little or no proliferation, fails to undergo outgrowth, and rapidly differentiates. Exogenous IGF‐I and insulin promote the outgrowth and proliferation and suppress the differentiation of distal mesodermal cells in posterior explants lacking an AER or limb ectoderm, thus mimicking at least to some extent the outgrowth promoting and antidifferentiative effects normally elicited on the subridge mesoderm by the AER. Furthermore, IGF‐I and insulin‐treated posterior explants exhibit high IGF‐I mRNA expression, indicating that IGF‐I and insulin maintain the expression of endogenous IGF‐I by the subridge mesoderm. We have also found IGF‐I and insulin can affect the morphology and activity of the AER. When the posterior portion of the wing bud tip is cultured with the AER intact in control medium, on day 4–5 the AER flattens, ceases expressing high amounts of the AER‐characteristic homeobox‐containing gene Msx2, and concomitantly an elongated cartilaginous element differentiates in the subridge mesoderm. In contrast, in the presence of exogenous IGF‐I or insulin the AER of such explants does not flatten, continues expressing high amounts of Msx2, and the subridge mesoderm remains undifferentiated and proliferative. Thus, exogenous IGF‐I and insulin maintain the thickness of the AER and sustain its expression of Msx2, while sustaining the anti‐differentiative effect normally elicited on the subridge mesoderm by a thickned functional AER. Notably, we have also found that exogenous IGF‐I and insulin induce the formation of a thickened ridge‐like structure that expresses high amounts of Msx2 from the normally thin distal anterior ectoderm of the limb bud, while promoting dramatic outgrowth and proliferation of the anterior mesoderm, which normally undergoes little outgrowth or proliferation. These studies provide support for the hypothesis that endogenous IGF‐I and insulin may be involved in promoting the outgrowth and suppressing the differentiation of limb mesoderm in response to the AER, and also in regulating and/or maintaining at least some aspects of AER activity. © 1995 Wiley‐Liss, Inc.
Mesenchymal stem cells reside within the connective tissues of many organs
Authors: Young H. E. , Mancini M. L. , Wright R. P. , Smith J. C. , Black A. C. , Reagan C. R. , Lucas P. A.
Published: February 2005

Previous studies have noted the presence of mesenchymal stem cells located within the connective tissue matrices of avian skeletal muscle, dermis, and heart. In these studies, clonal analysis coupled with dexamethasone treatment revealed the presence of multiple populations of stem cells composed of both lineage‐committed progenitor mesenchymal stem cells and lineage‐uncommitted pluripotent mesenchymal stem cells. The present study was undertaken to assess the distribution of these stem cells in the connective tissues throughout various regions of the body. Day 11 chick embryos were divided into 26 separate regions. Heart, limb skeletal muscle, and limb dermis were included as control tissues. Cells were harvested enzymatically and grown using conditions optimal for the isolation, cryopreservation, and propagation of avian mesenchymal stem cells. Cell aliquots were plated, incubated with various concentrations of dexamethasone, and examined for differentiated phenotypes. Four recurring phenotypes appeared in dexamethasone‐treated stem cells: skeletal muscle myotubes, fat cells, cartilage nodules, and bone nodules. These results suggest that progenitor mesenchymal stem cells and putative pluripotent mesenchymal stem cells with the potential to form at least four tissues of mesodermal origin have a widespread distribution throughout the body, being located within the connective tissue compartments of many organs and organ systems. © 1995 Wiley‐Liss, Inc.
Examination of fibronectin distribution and its sources in the regenerating newt limb by immunocytochemistry and in situ hybridization
Authors: Nace Jackie D. , Tassava Roy A.
Published: February 2005

Using monoclonal antibodies (mAbs) reactive to newt limb regenerates, we hope to gain insight into the identity and function of regeneration significant molecules. mAb MT4 (matrix 4) identifies an extracellular matrix (ECM) protein that is strongly up‐regulated first in the distal stump and then in the blastema during regeneration. Within the first 24 hr after amputation the MT4 antigen is localized to an acellular space beneath the wound epithelium, and first appears in the basal cells of the wound epithelium between days 5 and 7. At mid‐bud blastema stages, the MT4 antigen is homogeneously distributed as thin fibers in the blastema ECM, and is later largely restricted to the distal tip of the blastema and the areas of cartilage condensation. After extraction and immunoblotting, the MT4 antigen was observed as three reduced species of Mr 225, 250, and 260. Taken together, the immunoblot and immunocytochemistry results suggested that mAb MT4 recognized newt fibronectin (FN). Sequence from a cDNA (NvFN.10) obtained by screening a newt blastema cDNA expression library with mAb MT4 conclusively identified the MT4 antigen as FN. To further investigate the expression of FN in regeneration, cDNA NvFN.10 was used to construct a riboprobe and in situ hybridization was done. In the unamputated limb only a few scattered cells expressed the FN gene. Within the first 3 days after amputation strong hybridization signal was observed in the basal cells of the wound epithelium. Most of the stump cells that dedifferentiated and accumulated beneath the wound epithelium at 7 days expressed the FN gene, while the basal cells of the wound epithelium maintained their expression. At mid‐ and late‐bud blastema stages the vast majority of the blastema cells were strongly expressing the FN gene, but the wound epithelial cells now showed only weak FN transcription. Thus initially FN comes from the plasma. Then FN is synthesized by both the wound epithelium and mesenchyme. Finally, at blastema stages FN is produced primarily by the mesenchyme. The expression pattern of FN throughout regeneration suggests that this glycoprotein has roles in wound epithelial and mesenchymal cell migration and mesenchymal cell proliferation and differentiation. © 1995 Wiley‐Liss, Inc.
Angiogenic potential of the avian somite
Authors: Wilting Jörg , Brand‐Saberi Beate , Huang Ruijin , Zhi Qixia , Köntges Georgy , Ordahl Charles P. , Christ Bodo
Published: February 2005

We have studied the angiogenic potential of the unsegmented paraxial mesoderm and epithelial somites of the trunk with homotopical grafts between quail and chick embryos. Quail endothelial cells of the grafts were stained with the QH‐1 antibody after 1–6 days of reincubation. The unsegmented paraxial mesoderm and all parts of the epithelial somite were found to contain angioblasts which develop into QH‐1 positive endothelial cells. These cells are incorporated into the lining of the host's blood vessels such as the perineural vascular plexus and the dorsal branches of the aorta. There is a certain preference as concerns the location of endothelial cells derived from different parts of the somites. Angioblasts from ventral somite halves are mainly found in ventrolateral blood vessels. Those from dorsomedial quadrants form vessels in the dermis of the back, and those from dorsolateral quadrants can be found in the ventrolateral body wall and the wing. With the exception of the dorsal perineural vascular plexus, angioblasts do not cross the median plane of the body. This shows that, although angioblasts migrate extensively, there is bilaterality of the vascular system in the trunk. It remains to be studied whether the notochord plays a role in the establishment of this bilaterality. © 1995 Wiley‐Liss, Inc.
Experimental analysis of Msx‐1 and Msx‐2 gene expression during chick mandibular morphogenesis
Authors: Mina Mina , Gluhak Jelica , Upholt William B. , Kollar Edward J. , Rogers Barbara
Published: February 2005

Homeobox‐containing genes are thought to be involved in regulating pattern formation in a variety of tissues during embryogenesis. We have examined the expression of the homeobox‐related genes Msx‐1 and Msx‐2 during the development of the chick mandibular arch. Northern blot hybridization indicates that transcripts for both Msx‐1 (1.6 Kb) and Msx‐2 (3 Kb) are present in the mandibular arch as early as stage 18. The levels of both transcripts in the whole mandible decrease as cartilage is formed in vivo and in vitro. Using in situ hybridization, transcripts of Msx‐1 were localized in high amounts to the mesenchyme of the mesial tips of the arches. Msx‐2 transcripts were localized in high amounts to medial regions of the arches. Little or no hybridization of either probe was detected in the chondrogenic and myogenic regions of the arches. Transcripts of both genes were also excluded from calcified bone and cartilage. Our results further demonstrate that the mesial tip mesenchyme expressing Msx‐1 includes areas of highly proliferative cells and has in vitro chondrogenic potential. The region of mesenchymal cells expressing the Msx‐2 gene overlap with areas of developmentally programmed cell death which also contain very few proliferative cells and lack chondrogenic potential in vitro. These results are consistent with the possibility that Msx‐1 may be involved in the outgrowth of the mandibular arch and Msx‐2 may be involved in both developmentally programmed cell death and delineating the non‐chondrogenic region of the medial part of the mandibular arch. © 1995 Wiley‐Liss, Inc.
Transient and continuous expression of NADPH diaphorase in different neuronal populations of developing rat spinal cord
Authors: Wetts Richard , Phelps Patricia E. , Vaughn James E.
Published: February 2005

Nitric oxide is a novel intercellular messenger whose role in neuronal development is not yet known. As a first step toward elucidating its developmental function, we examined the pattern of NADPH diaphorase histochemical staining, an indicator of the presence of nitric oxide synthase, in the rat spinal cord at pre and postnatal ages. Some types of neurons expressed diaphorase activity transiently during development. For example, a subset of somatic motor neurons, located in the ventrolateral corner of a few caudal segments of the cervical spinal cord, were diaphorase‐positive beginning on E15, but gradually became diaphorase‐negative by birth. In contrast, other spinal neurons expressed diaphorase activity continuously from development into adulthood. Preganglionic autonomic motor neurons became diaphorase‐positive early in their development, as they were migrating toward their adult positions. Other spinal neurons, such as those in superficial dorsal horn, first expressed diaphorase relatively late in their development, after reaching their final location. The transient expression in some cell types, as well as the early expression in others, suggest that nitric oxide may have an important role(s) during development, which may differ from its functions in the adult nervous system. © 1995 Wiley‐Liss, Inc.
MyoD protein accumulates in satellite cells and is neurally regulated in regenerating myotubes and skeletal muscle fibers
Authors: Koishi Kyoko , Zhang Ming , McLennan Ian S. , Harris A. John
Published: February 2005

MyoD belongs to a family of helix‐loop‐helix proteins that control myogenic differentiation. Transfection of various non‐myogenic cell lines with MyoD transforms them into myogenic cells. In normal embryonic development MyoD is upregulated at the time when the hypaxial musculature begins to form, but its role in the function of adult muscle remains to be elucidated. In this study we examined the cellular locations of MyoD protein in normal and abnormal muscles to see whether the presence of MyoD protein is correlated with a particular cellular behaviour and to assess the usefulness of MyoD as a marker for satellite cells. Adult rats were anaesthetised and their tibialis anterior or soleus muscles either denervated, tenotomised, freeze lesioned, lesioned and denervated, or lesioned and tenotomised. At various intervals after the operations the rats were killed and their muscles removed, snap frozen, and sectioned with a cryostat along with muscles from unoperated neonatal and adult rats. The sections were processed for immunohistochemistry using a rabbit affinity‐purified antibody to recombinant MyoD. MyoD proved to be an excellent marker for active satellite cells; satellite cells in neonatal and regenerating muscles contained high levels of MyoD protein. MyoD positive cells were not observed in the muscles of old adults, in which the satellite cells are fully quiescent. MyoD immunoreactivity was rapidly lost from satellite cell nuclei after they fused into myotubes and was not detected in either sub‐synaptic or non‐synaptic nuclei of mature fibers. Denervation, and to a lesser extent tenotomy, of lesioned muscles induced expression of MyoD in myotubal nuclei. Denervation of normal muscles also upregulated MyoD in muscle fiber nuclei, an effect which was maximal after 3 days. We conclude that MyoD protein is neurally regulated in both myotubes and muscle fibers. © 1995 Wiley‐Liss, Inc.
Basal lamina development in chicken muscle spindles
Authors: Maier Alfred , Mayne Richard
Published: February 2005

The development of basal laminas was examined in immunohistochemical sections of chicken leg muscle spindles from embryonic day (E) 13 to 8 weeks postnatal. Fragments of basal laminas as seen with immunostaining for isoforms of laminin were already observed in E6 muscles. When clusters of intrafusal myotubes were first recognized at E13‐14, they were surrounded by basal laminas which were incomplete both in terms of coverage and molecular composition. More mature basal lamina tubes individually enclosed young myofibers at E18. Afterents made contact with myotubes, synaptic portions of basal laminas at myosensory junctions reacted strongly with antibodies against s‐laminin and chondroitin sulfate proteoglycan, while extrasynaptic portions were negative or reacted only weakly. At synaptic basal laminas of neuromuscular junctions heparin sulfate proteoglycan and s‐laminin became prominent after E16. Contrary to the early presence of basal lamina proteins around intrafusal fibers, initial deposition of basal lamina proteins in the outer spindle capsule was not recognized until E17‐18, and significant amounts were not detected until postnatal week 1. Unlike intrafusal basal laminas, capsular basal laminas developed no distinct specialized regions; however, molecular compositions of intrafusal and capsular basal laminas were similar. © 1995 Wiley‐Liss, Inc.
Clusterin expression within skin correlates with hair growth
Authors: Seiberg Miri , Marthinuss Jeffrey
Published: February 2005

Clusterin/TRPM‐2 is a sulfated glycoprotein that is expressed in many tissues. Independently cloned and isolated by several laboratories, it bears many names, and has been shown to be involved in many processes. These include cell‐cell adhesion and aggregation, inhibition of complement cytolysis, programmed cell death and apoptosis, tissue remodeling, and terminal differentiation. The hair follicle undergoes cycles of growth, regression, and rest, which involve both tissue remodeling and programmed cell death. To identify whether clusterin expression is involved in hair growth and cycling, we studied the expression of clusterin throughout the hair cycle. We demonstrate that clusterin is expressed during the growth phase of the hair cycle. We found no correlation between clusterin expression and the apoptotic regression of the hair follicle. Using immunohistochemistry we localized clusterin to the inner root sheath of the follicle. This suggests that clusterin might be involved in the morphogenesis and differentiation of the hair follicle. We propose that clusterin has a role in the maintenance of the layered structure of the hair follicle, and in the interactions between the inner root sheath and both the outer root sheath and the hair shaft. © 1995 Wiley‐Liss, Inc.
FGF‐2 mRNA and its antisense message are expressed in a developmentally specific manner in the chick limb bud and mesonephros
Authors: Savage Mary Pfann , Fallon John F.
Published: February 2005

FGF‐2 protein is present in the ectoderm and mesoderm of the developing chick limb bud. Its importance has been shown by the ability of ectopically applied FGF‐2 to replace the apical ectodermal ridge, allowing complete outgrowth and subsequent pattern formation of the limb bud. The first goal of this study was to determine whether FGF‐2 mRNA was present in the same ectodermal and mesodermal regions of the chick embryo as FGF‐2 protein. FGF‐2 also has an antisense message that is convergently transcribed from the opposite DNA strand (Kimelman and Kirschner [1989] Cell 59:687–696; Volk et al. [1989] EMBO J. 8:2983–2988). The second goal was to demonstrate the expression and distribution of the antisense message. Using RNAse protection assays we detected a full length protected fragment that corresponds to chick embryo FGF‐2 mRNA, and a partially protected fragment that corresponds to the antisense message. We used in situ hybridization to show that FGF‐2 mRNA was present in the ectoderm and subjacent mesoderm of the chick wing bud. FGF‐2 mRNA was also present in body ectoderm and undifferentiated mesoderm throughout the embryo, and in muscle cells, dorsal neural tube, and mesonephros. In situ hybridization also revealed evidence for the presence of the natural antisense message in the embryo in most, but not all, of the same regions as the FGF‐2 mRNA. FGF‐2 mRNA and its antisense message colocalized in undifferentiated limb mesoderm; however, antisense message was not detected in differentiated muscle or cartilage. It is important to note that FGF‐2 mRNA was always present in the mesonephros but that the antisense message was never observed in the mesonephros, thereby providing an internal control for non‐specific signal. Although little is known about its function, Kimelman and Kirschner ([1989] Cell 59:687–696) proposed that the antisense message may increase turnover of FGF‐2 mRNA. When we compared the in situ hybridization data of both mRNAs with levels of FGF‐2 protein (Savage et al. [1994] Dev. Dyn. 198:159–170), interesting tissue specific patterns emerged that support this hypothesis. © 1995 Wiley‐Liss, Inc.
Expression pattern of the murine LIM class homeobox gene Lhx3 in subsets of neural and neuroendocrine tissues
Authors: Zhadanov Alexander B. , Bertuzzi Stefano , Taira Masanori , Dawid Igor B. , Westphal Heiner
Published: February 2005

Murine Lhx3 cDNA isolated from the mouse pituitary cDNA library encodes a LIM‐type homeodomain protein that contains two tandemly repeated LIM domains and the homeodomain. The identities of predicted amino acid sequences between the mouse Lhx3 and Xenopus Xlim‐3 genes are 80, 95, and 97% in the LIM domains 1 and 2, and the homeodomain, respectively, and 84% in the entire protein. 5′‐RACE procedures and genomic cloning revealed that two distinct N‐terminal sequences arise from two different exons 1a and 1b. Exon 1a encodes a sequence similar to that of Xlim‐3, whereas exon 1b encodes a different N‐terminus. It is likely that there are two transcription initiation sites in the Lhx3 gene. The Lhx3 transcripts were detected by whole mount in situ hybridization as early as day E9.5 post coitum in Rathke's pouch and the closing neural tube. During subsequent development, Lhx3 expression was observed in the anterior and intermediate but not in the posterior lobes of the pituitary, and in the ventral hindbrain and spinal cord. Northern blot analysis of adult tissues showed that Lhx3 mRNA persists in the pituitary. The expression pattern of Lhx3 is well conserved between Xenopus and mouse, underscoring the functional importance of this gene as a regulator of development. A number of established cell lines of pituitary origin express Lhx3 and therefore constitute a useful tool for further study of Lhx3 gene function. © 1995 Wiley‐Liss, Inc.
Re‐programming of expression of the KGFR and bek variants of fibroblast growth factor receptor 2 during limb regeneration in newts (Notophthalmus viridescens)
Authors: Poulin Matthew L. , Chiu Ing‐Ming
Published: February 2005

We have previously shown, by in situ hybridization, that fibroblast growth factor receptor 2 (FGFR2) is present in the basal layer of wound epithelium during limb regeneration in newts (Notophthalmus viridescens ). In contrast, FGFR1 expression is observed throughout the blastema mesenchyme but is distinctly absent from the wound epithelium (Poulin et al. [1993] Development 119:353–361). Sequence analysis revealed that we have isolated both the KGFR and bek variants of FGFR2. These two variants differ only in the second half of the last of their three (or two) Ig‐like domains. In this report, we show the expression patterns of FGFR2 variants during limb regeneration by in situ hybridization. During the pre‐blastema stages of regeneration, FGFR2 expression was observed in the basal layer of the wound epithelium and in the cells of the periosteum. The wound epithelial hybridization was observed when the KGFR‐specific probe was used while the bek ‐specific probe hybridized to mRNA in the cells of the periosteum. As regeneration progresses to the blastema stages, KGFR expression continued to be observed in the basal layer of the wound epithelium with additional hybridization seen in the blastema mesenchyme closely associated with the bisected bones. The bek ‐specific hybridization pattern observed at this stage corresponds specifically to the mesenchymal hybridization. In the differentiation stages of regeneration, the mesenchymal expression of FGFR2 becomes restricted to the cells of the condensing cartilage and later to the perichondrium. Interestingly, there appears to be a dorsoventral gradient of the expression of both KGFR and bek variants of FGFR2, which are opposite each other at the later stages of regeneration. Thus, re‐programming of expression of the two FGFR2 variants is required during the initial wound closure of limb regeneration. Remarkably, the expression patterns of KGFR and bek mimic those observed in the mouse limb bud during early embryonic development (Orr‐Urtreger et al. [1993] Dev. Biol. 158:475–486). Moreover, our results suggest that the two FGFR2 variants have distinct roles in limb regeneration. Further investigation regarding the potential sources of the FGF ligands will help establish the roles that FGFs and FGFRs play in limb regeneration. © 1995 Wiley‐Liss, Inc.
α6 subunit of integrins in the development and sex differentiation of the mouse ovary
Authors: Fröjdman Kim , Pelliniemi Lauri J.
Published: February 2005

The localization of the α6 subunit of integrins in the ovary was studied by conventional and immunolabeling light and electron microscopy starting from the pregonadal embryonic phase until adulthood. The formation of gonadal blastema cells included an initial expression of the α6 subunit on the plasma membranes of all blastema cells. Subsequently the reaction for the α6 subunit became restricted in groups of these cells, which differentiated into gonadal cord cells, the precursors of follicular cells. The α6 subunit was also found in the cells of the mesonephric duct, mesonephric tubules, and the ovarian rete. Reorganization of the gonadal cords into follicles at birth was accompanied with strong and uniform re‐expression of the α6 subunit on the surface of the cord cells. Vascular endothelial cells and the cells of the postnatal surface epithelium remained positive for the α6 integrin subunit. In larger follicles, the intensity of the reaction for the integrin subunit varied. The theca cells of growing follicles contained the α6 subunit. The results show that this subunit of integrins is present in phases of increased adhesion and aggregation, and that its expression probably is involved in the regulation of ovarian epithelial differentiation. The distribution of α6 integrin in ovarian cells shows potentially important sex‐specific and developmental differences in epithelial organization when compared with respective changes found earlier by us in the male gonad. © 1995 Wiley‐Liss, Inc.
Epithelial cell polarity in early Xenopus development
Authors: Müller H.‐Arno J. , Hausen Peter
Published: February 2005

The Xenopus blastula consists of two morphologically distinct cell types. Polarized epithelial cells build up the embryonic surface and fence off an inner non‐polarized cell population. We examined the establishment of this early functional cell diversification in the embryo by single cell analysis, in vitro cell culture, and transplantation experiments. Single blastomeres from a 64‐cell embryo (1/64 cells) exhibit several features of polarized cells. The plasma membrane of 1/64 cells consists of an apical domain, which is inherited from the original egg membrane, and a basolateral domain derived from newly formed membrane during cleavage. These are inherent, cell‐autonomous properties of the blastomeres, as they form and are maintained in blastomeres raised in the absence of any cell interactions in calcium free medium. Upon in vitro culture a single 1/64 cell gives rise to an aggregate of two different cell types. Cells carrying a part of the former egg membrane domain differentiate into polarized epithelial cells, whereas cells lacking this membrane domain are not polarized. These results demonstrate that the inclusion of the egg membrane, rather than external signals related to the position of a cell in the intact embryo, is required for the apical/basolateral differentiation of the surface epithelium. This view is supported by cell transplantation studies. A single 1/64 cell was implanted into the blastocoel of a stage 8 blastula embryo. The progeny of the implanted cell proliferate within the host embryo and split into two morphologically distinct populations with different cell behaviours. Cells incorporating a part of the egg membrane form coherent patches of polarized epithelial cell sheets in the interior of the host embryo. In contrast, cells lacking egg membrane do not exhibit any characteristics of polarized cells and eventually spread into different regions of the host embryo. Our results show that the egg membrane and/or components of the submembrane cortex play a determinative role in the formation of the blastula epithelium. © 1995 Wiley‐Liss, Inc.
Involvement of extracellular matrix in the formation of the inner ear
Authors: Gerchman Eric , Hilfer S. Robert , Brown Joyce W.
Published: February 2005

Formation of the inner ear from the otic placode differs from invagination of other cup‐shaped organ primordia. Activation of the actin cytoskeleton seems to play a limited role because precocious invagination does not occur upon treatment with activators of a contractile event and cannot be prevented by inhibitors. In this study, the possibility that invagination is mediated by changes in the surrounding mesenchyme was tested by treating embryos with agents which interfere with the integrity of extracellular matrix. Enzymes degrading hyaluronate and/or chondroitin sulfate were microinjected into the otic region prior to folding. Synthesis of chondroitin sulfate proteoglycan was inhibited by microinjection of β‐xyloside. All treatment inhibited otic pit formation by interfering with fold formation within the placode. Immunocytochemical procedures showed depletion of the appropriate extracellular matrix components for a short time period after enzyme treatments and for up to 24 hr after β‐xyloside injection. Invagination of the otic primordium is concluded to be controlled in part by anchorage of the epithelium to adjacent structures and possibly by expansion of the mesenchymal extracellular matrix. © 1995 Wiley‐Liss, Inc.
Cell cycle of globose basal cells in rat olfactory epithelium
Authors: Huard Josée M. T. , Schwob James E.
Published: February 2005

The olfactory epithelium of adult mammals has the unique property of generating olfactory sensory neurons throughout life. Cells of the basal compartment, which include horizontal and globose basal cells, are responsible for the ongoing process of neurogenesis in this system. We report here that the globose basal cells in olfactory epithelium of rats, as in mice, are the predominant type of proliferating cell, and account for 97.6% of the actively dividing cells in the basal compartment of the normal epithelium. Globose basal cells have not been fully characterized in terms of their proliferative properties, and the dynamic aspects of neurogenesis are not well understood. As a consequence, it is uncertain whether cell kinetic properties are under any regulation that could affect the rate of neurogenesis. To address this gap in our knowledge, we have determined the duration of both the synthesis phase (S‐phase) and the full cell cycle of globose basal cells in adult rats. The duration of the S‐phase was found to be 9 hr in experiments utilizing sequential injections of either IdU followed by BrdU or 3H‐thy followed by BrdU. The duration of the cell cycle was determined by varying the time interval between the injections of 3H‐thy and BrdU and tracking the set of cells that exit S shortly after the first injection. With this paradigm, the interval required for these cells to traverse G2, M, G1, and a second S‐phase, is equivalent to the duration of one mitotic cycle and equals 17 hr. These observations serve as the foundation to assess whether the cell cycle duration is subject to regulation in response to experimental injury, and whether such regulation is partly responsible for changes in the rate of neurogenesis in such settings. ©1995 Wiley‐Liss, Inc.
Mechanics of cardiac looping
Authors: Taber Larry A. , Lin I.‐En , Clark Edward B.
Published: February 2005

During the early stages of embryonic development, the heart is a smooth‐walled, muscle‐wrapped tube that bends and rotates in a vital, but poorly understood, morphogenetic process called looping. Since looping involves biomechanical forces, this paper examines two mechanically based hypotheses for the bending component of cardiac looping. The first hypothesis is that an initial tension in or near the dorsal mesocardium (DM), a longitudinal structure along the outside of the ventricle, drives the deformation. To relieve the bending stresses in the tube, the myocytes change shape passively, and then they deform actively to continue the process to completion of a full loop. In the second hypothesis, contraction of circumferentially arranged actin macrofilaments produces circumferential compression and longitudinal expansion (due to incompressibility) of the myocytes. The DM locally constrains the longitudinal deformation, forcing the tube to bend. The feasibility of these hypotheses was evaluated using theoretical models and published experimental results. The models, which consist of beams composed of two layers representing the DM and the ventricular myocardium, show that the hypotheses are consistent with most of the known data, but further studies are necessary. In this regard, the models provide a conceptual framework for designing experiments to investigate the mechanics of looping. ©1995 Wiley‐Liss, Inc.
Vascularization of the mouse embryo: A study of flk‐1, tek, tie, and vascular endothelial growth factor expression during development
Authors: Dumont Daniel J. , Fong Guo‐Hua , Puri Mira C. , Gradwohl Gérard , Alitalo Kari , Breitman Martin L.
Published: February 2005

We report the detailed developmental expression profiles of three endothelial specific receptor tyrosine kinases (RTKs) flk‐1, tek, tie, as well as vascular endothelial growth factor (VEGF), the flk‐1 ligand. We also examined the expression of the other VEGF receptor, flt‐1, during placental development.flk‐1, tek, and tie transcripts were detected sequentially at one‐half day intervals starting at E7.0, suggesting that each of these RTKs play a unique role during vascularization of the mouse embryo. All three RTKs were expressed in the extraembryonic and embryonic mesoderm in regions that eventually give rise to the vasculature. Except for the expression of tek and flk‐1 in the mesoderm of the amnion, the expression of these RTKs from E8.5 onwards was virtually indistinguishable. An abundant amount of flt‐1 transcripts was found in the spongiotrophoblast cells of the developing placenta from E8.0 onwards. This cellular compartment is located between the maternal and labyrinthine layers of the placenta, which both express VEGF. VEGF transcripts were detected as early as E7.0 in the endoderm juxtaposed to the flk‐1 positive mesoderm, and later in development VEGF expression displayed an expression profile both contiguous with that of flk‐;1, and also in tissues found some distance from the flk‐1‐expressing endothelium. These results suggest a possible dual role for VEGF which includes a chemotactic and/or a cellular maintenance role for VEGF during vascularization of the mouse embryo. ©1995 Wiley‐Liss, Inc.
Sequential expression and differential localization of I‐, L‐, and T‐Fimbrin during differentiation of the mouse intestine and yolk sac
Authors: Chafel Mark M. , Shen Wenyan , Matsudaira Paul
Published: February 2005

During the differentiation of the intestine epithelium, three cytoskeletal proteins, villin, fimbrin, and myosin I, are sequentially expressed and localized to the apical membrane. Recently, we found that in the adult mouse and human, three fimbrin isoforms are expressed in a cell specific manner. I‐fimbrin is expressed by intestine and kidney epithelial cells, L‐fimbrin is expressed by leukocytes and many tumors, while T‐fimbrin is expressed by various cells and tissues. Because non‐intestinal isoforms of fimbrin could be expressed early in development, the expression of fimbrin isoforms during days 10.5 to 16.5 of intestine development was investigated. By immunofluorescence microscopy, T‐fimbrin was detected in the early stages of intestinal epithelial cell differentiation until day 14.5 and was localized predominantly at the apical surface. L‐fimbrin was also detected during this period but it was localized to the basal surface of the epithelium instead of the apical surface. By day 16.5 no L or T‐fimbrin was detected in the epithelium. I‐fimbrin was first detected at day 14.5 and a brush border‐like apical localization pattern was seen by day 16.5. Unlike the intestinal cells, the visceral endoderm expressed I, L, and T‐fimbrin throughout the period examined, with the level of I‐fimbrin increasing as time progresses. L‐fimbrin was more evident at the earlier stage than at the later stage of the development. Collectively, these results suggest that three fimbrin isoforms play different roles during epithelial cell differentiation. T‐ and I‐fimbrin expression could be critical for the formation and extension of the microvilli whereas L‐fimbrin may play a role in controlling cell adhesion. ©1995 Wiley‐Liss, Inc.
Proliferation pattern of capillary endothelial cells in chorioallantoic membrane development indicates local growth control, which is counteracted by vascular endothelial growth factor application
Authors: Kurz Haymo , Ambrosy Sonja , Wilting Jörg , Marmé Dieter , Christ Bodo
Published: February 2005

The density and distribution of whole mount BrdU‐anti‐BrdU labeled endothelial cells (days 6–15) in the chick chorioallantoic membrane (CAM) was analyzed with computer‐assisted microscopy. A significant loss of proliferative activity was noted after day 10: the density of labeled nuclei (in 10−2 mm−2) decreased from a median 7.78 (days 6, 8, 10) to 2.42 (days 12, 14, 15). CAMs initially showed random patterns of labeled endothelial cells, but changed to clearly focal patterns after day 12. A regular arrangement of labeled nuclei was never seen. After application of vascular endothelial growth factor (VEGF) to the day 13 CAM, a significant increase in proliferative activity (11.50) and a random distribution of labeled endothelial cells was observed on day 15. Development of CAM precapillary vessels was assessed in terms of length density (in mm−1, mean ± standard deviation), which was augmented threefold from day 6 (1.22 ± 0.05) to day 14 (3.54 ± 0.23) and then remained nearly constant. VEGF application from day 13 to 15 raised arterial length per unit area to 4.53 ± 0.77. It is concluded that normally a local regulation of endothelial proliferation and differentiation develops in the CAM, which doubles capillary endothelial cell density but simultaneously adapts to the decreasing need for endothelial cells, and thus maintains the quasi two‐dimensional vessel pattern. However, proliferative foci persist in the capillary layer after day 10, and precapillary vessel density continues to increase until day 14. VEGF enhances DNA synthesis in all capillary endothelial cells. This leads to the disappearance of proliferative foci, to multiple capillary layers, and to an excessive formation of precapillary vessels. We propose that CAM endothelial cell proliferation is regulated by factors like endothelial cell density and extension. The proliferative pattern in capillaries, and the length density of precapillary vessels should be used for the evaluation of angiogenesis in the CAM assay. We recommend the CAM after day 12 for evaluating putative angiogenic mitogens. ©1995 Wiley‐Liss, Inc.
Galectin‐3 is expressed in the notochord, developing bones, and skin of the postimplantation mouse embryo
Authors: Fowlis Deborah , Colnot Céline , Ripoche Marie‐Anne , Poirier Françoise
Published: February 2005

The galectins are a family of low molecular weight, calcium‐independent mammalian carbohydrate binding proteins that exhibit specificity for beta‐galactoside derivatives. We have examined the expression pattern of galectin‐3 in the developing mouse embryo by in situ hybridisation and immunohistochemistry. In the embryo proper, galectin‐3 message and protein are first detected in notochord, starting from 8.5 days post coitum (dpc), and persist until this structure disappears. Galectin‐3 is later found in cartilage primordia and in developing skin from 13.5 dpc. This very restricted and dynamic pattern suggests that galectin‐3 may participate in the establishment and/or maintenance of notochord as well as the formation of cartilage and differentiation of skin. Finally, we find that galectin‐3, which is identical to the macrophage marker Mac‐2, is also expressed in embryonic macrophages. © 1995 Wiley‐Liss, Inc.
Stages of embryonic development of the zebrafish
Authors: Kimmel Charles B. , Ballard William W. , Kimmel Seth R. , Ullmann Bonnie , Schilling Thomas F.
Published: February 2005

We describe a series of stages for development of the embryo of the zebrafish, Danio (Brachydanio) rerio. We define seven broad periods of embryogenesis—the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods. These divisions highlight the changing spectrum of major developmental processes that occur during the first 3 days after fertilization, and we review some of what is known about morphogenesis and other significant events that occur during each of the periods. Stages subdivide the periods. Stages are named, not numbered as in most other series, providing for flexibility and continued evolution of the staging series as we learn more about development in this species. The stages, and their names, are based on morphological features, generally readily identified by examination of the live embryo with the dissecting stereomicroscope. The descriptions also fully utilize the optical transparancy of the live embryo, which provides for visibility of even very deep structures when the embryo is examined with the compound microscope and Nomarski interference contrast illumination. Photomicrographs and composite camera lucida line drawings characterize the stages pictorially. Other figures chart the development of distinctive characters used as staging aid signposts. ©1995 Wiley‐Liss, Inc.
Gsh‐1: A novel murine homeobox gene expressed in the central nervous system
Authors: Valerius M. Todd , Li Hung , Stock Jeffrey L. , Weinstein Michael , Kaur Satbir , Singh Gurparkash , Potter S. Steven
Published: February 2005

We report the characterization of Gsh‐1, a novel murine homeobox gene. Northern blot analysis revealed a transcript of approximately 2 kb in size present at embryonic days 10.5, 11.5, and 12.5 of development. The cDNA sequence encoded a proline rich motif, a polyalanine tract, and a homeodomain with strong homology to those encoded by the clustered Hox genes. The Gsh‐1 expression pattern was determined for days E8.5 to E13.5 by whole mount and serial section in situ hybridizations. Gsh‐1 transcription was restricted to the central nervous system. Expression is present in the neural tube and hindbrain as two continuous, bilaterally symmetrical stripes within neural epithelial tissue. In the mesencephalon, expression is seen as a band across the most anterior portion. There is also diencephalon expression in the anlagen of the thalamus and the hypothalamus as well as in the optic stalk, optic recess, and the ganglionic eminence. Moreover, through the use of fusion proteins containing the Gsh‐1 homeodomain, we have determined the consensus DNA ninding site of the Gsh‐1 homeoprotein to be GCT/CA/CATTAG/A. ©1995 Wiley‐Liss, Inc.
Collagen gene expression during development of avian synovial joints: Transient expression of types II and XI collagen genes in the joint capsule
Authors: Nalin Andrew M. , Greenlee Theodore K. , Sandell Linda J.
Published: February 2005

The developmental sequence of the embryonic joint has been well studied morphologically. There are, however, no definitive studies of cell function during joint development. In order to begin to understand the differentiation events that contribute to joint formation, we examined the expression of collagen mRNAs encoding types I, IIA, IIB, and XI. In situ hybridization was performed on chicken embryo hind limb buds and digits from day 7 to day 18 (Hamburger and Hamilton stages 31–44). In the day 7 (stage 31) limb bud, there was a condensation of mesenchyme forming the primitive tarsal and metatarsal bones that showed abundant expression of type IIA procollagen message, but no type IIB or type α1(XI) message. By day 8 (stage 33), co‐expression of types IIA, and type XI procollagen mRNAs was observed in the condensations, with expression of IIB restricted to early chondrocytes with metachromatically staining matrix. At this stage, DNA fragmentation characteristic of apoptosis was observed in cells near the midline of the interzone region between the developing anlagen, and in areas between and around the individual digits of the paddle. The presumptive apoptotic cells were more numerous at day 9 (stage 35), and were not found in the developing joint at subsequent time points, including the initiation of spatial cavitation of the joint. From days 11–18, type IIA procollagen mRNA was expressed in flattened cells at the surface of the anlagen, and in the perichondrium and in the developing joint capsule; type IIB mRNA message was found only in chondrocytes. Type XI mRNA was expressed by all type II‐expressing cells. Alpha 1(I) mRNA was expressed early by cells of th8e interzone and capsule, but as cavitation progressed, the type I expressing cells of the interzone merged with the superficial layer of the articular surface. Thus, at the time of joint cavitation, there was a distinct pattern of expression of procollagen messages at the articular surface, with type I being outermost, followed by morphologically similar cells expressing type IIA, then chondrocytes expressing type IIB. The progenitor cells expressing type IIA message define a new population of cells. These cell populations contribute to the molecular heterogeneity of the articular cartilage, and these same populations likely exist in the developing joints of other species. The transient transcription of type II and type XI collagen genes, characteristic of chondrocytes, by cells in the joint capsule demonstrates that these cells may have chondrogenic potential. ©1995 Wiley‐Liss, Inc.
Homozygosity for theMinallele ofApcresults in disruption of mouse development prior to gastrulation
Authors: Moser Amy Rapaich , Shoemaker Alex R. , Connelly Camille S. , Clipson Linda , Gould Karen A. , Luongo Cindy , Dove William F. , Siggers Pamela H. , Gardner Richard L.
Published: February 2005

Mutation of theAPC(adenomatous polyposis coli) gene is an early event in colon tumor development in humans. Mice carryingMin(multiple intestinal neoplasia), a mutant allele ofApc, develop intestinal and mammary tumors as adults. To study the role of theApcgene in development, we have investigated the phenotype of embryos homozygous forApcMin(Min). Development of the primitive ectoderm fails prior to gastrulation in homozygousMinembryos. By midgestation, the presumed homozygotes consist of a mass of trophoblast giant cells with an additional cluster of much smaller embryonic cells. These results indicate that functional Apc is required for normal growth of inner cell mass derivatives. ©1995 Wiley‐Liss, Inc.