Developmental Dynamics

Winklbauer, Rudolf

doi: 10.1002/(SICI)1097-0177(199807)212:3<335::AID-AJA1>3.0.CO;2-Ipmid: 9671937

Fibronectin fibril formation on a multilayered cohesive cell sheet is studied in the Xenopus embryo. In the blastula, secreted fibronectin accumulates in the blastocoel, where it associates with mucous material. At the onset of gastrulation, a fibrillar fibronectin matrix develops on the blastocoel roof. Cells engage in this process stochastically within a 2‐hr period. Fibril network formation requires more than 60 μg/ml of fibronectin, but the timing of fibrillogenesis is not regulated through the availability of fibronectin. With the exception of a few isolated mesoderm cells, only the cells of the blastocoel roof are able to form fibronectin fibrils. However, this requires that cells are provided with a free surface and, at the same time, with lateral adhesive cell contacts, i.e. fibril assembly occurs only on the surface of cohesive cells aggregates. This explains the observed restriction of fibronectin matrix formation to the inner surface of the blastocoel roof in the embryo. In addition, a minimum blastocoel roof size is required for fibril formation. Dev. Dyn. 1998;212:335–345. © 1998 Wiley‐Liss, Inc.

Que, Benito G.; Wise, Gary E.

doi: 10.1002/(SICI)1097-0177(199807)212:3<346::AID-AJA2>3.0.CO;2-Hpmid: 9671938

Tooth eruption is a localized developmental event that requires the presence of the dental follicle, a loose connective tissue sac that surrounds each tooth. Early postnatally in the first mandibular molar of the rat there is an influx into the follicle of mononuclear cells (monocytes) which, in turn, fuse to form osteoclasts that resorb the bone to form an eruption pathway. The chemoattractant that may attract the mononuclear cells to the follicle to initiate the cellular events of eruption is monocyte chemotactic protein‐one (MCP‐1). MCP‐1 is secreted by the dental follicle cells and its gene is expressed maximally at an early postnatal age, correlating with the monocyte influx into the follicle. In this study, we show that other potential tooth eruption molecules—EGF, IL‐1α, TGF‐β1 and CSF‐1—all enhance the expression of the MCP‐1 gene in the cultured dental follicle cells. In vivo, injections of IL‐1α or EGF also enhance the gene expression of MCP‐1 in the follicle with maximal enhancement occurring in the early postnatal days. Thus, there appears to be a redundant function of the different tooth eruption genes to ensure that the MCP‐1 gene is expressed. In turn, expression of MCP‐1 may be critical for recruiting the monocytes to the dental follicle to initiate the cellular events of tooth eruption. Dev. Dyn. 1998;212:346–351. © 1998 Wiley‐Liss, Inc.

Stark, David R.; Gates, Phillip B.; Brockes, Jeremy P.; Ferretti, Patrizia

doi: 10.1002/(SICI)1097-0177(199807)212:3<352::AID-AJA3>3.0.CO;2-Gpmid: 9671939

Members of the hedgehog family have been shown to play a key role in many developmental processes, including limb patterning and chondrogenesis. We have therefore investigated whether members of this family are also expressed during regeneration of the adult urodele limb and are regulated by retinoic acid (RA), since this derivative induces proximodistal duplications in regenerating limbs, and has been shown to regulate sonic hedgehog (shh) in the developing limbs of birds and mammals. We report here that a newt homologue of Xenopus banded hedgehog, called N‐bhh, is uniformly expressed by mesenchymal blastemal cells from the initial stages of regeneration and is up‐regulated by RA. In addition, we show that N‐bhh is uniformly expressed in the early limb bud of the newt embryo. Since bhh has not been detected in developing limbs of higher vertebrates, its expression in developing and regenerating newt limbs may be related to the regenerative capability of urodeles. Dev. Dyn. 1998;212:352–363. © 1998 Wiley‐Liss, Inc.

Hoang, Bang H.; Thomas, J. Terrig; Abdul‐Karim, Fadi W.; Correia, Kristen M.; Conlon, Ronald A.; Luyten, Frank P.; Ballock, R. Tracy

doi: 10.1002/(SICI)1097-0177(199807)212:3<364::AID-AJA4>3.0.CO;2-Fpmid: 9671940

Wnt proteins have been implicated in regulating growth and pattern formation in a variety of tissues during embryonic development. We previously identified Frzb‐1, a gene which encodes a secreted protein with homology in the ligand binding domain to the Wnt receptor Frizzled, but lacking the domain encoding the putative seven transmembrane segments. Frzb‐1 has recently been shown to bind to Wnt proteins in vitro, and to inhibit the activity of Xenopus Wnt‐8 in vivo. We report now that mFrzb‐1 and Wnt transcripts display both complementary and overlapping expression patterns at multiple sites throughout embryonic development. By Northern analysis, the expression of mFrzb‐1 in the developing mouse embryo is greatest from 10.5 to 12.5 days postcoitum (dpc). In the early embryo, mFrzb‐1 is expressed in the primitive streak, presomitic mesoderm, somites, and brain. Later, mFrzb‐1 exhibits sharp boundaries of expression in the limb bud, branchial arches, facial mesenchyme, and in cartilaginous elements of the appendicular skeleton. We conclude from these experiments that Frzb‐1is expressed at a time and location to modulate the action of Wnt family members during development of the limbs and central nervous system. Dev. Dyn. 1998;212:364–372. © 1998 Wiley‐Liss, Inc.

Poelmann, R.E.; Mikawa, T.; Gittenberger‐De Groot, A.C.

doi: 10.1002/(SICI)1097-0177(199807)212:3<373::AID-AJA5>3.0.CO;2-Epmid: 9671941

The heart consists of cells deriving from the cardiogenic plate and also from extracardiac sources. One of the major extracardiac contributions is given by the neural crest. The differentiation pathway and fate of the neural crest cells in the outflow tract have been followed over a prolonged period during outflow tract septation. We studied the role of the neural crest in remodeling the outflow tract by long‐term cell tracing, differentiation markers and apoptosis. The pattern of neural crest cells migrating to the heart was investigated by heterospecific chicken quail chimeras and by retroviral infection of the reporter gene LacZ to the stemcells. The tagged neural crest cells move to areas that are morphogenetically active, such as the outflow tract, the semilunar valves, the wall of the arteries and the cardiac ganglia. Two differentiated subpopulations are discerned on the basis of immunohistochemical characterization with antibodies against smooth muscle cells in the arterial vessel wall and against ganglionic cells that were scattered around the vessels of the arterial pole and the heart. A third subpopulation did not stain with these antibodies, but presented locally with the phenomenon of apoptosis as shown with the TUNEL approach. In a developmental series of chicken embryos the populations were followed until stage 40. It was evident that the outflow tract septum in the early phase of development consisted mainly of mesenchymal neural crest cells. In a later phase neural crest cells were still detected at semilunar valve level, but nearly absent in the outflow tract septum below valve level. The septum at that time had become myocardialized. It is evident that neural crest cells are actually removed from this part of the heart by apoptosis. We are pursuing the hypothesis that an important function of apoptotic cells in heart development might be to activate the cardiomyocytes to muscularize the outflow tract septum through mobilizing or delivering growth factors at the time and place that septum formation is initiated. Dev. Dyn. 1998;212:373–384. © 1998 Wiley‐Liss, Inc.

Cowin, A.J.; Brosnan, M.P.; Holmes, T.M.; Ferguson, M.W.J.

doi: 10.1002/(SICI)1097-0177(199807)212:3<385::AID-AJA6>3.0.CO;2-Dpmid: 9671942

The recruitment of inflammatory cells to a wound may play an important role in the resulting cellular processes and ultimately the quality of the healing response in the fetus (scar‐free healing) or the adult (scar‐forming healing). Using a range of antibodies to monocytes and macrophages and also to different activation markers of activated macrophages, we have compared the inflammatory profile of scar‐free healing E16 mouse fetal wounds to those of scarring adult wounds. In the fetal wound, small numbers of monocyte derived cells (MOMA‐2 and F4/80 positive) are recruited to the wound by 3 hr post‐wounding. No Mac‐1 positive cells indicative of activated macrophages were observed until 18 hr post‐wounding. Eventually all types of macrophages studied were recruited to both adult and fetal wound sites but the numbers and persistence of these cells are lower in the fetus than in the adult. B cells were detected in healing adult wounds but not in the fetal wounds. This absence of H‐21‐A positive (B) cells in murine fetal wounds could be associated with the low levels of activated Mac‐1 positive macrophages at the murine fetal wound site. Activated macrophages in addition to releasing growth factors may also release signals to recruit B cells. Thus, the E16 mouse fetus can mount an inflammatory response to wounding. This response differs from that of the adult in the numbers of inflammatory cells recruited to the wound and the subpopulations of activated cells found at the wound site. This study indicates that there are complex differences between the inflammatory responses elicited in adult and fetal murine dermal wounds. These differences may determine the profile of growth factors and cytokines released at fetal and adult wound sites. Manipulation of either the numbers or the activation states of inflammatory cells at the adult wound site may be an approach to the control of scarring during adult wound healing. Dev. Dyn. 1998;212:385–393. © 1998 Wiley‐Liss, Inc.

Kadoya, Yuichi; Nomizu, Motoyoshi; Sorokin, Lydia M.; Yamashina, Shohei; Yamada, Yoshihiko

doi: 10.1002/(SICI)1097-0177(199807)212:3<394::AID-AJA7>3.0.CO;2-Cpmid: 9671943

Active sequences from the laminin α1 and α2 chain carboxyl‐terminal globular domains (G domain) have been identified by screening overlapping synthetic peptides in a number of biological assays (Nomizu et al. (1995) J. Biol. Chem. 270:20583–20590; Nomizu et al. (1996) FEBS Lett. 396:37–42). We have tested the activity of these peptides in submandibular gland explants of embryonic day 13 mice to determine the functional sites involved in organ development. The laminin α1 chain peptide, RKRLQVQLSIRT (residues 2719–2730 and designated AG‐73), significantly inhibited epithelial branching morphogenesis. In contrast, other cell adhesive laminin α1 chain peptides including the AASIKVAVSADR and NRWHSIYITRFG failed to inhibit the branching. MG‐73, a homologue of AG‐73 from the laminin α2 chain, did not inhibit the branching. The α2 chain peptide had no effect, which may be due to the low levels of this laminin chain in day 13 mice. Laminin α2 chain‐specific monoclonal antibodies strongly reacted with the basement membranes of developed acini but only weakly stained embryonic day 13 submandibular epithelium. The expression of E‐cadherin and α6 integrin, as detected by immunofluorescence, were unchanged in both AG‐73 and control scramble peptide‐treated epithelial cells of the explants. In contrast, immunostaining of nidogen/entactin showed that explants treated with AG‐73 for 3 days had a discontinuous basement membrane. Explants treated for 3 days with control peptide showed a normal basement membrane. These results suggest that the region containing the AG‐73 sequence of the laminin α1 chain is crucial for development of submandibular gland at early embryonic stages. The discontinuous basement membrane in AG‐73‐treated explants may indicate an important role for this region in basement membrane assembly. Dev. Dyn. 1998;212:394–402. © 1998 Wiley‐Liss, Inc.

Yang, Claire Q.; Friesel, Robert

doi: 10.1002/(SICI)1097-0177(199807)212:3<403::AID-AJA8>3.0.CO;2-Lpmid: 9671944

To begin to determine the role of receptor‐like tyrosine phosphatases during Xenopus development, we have isolated a cDNA predicted to encode receptor‐like tyrosine phosphatase with significant amino acid sequence identity to mouse and human protein tyrosine phosphatase α (PTPα). Xenopus PTPα (XPTPα) exists as a maternally expressed mRNA that decreases in expression during gastrulation and then maintains a constant lower level of expression through early tadpole stages. In situ hybridization reveals that XPTPα mRNA is expressed throughout the gastrula stage embryo. During subsequent development, XPTPα mRNA becomes restricted in its expression to various regions of the brain and the visceral arches. XPTPα mRNA is also expressed in several adult tissues and in Xenopus XTC cells. Immunoblot analysis demonstrates that XPTPα protein is expressed at relatively uniform levels throughout development. Expression of XPTPα protein in insect cells with a recombinant baculovirus results in a glycosylated polypeptide of 110–130 kDa with intrinsic phosphotyrosine phosphatase activity. The spatial and temporal patterns of expression of XPTPα indicate that it may play multiple roles during early development including development of the brain. Dev. Dyn. 1998;212:403–412. © 1998 Wiley‐Liss, Inc.

Barron, Matthew; McAllister, Donna; Smith, Susan M.; Lough, John

doi: 10.1002/(SICI)1097-0177(199807)212:3<413::AID-AJA9>3.0.CO;2-Kpmid: 9671945

Previous studies have shown that anterior lateral plate endoderm from stage 6 chicken embryos is necessary and sufficient to enable precardiac mesoderm to complete its cardiogenic program in vitro, culminating in a rhythmically contractile multicellular vesicle (Sugi and Lough (1994) Dev. Dyn. 200:155–162). To identify cardiogenic factors, we have begun to characterize proteins that are secreted by endoderm cell explants. Fluorography of proteins from endoderm‐conditioned medium revealed 1–2 dozen bands, the most prominent of which migrated at approximately 17 and 25 kD. The bulk of the 17‐kD band, which migrates near FGFs and subunits of the transforming growth factor‐β family, was identified by N‐terminal sequencing as transthyretin (TTR). A component of the 25‐kD band was identified by Western blotting as retinol binding protein (RBP). RT/PCR analysis revealed that mRNAs for both proteins are in the embryo as early as stage 3. In situ hybridization localized these mRNAs to the extraembryonic endoderm at stage 6, after which they were detected in endoderm overlying the embryo proper, including the developing heart. Later, RBP and TTR mRNA and protein were detected in cells associated with the developing heart. Western blotting of whole embryo proteins revealed the presence of RBP by stage 7, followed by sequential increases to stage 25; by contrast, content of RBP in isolated hearts peaked at stage 14, then declined. Immunohistochemistry revealed the presence of RBP protein in the extracellular matrix subjacent to lateral plate endoderm beginning at stage 8; upon formation of the definitive heart, intense staining was observed in the cardiac “jelly.” By contrast TTR was intracellular, first detected as subtle deposits in stage 6 embryonic endoderm, which by stage 8 were prominent in the dorsally invaginated endoderm subjacent to the precardiac splanchnic mesoderm. At stages 11–14, TTR was detected only in myocardial cells. Such localization of RBP and TTR may indicate a role in the transport and distribution of retinol and thyroid hormone, respectively, from yolk to embryo prior to establishment of the circulatory system, and is suggestive of a subsequent role in heart development. Dev. Dyn. 1998;212:413–422. © 1998 Wiley‐Liss, Inc.

Jacquemin, Patrick; Sapin, Vincent; Alsat, Eliane; Evain‐Brion, Danièle; Dollé, Pascal; Davidson, Irwin

doi: 10.1002/(SICI)1097-0177(199807)212:3<423::AID-AJA10>3.0.CO;2-1pmid: 9671946

We describe the molecular cloning of murine (m) Transcriptional Enhancer Factor (TEF)‐5 belonging to the TEF family of transcription factors. We show that mTEF‐5 is specifically expressed in trophoblast giant cells and other extra‐embryonic structures at early stages of development. At later stages, mTEF‐5 is specifically expressed in the labyrinthine region of the placenta and in several embryonic tissues. We further show that the other mTEFs are differentially expressed in extraembryonic structures and in the mature placenta. Interestingly, human (h)TEF‐5 is specifically expressed in the differentiated syncytiotrophoblast of the human term placenta and its expression is upregulated during the differentiation of cytotrophoblasts to syncytiotrophoblast in vitro, whereas that of hTEF‐1 is down‐regulated. Together with previous results describing hTEF‐binding sites in the human placental lactogen‐B gene enhancer, these novel observations support a role for hTEF‐5 in the regulation of this gene. We further propose that the hTEF factors may play a more general role in placental gene regulation and development. Dev. Dyn. 1998; 212:423–436. © 1998 Wiley‐Liss, Inc.