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In this communication, we describe the otologic and audiometric findings of a rare congenital syndrome associated with profound hearing loss. We offer a hypothesis of the possible inner ear pathologic condition. The major anomalies of this syndrome represent features of an ectodermal dysplasia syndrome.
Recently, we isolated a novel mouse gene, Etl-1 (Enhancer-trap-locus-1), whose deduced amino acid sequence shows in its C-terminal portion striking homology to the brahma protein (BRM), a transcriptional regulator of homeotic genes in Drosophila, and to SNF2/SWI2, a transcriptional regulator of various genes in Saccharomyces cerevisiae. Here we report the generation of antibodies against the Etl-1 gene product (ETL-1) and describe the subcellular localization as well as the expression and distribution of the ETL-1 protein during mouse pre- and early post-implantation development. ETL-1 is a nuclear protein and is expressed in a biphasic manner during early embryogenesis. Moderate levels of ETL-1 were detected in unfertilized and fertilized eggs but in the latter the protein was not concentrated in the pronuclei and seemed evenly distributed throughout the cytoplasm. In two-cell embryos nuclear ETL-1 protein accumulated transiently and levels decreased during subsequent cleavage development. After the morula stage, ETL-1 levels increased again; in blastocysts high levels of ETL-1 were present in inner cell mass cells whereas trophectoderm cells contained little or no ETL-1. During subsequent development essentially all cell types except parietal endoderm and trophoblast cells contained high levels of ETL-1. Our results imply that nuclear ETL-1 is dispensable for the progression to the two cell stage, and suggest that during cleavage ETL-1 might be needed at the onset of embryonic transcription. In blastocysts ETL-1 function might be specifically required in cells of the inner cell mass and later in most cells of the embryo proper and extraembryonic ectoderm lineage.
The development of mouse embryos in culture from the implantation to the head-fold stage was sequentially examined. Our goal was to compare the morphology of embryos grown in vitro to those developed in vivo, published in standard texts, and to delineate the stages involved in the process of tissue differentiation and organization. Mouse blastocysts (stage 6) were collected at 3.5 days p.c. and cultured. Attachment of the blastocysts occurred on the second day of culture (stage 8). Following the collapse of the blastocyst endoderm cells began to migrate and to encircle the inner cell mass. At 2 days in culture the embryonic and extra-embryonic ectoderm became distinguishable and the proamniotic cavity appeared (stage 9). Egg cylinders began to project above the substrate at 2.5 days in culture (stage 10) and to progress through the stages observed in vivo. At 4 days a posterior amniotic fold began to form (stage 11) and was followed at 5 days by the formation of the chorion, the appearance of mesoderm, exocoelom, and head fold (stage 12). At 6 days in culture the embryo had differentiated longitudinally and developed an allantois, blood islands, Reichert's membrane, head process, and primitive streak. At 7 days somites as well as the neural fold and heart were observed (stage 14) and were followed by further differentiation at 8 days (stage 15). These observations indicate that apparently normal embryo development can be maintained in vitro through the early stages of organogenesis, thus providing a unique opportunity for investigating the regulation of early mammalian development.
The Ovo gene family encodes a group of evolutionarily conserved transcription factors and includes members that reside downstream of key developmental signaling pathways such as Wg/Wnt and BMP/TGF-beta. In the current study, we explore the function of Ovol2, one of three Ovo paralogues in mice. We report that Ovol2 is expressed during early-mid embryogenesis, particularly in the inner cell mass at E3.5, in epiblast at E6.5, and at later stages in ectodermally derived tissues such as the rostral surface (epidermal) ectoderm. Embryos in which Ovol2 is ablated exhibit lethality by E10.5, prior to which they display severe defects including an open cranial neural tube. The neural defects are associated with improper Shh expression in the underlying rostral axial mesoderm and localized changes of neural marker expression along the dorsoventral axis, as well as with expanded cranial neural tissue and reduced cranial surface ectoderm culminating in a lateral shift of the neuroectoderm/surface ectoderm border. We propose that these defects reflect the involvement of Ovol2 in independent processes such as regionalized gene expression and neural/non-neural ectodermal patterning. Additionally, we present evidence that Ovol2 is required for efficient migration and survival of neural crest cells that arise at the neuroectoderm/surface ectoderm border, but not for their initial formation. Collectively, our studies indicate that Ovol2 is a key regulator of neural development and reveal a previously unexplored role for Ovo genes in mammalian embryogenesis.
Inner ear induction, like induction of other tissues examined in recent years, is likely to be comprised of several stages. The process begins during gastrulation when the ectoderm is competent to respond to induction. It appears that a signal from the endomesoderm underlying the otic area during gastrulation initiates induction complemented by a signal from presumptive neural tissue. By the neural plate stage, a region of ectoderm outside the neural plate is "biased" toward ear formation; this process may be part of a more general "placodal" bias shared by several sensory tissues. Induction continues during neurulation when a signal from neural tissue (possibly augmented by mesoderm underlying the otic area) results in ectoderm committed to otic vesicle formation at the time of neural tube closure. Studies on several gene families implicate them in the ear determination process. Fibroblast Growth Factor (FGF) family members are clearly involved in induction: FGFs are appropriately expressed for such a role, and have been shown to be essential for inner ear development. FGFs also have inductive activity, although it is not clear if they are sufficient for ear induction. Activation of transcription factors in the otic ectoderm, for example, by Pax gene family members, provides evidence for important changes in the responding ectoderm beginning during gastrulation and continuing through specification at the end of neurulation, although few functional tests have defined the role of these genes in determination. The challenge remains to merge embryologic data with gene function studies to develop a clear model for the molecular basis of inner ear induction.
The frontal region of the embryonic chick was studied to determine whether epithelial influences are necessary for frontal bone development. The frontal bone is a membrane bone, of neural crest and head mesodermal origin, which develops within mesenchyme sandwiched between two epithelia, neural ectoderm and epidermis. Rudiments were treated enzymatically to separate epithelial and mesenchymal tissues. Frontal mesenchyme then was grown as chorioallantoic membrane grafts either in the presence or absence of neural ectoderm and/or epidermis. The results indicate that neural ectoderm, though required during early stages of development to induce frontal bone development (Schowing, 1968), is not required during later stages (HH 22-30, the stages tested in this study) for osteogenesis. Epidermis, however, was shown to be required for frontal bone development during the stages tested. Frontal mesenchyme formed bone when epidermis was present on the outer aspect of the mesenchyme, and did not form bone when the epidermis had been removed prior to grafting, whether or not neural ectoderm was present. This dependence upon epidermis continues beyond the onset of meningeal differentiation. Once the outer ectomeninx-dermis is distinguishable from the inner endomeninx, osteogenic capabilities are confined to the ectomeninx-dermis layer. Furthermore, the ectomeninx-dermis layer attached to epidermis is able to form membrane bone in the absence of the endomeninx and neural ectoderm. The endomeninx, though normally nonchondrogenic, was shown to be capable of forming cartilage when the neural ectoderm is removed. Neural ectoderm, therefore, may have an inhibitory effect on chondrogenesis in the endomeninx.
The nervous system is ectodermal in origin. All nerve ganglia arise separately by proliferation and later delamination from the ectoderm, not by invagination. They become secondarily connected to one another by commissures and connectives developing as extensions from the peripheral layer of ganglionic nerve cells. Rudiments of the cerebral, pedal, pleural and intestinal (parietal) ganglia arise almost simultaneously at a relatively early stage (Stage V). The cerebral ganglia develop from the ectoderm of the head plates. Rudiments of the pedal and pleural ganglia are separate at their inception. They later fuse (Stage VI) to form a pleuro-pedal ganglionic mass on each side. The 2 intestinal ganglia are symmetrical at the beginning, but they soon lose their symmetry as a result of torsion. The right ganglion crosses to the left over the gut and persists as the supraintestinal ganglion. The left or subintestinal ganglion shifts to the right and forward, and fuses with the right pleural ganglion (Stage VIII), thus obscuring the chiastoneury. The paired buccal and single visceral (abdominal) ganglia start differentiating in Stage VII. The former develop from the ectodermal wall of the stomodaeum, while the visceral ganglion delaminates from the right wall of the visceral sac, then shifts to the left during torsion. The statocysts develop early (Stage V) from 2 ectodermal invaginations on either side of the rudimentary foot. They later separate from the overlying ectoderm and statoconi appear in their lumina. Contrary to earlier reports on related ampullariids, the osphradium proved to be ontogenetically older than the mantle and mantle cavity. It starts differentiating as a thickened ectodermal plate in the right wall of the visceral sac (Stage V). During torsion, it becomes engulfed in the mantle cavity and shifts to the left side, then is carried forward as the mantlegrow. The eyes develop late (Stage IX) as ectodermal invaginations which rapidly separate from the ectoderm to form closed vesicles. Their cells start differentiating before hatching to form the retina, in which pigment is deposited, and the inner cornea. The lens is secreted in the lumen of the eye and grows by addition of concentric layers of secretion.
In early amphibian development, the mesoderm is formed around the equator of the blastula in response to an inductive signal from the endoderm. A screen of candidate substances showed that a small group of heparin-binding growth factors (HBGFs) were active as mesoderm-inducing agents in vitro. The factors aFGF, bFGF, kFGF and ECDGF all show similar potency and can produce inductions at concentrations above about 100 pM. The product of the murine int-2 gene is also active, but with a lower specific activity. Above the induction threshold there is a progressive increase of muscle formation with dose. Single blastula ectoderm cells can be induced and will differentiate in a defined medium to form mesodermal tissues. All inner blastula cells are competent to respond to the factors but outer cells, bearing oocyte-derived membrane, are not. Inducing activity can be extracted from Xenopus blastulae and binds to heparin like the previously described HBGFs. Antibody neutralization and Western blotting experiments identify this activity as bFGF. The amounts present are small but would be sufficient to evoke inductions in vivo. It is not yet known whether the bFGF is localized to the endoderm, although it is known that inducing activity secreted by endodermal cells can be neutralized by heparin. The competence of ectoderm to respond to HBGFs rises from about the 128-cell stage and falls again by the onset of gastrulation. This change is paralleled by a rise and fall of binding of 125I-aFGF. Chemical cross-linking reveals that this binding is attributable to a receptor of relative molecular mass about 130 x 10(3).(ABSTRACT TRUNCATED AT 250 WORDS)
In mammals, the permanence of acquired hearing loss is mostly due to the incapacity of the cochlea to replace lost mechanoreceptor cells, or hair cells. In contrast, damaged vestibular organs can generate new hair cells, albeit in limited numbers. Here we show that the adult utricular sensory epithelium contains cells that display the characteristic features of stem cells. These inner ear stem cells have the capacity for self-renewal, and form spheres that express marker genes of the developing inner ear and the nervous system. Inner ear stem cells are pluripotent and can give rise to a variety of cell types in vitro and in vivo, including cells representative of ectodermal, endodermal and mesodermal lineages. Our observation that these stem cells are capable of differentiating into hair cell-like cells implies a possible use of such cells for the replacement of lost inner-ear sensory cells.
In early amphibian development the mesoderm is formed around the equator of the blastula in response to inductive signals from the endoderm. At the time of its formation the mesoderm consists of a large 'ventral type' zone and a small 'organizer' zone. A screen of candidate substances showed that a small group of heparin binding growth factors (HBGFs) were active as mesoderm inducing agents in vitro. The fibroblast growth factors (aFGF and bFGF) and embryonal carcinoma derived growth factor (ECDGF) all show similar potency and can produce ventral inductions at concentrations above about 100 pm. Single blastula ectoderm cells can be induced and will differentiate in a defined medium to form mesodermal tissues and all inner blastula cells are competent to respond to the factors. Inducing activity can be extracted from Xenopus blastulae and can be purified by heparin affinity chromatography. Antibody neutralization and Western blotting experiments identify this activity as bFGF. The amounts present are small but would be sufficient to evoke ventral inductions in vivo. It is not yet known whether the bFGF is localized to the endoderm, although it is known that inducing activity secreted by endodermal cells can be neutralized by heparin. The competence of ectoderm to respond to FGF rises from about the 128-cell-stage and falls again by the onset of gastrulation. This change is paralleled by a rise and fall of binding of 125I-labelled aFGF. Chemical cross-linking reveals that this binding is attributable to a receptor of molecular mass about 130 kilodaltons (kDa). The receptor is present both in the marginal zone, which responds to the signal in vivo, and in the animal pole region, which is not induced in vivo but which will respond to HBGFs in vitro. In intact embryos we believe that the ventral type mesoderm forms the somites, kidney and other intermediate structures as well as the blood islands of the ventral midline. These intermediate structures are induced as a function of distance from the organizer in a process called 'dorsalization'. Lithium salts have a dorsalizing effect on whole embryos and also on explants from the ventral marginal zone, causing them to form large blocks of muscle. Lithium will also cause large muscle blocks to form when applied to ectoderm explants together with FGF. It is difficult to extend these results directly to mammalian embryos, but we have shown that the products of the murine int-2 gene and of the human k-fgf genes are active as mesoderm inducing factors.
Embryonic stem (ES) cells are derived from preimplantation stage mouse embryos at the time when they have reached the blastocyst stage. It is at this point that the first steps of differentiation take place during mammalian embryonic development. The individual blastomeres now start to organize themselves into three distinct locations, each encompassing a different cell type: outside epithelial cells, trophectoderm; cells at the blastocele surface of the inner cell mass (ICM), the primitive endoderm; and inside cells of the ICM, the primitive ectoderm. ES cells originate from the third population, the primitive ectoderm, which is a transiently existing group of cells in the embryo. Primitive ectoderm cells diminish within a day as the embryo is entering into the next steps of differentiation. ES cells, however, while retaining the property of their origin in terms of developmental potential, also have the ability to self-renew. It is hence important to realize that ES cells do not exist in vivo; they should be regarded simply as tissue culture artifact. Nevertheless, these powerful cells have the potential to differentiate into all the cells of the embryo proper and postnatal animal. Furthermore, they retain the limitation of their origin through their inability to contribute to the trophectoderm lineage (the trophoblast of the placenta) and the lineages of the primitive endoderm, the visceral and parietal endoderm. Due to these unique features, we must admit that even if we regard ES cells as products of in vitro culture and should not compare them to true somatic stem cells found in the adult organism, they certainly offer us a fantastic tool for genetic, developmental, and disease studies.
Cardiac morphogenesis proceeds from a sequential series of epithelial-mesenchymal transitions which begins by establishing bipotential heart-forming cells and later their segregation into endocardial and myocardial lineages. Cells within each lineage integrate to form two concentric epithelia which inductively interact to transform cells of the inner epithelium, the endocardium, into mesenchymal or 'cushion' cells. Noncardiogenic epithelia (dorsal mesocardium, epicardium, neural ectoderm and coelomic mesothelium) undergo transition into populations of extracardiac mesenchyme that combine over time with cushion tissue to remodel the simple tubular heart into a four-chambered organ. Model systems are described for studying the mechanisms of cardiac-related transformations including primary cultures of precardiac epithelia and a differentiation-inducible, avian stem cell line called QCE-6. Focus is centered on the molecular mechanism by which endocardial epithelium transforms into cushion mesenchyme. Experimental findings are reviewed and interpreted in the context of a hypothetical model that seeks to answer why only some cells within an epithelium transform and whether the transformation process is regulated by intrinsic or extrinsic mechanisms. The model proposes that epithelial cells competent to transform to mesenchyme express characteristic markers including receptors for extrinsic signals secreted by stimulator cells (e.g. myocardium). Candidate extrinsic signals include multicomponent complexes called adherons. If applied directly to cultured endocardium, myocardial adherons but not those secreted by L6 myoblasts, induce changes in gene expression within target endocardial cells for proteases and cell:cell and cell:matrix adhesion molecules that accompanied transformation to mesenchyme. A main component of myocardial adherons has been identified as ES antigens, one of which, ES/130, has been cloned, found to have a novel sequence and in culture assays shown to be required for endocardium to transform to mesenchyme. The spatiotemporal pattern of ES protein expression within the embryo suggests that common mechanisms may exist for embryonic epithelial-mesenchymal transformations.
The inner ear, which contains the sensory organs specialised for audition and balance, develops from an ectodermal placode adjacent to the developing hindbrain. Tissue grafting and recombination experiments suggest that placodal development is directed by signals arising from the underlying mesoderm and adjacent neurectoderm. In mice, Fgf3 is expressed in the neurectoderm prior to and concomitant with placode induction and otic vesicle formation, but its absence affects only the later stages of otic vesicle morphogenesis. We show here that mouse Fgf10 is expressed in the mesenchyme underlying the prospective otic placode. Embryos lacking both Fgf3 and Fgf10 fail to form otic vesicles and have aberrant patterns of otic marker gene expression, suggesting that FGF signals are required for otic placode induction and that these signals emanate from both the hindbrain and mesenchyme. These signals are likely to act directly on the ectoderm, as double mutant embryos showed normal patterns of gene expression in the hindbrain. Cell proliferation and survival were not markedly affected in double mutant embryos, suggesting that the major role of FGF signals in otic induction is to establish normal patterns of gene expression in the prospective placode. Finally, examination of embryos carrying three out of the four mutant Fgf alleles revealed intermediate phenotypes, suggesting a quantitative requirement for FGF signalling in otic vesicle formation.
We studied the roles of beta-catenin in inner ear development in rat embryos using immunostaining and antisense experiments. High levels of beta-catenin appeared transiently in the otic cup during inner ear development. While beta-catenin accumulation was not yet observed in the thickened surface ectoderm at the otic placode, it became to be detected at the apical surface of the otic cup. Then it disappeared from the otocyst. When embryos were treated with the beta-catenin antisense oligodeoxynucleotide (ODN), accumulation of beta-catenin in the otic cup was suppressed and the beta-catenin protein level was significantly less in treated embryos than in controls. The number of cells in the otic cup in treated embryos was smaller than in control embryos. Cells that incorporated bromodeoxyuridine (BrdU) in the otic placode were fewer in number in treated embryos than in controls. In control embryos, acoustic neurons were detected by 2H3 (anti-neurofilament 165 kDa antibody) staining within the acoustic neural crest complex, while only a little staining of 2H3 was observed in the complex of the treated embryos. These results suggested that beta-catenin plays a role in cell proliferation in the otic placodes and in differentiation of acoustic neurons within the acoustic neural crest complex.
At the blastocyst stage of pre-implantation mouse development, close contact of polar trophectoderm with the inner cell mass (ICM) promotes proliferation of undifferentiated diploid trophoblast. However, ICM/polar trophectoderm intimacy is not maintained during post-implantation development, raising the question of how growth of undifferentiated trophoblast is controlled during this time. The search for the cellular basis of trophoblast proliferation in post-implantation development was addressed with an in vitro spatial and temporal analysis of fibroblast growth factor 4-dependent trophoblast stem cell potential. Two post-implantation derivatives of the polar trophectoderm - early-streak extra-embryonic ectoderm and late-streak chorionic ectoderm - were microdissected into fractions along their proximodistal axis and thoroughly dissociated for trophoblast stem cell culture. Results indicated that cells with trophoblast stem cell potential were distributed throughout the extra-embryonic/chorionic ectoderm, an observation that is probably attributable to non-coherent growth patterns exhibited by single extra-embryonic ectoderm cells at the onset of gastrulation. Furthermore, the frequency of cells with trophoblast stem cell potential increased steadily in extra-embryonic/chorionic ectoderm until the first somite pairs formed, decreasing thereafter in a manner independent of proximity to the allantois. Coincident with occlusion of the ectoplacental cavity via union between chorionic ectoderm and the ectoplacental cone, a decline in the frequency of mitotic chorionic ectoderm cells in vivo, and of trophoblast stem cell potential in vitro, was observed. These findings suggest that the ectoplacental cavity may participate in maintaining proliferation throughout the developing chorionic ectoderm and, thus, in supporting its stem cell potential. Together with previous observations, we discuss the possibility that fluid-filled cavities may play a general role in the development of tissues that border them.
X-chromosome activity in female mouse embryos was studied at the cellular level using an X-linked lacZ transgene which encodes beta-galactosidase (beta-Gal). Translation of maternal RNA in oocytes is seen as beta-Gal activity that persists into early cleavage-stages. Zygotic transcription of the transgene from the maternal X chromosome (Xm) is first found at about the 8-cell stage. By contrast, expression of the lacZ transgene on the paternal X chromosome (Xp) is not seen until later at the 16-32-cell stage. Preferential inactivation of Xp occurs in the mural trophectoderm, the primitive endoderm, and derivatives of the polar trophectoderm, but a small number of cells in these lineages may still retain an active paternal X chromosome. X inactivation begins at 3.5 days in the inner cell mass but contrary to previous findings the process is not completed in the embryonic ectoderm by 5.5 to 6.0 days. Regional variation in beta-Gal activity is also observed in the embryonic ectoderm during gastrulation which may be related to the specification of cell fates. Random inactivation of Xp and Xm ensues in all somatic tissues but the process is completed at different times in different tissues. The slower progression of X inactivation in tissues such as the notochord, the heart, and the embryonic gut is primarily due to the persistent maintenance of two active X chromosomes in a significant fraction of cells in these tissues. Recent findings on the methylation of endogenous X-linked genes suggest that the prolonged expression of beta-Gal might also be due to the different rate of spreading of inactivation along the X chromosome to the lacZ transgene locus in different tissues.
A qualitative bioassay was adapted and used to determine the ability of the early post-implantation mouse embryo to synthesise interferon. Interferon production was not seen in any embryo tissue in the absence of an inducer and could only be detected in virus-induced tissue from the early 7th day of development. This induced interferon synthesis was initially confined to the trophoblast of the early 7th day embryo. It was then found in tissues of both trophoblast and inner cell mass origin in the early 8th day, and subsequently, in derivatives of the embryonic ectoderm in the 13th-day embryo.
Rat-mouse hybridoma antibodies were produced against mouse teratocarcinoma F9 or PCC4 aza1 cells, and four clones were established. Both the F11 (IgM) and F20 (IgG2c) antibodies showed a similar specificity, reacting only with nullipotential teratocarcinoma cells. They were also found to agglutinate sheep red blood cells. Solid-phase enzyme-linked immunofluorescence assay showed that, among the neutral glycolipids studied, they only reacted with the Forssman antigen. P2 antibody (IgG2b) reacted with the undifferentiated-type and embryonal endodermtype teratocarcinoma cells. During the preimplantation stage, this antibody did not stain mouse embryos, but it reacted very weakly with the inner cell mass of blastocysts cultured in vitro. In the 5th-day embryo, the embryonic ectoderm as well as the visceral and parietal endoderm were positive, but the extraembryonic ectoderm was not. Mesoderm of the 7.5th-day embryo also reacted with this antibody. However, P2 antigen was not observed in the 16th-day embryo or in adult tissues. F2 antibody (IgG2a), which was reactive with all of the cultured cell lines tested, showed an immunoreaction with mouse embryos throughout the preimplantation stage. However, in the 7.5th-day embryo, the presence of F2 was limited to the cells forming the parietal endoderm. This antigen was present in some epithelial tissues of the 16th-day embryo and adult mouse. Of these antigens, P2 and F2 are probably novel differentiation antigens of the early mouse embryo. Together with the Forssman antigen, these will be important markers for analyzing cell-surface antigens of mouse teratocarcinoma cells as well as embryos.