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A conserved developmental program for sensory organ formation in Drosophila melanogaster.

Different sensory organs, such the eye and ear, are widely thought to have separate origins, guided by distinct organ-specific factors that direct all aspects of their development. Previous studies of the D. melanogaster gene eyeless (ey) and its vertebrate homolog Pax6 suggested that this gene acts in such a manner and specifically drives eye development. But diverse sensory organs might instead arise by segment-specific modification of a developmental program that is involved more generally in sensory organ formation. In D. melanogaster, a common proneural gene called atonal (ato) functions in the initial process of development of a number of segment-specific organs, including the compound eye, the auditory organ and the stretch receptor, suggesting that these organs share an evolutionary origin. Here we show that D. melanogaster segment-specific sensory organs form through the integration of decapentaplegic (dpp), wingless (wg) and ecdysone signals into a single cis-regulatory element of ato. The induction of ectopic eyes by ey also depends on these signals for ato expression, and the ey mutant eye imaginal disc allows ato expression if cell death is blocked. These results imply that ey does not induce the entire eye morphogenetic program but rather modifies ato-dependent neuronal development. Our findings strongly suggest that various sensory organs evolved from an ato-dependent protosensory organ through segment specification by ey and Hox genes.

Animals↗

Regional repression of a Drosophila POU box gene in the endoderm involves inductive interactions between germ layers.

An induction process occurring between the mesodermal and the endodermal germ layers has recently been described in the regulation of the Drosophila homeotic gene labial (lab). We report here that proper spatial regulation of the Drosophila POU box gene pdm-1 products also involves interaction between these two germ layers. pdm-1 transcripts are initially present in both the anterior and the posterior endodermal midgut primordia. Upon fusion of the two primordia, transcripts disappear from two regions in the endoderm, a central domain and an anterior domain. The anterior repression domain of pdm-1 is independent of the expression of known homeotic genes and genes encoding secreted signalling molecules in the visceral mesoderm, both for its positioning and its repression. Repression in the central domain requires both the homeotic gene Ultrabithorax (Ubx) and the decapentaplegic (dpp) gene, which encodes a secreted protein. Both of these genes are also required for lab induction. However, the analysis of pdm-1 expression in various mutant backgrounds indicates that the regulation of lab and pdm-1 across germ layers is controlled by different genetic cascades. Our study indicates that dpp is not the signal that dictates central pdm-1 repression across germ layers and suggests that in the same midgut region, different signalling pathways result in the differential activation or repression of potential transcription factors.

Animals↗

Morphogenesis of the C. elegans hermaphrodite uterus.

We have undertaken electron micrographic reconstruction of the Caenorhabditis elegans hermaphrodite uterus and determined the correspondence between cells defined by their lineage history and differentiated cell types. In this organ, many cells do not move during morphogenesis and the cell lineage may function to put cells where they are needed. Differentiated uterine cell types include the toroidal ut cells that make structural epithelium, and specialized utse and uv cells that make the connection between the uterus and the vulva. A cell fate decision in which the anchor cell (AC) induces adjacent ventral uterine intermediate precursor cells to adopt the pi fate, rather than the ground state rho, has profound consequences for terminal differentiation: all pi progeny are directly involved in making the uterine-vulval connection whereas all rho progeny contribute to ut toroids or the uterine-spermathecal valve. In addition to specifying certain uterine cell fates, the AC also induces the vulva. Its multiple inductions thereby function to coordinate the connection of an internal to an external epithelium. The AC induces the pi cells and ultimately fuses with a subset of their progeny. This is an example of reciprocal cell-cell interaction that can be studied at single cell resolution. The AC is thus a transitory cell type that plays a pivotal role in organizing the morphogenesis of the uterine-vulval connection.

Animals↗

Epidermal induction and inhibition of neural fate by translation initiation factor 4AIII.

Bone Morphogenetic Protein-4 (BMP-4) is a potent epidermal inducer and inhibitor of neural fate. We have used differential screening to identify genes involved in epidermal induction downstream of BMP-4 and report here evidence of a novel translational mechanism that regulates the division of the vertebrate ectoderm into regions of neural and epidermal fate. In dissociated Xenopus ectoderm, addition of ectopic BMP-4 leads to an increase in the expression of translation initiation factor 4AIII (eIF-4AIII), a divergent member of the eIF-4A gene family until now characterized only in plants. In the gastrula embryo, Xenopus eIF-4AIII (XeIF-4AIII) expression is elevated in the ventral ectoderm, a site of active BMP signal transduction. Moreover, overexpression of XeIF-4AIII induces epidermis in dissociated cells that would otherwise adopt a neural fate, mimicking the effects of BMP-4. Epidermal induction by XeIF-4AIII requires both an active BMP signaling pathway and an extracellular intermediate. Our results suggest that XeIF-4AIII can regulate changes in cell fate through selective mRNA translation. We propose that BMPs and XeIF-4AIII interact through a positive feedback loop in the ventral ectoderm of the vertebrate gastrula.

Amino Acid Sequence↗

[Induction of the mesoderm and primordial germ cells by the endoderm of Pleurodeles waltlii (Amphibia, Urodele): development during gastrulation].

Blastulae ectoderm is combined with dorsal or ventral endoderm from blastulae, gastrulae and early neurulae. In vitro culture reveals the presence of different mesodermal structures whose nature is connected with the endoderm origin site. Primordial germ cells differentiate essentially in the recombinates including ventral endoderm. The inducing capacity of this latter concerning germ cells is maximum at the beginning of gastrulation, then decreases during it and finally disappears at the onset of neurulation.

Age Factors↗

Dorsal differentiation of neural plate cells induced by BMP-mediated signals from epidermal ectoderm.

The cellular interactions that control the differentiation of dorsal cell types from neural progenitors have been examined in neural plate explants. Certain genes that are expressed in the dorsal neural tube are initially expressed uniformly within the neural plate and appear to achieve their dorsal restriction through a Sonic hedgehog (SHH)-mediated repressive signal from the notochord. The acquisition of definitive dorsal cell fates, however, requires a contact-dependent signal from the epidermal ectoderm. BMP4 and BMP7 are expressed in the epidermal ectoderm, and both proteins mimic its inductive activity. BMP4 and a related gene, DSL1, are subsequently expressed by cells in the dorsal neural tube. The differentiation of dorsal cell types, therefore, appears to be initiated at the neural plate stage and to involve the opponent activities of a BMP-mediated dorsalizing signal from the epidermal ectoderm and a SHH-mediated ventralizing signal from the notochord.

Animals↗

The C. elegans ksr-1 gene encodes a novel Raf-related kinase involved in Ras-mediated signal transduction.

Vulval induction in C. elegans is controlled by a highly conserved signaling pathway similar to the RTK-Ras-MAPK cascade in mammals. By screening for suppressors of the Multivulva phenotype caused by an activated let-60 ras allele, we isolated mutations in a gene, ksr-1, that acts as a positive modifier of vulval induction and is required for at least two other let-60 ras-mediated processes. Although ksr-1 mutations do not perturb vulval induction in an otherwise wild-type background, they have very strong effects on vulval induction in genetic backgrounds where Ras pathway activity is constitutively activated or compromised, suggesting that ksr-1 activity is required for maximal stimulation of vulval fates by the Ras pathway. Genetic epistasis analysis suggests that ksr-1 acts downstream of or in parallel to let-60 ras. We cloned ksr-1 and have shown that it encodes a novel putative protein kinase related to the Raf family of Ser/Thr kinases.

Amino Acid Sequence↗

XTcf-3 transcription factor mediates beta-catenin-induced axis formation in Xenopus embryos.

XTcf-3 is a maternally expressed Xenopus homolog of the mammalian HMG box factors Tcf-1 and Lef-1. The N-terminus of XTcf-3 binds to beta-catenin. Microinjection of XTcf-3 mRNA in embryos results in nuclear translocation of beta-catenin. The beta-catenin-XTcf-3 complex activates transcription in a transient reporter gene assay, while XTcf-3 by itself is silent. N-terminal deletion of XTcf-3 (delta N) abrogates the interaction with beta-catenin, as well as the consequent transcription activation. This dominant-negative delta N mutant suppresses the induction of axis duplication by microinjected beta-catenin. It also suppresses endogenous axis specification upon injection into the dorsal blastomeres of a 4-cell-stage embryo. We propose that signaling by beta-catenin involves complex formation with XTcf-3, followed by nuclear translocation and activation of specific XTcf-3 target genes.

Amino Acid Sequence↗

Neuronal patterning by BMPs: a requirement for GDF7 in the generation of a discrete class of commissural interneurons in the mouse spinal cord.

Inductive factors are known to direct the regional differentiation of the vertebrate central nervous system (CNS) but their role in the specification of individual neuronal cell types is less clear. We have examined the function of GDF7, a BMP family member expressed selectively by roof plate cells, in the generation of neuronal cell types in the dorsal spinal cord. We find that GDF7 can promote the differentiation in vitro of two dorsal sensory interneuron classes, D1A and D1B neurons. In Gdf7-null mutant embryos, the generation of D1A neurons is eliminated but D1B neurons and other identified dorsal interneurons are unaffected. These findings show that GDF7 is an inductive signal from the roof plate required for the specification of neuronal identity in the dorsal spinal cord and that GDF7 and other BMP family members expressed by the roof plate have non-redundant functions in vivo. More generally, these results suggest that BMP signaling may have a prominent role in the assignment of neuronal identity within the mammalian CNS.

Amino Acid Sequence↗

Heart induction by Wnt antagonists depends on the homeodomain transcription factor Hex.

Inhibition of canonical Wnt/beta-catenin signaling by Dickkopf-1 (Dkk-1) or Crescent initiates cardiogenesis in vertebrate embryos. However, nearly nothing is known about the downstream effectors of these secreted Wnt antagonists or the mechanism by which they activate heart formation. Here we show that Wnt antagonists in Xenopus stimulate cardiogenesis non-cell-autonomously, up to several cells away from those in which canonical Wnt/beta-catenin signaling is blocked, indicative of an indirect role in heart induction. A screen for downstream mediators revealed that Dkk-1 and other inhibitors of the canonical Wnt pathway induce the homeodomain transcription factor Hex, which is normally expressed in endoderm underlying the presumptive cardiac mesoderm in amphibian, bird, and mammalian embryos. Loss of Hex function blocks both endogenous heart development and ectopic heart induction by Dkk-1. As with the canonical Wnt pathway antagonists, ectopic Hex induces expression of cardiac markers non-cell-autonomously. Thus, to initiate cardiogenesis, Wnt antagonists act on endoderm to up-regulate Hex, which, in turn, controls production of a diffusible heart-inducing factor. This novel function for Hex suggests an etiology for the cardiac malformations in Hex mutant mice and will make possible the isolation of factors that induce heart directly in the mesoderm.

Animals↗

Thalamic development induced by Shh in the chick embryo.

Patterning of the early neural tube is achieved in part by the inductive signals, which arise from neuroepithelial signaling centers. The zona limitans intrathalamica (ZLI) is a neuroepithelial domain in the alar plate of the diencephalon which separates the prethalamus from the thalamus. The ZLI has recently been considered to be a possible secondary organizer, effecting its inductions via sonic hedgehog (Shh), a signaling molecule which drives morphogenetic information for the thalamus. Using experimental embryological techniques involving the generation of chimeric embryos, we show that the formation of the ZLI in the diencephalic alar plate is due to an interaction between the prechordal and epichordal plate neuroepithelia. We also provide evidence that Shh expression in the ZLI underlies the morphogenetic activity of this putative diencephalic organizer. Ectopic Shh led to the auto-induction of its own gene expression in host cells, as well as to the expression of other genes involved in diencephalic regionalization and histogenesis. Analysis of long-term surviving embryos after Shh ectopic expression demonstrated that Shh was able to induce thalamic structures and local overgrowth. Overall, these results indicate that Shh expressed in the ZLI plays an important role in diencephalic growth and in the development of the thalamus.

Acetylcholinesterase↗

Partnership between DPC4 and SMAD proteins in TGF-beta signalling pathways.

The TGF-beta/activin/BMP superfamily of growth factors signals through heteromeric receptor complexes of type I and type II serine/threonine kinase receptors. The signal originated by TGF-beta-like molecules appears to be transduced by a set of evolutionarily conserved proteins known as SMADs, which upon activation directly translocate to the nucleus where they may activate transcription. Five SMAD proteins have so far been characterized in vertebrates. These factors are related to the mediator of decapentaplegic (dpp) signalling, mothers against dpp (Mad), in Drosophila and to the Sma genes from Caenorhabditis elegans. Smad1 and Smad2 have been shown to mimic the effects of BMP and activin, respectively, both in Xenopus and in mammalian cells, whereas Smad3 (a close homologue of Smad2) and the related protein DPC4, a tumour-suppressor gene product, mediate TGF-beta actions. We report here that DPC4 is essential for the function of Smad1 and Smad2 in pathways that signal mesoderm induction and patterning in Xenopus embryos, as well as antimitogenic and transcriptional responses in breast epithelial cells. DPC4 associates with Smad1 in response to BMP and with Smad2 in response to activin or TGF-beta. DPC4 is therefore a regulated partner of SMADs that function in different signalling pathways of the TGF-beta family.

Activins↗

Xenopus Sprouty2 inhibits FGF-mediated gastrulation movements but does not affect mesoderm induction and patterning.

Signal transduction through the FGF receptor is essential for the specification of the vertebrate body plan. Blocking the FGF pathway in early Xenopus embryos inhibits mesoderm induction and results in truncation of the anterior-posterior axis. The Drosophila gene sprouty encodes an antagonist of FGF signaling, which is transcriptionally induced by the pathway, but whose molecular functions are poorly characterized. We have cloned Xenopus sprouty2 and show that it is expressed in a similar pattern to known FGFs and is dependent on the FGF/Ras/MAPK pathway for its expression. Overexpression of Xsprouty2 in both embryos and explant assays results in the inhibition of the cell movements of convergent extension. Although blocking FGF/Ras/MAPK signaling leads to an inhibition of mesodermal gene expression, these markers are unaffected by Xsprouty2, indicating that mesoderm induction and patterning occurs normally in these embryos. Finally, using Xenopus oocytes we show that Xsprouty2 is an intracellular antagonist of FGF-dependent calcium signaling. These results provide evidence for at least two distinct FGF-dependent signal transduction pathways: a Sprouty-insensitive Ras/MAPK pathway required for the transcription of most mesodermal genes, and a Sprouty-sensitive pathway required for coordination of cellular morphogenesis.

Amino Acid Sequence↗

Induction across germ layers in Drosophila mediated by a genetic cascade.

We report an induction process occurring between two germ layers in the Drosophila embryo that involves a cascade of five interacting genes. Two of these, Ultrabithorax and abdominal-A, encode nuclear homeobox proteins; each of them is expressed in one of two adjacent parasegments in the visceral mesoderm and directs expression in its parasegment of a separate target gene, decapentaplegic in parasegment 7 and wingless in parasegment 8. The activity of both target genes is required for normal expression of another homeotic gene, labial, in cells of the adhering midgut epithelium. Their products are putative extracellular proteins, which presumably act as signals between the two germ layers. Positional instruction of this kind may be needed since the endoderm, unlike the mesoderm, appears unsegmented at first as it originates from two primordia near the embryonic poles, outside the realm of segmentation genes.

Animals↗

Early specification for body position in mes-endodermal regions of an amphibian embryo.

Specification for development of the body pattern in the amphibian embryo has usually been thought of as a prolonged process, initiated from an ooplasmic localisation of some kind in what will become the dorsal-anterior midline. The evidence has been interpreted as suggesting that this initial localisation is centred in what will become anterior endoderm, but gives rise by an inductive process in early blastula stages to an overlying organising centre which eventually controls the genesis of mesodermal pattern. Neurectodermal development (especially, the position and pattern of the central nervous system) is seen as controlled considerably later, by inductive signals from submigrating mesoderm at gastrulation. Current work tends to confirm that this sequence of inductive influences can occur at least in experimental situations. It also suggests, however, that in the normal development of the rather small egg of Xenopus, genesis of positional cues that specify the body pattern contributions within the more vegetal material (mes-endoderm) is a rather rapid, widespread and direct consequence of events occurring in the interval between fertilisation and cleavage. Possible molecular bases of early nuclear responses to position within egg material, and the more problematic nature of the positional system itself, are discussed.

Animals↗

Markers of vertebrate mesoderm induction.

Mesoderm formation is the first major differentiative event in vertebrate development. Many new mesoderm-specific genes have recently been described in the mouse, chick, frog and fish and belong to classes comprising T-domain genes, homeobox genes and those encoding secreted proteins. The T-domain genes have different but overlapping expression patterns and, in Xenopus, can ectopically activate nearly all other mesodermal genes. Several new homebox genes seem to mediate the ventralising activity of bone morphogenetic protein. New genes encoding secreted proteins induce dorsal mesoderm, in some cases by antagonizing ventralising factors.

Animals↗

Early head specification in Xenopus laevis.

The head represents the most dorsal and anterior extent of the body axis. In Xenopus, the progressive determination of the head is an extremely complex process involving the activation and localized antagonism of a number of interdependent intracellular signaling pathways including the Wingless/Int-1 (Wnt), bone morphogenetic protein (BMP), and nodal-related pathways. The sequence of events that specify the head are: dorsal-ventral polarization and head organizer specification in the blastula; gastrulation; neural induction; and patterning of the anterior-posterior and dorsal-ventral neuraxes. Wnt signaling is required for the specification of the dorsal side initially but is then inhibited within the organizer once it has formed. Similarly, Wnt signaling is required along the length of the neural tube, but must be suppressed at its rostral end for normal brain development. Nodal signaling is also necessary for induction of the mesendoderm, but is subsequently suppressed in its dorsal-anterior extreme to specify head organizer. BMP signaling is required for ventral mesoderm and non-neural ectoderm, and must also be suppressed in the head organizer region and for the differentiation of the ventral midline of the neural tube. Thus, development of the head, and indeed the body plan in general, requires precisely timed and spatially restricted activation and repression of these signaling pathways.

Animals↗

The initiation of new gene transcription during Xenopus gastrulation requires immediately preceding protein synthesis.

The incubation of Xenopus embryo fragments in cycloheximide at 5 or 10 micrograms ml-1 rapidly inhibits protein synthesis to 10% or less of control levels. In most batches of embryos, treatment with cycloheximide for up to 1 h causes no obvious cellular damage and protein synthesis is fully restored to normal levels 5 h later. Transcript analysis with RNA probes shows that the inhibition of protein synthesis at late blastula or early gastrula stages completely suppresses the normal initiation of actin gene transcription at the mid-late gastrula stage. This applies to muscle-specific actin genes, whose transcription is initiated by induction, as well as to cytoskeletal actin genes not activated by induction. Two-dimensional gel protein analysis shows that cycloheximide irreversibly inhibits only 10% of all genes normally expressed at a postneurula stage and that all of these are genes whose expression is normally initiated during or soon after gastrulation. Cycloheximide treatment causes a limited reduction of DNA synthesis, and no reduction of overall RNA synthesis. We conclude that the initiation of new gene transcription during gastrulation in Xenopus is dependent on the immediately preceding synthesis of certain proteins.

Actins↗