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TGFbeta2 acts as an "activator" molecule in reaction-diffusion model and is involved in cell sorting phenomenon in mouse limb micromass culture.

It was previously speculated that TGFbeta acts as an "activator"-molecule in chondrogenic pattern formation in the limb micromass culture system, but its precise role and relationship with the cell sorting phenomenon have not been properly studied. In the present study, we examined whether the TGFbeta2 molecule satisfies the necessary conditions for an "activator"-molecule in the reaction-diffusion model. Firstly, we showed that TGFbeta2 became localized at chondrogenic sites during the establishment of a chondrogenic pattern, and exogenous TGFbeta2 promoted chondrogenesis when added in the culture medium. Secondly, TGFbeta2 protein was shown to promote the production of its own mRNA after 3 hr, indicating that a positive feedback mechanism exists which may be responsible for the emergence of the chondrogenic pattern. We then found that when locally applied with beads, TGFbeta2 suppressed chondrogenesis around the beads, indicating it induces the lateral inhibitory mechanism, which is a key element for the formation of the periodic pattern. We also examined the possible effects of TGFbeta2 on the cell sorting phenomenon and found that TGFbeta2 exerts differential chemotactic activity on proximal and distal mesenchyme cells of the limb bud, and at very early phases of differentiation TGFbeta2 promotes the expression of N-cadherin protein which is known to be involved in pattern formation in this culture system. These findings suggest that TGFbeta2 acts as an "activator"-like molecule in chondrogenic pattern formation in vitro, and is possibly responsible for the cell sorting phenomenon.

Animals↗

Can't get no SMADisfaction: Smad proteins as positive and negative regulators of TGF-beta family signals.

The identification of Smad proteins as molecular components of the transforming growth factor-beta (TGF-beta) signaling cascade has enhanced our understanding of how ligand-mediated activation of TGF-beta receptors leads to modulation of target gene transcription. Recent studies have identified a distinct, structurally related class of Smads which inhibits, rather than transduces, TGF-beta family signals. The molecular mechanism of action and the exact signaling pathways that are targeted by antagonistic Smads are not completely understood. These proteins appear to participate in autoregulatory negative feedback loops in which signaling initiated by specific TGF-beta family ligands induces the expression of an inhibitory Smad that then functions to modulate the amplitude or duration of signaling. Negative feedback circuits such as these play important roles in fine-tuning the activity of multifunctional signaling molecules during embryonic patterning and in response to pathologic stimuli in adults.

Amino Acid Sequence↗

Expression of the zebrafish Iroquois genes during early nervous system formation and patterning.

Iroquois genes are involved in many patterning processes during development. In particular, they act as prepattern genes to control proneural gene expression both in Drosophila and in vertebrates. In this paper, we have analyzed the expression during embryogenesis of the 11 zebrafish Iroquois genes, with special interest for nervous system formation and patterning. During the first 2 days of development, Iroquois genes are expressed in distinct domains in the neuroepithelium, as well as in groups of neuronal progenitors and neurons. They are also expressed at different stages of placodal development. These expression patterns are similar to the patterns of the murine irx genes and also show features specific to teleosts. For the zebrafish Iroquois gene family, we find both specific patterns and patterns conserved within a cluster, between paralogues, or in most genes of the family. Overall, these expression data suggest functions for the Iroquois family of transcription factors in neural and placodal patterning, neurogenesis, and neuronal specification.

Animals↗

Villin: A marker for development of the epithelial pyloric border.

In the adult gastrointestinal tract, the morphologic borders between esophagus and stomach and between stomach and small intestine are literally one cell thick. The patterning mechanisms that underlie the development of these sharp regional divisions from a once continuous endodermal tube are still obscure. In the embryonic endoderm of the developing gut, region-specific expression of certain genes (e.g., intestine-specific expression of the actin bundling protein villin) can be detected as early as 9.0 days post coitum, although the morphologic differentiation of the gut epithelium proper does not begin until 4 to 5 days later. By using a mouse model in which a beta-galactosidase marker has been inserted into the endogenous villin locus, we examined the development of the stomach/intestinal (pyloric) border during gut organogenesis. The data indicate that the border is not sharp from the outset. Rather, the initial border region is characterized by a decreasing gradient of villin/beta-galactosidase expression that extends into the distal stomach. A sharp epithelial border of villin/beta-galactosidase expression appears abruptly at day 16 and is further refined over the next 3 weeks to form the distinct one-cell-thick border characteristic of the adult. These results indicate that an important previously unrecognized patterning event occurs in the gut epithelium at 16 days; this event may define an epithelial compartment boundary between the stomach and the intestine. The villin/beta-galactosidase mouse model characterized here provides an excellent substrate with which to further dissect the mechanisms involved in this patterning process.

Animals↗

Vertebrate limb development: from Harrison's limb disk transplantations to targeted disruption of Hox genes.

Various animal organs have long been used to investigate the cellular and molecular nature of embryonic growth and morphogenesis. Among those organs, the tetrapod limb has been preferentially used as a model system for elucidating general patterning mechanisms. At the appropriate time during the embryonic period, the limb territories are first determined at the right positions along the cephalocaudal axis of the animal body, and soon the limb buds grow out from the flanks as mesenchymal cell masses covered by simple ectoderm. The position, number, and identity of the limbs depend on the expression of specific Hox genes. Limb morphogenesis occurs along three axes, which become gradually fixed: first the anteroposterior axis, then the dorsoventral, and finally the proximodistal axis, along which the bulk of limb growth occurs. Growth of the limb in amniotes depends on the formation of the apical ectodermal ridge, which, by secreting many members of the fibroblast growth factors family, attracts lateral plate and somitic mesodermal cells, keeps these cells in the progress zone proliferating, and prevents their differentiation until an appropriate time period. Mutual interactions between mesoderm and ectoderm are important in the growth process, and signaling regions have been identified, such as the zone of polarizing activity, the dorsal limb ectoderm, and the apical ectodermal ridge. Several molecules have been found to play leading roles in various biological processes relevant to morphogenesis. Besides its intrinsic merit as a model for unraveling the mechanisms of development, the limb deserves considerable clinical interest because defects of limb development are the most common single category of congenital abnormalities.

Animals↗

Axial patterning and diversification in the cnidaria predate the Hox system.

Across the animal kingdom, Hox genes are organized in clusters whose genomic organization reflects their central roles in patterning along the anterior/posterior (A/P) axis . While a cluster of Hox genes was present in the bilaterian common ancestor, the origins of this system remain unclear (cf. ). With new data for two representatives of the closest extant phylum to the Bilateria, the sea anemone Nematostella and the hydromedusa Eleutheria, we argue here that the Cnidaria predate the evolution of the Hox system. Although Hox-like genes are present in a range of cnidarians, many of these are paralogs and in neither Nematostella nor Eleutheria is an equivalent of the Hox cluster present. With the exception of independently duplicated genes, the cnidarian genes are unlinked and in several cases are flanked by non-Hox genes. Furthermore, the cnidarian genes are expressed in patterns that are inconsistent with the Hox paradigm. We conclude that the Cnidaria/Bilateria split occurred before a definitive Hox system developed. The spectacular variety in morphological and developmental characteristics shown by extant cnidarians demonstrates that there is no obligate link between the Hox system and morphological diversity in the animal kingdom and that a canonical Hox system is not mandatory for axial patterning.

Animals↗

Simulated morphogenesis of developmental folds due to proliferative pressure.

We present a simulation that models individual cells as spherical particles that can migrate, interact, divide and differentiate. We simulate the evolution of a progenitor layer of cells that reproduce, leading either to more progenitors or to differentiated daughters. We find that this simplified model produces spontaneous folds whose lengths depend linearly on the ratio of rates of production of progenitors to differentiated daughters. We also find that folds grow approximately exponentially in time, and that larger folds can be placed via patterning events that perturb the positions of selected progenitor cells early in the developmental process.

Animals↗

Fgf19 expression patterns in the developing chick inner ear.

The inner ear is a complex sensorial structure with hearing and balance functions. A key aim of developmental biology is to understand the molecular and cellular mechanisms involved in the induction, patterning and innervation of the vertebrate inner ear. These developmental events could be mediated by the expression of regulating genes, such as the members of the family of Fibroblast Growth Factors (Fgfs). This work reports the detailed spatial and temporal patterns of Fgf19 expression in the developing inner ear from otic cup (stage 14) to 8 embryonic days (stage 34). In the earliest stages, Fgf19 and Fgf8 expressions determine two subdomains within the Fgf10-positive proneural-sensory territory. We show that, from the earliest stages, the Fgf19 expression was detected in the acoustic-vestibular ganglion and the macula utriculi. The Fgf19 gene was also strongly, but transiently, expressed in the macula lagena, whereas the macula neglecta never expressed this gene in the period analysed. The Fgf19 expression was also clearly observed in some borders of various sensory elements. These results could be useful from further investigations into the role of FGF19 in otic patterning.

Animals↗

EphA7 receptor is expressed differentially at chicken prosomeric boundaries.

We reexamined tyrosine-kinase receptor EphA7 RNA signal in embryonic chicken forebrain, to clarify its topographic relationships with early regionalization processes, such as establishment of prosomeric boundaries. After neurulation, uniform alar expression appears across prospective prosomeres prosomere 1, prosomere 2 and prosomere 3 (prethalamus, thalamus and pretectum). This pattern soon changes by differential downregulation at or in between some of the prosomeric boundaries in an individual pattern for each limit, and by expansion of expression into the rostral midbrain. The secondary distribution highlights various transversal and longitudinal domains, notably the zona limitans intrathalamica and the pretectum limits, as well as two longitudinal bands in the basal plate, termed paramedian and parabasal. Strong expression of EphA7 appears at the mammillary pouch and a supramammillary tegmental arch from stage Hamburger and Hamilton stages 14-15 onwards. At the end of the developmental period examined, expression of EphA7 in the ventricular zone decreases generally (with some exceptions) and novel expression domains start to appear in the mantle layer, initiating a third phase of differential expression. Thus, while the expression of EphA7 does not show a fixed functional or topographic relationship to prosomeric boundaries, sequential transcription changes during chicken development are consistent with a differential involvement of the diverse interprosomeric boundaries, as well as dorsoventral patterning organizers, in the regulation of EphA7 expression.

Animals↗

Neonatal exposure to short days and low temperatures blunts stress response and yields low fluctuating asymmetry in Siberian hamsters.

Fluctuating asymmetry (FA) refers to small, non-directional deviations from perfect bilateral symmetry in morphological characters. Individuals with low FA presumably either developed in a relatively stable environment and/or were better able to buffer against developmental stressors. The present study investigated the effects of seasonal factors measured by day length and ambient temperature manipulations on the development of bilateral characters and concomitant changes in stress responses. Siberian hamsters were exposed to either long days (16 h of light per day) or short days (8 h of light per day) combined with either standard temperatures (21+/-2 degrees C) or low temperatures (8+/-2 degrees C) on the day of birth until weaning. Cortisol concentrations at baseline and following acute restraint stress, and FA values were measured in adulthood. Females reared in winter-like conditions with short day lengths and low temperatures had low FA and low cortisol concentrations following restraint stress compared to other females. Females reared in long day lengths and standard temperatures had the highest rate of increase in cortisol concentrations after restraint among other female groups. No group effects were observed in males regarding day length and temperature manipulations. Baseline and post-restraint cortisol concentrations were higher in females than males for all groups except in animals reared in short day lengths and low temperatures. Our results suggest that winter-like conditions during neonatal period evoke hyposensitivity to stress in adult females and this blunted response to stress is a key factor in achieving ideal growth patterns.

Adaptation, Physiological↗

Gradients and thresholds: BMP response gradients unveiled in Drosophila embryos.

Bone morphogenetic proteins (BMP) direct dorsal-ventral patterning in both invertebrate and vertebrate embryos, with strong evolutionary conservation of molecular components of the pathway. Dorsal-ventral patterning of the early Drosophila embryo is a powerful experimental system to probe mechanisms of BMP gradient formation and interpretation. Recent studies have found that spatial patterns of activated BMP signal transducers in Drosophila go through an unexpected transition: a shallow gradient of weak responses at mid-cellularization changes to a step gradient of stronger responses in cellularized embryos. The transition between two gradients of different shape yields new insights into the progression of Drosophila dorsal-ventral patterning and raises new issues about the mechanisms of gradient formation.

Animals↗

Temporal analysis of the early BMP functions identifies distinct anti-organizer and mesoderm patterning phases.

BMP signaling performs multiple important roles during early embryogenesis. Signaling through the BMP pathway is mediated by different BMP ligands expressed in partially overlapping temporal and spatial patterns. Assignment of different BMP-dependent activities to the individual ligands has relied on the patterns of expression of the various BMP genes. Temporal analysis of BMP signaling prior to and during gastrulation was performed using glucocorticoid-controlled Smad proteins. Overexpression of the BMP-specific Smad1 and Smad5 revealed that suppression of Spemann's organizer formation in Xenopus embryos can only take place by activating the BMP pathway prior to the onset of gastrulation. Blocking BMP signaling with the inhibitory Smad, Smad6, results in dorsalized embryos or secondary axis induction, only when activated up to early gastrula stages. BMP2 efficiently represses organizer-specific transcription from the midblastula transition onwards while BMP4 is unable to prevent the early activation of organizer-specific genes. Manipulation of the BMP pathway during mid/late gastrula affects mesodermal patterning with no external phenotypic effects. These observations suggest that the malformations resulting from inhibition or promotion of organizer formation, ventralized or dorsalized, respectively, are the result of a very early BMP function, through its antagonism of organizer formation. This function is apparently fulfilled by BMP2 and only at its latest phase by BMP4. Subsequently, BMP functions in the patterning of the mesoderm with no apparent phenotypic effects.

Animals↗

tortuga refines Notch pathway gene expression in the zebrafish presomitic mesoderm at the post-transcriptional level.

We have identified the zebrafish tortuga (tor) gene by an ENU-induced mutation that disrupts the presomitic mesoderm (PSM) expression of Notch pathway genes. In tor mutants, Notch pathway gene expression persists in regions of the PSM where expression is normally off in wild type embryos. The expression of hairy/Enhancer of split-related 1 (her1) is affected first, followed by the delta genes deltaC and deltaD, and finally, by another hairy/Enhancer of split-related gene, her7. In situ hybridization with intron-specific probes for her1 and deltaC indicates that transcriptional bursts of expression are normal in tor mutants, suggesting that tor normally functions to refine her1 and deltaC message levels downstream of transcription. Despite the striking defects in Notch pathway gene expression, somite boundaries form normally in tor mutant embryos, although somitic mesoderm defects are apparent later, when cells mature to form muscle fibers. Thus, while the function of Notch pathway genes is required for proper somite formation, the tor mutant phenotype suggests that precise oscillations of Notch pathway transcripts are not essential for establishing segmental pattern in the presomitic mesoderm.

Animals↗

Bone morphogenetic proteins and noggin: inhibiting and inducing fungiform taste papilla development.

Fungiform papillae are epithelial specializations that develop in a linear pattern on the anterior mammalian tongue and differentiate to eventually contain taste buds. Little is known about morphogenetic and pattern regulation of these crucial taste organs. We used embryonic rat tongue, organ cultures to test roles for bone morphogenetic proteins, BMP2, 4 and 7, and antagonists noggin and follistatin, in development of papillae from a stage before morphological initiation (E13) or from a stage after the pre-papilla placodes have formed (E14). BMPs and noggin proteins become progressively restricted to papilla locations during tongue development. In E13 cultures, exogenous BMPs or noggin induce increased numbers of fungiform papillae, in a concentration-dependent manner, compared to standard tongue cultures; BMPs, but not noggin, lead to a decreased tongue size at this stage. In E14 cultures, however, exogenous BMP2, 4 or 7 each inhibits papilla formation so that there is a decrease in papilla number. Noggin substantially increases number of papillae in E14 cultures. Using beads for a highly localized protein delivery, papillae are inhibited in the surround of BMP-soaked beads and induced in large clusters around noggin-soaked beads. Follistatin, presented in culture medium or by bead, does not alter papilla formation or number. In all fungiform papillae that form under various culture conditions, the molecular marker, sonic hedgehog, is within each papilla. However, the BMP inhibitory effect on papillae is not prevented by disrupting sonic hedgehog signaling through addition of cyclopamine to cultures. BMPs and noggin alter cell proliferation in tongue epithelium in opposite ways, demonstrated with Ki67 immunostaining. We propose that the BMPs and noggin, colocalized within papilla placodes and the fungiform papillae per se, have opposing inhibitory and activating or inducing roles in papilla development in linear patterns. We present a model for these effects.

Animals↗

Dmbx1, a novel evolutionarily conserved paired-like homeobox gene expressed in the brain of mouse embryos.

To identify novel homeobox genes expressed during mouse embryogenesis, we searched the databases and found a novel mouse paired-like homeobox gene, Dmbx1(diencephalon/mesencephalon-expressed brain homeobox gene 1), that is also conserved in zebrafish and human. Linkage analysis mapped mouse Dmbx1 to the mid-portion of chromosome 4 that is the homologous gene cluster region of human chromosome 1, where human DMBX1 is located. Both mouse and human Dmbx1/DMBX1 have four coding exons and their gene structures are conserved. Whole-mount in situ hybridization revealed that Dmbx1 expression is detected in 7.5-9.5 dpc mouse embryos. At 7.5 and 8.5 dpc, Dmbx1 is expressed in a sub-region of the anterior head folds. At 9.5 dpc, expression is observed in the caudal diencephalon as well as in the mesencephalon and is restricted to the neuroepithelium. Expression in adult tissues was detected in brain, stomach, and testis. Dmbx1 provides a unique marker of the developing anterior nervous system and should provide a useful molecular resource to elucidate the mechanisms that pattern the vertebrate brain.

Amino Acid Sequence↗

Gene expression screening in Xenopus identifies molecular pathways, predicts gene function and provides a global view of embryonic patterning.

In a large-scale gene expression screen 1765 randomly picked cDNAs were analyzed by whole-mount in situ hybridization in Xenopus embryos. Two hundred and seventy three unique, differentially expressed genes were identified, 204 of which are novel in Xenopus. Partial DNA sequences and expression patterns were documented and assembled into a database, 'AXelDB'. Approximately 30% of cDNAs analyzed represent differentially expressed genes and about 5% show highly regionalized expression. Novel marker genes and potential developmental regulators were found. Differential expression of mitochondrial genes was observed. Marker genes were used to study regionalization of the entire gastrula as well as the tail forming region and the epidermis of the tailbud embryo. Four 'synexpression' groups representing genes with shared, complex expression pattern that predict molecular pathways involved in patterning and differentiation were identified. According to their probable functional significance these groups are designated as Delta1, Bmp4, ER-import and Chromatin group. Within synexpression groups, a likely function of genes without sequence similarity can be predicted. The results indicate that synexpression groups have strong prognostic value. A cluster analysis was made by comparing gene expression patterns to derive a novel parameter, 'tissue relatedness'. In conclusion, this study describes a semi-functional approach to investigate genes expressed during early development and provides global insight into embryonic patterning.

Animals↗

The cytoplasmic dynein and kinesin motors have interdependent roles in patterning the Drosophila oocyte.

BACKGROUND: Motor proteins of the minus end-directed cytoplasmic dynein and plus end-directed kinesin families provide the principal means for microtubule-based transport in eukaryotic cells. Despite their opposing polarity, these two classes of motors may cooperate in vivo. In Drosophila circumstantial evidence suggests that dynein acts in the localization of determinants and signaling factors during oogenesis. However, the pleiotropic requirement for dynein throughout development has made it difficult to establish its specific role. RESULTS: We analyzed dynein function in the oocyte by disrupting motor activity through temporally restricted expression of the dynactin subunit, dynamitin. Our results indicate that dynein is required for several processes that impact patterning; such processes include localization of bicoid (bcd) and gurken (grk) mRNAs and anchoring of the oocyte nucleus to the cell cortex. Surprisingly, dynein function is sensitive to reduction in kinesin levels, and germ line clones lacking kinesin show defects in dorsal follicle cell fate, grk mRNA localization, and nuclear attachment that are similar to those resulting from the loss of dynein. Significantly, dynein and dynactin localization is perturbed in these animals. Conversely, kinesin localization also depends on dynein activity. CONCLUSIONS: We demonstrate that dynein is required for nuclear anchoring and localization of cellular determinants during oogenesis. Strikingly, mutations in the kinesin motor also disrupt these processes and perturb dynein and dynactin localization. These results indicate that the activity of the two motors is interdependent and suggest a model in which kinesin affects patterning indirectly through its role in the localization and recycling of dynein.

Animals↗

Coordinating proliferation and tissue specification to promote regional identity in the Drosophila head.

The Decapentaplegic and Notch signaling pathways are thought to direct regional specification in the Drosophila eye-antennal epithelium by controlling the expression of selector genes for the eye (Eyeless/Pax6, Eyes absent) and/or antenna (Distal-less). Here, we investigate the function of these signaling pathways in this process. We find that organ primordia formation is indeed controlled at the level of Decapentaplegic expression but critical steps in regional specification occur earlier than previously proposed. Contrary to previous findings, Notch does not specify eye field identity by promoting Eyeless expression but it influences eye primordium formation through its control of proliferation. Our analysis of Notch function reveals an important connection between proliferation, field size, and regional specification. We propose that field size modulates the interaction between the Decapentaplegic and Wingless pathways, thereby linking proliferation and patterning in eye primordium development.

Animals↗