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Concerted action of two dlx paralogs in sensory placode formation.

Sensory placodes are ectodermal thickenings that give rise to elements of the vertebrate cranial sensory nervous system, including the inner ear and nose. Although mutations have been described in humans, mice and zebrafish that perturb ear and nose development, no mutation is known to prevent sensory placode formation. Thus, it has been postulated that a functional redundancy exists in the genetic mechanisms that govern sensory placode development. We describe a zebrafish deletion mutation, b380, which results in a lack of both otic and olfactory placodes. The b380 deletion removes several known genes and expressed sequence tags, including dlx3 and dlx7, two transcription factors that share a homoeobox domain similar in sequence to the Drosophila Distal-less gene. dlx3 and dlx7 are expressed in an overlapping pattern in the regions that produce the otic and olfactory placodes in zebrafish. We present evidence suggesting that it is specifically the removal of these two genes that leads to the otic and olfactory phenotype of b380 mutants. Using morpholinos, antisense oligonucleotides that effectively block translation of target genes, we find that functional reduction of both dlx genes contributes to placode loss. Expression patterns of the otic marker pax2.1, olfactory marker anxV and eya1, a marker of both placodes, in morpholino-injected embryos recapitulate the reduced expression of these genes seen in b380 mutants. We also examine expression of dlx3 and dlx7 in the morpholino-injected embryos and present evidence for existence of auto- and cross-regulatory control of expression among these genes. We demonstrate that dlx3 is necessary and sufficient for proper otic and olfactory placode development. However, our results indicate that dlx3 and dlx7 act in concert and their importance in placode formation is only revealed by inactivating both paralogs.

Animals↗

The role of FGF-3 in early inner ear development: an analysis in normal and kreisler mutant mice.

The development of the otic placode is believed to depend on an inductive signal from the adjacent hindbrain. A candidate for this signal is FGF-3 (Int-2), which is expressed in the hindbrain adjacent to the future ear in rhombomeres 5 and 6 (r5 and r6). However, in vitro tests (Represa et al. (1991), Nature 353, 561-563) conflict with findings from FGF-3 knockout mice (Mansour et al. (1993), Development 117, 13-28). The former suggest that FGF-3 from the hindbrain is required to induce formation of the otocyst, while the latter imply that FGF-3 is required only in the later process of otocyst differentiation. We find that in normal embryos at early stages the gene is expressed not only in r5 and r6, but also in most of the hindbrain anterior to this and in the head ectoderm in the prospective otic placode region. In kreisler mutant embryos, however, there is no heightened expression in r5 and r6, but the early patch of expression in the prospective otic placode ectoderm is still seen and the otic vesicle still forms at nearly the normal place. Subsequent malformations of the inner ear in kreisler and in FGF-3 knockout mice are similar, involving failure of the development of the endolymphatic appendage. These findings argue that FGF-3 is not required as an inductive signal for invagination of the otic placode to form a vesicle, whose future site is already marked out independently of any localized FGF-3 signal from r5 and r6. FGF-3 does, however, appear to be required for a correct pattern of differentiation within the vesicle.

Animals↗

A scanning electron-microscopic observation on the rat embryo with the frozen resin-cracking method.

The early embryos at the embryonal cylinder stage in rats were studied by scanning electron microscopy, employing the frozen resin-cracking method. An embryo at 15:00 o'clock on day 7 of pregnancy (L6-15) was exposed in the frozen resin-cracked surface. It was located at the antimesometrial side of the uterus and constructed of the embryonic node and trophoblasts, enclosing the blastocele. The embryonic node consisted of the primary ectodermal cell mass and the primary entodermal cells, surrounding the former. The ectodermal cells, forming a solid mass, are polygonal in shape and contain each a comparatively very large nucleus having a significant nucleolus. The entodermal cells are simple columnar or cuboidal in shape, contain numerous vacuoles of various sizes, in which threadlike structures forming a network are seen, and surround closely the ectodermal cell mass; they continue at the ectoplacental cone to the distal entodermal cells, which are very flattened, simple squamous in shape and line the inner surface of Reichert's membrane. In this specimen, the endometrial luminal epithelium has disappeared completely; the narrow blastocele is observed, but the primary amniotic cavity is not.

Animals↗

In vitro development of core cells of the inner cell mass of the mouse blastocyst: effects of conditioned medium.

Blastocysts submitted to two rounds of immunosurgery give rise to cores of presumptive ectoderm cells, many of which do not survive for more than 48 hours when cultured individually. Precoating of the culture plates with conditioned medium (CM) from PYS-2 cells increases the incidence with which cores regenerate an outer layer. This procedure also improves the survival frequency of the cores, but only for a limited period of time. The small number of cores which survive for two weeks or more, either in uncoated or CM-coated plates, give rise to any array of cell types, including giant cells resembling trophoblast.

Animals↗

Derivation of pluripotent, embryonic cell lines from the pig and sheep.

As previously described for the establishment of stable, pluripotent cell lines from pig blastocysts, an analogous cell line was isolated from a sheep blastocyst. There are common features in the morphologies and growth characteristics of the pig and sheep cells in culture; in particular, pig and sheep cells display large nuclei and relatively sparse cytoplasm, as is observed in mouse embryonic stem cells. Furthermore, the morphology of the sheep cells closely resembles that of cells in primary cultures of inner cell masses isolated immunosurgically from sheep blastocysts. This suggests that the sheep cell line represents a primary ectodermal lineage.

Animals↗

[New aspects in the genesis of inverted papillomas].

In the last 15 years papers dealing with the inverting papilloma (IP) of the nose and the paranasal sinuses mainly described the clinical appearance and behaviour and/or discussed treatment modalities. Little is known about the aetiology of the IP. Comparisons between the histological appearance of the IP and the different stages of the embryonic development of Schneider's membrane, i.e. the lamina mucosa nasi derived from the ectoderm, show strikingly similar growth patterns. The main task of Schneider's membrane is to actively form the inner nose and the paranasal sinuses during development of the foetus. Hence, it employs the system of "asymmetric dichotomy" when growing into the embryonic tissue of the viscerocranium. As the IP originates exclusively in the area equivalent to Schneider's membrane and moreover presents some unique features which are histologically and morphologically identical with this membrane, the author believes that the embryonic determination to build up the sinuses and the meatus of the nose is responsible for the histological appearance of the inverting papilloma.

Humans↗

Temporal and spatial patterns of gene expression for the hatching enzyme in the teleost embryo, Oryzias latipes.

The hatching enzyme of the medaka, Oryzias latipes, consists of two proteases, high choriolytic enzyme (HCE) and low choriolytic enzyme (LCE). They are synthesized and accumulated in the same unicellular hatching glands and are secreted from them at the end of embryonic development to digest the egg envelope. Recently, these enzymes were purified, and their cDNA clones were isolated. In the present study, we examined temporal and spatial patterns of expression of the hatching enzyme genes during embryogenesis using cDNAs for HCE and LCE as probes. According to Northern blotting analysis, the expression of both genes started at the same time (stage 21-22 embryos: brain differentiation and lens formation) and the patterns of expression changed in parallel during development. In situ hybridization to whole embryo and the sections revealed that the expression of the HCE genes was detected first in the anterior end of the hypoblast layer in stage 16-17 (late gastrula) embryos. Distinct signals of the HCE gene expression were then detected in a group of cells located at the front of the head rudiment of embryos at stage 18-19 (1 somite). Treatment of the embryos with retinoic acid, which is known to affect the anterior differentiation of embryos, suppressed the hatching gland cell differentiation in accordance with the result of in situ hybridization. In stage 22 embryos, the HCE-positive cells dispersed in an ectodermal layer under the forebrain and optic vesicles. Thereafter, the hatching gland cells expressing the HCE mRNA were aligned along the branchial arches and finally rearranged to the inner wall of the pharyngeal cavity, following a marked elongation of the lower jaw. The results of in situ hybridization to whole embryos at consecutive developmental stages demonstrated that the hatching gland cells located at the most anterior portion of the hypoblast migrated posteriorward to endoderm (pharyngeal endoderm) by way of ectoderm, while they were expressing mRNA for the hatching enzyme. Retinoic acid treatment of embryos gave rise to aberrations in the final location of the hatching gland cells probably by disturbing their migration. Moreover, the number of hatching gland cells increased markedly during their migration. This fact strongly suggested a concurrence of gene expression and mitosis of a gland cell and/or a successive initiation of gene expression in maturing gland cells during migration.

Animals↗

FGF is an essential regulator of the fifth cell division in preimplantation mouse embryos.

Fibroblast growth factor (FGF) signaling is required prior to gastrulation in the mouse embryo. To test for the spatial and temporal requirements of FGF signaling, a dominant negative FGF receptor (dnFGFR) was used to make transgenic mouse embryos. In mosaic embryos, cell division ceased at the fifth cell division in all cells that expressed the mutant receptor, but cell death did not increase. After the fifth cell division, the progeny of unaltered cells and cells expressing lacZ continued to accumulate at the same rate, suggesting that the FGF requirement is cell autonomous. In mosaic embryos, lacZ, but not dnFGFR expression was detected in mitotic trophoblasts adjacent to the ICM. Conversely, dnFGFR-expressing extraembryonic ectoderm cells were detected at the abembryonic pole in postmitotic cells. In blastocysts expressing the dnFGFR in all cells, the morphology appeared normal and inner cell masses (ICMs) formed, but resultant embryos had only one-third the number of cells as control embryos. In these blastocysts, cell division had also ceased at the fifth cell division, but cavitation, a concurrent morphogenetic event, initiated and progressed normally. To test for the continuing requirement of FGF, FGFR-3 was overexpressed in all cells and resulted in an increase in cell numbers after the fifth cell cycle. In a model for postimplantation development, addition of FGF-4 to blastocyst outgrowths increased the number of extraembryonic ectoderm cells, suggesting a continuing role for FGF. Thus, FGF signaling induces the cell division of embryonic and extraembryonic cells in the preimplantation mouse embryo starting at the fifth cell division. The signal requirement for FGF is cell autonomous, but is not required to prevent cell death. This provides the first evidence for the necessity of a growth factor before implantation.

Animals↗

In vitro differentiation of mouse embryonic stem cells after activation by retinoic acid.

Embryonic stem (ES) cells are pluripotent cells isolated from the inner cell mass of blastocysts. ES cells are able to differentiate into the three primitive layers (endoderm, mesoderm, and ectoderm) of the organism, including the germline. In recent reports mouse ES cells have been successfully applied in the treatment of spinal cord injury, hereditary myelin disorder of the central nervous system, and diabetes mellitus. In this study, we investigated the induction of mouse ES cell differentiation, using culture of embryoid bodies (EBs) into the diverse tissues. EBs were formed by culturing ES cells (129/SV strain) in DMEM supplemented with 10% FBS, in the absence of feeder cells and leukemia inhibitory factor (LF). EBs were induced to differentiate by treatment with retinoic acid (RA). In control medium (non-RA medium) beating muscles, blood vessels, hemocytes, and cartilages were frequently observed in EBs. Moreover, when EBs were cultured in medium including RA (5 x 10(-8) M, and 5 x 10(-9) M), differentiation of the optic vesicle, lens, retina, and neural groove was observed. In this study we demonstrated that an efficient system for inducing the differentiation of ES cells using EBs.

Animals↗

Embryoid bodies: an in vitro model of mouse embryogenesis.

Embryonic stem (ES) cells are pluripotent cells isolated from the inner cell mass of blastocysts. ES cells are able to differentiate into the three primitive layers (endoderm, mesoderm and ectoderm) of the organism, including the germline. To study early stages of development, as well as to investigate the impact of a gene knock-out in vitro, ES cells are differentiated into three-dimensional structures called embryoid bodies, because of their ability to mimick post-implantation embryonic tissues. This review summarises the work on ES cell differentiation into haematopoietic and vascular cells, neuronal and glial cells, myocytes, and adipocytes, using this in vitro model of early embryogenesis. We also present the potential of this method to analyse the impact of genetic alterations in vitro.

Animals↗

Inner ridge cells may be the main source of tectorial membrane type II collagen: evidence from quantitative mRNA in situ hybridization.

In a previous study we showed that COL2A1 mRNA is expressed in both ectodermally and mesodermally derived structures of second trimester human fetal cochlea, whereas type II collagen is present in mesodermally derived structures and in tectorial and basilar membranes. Because the tectorial membrane is acellular and therefore does not make its own proteins, the source of type II collagen and proteoglycans in this membrane has been of interest. We have attempted to address this issue, at least in part, by performing quantitative cRNA mRNA in situ hybridization on second trimester human fetal cochlear sections using a COL2A1 probe. By counting the number of silver grains cell in the interdental cells, inner sulcus cells and inner ridge Kolliker organ cells and by an analysis of variance of these quantitative data. inner ridge cells were found to have significantly higher levels of COL2A1 mRNA than interdental and inner sulcus cells (p < 0.0001). On the basis of significantly higher COL2A1 mRNA levels in inner ridge cells and their higher numbers than interdental and inner sulcus cells we postulate that type II collagen for human fetal tectorial membrane is derived mostly from inner ridge Kolliker organ cells. The lower COL2A1 mRNA in interdental cells appears to provide type II collagen for the spiral limbus and the tectorial membrane. The inner sulcus cells, hair cells. Deiter's and Hensen's cells also appear to contribute lesser amounts of type II collagen to the tectorial membrane. In analogy to these findings it is possible that other tectorial membrane proteins, including proteoglycans and other collagens, are also largely derived from these cells during human fetal development.

Cochlea↗

Expression of cytokeratin polypeptides during development of the rat inner ear.

The expression of cytokeratin polypeptides in the different epithelia of the developing inner ear of the rat from 12 days post conception to 20 days after birth was analysed immunohistochemically, using a panel of monoclonal antibodies. Throughout the development of the complex epithelial lining of the inner ear originating from the otocyst epithelium, only cytokeratins which are typical of simple epithelia were expressed. Cytokeratins 8, 18, and 19 were detectable shortly after the formation of the otocyst from the ectoderm (12 dpc), whereas cytokeratin 7 expression was delayed and first appeared in the vestibular portion and subsequently in the developing cochlear duct. During the development of the different types of specialized cells, differentiation-dependent modulation of the cytokeratin expression patterns was observed. In the mature inner ear, the specialized cell types displayed a function-related cytokeratin expression profile, both in the cochlear and vestibular portion. Cytokeratin expression in the flat epithelium of the vestibular portion suggests a more complex composition of this epithelium than has been established from routine morphology. Remarkably, the cochlear sensory cells were apparently devoid of cytokeratins, but no final conclusion could be drawn on the presence of cytokeratins in the sensory cells of the vestibular portion, because of the difficulty to delineate the cell borders between sensory cells and supporting cells.

Animals↗

Spatial and temporal pattern of Wnt-6 expression during chick development.

The WNT family of proteins is composed of several members. In the present study we isolated the full length chick Wnt-6 cDNA and analyzed its expression pattern by in situ hybridization during chick development. Wnt-6 expression is observed in the ectoderm from HH-stage 4 onwards. At HH-stages, 7-16 expression can be seen in the ectoderm overlying the segmental plate and the epithelial somite, while the ectoderm overlying the compartmentalized somite is Wnt-6 negative. Expression is also observed at the heart outflow tract and in the ectoderm overlying the pharyngeal arches. From HH-stages 17 to 27, expression is also observed at limb level, both in the dorsal and ventral ectoderm and a stronger expression in the dorsoventral boundary. Furthermore, expression in the ectoderm delimiting the somitic boundaries in the anteroposterior and mediolateral axis at limb level was observed, as well as in the ventral body wall. Expression becomes evident in the inner ear. From HH-stage 30 onwards, expression is restricted to the feather buds and to the gastrointestinal tract.

Amino Acid Sequence↗

Ventrally emigrating neural tube cells migrate into the developing vestibulocochlear nerve and otic vesicle.

Virtually all cell types in the inner ear develop from the cells of the otic vesicle. The otic vesicle is formed by the invagination of non-neural ectodermal cells known as the otic placode. We investigated whether a recently described cell population, originating from the ventral part of the hindbrain neural tube known as the ventrally emigrating neural tube (VENT) cells, also contributes cells to the otic vesicle. The ventral hindbrain neural tube cells were labeled with the fluorescent vital dye DiI or replication-deficient retroviruses containing the LacZ gene in chick embryos on embryonic day 2, after the emigration of neural crest from this region. One day later, the labeled cells were detected only in the hindbrain neural tube. Shortly thereafter, the labeled cells began to appear in the eighth (vestibulocochlear) cranial nerve and otic vesicle. From embryonic day 3.5-5, the labeled cells were detected in the major derivatives of the otic vesicle, i.e. the endolymphatic duct, semicircular canals, utricle, saccule, cochlea, and vestibulocochlear ganglion. That the emigrated cells originated from the ventral part of the hindbrain neural tube was confirmed by focal application of DiI impregnated filter paper and with quail chimeras. It is concluded that, in addition to the otic placode cells, the otic vesicle also contains the ventrally emigrating neural tube cells, and that both cell populations contribute to the structures and cell types in the inner ear. It is well known that inductive signals from the hindbrain are required for the morphogenesis of the inner ear. The migration of the hindbrain neural tube cells into the otic vesicle raises the possibility that the inductive effect of the hindbrain might be mediated, at least in part, by the ventrally emigrating neural tube cells and that, therefore, a mechanism exists that involves cells rather than diffusible molecules only.

Animals↗

Spatiotemporal distribution of SPARC/osteonectin in developing and mature chicken retina.

Expression of SPARC (Secreted Protein, Acidic, Rich in Cysteine), a counteradhesive, calcium-binding extracellular matrix (ECM) glycoprotein, is associated with several morphogenetic events during early development. In this study, changes in the spatiotemporal distribution of SPARC transcripts and the protein during chicken retinal development were documented by in situ hybridization and indirect immunofluorescence microscopy. SPARC transcripts were first detected within the proliferating neural ectoderm at embryonic day 4. 5 (E4.5), followed short thereafter (E5) by appearance of SPARC. SPARC was enriched within the inner plexiform layer (IPL) by E10 and within the outer plexiform layer (OPL) by E14, several days after these layers became morphologically distinct. Significant levels of SPARC transcripts were first observed within the ganglion cell layer (GCL) at E17 prior to accumulation of SPARC within the nerve fiber layer, seen first at E20. SPARC protein was first detected within the developing retinal pigment epithelium (RPE) at E10 and increased significantly at RPE cells ceased to proliferate and continued differentiating. Of special note was the restriction of SPARC to the basal-half of the RPE cells. SPARC transcripts were similarly distributed in the adult retina, but at lower levels than in the period just prior to hatching. In the adult retina SPARC was retained in the nerve fiber layer and present in the inner nuclear layer (INL) and outer nuclear layer (ONL), but lost from the IPL and OPL. These changes in expression pattern with time indicate that SPARC is developmentally regulated and therefore may have important function(s) in both morphological development of the retina and functioning of the mature eye.

Amino Acid Sequence↗

A monoclonal antibody, MA21, recognizes a surface component that is present on F9 teratocarcinoma cells and that appears vectorially on the trophectoderm of peri-implantation-stage mouse blastocysts.

A monoclonal antibody (MAb) "MA21", derived from lymphoid tissue of a multiparous mouse and selected for binding to mouse teratocarcinoma cell line F9, recognizes a surface antigen that appears on peri-implantation-stage mouse blastocysts. In indirect immunofluorescence assays, MAb MA21 does not bind to 1-cell-through morula-stage embryos, nor to early, 3.5-day post-coitum (p.c.) blastocysts. When 3.5-day p.c. blastocysts are maintained 17 h in vitro and then assayed, MAb MA21 binds to a limited number of trophectoderm cells that are centered at the embryonic pole. As culture time lengthens, the number of antigen-expressing trophectoderm cells increases, forming a cap that spreads from the embryonic pole into the abembryonic region. Embryos maintained 48 h in vitro bind MAb MA21 over as much as 100% of the trophectoderm surface. MAb MA21 does not bind to the inner cell mass. When mouse pregnancy uteri are assayed by the immunoperoxidase method, MAb MA21 binds to extra-embryonic ectoderm and trophectoderm of 5-day p.c. implanted blastocysts, but does not bind to 6-day p.c. blastocysts. MAb MA21 recognizes a component with an estimated mol. wt of 44,000 from NP-40 detergent extracts of F9 cells and peri-implantation-stage mouse blastocysts. The component appears to be firmly associated with the plasma membrane; it is resistant to removal by high salt or moderate concentrations of non-ionic detergent.

Animals↗