bluuebunny/crossref_metadata_2025 huggingface dataset:
Uncoupling histogenesis from morphogenesis in the vertebrate embryo by collapse of the transneural tube potential
Authors: Borgens Richard B. , Shi Riyi
Published: February 2005

We have shown that unidirectional pumping of Na+out of the neural tube's luminal fluids in amphibian embryos produces a large potential difference (40–90 mV, lumen negative to the abluminal surface). This transneural tube potential (TNTP) is analogous to the Na+dependent transepithelial potential (TEP) that exists across surface ectoderm. This TEP is retained in ectoderm after it is internalized when the neural folds fuse to form the neural tube. The TNTP can be markedly reduced for several hours by injection of the Na+channel blockers amiloride or benzamil into the lumen by iontophoresis through microelectrodes. Here we describe the effect of TNTP modification on developmental anatomy. Axolotl embryos possessing a fused and closed neural tube (stage 21–23) were injected with either amiloride or benzamil and allowed to continue development for 36–52 hr. These were compared to control embryos injected with vehicle alone, or to embryos in which amiloride or benzamil was iontophoresed just beneath surface ectoderm. All embryos in which the TNTP was reduced were grossly defective. These were characterized by a disaggregation of the cells comprising the structures that had already begun to form (otic primordia, brain, spinal cord, notochord) as well as a failure in the development of new structures. Remarkably, some of these embryos displayed continuing development of external form in the complete absence of concomitant internal histogenesis. We discuss the ways in which a large endogenous voltage gradient associated with an epithelial potential difference (the TNTP) may be required both for the structural integrity of the early neuroepithelium, and a prerequisite for normal morphogenesis. ©1995 Wiley‐Liss, Inc.
Chondrocyte apoptosis in endochondral ossification of chick sterna
Authors: Gibson Gary J. , Kohler William J. , Schaffler Mitchell B.
Published: February 2005

In the process of endochondral ossification, chondrocytes progress through a series of maturational changes, including division and hypertrophy, that culminate in chondrocyte loss and cartilage resorption. From an investigation of morphology, DNA fragmentation, and collagen synthesis in the developing chick sterna we have characterized chondrocytes death in this process. Light microscopy of resorbing sterna demonstrated chondrocyte condensation at the interface with the invading vasculature and electron microscopy demonstrated a range of chondrocyte morphologies, including retraction from the pericellular matrix, cytoplasmic and nuclear condensation, and vesiculation suggestive of sequential changes characteristic of apoptosis. Isolation and end‐labeling of DNA from chick primary ossification centers demonstrated fragmentation to nucleosome sized units, only in primary ossification centers exhibiting active resorption, and in situ detection of DNA fragmentation showed a restriction to chondrocytes at the interface with invading blood. We conclude that terminal differentiation of chondrocytes results in death by an apoptotic process prior to resorption of the tissue and invasion by blood vessels. The extent of DNA fragmentation correlated closely with the proportion of cells displaying a condensed phenotype in contralateral primary ossification centers and peaked at an early stage of resorption, suggesting that chondrocyte apoptosis may be an initiating event in tissue resorption and vascular invasion. Comparison of DNA fragmentation with expression of the hypertrophic chondrocyte phenotype, as indicated by type X collagen synthesis, suggested that DNA fragmentation was a late event in the process of chondrocyte hypertrophy and probably corresponded with chondrocyte condensation. ©1995 Wiley‐Liss, Inc.
Thrombospondin‐4 is expressed by early osteogenic tissues in the chick embryo
Authors: Tucker Richard P. , Adams Josephine C. , Lawler Jack
Published: February 2005

The thrombospondins are a family of related glycoproteins found in the embryonic extracellular matrix. To date, five members of this family have been identified. Thrombospondin‐1 and thrombospondin‐2 have similar primary structure, but are expressed in different tissues at different times during development. Thrombospondins‐3, ‐4, and cartilage oligomeric protein belong to a second thrombospondin subgroup in which the carboxyl‐half of each molecule is most similar to thrombospondin‐1 and ‐2. Here, we report the cloning and sequencing of a novel probe to avian thrombospondin‐4. We have used this probe to determine the origins of thrombospondin‐4 in the chick embryo by in situ hybridization. Thrombospondin‐4 transcripts first appear in the mesenchyme surrounding bone anlage coinciding with the initial stages of osteogenesis. The expression in osteogenic tissues is transient: thrombospondin‐4 mRNAs are not seen in the osteoblasts of bone collars in developing long bones. This pattern is distinct from avian thrombospondin‐2, which is expressed in perichondrium and embryonic fibrous connective tissues. Our observations indicate that connective tissues are the principal site of thrombospondin‐4 expression in the chick. The diverse origins of different thrombospondin gene family members imply distinctive roles for these proteins related to the growth and differentiation of cartilage, tendons, and bone. ©1995 Wiley‐Liss, Inc.
Retinoid signaling and the generation of regional and cellular diversity in the embryonic mouse spinal cord
Authors: Colbert Melissa C. , Rubin William W. , Linney Elwood , LaMantia Anthony‐Samuel
Published: February 2005

Retinoid‐dependent gene expression accompanies the emergence of distinct regions and cell classes in the mouse spinal cord around midgestation. We asked whether changes in the expression of retinoid signaling molecules and retinoid‐responsive genes reflect the establishment of this regional and cellular diversity. At E10.5, retinoic acid (RA) receptors (RAR)α, RARβ, the retinoid X receptor (RXR)γ, cellular RA binding protein (CRABP)I, CRAPBII, and cellular retinol binding protein (CRBP)I mRNAs are found throughout the entire anterior‐posterior (AP) axis of the cord, as is RA (Colbert et al. [1993] Proc. Natl. Acad. Sci. U.S.A. 90:6572–6576) and RA‐sensitive transgene expression (Balkan et al. [1992] Proc. Natl. Acad. Sci. U.S.A. 89:3347–3351). At E12.5, RA, transgene expression, and RARβ become restricted to the cervical and lumbar cord. RARα, CRABPI, and RXRγ, however, are found throughout the AP extent. CRABPII and CRBPI, although expanded within the cervical and lumbar regions, are also found throughout the AP axis. Thus, several retinoid signaling molecules continue to be expressed beyond distinct regions of the spinal cord where RA is available and some RA‐responsive genes are either restricted or enhanced. Exogenous RA can activate a more widespread response resulting in ectopic transgene and RARβ expression in the thoracic and sacral cord. Not all RA‐sensitive genes, however, respond; CRABPII and CRBPI expression patterns are unchanged. Finally, not every cell within the normal or exogenously induced domains of RA‐dependent gene expression responds to RA, nor does every cell express RA receptors or binding proteins. Thus, regional and cellular differences in the distribution of the known retinoid receptors and binding proteins do not predict absolutely where or whether retinoid sensitive genes will be expressed or where retinoids will be available in the developing spinal cord. Instead, retinoid‐mediated gene expression in the cervical and lumbar cord seems to reflect retinoid responses that rely both on the local availability of retinoids, the identity of the responding gene, and an indeterminate array of retinoid signaling molecules. © 1995 wiley‐Liss, Inc.
Developmental expression of MMP‐9 (gelatinase B) mRNA in mouse embryos
Authors: Cañete‐soler Rafaela , Gui Yong‐Hao , Linask Kersti K. , Muschel Ruth J.
Published: February 2005

Considerable remodeling of the extracellular matrix as well as cellular migration takes place during embryogenesis. Since the metalloproteinase MMP‐9 is implicated in these functions in cancer cells, we studied the patterns of expression of MMP‐9 mRNA during the development of post‐implantation mouse embryos. MMP‐9 mRNA was detected using the ribonuclease protection assay in poly A + RNA from 13 to 17 day embryos, but not at 11 days. In order to localize these transcripts, in situ hybridization was performed on sections of murine embryos from 7.5 to 15 days of gestation. At the time of implantation, MMP‐9 mRNA was localized to the invading trophoblast cells. Strong signals were also seen in the yolk sac. No signal for MMP‐9 mRNA was seen by in situ hybridization in the embryo until day 11 when detectable reaction was seen in the central nervous system. By day 15 strong signals were seen in the liver, in the developing bronchial epithelium of the lungs and in the primordial alveoli, in the epithelium of the thyroid gland, in the thymus, in the endochondrial plates of the bone, and in neural cells. The liver from day 15 embryos contained gelatinase activity at 105 kDa consistent with MMP‐9. Thus, MMP‐9 expression appears to be expressed in specific organs in a precise temporal sequence during development. © 1995 wiley‐Liss, Inc.
Developmental pattern of expression of the mouse α1(XI) collagen gene (Col11a1)
Authors: Yoshioka Hidekatsu , Iyama Ken‐Ichi , Inoguchi Kazuhito , Khaleduzzaman Mohammed , Ninomiya Yoshifumi , Ramirez Francesco
Published: February 2005

Fibrillar networks are intimately involved in several morphogenetic processes which underlie the harmonious development of the vertebrate embryo. Recent genetic evidence has demonstrated that the minor types V and XI collagen are key regulators of types I and II fibrillogenesis in non‐cartilaginous and cartilaginous matrices, respectively. A comprehensive understanding of the expression and regulation of the genes coding for the chains of the minor collagen types is therefore relevant to animal morphogenesis and development. The present study was undertaken to elucidate the embryonic pattern of expression of the gene coding for the mouse α1 chain of type XI colagen (Col11α1) using the technique of in situ hybridization. Transcripts of the Col11α1 gene were detected as early as 11 days of gestation. The α1(XI) transcripts were found to accumulate mostly in cartilaginous tissues, such as the chondrocranium and the developing limbs. Like the major cartilage‐specific collagen (type II), Col11α1 expression was also noted in the neuro‐epithelium of the brain. However, α1(XI) transcripts accumulated in several other non‐cartilaginous sites. They include odontoblasts, trabecular bones, atrioventricular valve of the heart, the tongue, the intestine, and the otic vesicle. Altogether, the data confirm that Col11α1 has a broader spectrum of expression than previously thought. This finding raises the possibility that the α1(XI) chain may participate in the formation of stage‐ and tissue‐specific trimers with distinct functional properties. © 1995 wiley‐Liss, Inc.
Tissue‐nonspecific alkaline phosphatase participates in the establishment and growth of feather germs in embryonic chick skin cultures
Authors: Crawford Karen , Weissig Helge , Binette François , Millán José Luis , Goetinck Paul F.
Published: February 2005

Alkaline phosphatase activity is present in the mesoderm of embryonic chick skin and becomes spatially restricted to the dermal condensation of the developing feather germs. Inhibitors to tissue‐nonspecific (liver/bone/kidney), but not intestinal, alkaline phosphatase inhibit the establishment and growth of feather germs in cultured skins. A window of maximum sensitivity to the inhibitor was observed to be the first day of culture when early development and establishment of pattern takes place. The cDNA for the avian tissue‐nonspecific alkaline phosphatase was cloned and sequenced, and Southern analysis revealed a single copy of this gene in the avian genome. Northern analysis revealed that a 2.8 kb transcript for this form of alkaline phosphatase is present in developing skin. © 1995 wiley‐Liss, Inc.
Up‐regulation of a novel integrin α‐chain (αmt) on human fetal myotubes
Authors: Gullberg Donald , Velling Teet , Sjöberg Gunnar , Sejersen Thomas
Published: February 2005

Integrin expression and distribution was studied in cloned human fetal G6 myoblasts and myotubes. Immunoprecipitation of β1 integrins from surface iodinated and metabolically labeled G6 cells typically showed a five‐fold induction of a β1 integrin associated protein upon differentiation. Under non‐reducing conditions this β1 associated protein migrated as 145 kD. No such β1 associated protein was observed in the myogenic L8 rat cell line, before or after differentiation. The β1 integrin associated cell surface protein present in G6 myotubes remained associated with the β1 subunit in the presence of 1% Triton X‐100 and 0.5 M NaCl. Like integrin α‐chains, the protein dissociated from the β1 integrin subunit at low pH. Immunoprecipitation of G6 myotubes further indicated the presence of α, α, α5, and αa integrins, and small amounts of α4 and α6 integrins. Immunodepletion with integrin α‐chain antibodies to α1, α3, α4, α5, α6, and αv integrin chains could not deplete the β1 integrin associated protein, indicating that it did not interact with any of these known integrin heterodimers. Upon treatment with reducing agents, the β1 integrin associated protein migrated in SDS‐PAGE as a 155 kD protein. The decreased mobility in SDS‐PAGE upon reduction is a feature shared with α1, α2, and α9 integrin α‐chains. Antibodies to α1 immunoprecipitated an integrin heterodimer distinct from the 155 kD protein. Antibodies to α2 and α9 failed to immunoprecipitate proteins from G6 myotubes and Northern blot analysis likewise failed to detect messages for these two integrin α‐chains. Immunohistochemistry with antibodies to different integrins of fetal thigh muscles showed staining of α6 integrin at the sarcolemma. Antibodies to α2 and α9 stained keratinocytes and other non‐muscle cells in the fetal leg, but no signal was detected for α2 and α9 integrin on fetal myotubes. Based on these characteristics, we propose that the induced protein is a hitherto unidentified integrin α‐chain on myotubes that we name αmt. Our finding of a novel integrin α‐chain that is induced upon myogenic differentiation supports the idea that different integrins are important for myogenesis during different developmental stages, and we suggest that αmt takes part in early human myotube formation. © 1995 wiley‐Liss, Inc.
CCAAT/Enhancer binding protein isoforms expression in the colon of neonatal mice
Authors: Blais Sylvie , Boudreau François , Beaulieu Jean‐françois , Asselin Claude
Published: February 2005

Development of murine proximal colon follows a complex pattern of morphological and functional differentiation. Molecular mechanisms and factors responsible for colonspecific gene expression remain to be established. In an attempt to identify some of these factors, we examined the expression of the α, β, and δ isoforms of the CCAAT/enhancer binding protein (C/EBP) transcription factor gene family during murine colon development. Whereas C/EBPα mRNA levels are reduced during the third post‐natal week, C/EBPα 42 and 30 kD proteins levels decrease between post‐natal days 8 and 21. C/EBPβ mRNA levels increase between post‐natal days 4 and 8 and remain constant subsequently, in contrast to a decrease in C/EBPβ protein levels between post‐natal days 11 and 15. C/EBPδ mRNA levels increase gradually while C/EBPδ protein levels show variations during post‐natal development. Changes in C/EBP DNA binding activity coincides with modifications in C/EBP isoforms expression. By indirect immunofluorescence, we show restriction of C/EBPα expression to differentiated surface epithelial cells during crypt formation. C/EBPβ is predominantly nuclear with some cytoplasmic staining at all developmental stages. C/EBPβ and δ are both predominantly nuclear in crypt and differentiated surface epithelial cells, as well as in various cells of the lamina propria and muscular layers. Thus, specific C/EBP isoforms are differentially regulated during murine colon post‐natal development. Differential C/EBP isoforms pattern of expression suggests a role for these transcription factors in colon‐specific gene expression during development. © 1995 wiley‐Liss, Inc.
Role for platelet‐derived growth factor‐like and epidermal growth factor‐like signaling pathways in gastrulation and spiculogenesis in the Lytechinus sea urchin embryo
Authors: Ramachandran Ravi K. , Govindarajan Venkatesh , Seid Christopher A. , Patil Sonali , Tomlinson Craig R.
Published: February 2005

The mechanisms underlying sea urchin gastrulation and spiculogenesis have been sought for decades. We have identified two growth factor signaling pathways that are involved in these developmental events. Antibodies against mammalian platelet‐derived growth factor (PDGF) receptor‐β inhibited gastrulation and spiculogenesis, and antibodies against human epidermal growth factor (EGF) receptor disrupted gastrulation and spicule placement in Lytechinus pictus and L. variegatus embryos. Our studies suggested that the antibodies affect development by inhibiting rather than activating the signaling pathways. Polyclonal and monoclonal antibodies against the mammalian receptors recognized specifically Lytechinus proteins of the expected size of 170–180 × 103 Mr. Growth factor binding assays indicated that there are approximately 1.25 × 104 platelet‐derived growth factor‐like receptors per cell at the mesenchyme blastula stage of L. pictus, and human platelet‐derived growth factor bound with an apparent affinity of KD = 4.4 nM to dissociated cells at the mesenchyme blastula stage. Immunolabelling experiments showed that at the gastrula stage, the Lytechinus platelet‐derived growth factor‐like receptors are located on the primary mesenchyme cells, the gut, and most prominently on the secondary mesenchyme cells and the stomodeum. The epidermal growth factor‐like receptors stained less intensely on the gut and primary and secondary mesenchyme cells. Both receptors are expressed on the ciliary band and the gut of the pluteus larva but only the PDGF‐like receptors is expressed on the primary mesenchyme cells. Pulse studies showed that the embryos are sensitive to the platelet‐derived growth factor receptor‐β and epidermal growth factor receptor antibodies from the blastula to sometime between the mesenchyme blastula and midgastrula stages. We show that antibodies enter the blastocoel as late as the gastrula stage. Our results suggest that platelet‐derived growth factor‐like and epidermal growth factor‐like signaling pathways are involved in the early differentiation and morphogenesis of the sea urchin gut and spicules. © 1995 wiley‐Liss, Inc.
Developmental expression of H‐2K major histocompatibility complex class I transgenes requires the presence of proximal introns
Authors: Drezen J. M. , Cohen‐Tannoudji M. , Pournin S. , Babinet C. , Morello D.
Published: February 2005

The pattern of expression of the H‐2K major histocompatibility complex (MHC) class I gene is complex. During embryonic development H‐2K mRNA, detectable from midgestation, is poorly expressed. In the adult, H‐2K expression is nearly ubiquitous but transcriptional regulation occurs leading to different mRNA levels in the different organs of the mouse. In vitro studies have shown that most of the regulatory elements controlling H‐2K gene transcription are located in the 5′ region of the gene. However, using fusion transgenes in which reporter genes were under the control of 2 kb of H‐2K 5′ regulatory region, we have previously shown that this region, was not sufficient to ensure correct developmental transgene expression. By contrast, a native 9.25 kb H‐2K transgene was expressed appropriately both in the adult and in the embryo. In order to localise more precisely the cis‐acting regulatory sequences involved in H‐2K developmental expression, we have now constructed new transgenic lines containing H‐2Kb transgenes that were deleted from specific parts of the H‐2Kb gene. We show that deletion of either the H‐2K 3′ flanking region or the 5 (out of 7) distal introns results in an expression of the transgenes which is similar to that of the endogenous H‐2K gene, both in the adult and during embryonic development. By contrast, deletion of all the introns or of the two proximal ones abrogates H‐2K transgene expression. Our data reveal the complexity of H‐2K regulation and highlight the crucial role of proximal introns in H‐2K expression in the living organism. © 1995 wiley‐Liss, Inc.
Ubiquitin‐protein conjugates selectively distribute during early chicken embryogenesis
Authors: Wunsch Ann M. , Haas Arthur L.
Published: February 2005

The major mechanism for proteloysis in eucaryotes involves an ATP‐dependent pathway for which the covalent attachment of ubiquitin targets proteins for degradation. The involvement of ubiquitin conjugation in early embryonic vertebrate development was investigated by examinining the amounts and localization of ubiquitin conjugates at different stages of development in the chicken using an affinity‐purified antibody specific for conjugated ubiquitin. Solid phase immunochemical assays measuring whole embryo pools of free and conjugated ubiquitin demonstrated a progressive increase increase in conjugate pools to stage 18, followed by a decline to stage 24. In contrast, levels of free polypeptide showed a dramatic increase after stage 5, indicating a change in the dynamics of the two pools during develoment. Immunohistochemistry revealed that the distribution of ubiquitin adducts between stages 3 and 22 was pronounced in regions undergoing extensive cellular remodeling. Ubiquitin conjugates were detected in the primitive streak where cells ingress during gastrulastion. The presence of these degradative intermediate in both neuroectodermal cells of the neural folds and subsquent neural crest cells migrating from the dorsum of the neural tube is consistent with an involvement in key morphogenetic events. The localization of ubiquitin conjugates at other selected tissue interfaces including limb bud ectoderm/mesoderm, and cardiac atrioventricular myocardium/endothelium suggests an active role for ubiquitin‐mediated protein modification in similar developmental interactions. Conjugates were distributed first between somites, then in myotomes with a pattern spatially identical to that of the ubiquitin conjugating enzyme, E214K, the major cognate isozyme for isopeptide ligase (E3)‐dependent degradation. The potential involvement of ubiquitin conjugation at sites of epithelialmesenchymal associations was further analyzed ion culture using atrioventricular canal (AV) endothelium. Immunoreactivity was abundant in cells immediately prior to and during their transformation into mesenchyme. Collectively, the specific temporal and, spatial changes in ubiquitin conjugates during early vertebrate development suggest a regulatory role for this degradative pathway in the cellular remodeling accompanying embryonic growth and differentiation. © 1995 wiley‐Liss, Inc.
Developmental expression of two members of a new class of transcription factors: I. Expression of aryl hydrocarbon receptor in the C57BL/6N mouse embryo
Authors: Abbott B. D. , Birnbaum L. S. , Perdew G. H.
Published: February 2005

The aryl hydrocarbon receptor (AhR) is a ligand‐activated transcription factor with a basic region/helix‐loop‐helix (bHLH) motif. AhR has been sequenced and the functional domains defined and there is information on the formation of complexes with other peptides and interactions with DNA, although these areas continue to be investigated. AhR mediates many biological effects such as developmental toxicity, including induction of cleft palate and hydronephrosis. This regulatory protein is expressed in embryonic liver and has been immunohistochemically localized in cells of human and mouse secondary palate. The expression of AhR in embryonic tissues and its ability to disrupt development suggests a significant role for this protein in development. The present study examines the pattern of AhR expression in the C57BL/6N mouse embryo from gestation days (GD) 10–16, using in situ hybridization and immunohistochemical analysis. AhR mRNA was localized with 35S‐RNA antisense riboprobe (cAh1 probe, 1.8 Kb amino terminal DNA). AhR protein was localized with purified monoclonal antibody (RPT‐9) raised against the N‐terminal peptide sequence. AhR mRNA and protein were expressed in GD 10–13 neuroepithelium, and as development progressed the levels in brain decreased. GD 10–12 embryos also showed AhR in branchial arches, heart, somites, and liver. AhR protein and mRNA in heart were highest at GD 10–11 and decreased with age. In liver, AhR mRNA and protein levels increased and nuclear localization became more pronounced with gestational age. In GD 14–16 embryos levels in liver and adrenal were highest, but AhR was present in ectoderm, bone, and muscle. AhR expression was specific for both cell type, organ/tissue, and developmental stage, suggesting that this novel ligand‐activated transcriptional regulator may be important in normal embryonic development. © 1995 wiley‐Liss, Inc.

This article is a US Government work and, as such, is in the public domain in the United States of America.
Developmental expression of two members of a new class of transcription factors: II. Expression of aryl hydrocarbon receptor nuclear translocator in the C57BL/6N mouse embryo
Authors: Abbott B. D. , Probst M. R.
Published: February 2005

The aryl hydrocarbon receptor (AhR) and the AhR nuclear translocator protein (ARNT) are basic‐helix‐loop‐helix (bHLH) proteins involved in transcriptional regulation. The AhR is a ligand‐activated partner of the ARNT protein. Both proteins are required to transcriptionally regulate gene expression. ARNT must be complexed to AhR to permit binding to the regulatory DNA sequence. The AhR‐ligand complex is known to mediate a range of biological responses, such as developmental toxicity, induction of cleft palate, and hydronephrosis. AhR and ARNT are expressed in human embryonic palatal cells and AhR was recently shown to have a specific developmental pattern of expression in the mouse embryo. In the present study, expression of ARNT is characterized in C57BL/6N mouse embryos from gestation day (GD) 10–16 using immunohistochemistry and in situ hybridization. An affinity purified antibody against human ARNT (1.1 μg/ml) was detected with an avidinbiotin‐peroxidase complex. ARNT mRNA was localized with a 35S‐RNA probe from pBM5/NEOM1–1. Specific spatial and temporal patterns of ANRT expression emerged and mRNA and protein expression correlated. The GD 10–11 embryos showed highest levels of ARNT in neuroepithelial cells of the neural tube, visceral arches, otic and optic placodes, and preganglionic complexes. The heart also had significant expression of ARNT with strong nuclear localization. After GD11, expression in heart and brain declined. In GD 12–13 embryos expression was highest in the liver where expression increased from GD 12–16. At GD 15–16 the highest levels of ARNT occurred in adrenal gland and liver, although ARNT was also detected in submandibular gland, ectoderm, tongue, bone, and muscle. In all of these tissues ARNT was cytoplasmic as well as nuclear, except in some of the cortical adrenal cells in which ARNT was strongly cytoplasmic with little or no nuclear localization. These specific patterns of ARNT expression, which differ in certain tissues from the expression of AhR, suggest that ARNT may have additional roles in normal embryonic development. © 1995 wiley‐Liss, Inc.
Limb deformity proteins during avian neurulation and sense organ development
Authors: De La Pompa José Luis , James Dominic , Zeller Rolf
Published: February 2005

The nuclear Limb deformity (Ld) proteins (formins) are expressed during the avian primitive streak stages. Initially, they are detected predominantly in cells of the forming notochord, scattered mesodermal precursors and the induced neural plate. No expression is detected in endodermal cells. The subsequent graded distribution of Ld positive cells along the anterior‐posterior axis of the neural tube follows the antero‐posterior progression of its differentiation. The Ld proteins are also differentially expressed during induction and development of both the inner ear and eye. An unequal distribution of Ld proteins along the dorso‐ventral axis of the otic vesicle is observed during its initial patterning. In the eye, the Ld proteins are expressed by the optic vesicle during secondary induction of the lens placode. Following induction, the proteins are also expressed by the newly formed lens placode, a process which is reminiscent of homeogenetic induction. During differentiation of the retina and lens, the Ld domains seem to demarcate territories, giving rise to specific eye structures. A comparative analysis of the Ld distribution and BrdU incorporation in the two sense organs indicates that the proteins are predominantly expressed by committed and/or differentiating (post‐mitotic) cells. In general, expression of Ld proteins is induced during determination and remains during differentiation of particular cell‐types. This study implies that the nuclear Ld rproteins are involved in morphogenesis of both neuro‐ectodermal and mesodermal structures. © 1995 wiley‐Liss, Inc.
During secondary myotube formation, primary myotubes preferentially absorb new nuclei at their ends
Authors: Zhang Ming , McLennan Ian S.
Published: February 2005

Developing muscles contain at least two types of myoblasts. Early myoblasts are the first myoblast to form and are the only myoblasts present during primary myotube formation. By the time secondary myotube formation begins, early myoblasts are rare and late myoblasts are common. The late myoblasts have been postulated to give rise to secondary myotubes. While this is generally accepted, it is unclear whether late myoblasts also contribute to the growth of primary myotubes. One study has produced evidence that myoblasts present during secondary myogenesis selectively fuse with each other or with secondary myotubes, but not with primary myotubes (Harris et al. [1989a] Development 107:771–784). However, the sizes of primary myotubes increase during secondary myotube formation. We have therefore re‐examined the question of whether primary myotubes absorb new nuclei during secondary myotube formation. Pregnant rats were given a single intraperitoneal injection of 5 mg of 5‐bromodeoxyuridine (BrdU) on one embryonic day (from E13 to E19) and their embryos removed on E20. The brominated‐nuclei were labelled with an antibody to BrdU and the myotubes were marked with anti‐myosin antibodies. Double labelled sections from the soleus, tibialis anterior, and extensor digitorum longus muscles were examined with a confocal microscope. The numbers and locations of labelled nuclear profiles in primary and secondary myotubes were counted and recorded. The results show: (1) that primary myotubes absorb nuclei at all stages of development, including the period of secondary myotube formation; (2) that in the early stages of secondary myotube formation, more myoblasts fuse with primary than secondary myotubes whereas this situation is reversed by the end of secondary myotube formation; and (3) that the nuclei added to primary and secondary myotubes during the early stages of their formation are located within the middle of E20 muscles. The nuclei added to growing myotubes are preferentially located at the ends of the muscles. © 1995 wiley‐Liss, Inc.
Ectodermal stimulation of the production of hyaluronan‐dependent pericellular matrix by embryonic limb mesodermal cells
Authors: Knudson Cheryl B. , Munaim Syeda I. , Toole Bryan P.
Published: February 2005

Interaction of ectoderm and underlying mesoderm is essential for normal vertebrate limb morphogenesis. One of the functions of limb bud ectoderm is its influence on the composition of extracellular matrix in subectodermal mesoderm, which in turn participates in morphogenesis of this region of the limb. This matrix is highly enriched in hyaluronan, even at the time when the level of hyaluronan in the chondrogenic and myogenic regions of the limb decreases, due to secretion of a stimulatory factor by the ectoderm. In this study we show that limb bud ectoderm not only stimulates hyaluronan synthesis but induces formation of large hyaluronan‐dependent, pericellular matrices around cultured limb bud mesodermal cells. The ectodermal activity is mimicked in great part by fibroblast growth factor‐2 and transforming growth factor‐β, and antibodies to these proteins inhibit induction of mesodermal pericellular matrix by the ectodermal factor. It has been shown by other investigators that fibroblast growth factor‐2 is produced by limb ectoderm whereas transforming growth factor‐β is present in limb mesodermal tissues. Thus we conclude that the unique properties of mesodermally produced matrix underlying limb bud ectoderm are regulated, at least in part, by ectodermal fibroblast growth factor‐2, probably in concert with mesodermal transforming growth factor‐β. © 1995 wiley‐Liss, Inc.
Developmental expression of a type II collagen/β‐galactosidase fusion gene in transgenic mice
Authors: Metsäranta Marjo , Garofalo Silvio , Smith Chad , Niederreither Karen , De Crombrugghe Benoit , Vuorio Eero
Published: February 2005

The correct temporal and spatial expression of the type II collagen gene is believed to be important for normal development and growth of the skeleton and the eye, i.e., tissues where the protein product is predominantly found. To study transcriptinal activation of type II collagen gene in skeletal and nonskeletal tissues we produced transgenic mice carrying murine proα1(II) collagen/β‐galactosidase fusion gene constructs. The expression of the fusion gene was found to depend on the presence of intron 1 sequences: constructs with most of intron 1 deleted failed to reveal any β‐galactosidase activity confirming the important role of regulatory sequences within intron 1 of the gene. High‐level expression of the functional construct was clearly confined to cartilaginous tissues but transient low‐level expression was also observed in extraskeletal locations, such as the developing brain and the notochord. The results demonstrate that the regulatory elements in the proα1(II) collagen/β‐galactosidase fusion gene construct confer both temporal and spatial specificity indistinguishable from that of the endogenous proα1(II) collagen gene as determined by the presence of the corresponding mRNA by in situ hybridization. Furthermore the β‐galactosidase activity correlated well with the progression of chondrogenesis as seen by staining of whole mouse embryos with Alizarin red S and Alcian blue in the hybrid mouse strain used for microinjections. The transgenic mouse line produced should prove useful for studies on various aspects of chondrogenesis. Furthermore, the data shows that the regulatory elements present in the construct are sufficient for targetting the expression of other genes in cartilage. © 1995 wiley‐Liss, Inc.
Role of retinoic acid in mouse neural crest cell development in vitro
Authors: Ito Kazuo , Morita Toshiteru
Published: February 2005

This study examines the role of retinoic acid (RA) in mouse neural crest cell development in culture. We have compared the effects of RA on the developmental behavior of mouse neural crest cells from different axial leveles, that is the mesencephalic (cranial) and sympathoadrenal (trunk) levels by colony assay using antibodies against cell‐type‐specific markers. In control cultures in the absence of RA, the efficiency of colony formation by cranial neural crest cells was considerably lower than in colony cultures of trunk neural crest cells. Pulse‐labelling experiments using 5‐bromo‐2′‐deoxyuridine (BrdU) showed that the proportion of neural crest cells that incorporated BrdU was significantly smaller in day 5 cultures of cranial neural crest cells in the absence of RA compared to cultures from the trunk level. However, BrdU‐incorporation matched the labelling observed in trunk crest cell cultures when RA was added to the culture medium. The efficiency of colony formation and the proportion of BrdU‐incorporated cells in trunk crest cell cultures was similar in the presence and absence of RA. The results suggest that in the early stages of in vitro development, RA has a larger impact on proliferation and/or survival of cranial neural crest cells than of trunk neural crest cells. Moreover, the data indicate that RA significantly affects melanogenesis and the differentiation of serotonergic neurons in mouse neural crest cell cultures from both axial levels. Whereas melanogenesis was suppressed by RA treatment, serotonergic neurogenesis was promoted. Doublelabelling experiments with antibodies against BrdU and serotonin (5‐HT) indicated that RA selectively promoted proliferation of these neurons at a later stage of in vitro development. Furthermore, RA acted upon both committed cells and multipotent cells. Based on the results, we conclude that RA plays multiple critical roles in mouse neural crest cell development. © 1995 wiley‐Liss, Inc.
Expression of Cdx‐2 in the mouse embryo and placenta: Possible role in patterning of the extra‐embryonic membranes
Authors: Beck F. , Erler T. , Russell A. , James R.
Published: February 2005

Three mouse homologues of the Drosophila homeotic gene Caudal (Cad) have been described. They are currently designated Cdx‐1, Cdx‐2, and Cdx‐4. Cdx‐1 and 2 are both strongly expressed in the adult mid‐ and hindgut, while Cdx‐1 and 4 have been shown to be activated in the embryonic primitive streak. Using a polyclonal antibody against a fusion protein containing the amino terminal 109 amino acids of murine Cdx‐2, we here describe the topographical location of the gene product from early cleavage to 12.5 days of embryonic development. Cdx‐2 expression begins at 3.5 days and is confined to the trophectoderm, being absent from the inner cell mass. Subsequently, staining is located in the extra‐embryonic ectoderm adjacent to the epiblast, but sparing the more superficially placed polar, as well as the mural trophoblastic cells. Continuing expression in the fetal membranes involves the chorion, the allantoic bud, and, at even later stages, the spongiotrophoblast. From 8.5 days, Cdx‐2 begins to be expressed in embryonic tissues, principally (unlike Cdx‐1) in the posterior part of the gut from its earliest formation, as well as in the tail bud and in the caudal part of the neural tube. Cdx‐2 is, therefore, transcribed well before any other membrane of the Cad homologue group and of the related Hox‐C group; its expression in the extra‐embryonic membranes and in the hindgut reflects the phylogenetic relationship between the cloaca and the chorio‐allantois and suggests the possibility that homeobox genes may be involved in placental development and/or patterning. © 1995 wiley‐Liss, Inc.
Expression patterns of laminin receptor splice variants α6Aβ1 and α6Bβ1 suggest different roles in mouse development
Authors: Thorsteinsdóttir Sólveig , Roelen Bernard A. J. , Freund Eric , Gaspar Ana C. , Sonnenberg Arnoud , Mummery Christine L.
Published: February 2005

The α6β1 integrin is a receptor for laminins and is present from early stages of mouse embryogenesis. In the present study determined the temporal and spatial expression of the two cytoplasmic splice variants of the α6 integrin subunit, α6A and α6B, in the early‐ and midgestation mouse postimplantation embryo using RT‐PCR, in situ hybridization, and immunofluorescence. Our results show that α6B is present in the embryo at all stages studied and is expressed before α6A. α6A expression begins in 8.5 day p.c. embryos and is initially exclusively localized to the developing heart. In 8.5 (and 9.5) day p.c. embryos α6A mRNA and protein are present in a gradient in the myocardium of the heart tube from strongest expression in the sinus venosus and in the common atrial chamber to a weakening expression along the ventricle and bulbus cordis. In 10.5 day p.c. embryos this gradient is less evident and in 12.5 day p.c. embryos α6A mRNA and protein are present in comparable amounts between atria and ventricles. Neither α6A nor α6B is present in endocardial cushion tissue. By day 12.5 p.c. α6A expression is also present in the developing epidermis, dental primordia, lens, gonads, and in a few epithelia such as those of the digestive tract. α6B expression is always much more widespread than α6A expression. For example, only α6B is present in the myotome of the somites of 9.5 day p.c. embryos, in the developing central and peripheral nervous systems, and in the nephrogenic system at all stages studied, except after the differentiation of the gonads when α6A is also present. Furthermore, α6B is the only splice variant present on endothelial cells. We also examined the distribution of the β4 integrin subunit to determine whether the α6β4 integrin was present during these stages of development. β4 protein was absent in early postimplantation stages but was present in the epidermis and digestive tract of 12.5 day p.c. embryos. These results show a differential distribution of α6A and α6B during mouse development and thus strongly suggest a different function of these splice variants during embryogenesis. Our results point to a possible role for the α6Aβ1 integrin in the development of the myocardium of the developing heart, but not in the migration of endocardial cushion cells, while α6Bβ1 could be important in the developing nephrogenic and nervous systems. © 1995 wiley‐Liss, Inc.
Relationship between fibronectin expression during gastrulation and heart formation in the rat embryo
Authors: Suzuki Hiroaki R. , Solursh Michael , Baldwin H. Scott
Published: February 2005

By utilizing myosin immunostaining, we were able to identify early rat myocardium as a thin epithelial sheet and realized that its cohesive movement toward the midline leads to the straight heart tube formation. Localization study of fibronectin mRNA and protein was, therefore, carried out to investigate its tissue origin and possible roles in facilitating mesoderm migration and heart formation. Fibronectin mRNAs were first detected throughout the mesoderm during the early primitive streak stage, suggesting that the mesoderm is the source of fibronectin. By pre‐head fold (pre‐somite) and head fold (early somite) stages, the mesoderm became largely down‐regulated for fibronectin mRNAs, while it was also at these stages when myosin‐positive myocardium formed itself into the epithelium and was subsequently folding toward the midline. Thus, there appears to be little fibronectin synthesis during and directly relevant to early heart tube formation. Later, during the early straight heart tube stage (5 somite and older), endocardium became highly positive for fibronectin mRNAs, suggesting that the endocardium is the major source of fibronectin for the cardiac jelly. Based on the results, we present a map for the early mammalian heart in which the heart is a single crescentic band lying in front of the prechordal plate. We also suggest a process for heart tube formation based on the cohesive movement of the myocardial epithelium. During heart tube formation, fibronectin protein had been deposited previously by the mesoderm and was found uniformly in the ECM and not newly produced by any adjacent tissue. The data contradict the endodermal guidance of heart migration by fibronectin gradient and suggest, instead, a permissive role for the fibronectin substrate. © 1995 wiley‐Liss, Inc.
Molecular analysis of smooth muscle development in the mouse
Authors: McHugh Kirk M.
Published: February 2005

Little is currently known regarding the ontogeny of smooth muscle tissues during normal mammalian development. The α‐smooth muscle and γ‐smooth muscle isoactins have been shown to be excellent molecular markers of smooth muscle cell phenotype. This study characterizes both the temporal and spatial patterns of α‐smooth muscle and γ‐smooth muscle isoactin expression in the developing mouse. In situ analysis was performed on serial sections of whole mouse embryos on embryonic day 9, 11, 13, 15, and 17 using α‐smooth muscle and γ‐smooth muscle isoactin‐specific riboprobes. Distinct temporal and spatial patterns of α‐smooth muscle and γ‐smooth muscle isoactin gene expression were observed in the developing gastrointestinal tract, urogenital tract, respiratory tract, and vascular system. Independent expression of the α‐smooth muscle isoactin was observed during the early stages of skeletal, cardiac, and smooth muscle myogenesis as well as in a novel subset of distinct organs including the postanal component of the hindgut, allantois, and primitive placenta. The results of this study indicate that distinct cellular phenotypes are involved in smooth muscle myogenesis and suggest that organ‐specific mechanisms might exist for the initiation of smooth muscle development in vivo. In addition, the pattern of independent α‐smooth muscle isoactin expression observed in this study provides novel information regarding the early stages of hindgut and placental development, and suggests that a common functional phenotype may be associated with the early stages of skeletal, cardiac, and smooth muscle myogenesis. © 1995 wiley‐Liss, Inc.
Cell and matrix specialisations of rhombomere boundaries
Authors: Heyman Isobel , Faissner Andreas , Lumsden Andrew
Published: February 2005

Hindbrain segments, rhom‐bomeres, define distinct cellular and molecular domains which furnish the ground plan for important aspects of neural and cranial development. In this study, further evidence is presented that the interfaces between rhombomeres, rhombomere boundaries, contain both cells and extracellular matrix with specialised characteristics. Cells at rhombomere boundaries show temporally and spatially distinct expression patterns of developmentally important genes. Towards the end of the developmental period when rhombomeres are present, a fan‐shaped array of cells at rhom‐bomere boundaries, that constitute the ventricular ridge, shows decreased expression of two genes (Hoxb‐1 and Krox‐20), which earlier in development were expressed in all cells of specific rhombomeres. In contrast, these boundary cells show increased expression of another gene, Pax‐6, which earlier in development has a rhombomere‐specific expression pattern. A specialised identity for boundary cells is further suggested by increased labelling with an anti‐vimentin antibody at rhombomere boundaries, indicating that at least some boundary cells are radial glia or glial precursors. In addition to distinct cellular properties, the extracellular domain at rhombomere boundaries is also specialised. Chondroitin sulphate proteoglycan (CSPG) immunoreactivity is increased and, as revealed by immuno‐electron microscopy, localised to extracellular spaces. CSPG is also enriched in boundaries regenerated after ablation, or boundaries generated ectopically by rhombomere transplantation. We propose that rhombomere boundaries form their characteristic morphology at the interface between groups of cells with differing molecular characteristics, representing different cell states. A specialised band of cells then develops at the interface. Both the boundary cells and extracellular matrix have characteristics which could be important in later events of neural development such as axon guidance and cell migration. © 1995 wiley‐Liss, Inc.