Activation of a protease cascade involved in patterning the Drosophila embryo

Dorsoventral patterning of the Drosophila embryo is initiated by a ventralizing signal. Production of this signal requires the serine proteases Gastrulation Defective (GD), Snake, and Easter, which genetic studies suggest act sequentially in a cascade that is activated locally in response to a ventral cue provided by the pipe gene. Here, we demonstrate biochemically that GD activates Snake, which in turn activates Easter. We also provide evidence that GD zymogen cleavage is important for triggering this cascade but is not spatially localized by pipe. Our results suggest that a broadly, rather than locally, activated protease cascade produces the ventralizing signal, so a distinct downstream step in this cascade must be spatially regulated to restrict signaling to the ventral side of the embryo.

β-Trcp couples β-catenin phosphorylation-degradation and regulates Xenopus axis formation

Regulation of β-catenin stability is essential for Wnt signal transduction during development and tumorigenesis. It is well known that serine-phosphorylation of β-catenin by the Axin–glycogen synthase kinase (GSK)–3β complex targets β-catenin for ubiquitination–degradation, and mutations at critical phosphoserine residues stabilize β-catenin and cause human cancers. How β-catenin phosphorylation results in its degradation is undefined. Here we show that phosphorylated β-catenin is specifically recognized by β-Trcp, an F-box/WD40-repeat protein that also associates with Skp1, an essential component of the ubiquitination apparatus. β-catenin harboring mutations at the critical phosphoserine residues escapes recognition by β-Trcp, thus providing a molecular explanation for why these mutations cause β-catenin accumulation that leads to cancer. Inhibition of endogenous β-Trcp function by a dominant negative mutant stabilizes β-catenin, activates Wnt/β-catenin signaling, and induces axis formation in Xenopus embryos. Therefore, β-Trcp plays a central role in recruiting phosphorylated β-catenin for degradation and in dorsoventral patterning of the Xenopus embryo.

Activin A inhibits Pax-6 expression and perturbs cell differentiation in the developing spinal cord in vitro.

We have developed an in vitro model of the isolated chicken neural plate. Here we demonstrate that even in the absence of notochord, the neural plate rapidly develops a typical dorsoventral patterning. This observation suggests that the ventral cell types are specified or at least predetermined prior to notochord formation and that permissive conditions are sufficient for differentiation of ventral structures. Treatment of the neural plate with activin A extinguishes Pax-6 gene expression, whereas the dorsal markers Pax-3 and Pax-7 are still expressed. The absence of Pax-6 transcripts can be correlated with an impeded differentiation of the motor neurons, whereas the floor plate seems to be enlarged. We propose that the region-specific expression of Pax-6 in the spinal cord is under the control of activin-like molecules.

Anteroposterior neural tissue specification by activin-induced mesoderm

The transforming growth factor β superfamily member, activin, is able to induce mesodermal tissues in animal cap explants from Xenopus laevis blastula stage embryos. Activin can act like a morphogen of the dorsoventral axis in that lower doses induce more ventral, and higher doses more dorsal, tissue types. Activin has also previously been reported to induce neural tissues in animal caps. From cell mixing experiments it was inferred that this might be an indirect effect of induced mesoderm signaling to uninduced ectoderm. Here we demonstrate directly that neural tissues do indeed arise by the action of induced mesoderm on uninduced ectoderm. Dorsal mesoderm is itself subdivided into posterior and anterior domains in vivo, but this had not been demonstrated for induced mesoderm. We therefore tested whether different concentrations of activin recreate these different anteroposterior properties as well. We show that the anteroposterior positional value of induced mesoderm, including its neuroinductive properties, depends on the dose of activin applied to the mesoderm, with lower doses inducing more posterior and higher doses giving more anterior markers. We discuss the implications of these results for patterning signals and the relationship between anteroposterior and dorsoventral axes.

Abnormal skeletal patterning in embryos lacking a single Cbp allele: A partial similarity with Rubinstein–Taybi syndrome

CBP is a transcriptional coactivator required by many transcription factors for transactivation. Rubinstein–Taybi syndrome, which is an autosomal dominant syndrome characterized by abnormal pattern formation, has been shown to be associated with mutations in the Cbp gene. Furthermore, Drosophila CBP is required in hedgehog signaling for the expression of decapentapleigic, the Drosophila homologue of bone morphogenetic protein. However, no direct evidence exists to indicate that loss of one copy of the mammalian Cbp gene affects pattern formation. Here, we show that various abnormalities occur at high frequency in the skeletal system of heterozygous Cbp-deficient mice resulting from a C57BL/6-CBA × BALB/c cross. In support of a conserved signaling pathway for pattern formation in insects and mammals, the expression of Bmp7 was found to be reduced in the heterozygous mutants. The frequency of the different abnormalities was significantly lower in a C57BL/6-CBA background, suggesting that the genetic background is an important determinant of the variability and severity of the anomalies seen in Rubinstein–Taybi syndrome patients.

Regulation of dorsal fate in the neuraxis by Wnt-1 and Wnt-3a

Members of the Wnt family of signaling molecules are expressed differentially along the dorsal–ventral axis of the developing neural tube. Thus we asked whether Wnt factors are involved in patterning of the nervous system along this axis. We show that Wnt-1 and Wnt-3a, both of which are expressed in the dorsal portion of the neural tube, could synergize with the neural inducers noggin and chordin in Xenopus animal explants to generate the most dorsal neural structure, the neural crest, as determined by the expression of Krox-20, AP-2, and slug. Overexpression of Wnt-1 or Wnt-3a in the neuroectoderm of whole embryos led to a dramatic increase of slug and Krox-20-expressing cells, but the hindbrain expression of Krox-20 remained unaffected. Enlargement in the neural crest population could occur even when cell proliferation was inhibited. Wnt-5A and Wnt-8, neither of which is expressed in the dorsal neuroectoderm, failed to induce neural crest markers. Overexpression of glycogen synthase kinase 3, known to antagonize Wnt signaling, blocked the neural-crest-inducing activity of Wnt-3a in animal explants and inhibited neural crest formation in whole embryos. We suggest that Wnt-1 and Wnt-3a have a role in patterning the neural tube along its dorsoventral axis and function in the differentiation of the neural crest.

CA1-specific N-methyl-d-aspartate receptor knockout mice are deficient in solving a nonspatial transverse patterning task

In both humans and animals, the hippocampus is critical to memory across modalities of information (e.g., spatial and nonspatial memory) and plays a critical role in the organization and flexible expression of memories. Recent studies have advanced our understanding of cellular basis of hippocampal function, showing that N-methyl-d-aspartate (NMDA) receptors in area CA1 are required in both the spatial and nonspatial domains of learning. Here we examined whether CA1 NMDA receptors are specifically required for the acquisition and flexible expression of nonspatial memory. Mice lacking CA1 NMDA receptors were impaired in solving a transverse patterning problem that required the simultaneous acquisition of three overlapping odor discriminations, and their impairment was related to an abnormal strategy by which they failed to adequately sample and compare the critical odor stimuli. By contrast, they performed normally, and used normal stimulus sampling strategies, in the concurrent learning of three nonoverlapping concurrent odor discriminations. These results suggest that CA1 NMDA receptors play a crucial role in the encoding and flexible expression of stimulus relations in nonspatial memory.

Polaris, a Protein Involved in Left-Right Axis Patterning, Localizes to Basal Bodies and Cilia

Mutations in Tg737 cause a wide spectrum of phenotypes, including random left-right axis specification, polycystic kidney disease, liver and pancreatic defects, hydrocephalus, and skeletal patterning abnormalities. To further assess the biological function of Tg737 and its role in the mutant pathology, we identified the cell population expressing Tg737 and determined the subcellular localization of its protein product called Polaris. Tg737 expression is associated with cells possessing either motile or immotile cilia and sperm. Similarly, Polaris concentrated just below the apical membrane in the region of the basal bodies and within the cilia or flagellar axoneme. The data suggest that Polaris functions in a ciliogenic pathway or in cilia maintenance, a role supported by the loss of cilia on the ependymal cell layer in ventricles of Tg737orpk brains and by the lack of node cilia in Tg737Δ2-3βGal mutants.

Vascular patterning defects associated with expression of activated Notch4 in embryonic endothelium

Notch proteins function as receptors for membrane-bound ligands (Jagged and Delta-like) to regulate cell-fate determination. We have investigated the role of Notch signaling in embryonic endothelium of the mouse by expressing an activated form of the Notch4 protein in vasculature under the regulation of the Flk1 (VEGFR) locus. Expression of activated Notch4 results in a growth and developmental delay and embryonic lethality at about 10 days postcoitum. The extent of the developing vasculature in mutant embryos was restricted, fewer small vessels were seen, and vascular networks were disorganized. The brain periphery of mutant embryos contained large dilated vessels with evidence of compromised vessel-wall integrity and large areas of necrosis; yolk-sac vasculature was abnormal. Expression of an activated form of Notch4 in embryonic vasculature leads to abnormal vessel structure and patterning, implicating the Notch pathway in phases of vascular development associated with vessel patterning and remodeling.

Patterning of the mammalian cochlea

The mammalian cochlea is sophisticated in its function and highly organized in its structure. Although the anatomy of this sense organ has been well documented, the molecular mechanisms underlying its development have remained elusive. Information generated from mutant and knockout mice in recent years has increased our understanding of cochlear development and physiology. This article discusses factors important for the development of the inner ear and summarizes cochlear phenotypes of mutant and knockout mice, particularly Otx and Otx2. We also present data on gross development of the mouse cochlea.

A Common Position-Dependent Mechanism Controls Cell-Type Patterning and GLABRA2 Regulation in the Root and Hypocotyl Epidermis of Arabidopsis1

A position-dependent pattern of epidermal cell types is produced during root development in Arabidopsis thaliana. This pattern is reflected in the expression pattern of GLABRA2 (GL2), a homeobox gene that regulates cell differentiation in the root epidermis. GL2 promoter::GUS fusions were used to show that the TTG gene, a regulator of root epidermis development, is necessary for maximal GL2 activity but is not required for the pattern of GL2 expression. Furthermore, GL2-promoter activity is influenced by expression of the myc-like maize R gene (35S::R) in Arabidopsis but is not affected by gl2 mutations. A position-dependent pattern of cell differentiation and GL2-promoter activity was also discovered in the hypocotyl epidermis that was analogous to the pattern in the root. Non-GL2-expressing cell files in the hypocotyl epidermis located outside anticlinal cortical cell walls exhibit reduced cell length and form stomata. Like the root, the hypocotyl GL2 activity was shown to be influenced by ttg and 35S::R but not by gl2. The parallel pattern of cell differentiation in the root and hypocotyl indicates that TTG and GL2 participate in a common position-dependent mechanism to control cell-type patterning throughout the apical-basal axis of the Arabidopsis seedling.

Patterning of functional antibodies and other proteins by photolithography of silane monolayers.

We have demonstrated the assembly of two-dimensional patterns of functional antibodies on a surface. In particular, we have selectively adsorbed micrometer-scale regions of biotinylated immunoglobulin that exhibit specific antigen binding after adsorption. The advantage of this technique is its potential adaptability to adsorbing arbitrary proteins in tightly packed monolayers while retaining functionality. The procedure begins with the formation of a self-assembled monolayer of n-octadecyltrimethoxysilane (OTMS) on a silicon dioxide surface. This monolayer can then be selectively removed by UV photolithography. Under appropriate solution conditions, the OTMS regions will adsorb a monolayer of bovine serum albumin (BSA), while the silicon dioxide regions where the OTMS has been removed by UV light will adsorb less than 2% of a monolayer, thus creating high contrast patterned adsorption of BSA. The attachment of the molecule biotin to the BSA allows the pattern to be replicated in a layer of streptavidin, which bonds to the biotinylated BSA and in turn will bond an additional layer of an arbitrary biotinylated protein. In our test case, functionality of the biotinylated goat antibodies raised against mouse immunoglobulin was demonstrated by the specific binding of fluorescently labeled mouse IgG.

Functional specificity of Hoxa-4 in vertebral patterning lies outside of the homeodomain.

The Hox family of proteins plays a central role in establishing the body plan of a wide range of metazoan organisms. Each member of this family of transcriptional regulators has a distinct functional specificity, yet they bind to similar DNA target sequences through their conserved homeodomain. The mechanisms whereby Hox proteins achieve their diverse specificities in vivo remain undefined. Using the opposing effects of Hoxa-4 and Hoxc-8 in vertebral patterning, we demonstrate by replacing the homeodomain of Hoxa-4 with that of Hoxc-8 that the functional specificity of Hoxa-4 does not track with the homeodomain. These observations provide evidence that other regions of Hox proteins play an important role in mediating functional specificity during mammalian embryogenesis.

Role of glycogen synthase kinase 3 beta as a negative regulator of dorsoventral axis formation in Xenopus embryos.

The dorsoventral axis is established early in Xenopus development and may involve signaling by Wnts, a family of Wnt1-protooncogene-related proteins. The protein kinase shaggy functions in the wingless/Wnt signaling pathway, which operates during Drosophila development. To assess the role of a closely related kinase, glycogen synthase kinase 3 beta (GSK-3 beta), in vertebrate embryogenesis, we cloned a cDNA encoding a Xenopus homolog of GSK-3 beta (XGSK-3 beta). XGSK-3 beta-specific transcripts were detected by Northern analysis in Xenopus eggs and early embryos. Microinjection of the mRNA encoding a catalytically inactive form of rat GSK-3 beta into a ventrovegetal blastomere of eight-cell embryos caused ectopic formation of a secondary body axis containing a complete set of dorsal and anterior structures. Furthermore, in isolated ectodermal explants, the mutant GSK-3 beta mRNA activated the expression of neural tissue markers. Wild-type XGSK-3 beta mRNA suppressed the dorsalizing effects of both the mutated GSK-3 beta and Xenopus dishevelled, a proposed upstream signaling component of the same pathway. These results strongly suggest that XGSK-3 beta functions to inhibit dorsoventral axis formation in the embryo and provide evidence for conservation of the Wnt signaling pathway in Drosophila and vertebrates.

Control of somite patterning by signals from the lateral plate.

The body musculature of higher vertebrates is composed of the epaxial muscles, associated with the vertebral column, and of the hypaxial muscles of the limbs and ventro-lateral body wall. Both sets of muscles arise from different cell populations within the dermomyotomal component of the somite. Myogenesis first occurs in the medial somitic cells that will form the epaxial muscles and starts with a significant delay in cells derived from the lateral somitic moiety that migrate to yield the hypaxial muscles. The newly formed somite is mostly composed of unspecified cells, and the determination of somitic compartments toward specific lineages is controlled by environmental cues. In this report, we show that determinant signals for lateral somite specification are provided by the lateral plate. They result in a blockade of the myogenic program, which maintains the lateral somitic cells as undifferentiated muscle progenitors expressing the Pax-3 gene, and represses the activation of the MyoD family genes. In vivo, this mechanism could account for the delay observed in the onset of myogenesis between muscles of the epaxial and hypaxial domains.