Muscle LIM Proteins Are Associated with Muscle Sarcomeres and Require dMEF2 for Their Expression during Drosophila Myogenesis

A genetic hierarchy of interactions, involving myogenic regulatory factors of the MyoD and myocyte enhancer-binding 2 (MEF2) families, serves to elaborate and maintain the differentiated muscle phenotype through transcriptional regulation of muscle-specific target genes. Much work suggests that members of the cysteine-rich protein (CRP) family of LIM domain proteins also play a role in muscle differentiation; however, the specific functions of CRPs in this process remain undefined. Previously, we characterized two members of the Drosophila CRP family, the muscle LIM proteins Mlp60A and Mlp84B, which show restricted expression in differentiating muscle lineages. To extend our analysis of Drosophila Mlps, we characterized the expression of Mlps in mutant backgrounds that disrupt specific aspects of muscle development. We show a genetic requirement for the transcription factor dMEF2 in regulating Mlp expression and an ability of dMEF2 to bind, in vitro, to consensus MEF2 sites derived from those present in Mlp genomic sequences. These data suggest that the Mlp genes may be direct targets of dMEF2 within the genetic hierarchy controlling muscle differentiation. Mutations that disrupt myoblast fusion fail to affect Mlp expression. In later stages of myogenic differentiation, which are dedicated primarily to assembly of the contractile apparatus, we analyzed the subcellular distribution of Mlp84B in detail. Immunofluorescent studies revealed the localization of Mlp84B to muscle attachment sites and the periphery of Z-bands of striated muscle. Analysis of mutations that affect expression of integrins and α-actinin, key components of these structures, also failed to perturb Mlp84B distribution. In conclusion, we have used molecular epistasis analysis to position Mlp function downstream of events involving mesoderm specification and patterning and concomitant with terminal muscle differentiation. Furthermore, our results are consistent with a structural role for Mlps as components of muscle cytoarchitecture.

Conservation of the expression and function of apterous orthologs in Drosophila and mammals

The Drosophila apterous (ap) gene encodes a protein of the LIM-homeodomain family. Many transcription factors of this class have been conserved during evolution; however, the functional significance of their structural conservation is generally not known. ap is best known for its fundamental role as a dorsal selector gene required for patterning and growth of the wing, but it also has other important functions required for neuronal fasciculation, fertility, and normal viability. We isolated mouse (mLhx2) and human (hLhx2) ap orthologs, and we used transgenic animals and rescue assays to investigate the conservation of the Ap protein during evolution. We found that the human protein LHX2 is able to regulate correctly ap target genes in the fly, causes the same phenotypes as Ap when ectopically produced, and most importantly rescues ap mutant phenotypes as efficiently as the fly protein. In addition, we found striking similarities in the expression patterns of the Drosophila and murine genes. Both mLhx2 and ap are expressed in the respective nerve cords, eyes, olfactory organs, brain, and limbs. These results demonstrate the conservation of Ap protein function across phyla and argue that aspects of its expression pattern have also been conserved from a common ancestor of insects and vertebrates.

Ectopic bone morphogenetic proteins 5 and 4 in the chicken forebrain lead to cyclopia and holoprosencephaly

Proper dorsal–ventral patterning in the developing central nervous system requires signals from both the dorsal and ventral portions of the neural tube. Data from multiple studies have demonstrated that bone morphogenetic proteins (BMPs) and Sonic hedgehog protein are secreted factors that regulate dorsal and ventral specification, respectively, within the caudal neural tube. In the developing rostral central nervous system Sonic hedgehog protein also participates in ventral regionalization; however, the roles of BMPs in the developing brain are less clear. We hypothesized that BMPs also play a role in dorsal specification of the vertebrate forebrain. To test our hypothesis we implanted beads soaked in recombinant BMP5 or BMP4 into the neural tube of the chicken forebrain. Experimental embryos showed a loss of the basal telencephalon that resulted in holoprosencephaly (a single cerebral hemisphere), cyclopia (a single midline eye), and loss of ventral midline structures. In situ hybridization using a panel of probes to genes expressed in the dorsal and ventral forebrain revealed the loss of ventral markers with the maintenance of dorsal markers. Furthermore, we found that the loss of the basal telencephalon was the result of excessive cell death and not a change in cell fates. These data provide evidence that BMP signaling participates in dorsal–ventral patterning of the developing brain in vivo, and disturbances in dorsal–ventral signaling result in specific malformations of the forebrain.

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.

sqv-3, -7, and -8, a set of genes affecting morphogenesis in Caenorhabditis elegans, encode enzymes required for glycosaminoglycan biosynthesis

sqv (squashed vulva) genes comprise a set of eight independent loci in Caenorhabditis elegans required zygotically for the invagination of vulval epithelial cells and maternally for normal oocyte formation and embryogenesis. Sequencing of sqv-3, sqv-7, and sqv-8 suggested a role for the encoded proteins in glycolipid or glycoprotein biosynthesis. Using a combination of in vitro analysis of SQV enzymatic activities, sqv+-mediated rescue of vertebrate cell lines, and biochemical characterization of sqv mutants, we show that sqv-3, -7, and -8 all affect the biosynthesis of glycosaminoglycans and therefore compromise the function of one specific class of glycoconjugates, proteoglycans. These findings establish the importance of proteoglycans and their associated glycosaminoglycans in epithelial morphogenesis and patterning during C. elegans development.

The Drosophila LIM-only gene, dLMO, is mutated in Beadex alleles and might represent an evolutionarily conserved function in appendage development

The process of wing patterning involves precise molecular mechanisms to establish an organizing center at the dorsal–ventral boundary, which functions to direct the development of the Drosophila wing. We report that misexpression of dLMO, a Drosophila LIM-only protein, in specific patterns in the developing wing imaginal disc, disrupts the dorsal–ventral (D-V) boundary and causes errors in wing patterning. When dLMO is misexpressed along the anterior–posterior boundary, extra wing outgrowth occurs, similar to the phenotype seen when mutant clones lacking Apterous, a LIM homeodomain protein known to be essential for normal D-V patterning of the wing, are made in the wing disc. When dLMO is misexpressed along the D-V boundary in third instar larvae, loss of the wing margin is observed. This phenotype is very similar to the phenotype of Beadex, a long-studied dominant mutation that we show disrupts the dLMO transcript in the 3′ untranslated region. dLMO normally is expressed in the wing pouch of the third instar wing imaginal disc during patterning. A mammalian homolog of dLMO is expressed in the developing limb bud of the mouse. This indicates that LMO proteins might function in an evolutionarily conserved mechanism involved in patterning the appendages.

Regulation of number and size of digits by posterior Hox genes: A dose-dependent mechanism with potential evolutionary implications

The proper development of digits, in tetrapods, requires the activity of several genes of the HoxA and HoxD homeobox gene complexes. By using a variety of loss-of-function alleles involving the five Hox genes that have been described to affect digit patterning, we report here that the group 11, 12, and 13 genes control both the size and number of murine digits in a dose-dependent fashion, rather than through a Hox code involving differential qualitative functions. A similar dose–response is observed in the morphogenesis of the penian bone, the baculum, which further suggests that digits and external genitalia share this genetic control mechanism. A progressive reduction in the dose of Hox gene products led first to ectrodactyly, then to olygodactyly and adactyly. Interestingly, this transition between the pentadactyl to the adactyl formula went through a step of polydactyly. We propose that in the distal appendage of polydactylous short-digited ancestral tetrapods, such as Acanthostega, the HoxA complex was predominantly active. Subsequent recruitment of the HoxD complex contributed to both reductions in digit number and increase in digit length. Thus, transition through a polydactylous limb before reaching and stabilizing the pentadactyl pattern may have relied, at least in part, on asynchronous and independent changes in the regulation of HoxA and HoxD gene complexes.

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.

Self-assembled monolayers from a designed combinatorial library of de novo β-sheet proteins

A variety of naturally occurring biomaterials owe their unusual structural and mechanical properties to layers of β-sheet proteins laminated between layers of inorganic mineral. To explore the possibility of fabricating novel two-dimensional protein layers, we studied the self-assembly properties of de novo proteins from a designed combinatorial library. Each protein in the library has a distinct 63 amino acid sequence, yet they all share an identical binary pattern of polar and nonpolar residues, which was designed to favor the formation of six-stranded amphiphilic β-sheets. Characterization of proteins isolated from the library demonstrates that (i) they self assemble into monolayers at an air/water interface; (ii) the monolayers are dominated by β-sheet secondary structure, as shown by both circular dichroism and infrared spectroscopies; and (iii) the measured areas (500- 600 Å2) of individual protein molecules in the monolayers match those expected for proteins folded into amphiphilic β-sheets. The finding that similar structures are formed by distinctly different protein sequences suggests that assembly into β-sheet monolayers can be encoded by binary patterning of polar and nonpolar amino acids. Moreover, because the designed binary pattern is compatible with a wide variety of different sequences, it may be possible to fabricate β-sheet monolayers by using combinations of side chains that are explicitly designed to favor particular applications of novel biomaterials.

F-spondin is a contact-repellent molecule for embryonic motor neurons

The floor plate plays a key role in patterning axonal trajectory in the embryonic spinal cord by providing both long-range and local guidance cues that promote or inhibit axonal growth toward and across the ventral midline of the spinal cord, thus acting as an intermediate target for a number of crossing (commissural) and noncrossing (motor) axons. F-spondin, a secreted adhesion molecule expressed in the embryonic floor plate and the caudal somite of birds, plays a dual role in patterning the nervous system. It promotes adhesion and outgrowth of commissural axons and inhibits adhesion of neural crest cells. In the current study, we demonstrate that outgrowth of embryonic motor axons also is inhibited by F-spondin protein in a contact-repulsion fashion. Three independent lines of evidence support our hypothesis: substrate-attached F-spondin inhibits outgrowth of dissociated motor neurons in an outgrowth assay; F-spondin elicits acute growth cone collapse when applied to cultured motor neurons; and challenging ventral spinal cord explants with aggregates of HEK 293 cells expressing F-spondin, causes contact-repulsion of motor neurites. Structural–functional studies demonstrate that the processed carboxyl-half protein that contains the thrombospondin type 1 repeats is more prominent in inhibiting outgrowth, suggesting that the processing of F-spondin is important for enhancing its inhibitory activity.

Paper-like electronic displays: Large-area rubber-stamped plastic sheets of electronics and microencapsulated electrophoretic inks

Electronic systems that use rugged lightweight plastics potentially offer attractive characteristics (low-cost processing, mechanical flexibility, large area coverage, etc.) that are not easily achieved with established silicon technologies. This paper summarizes work that demonstrates many of these characteristics in a realistic system: organic active matrix backplane circuits (256 transistors) for large (≈5 × 5-inch) mechanically flexible sheets of electronic paper, an emerging type of display. The success of this effort relies on new or improved processing techniques and materials for plastic electronics, including methods for (i) rubber stamping (microcontact printing) high-resolution (≈1 μm) circuits with low levels of defects and good registration over large areas, (ii) achieving low leakage with thin dielectrics deposited onto surfaces with relief, (iii) constructing high-performance organic transistors with bottom contact geometries, (iv) encapsulating these transistors, (v) depositing, in a repeatable way, organic semiconductors with uniform electrical characteristics over large areas, and (vi) low-temperature (≈100°C) annealing to increase the on/off ratios of the transistors and to improve the uniformity of their characteristics. The sophistication and flexibility of the patterning procedures, high level of integration on plastic substrates, large area coverage, and good performance of the transistors are all important features of this work. We successfully integrate these circuits with microencapsulated electrophoretic “inks” to form sheets of electronic paper.

Using electroactive substrates to pattern the attachment of two different cell populations

This report describes the development of an electroactive mask that permits the patterning of two different cell populations to a single substrate. This mask is based on a self-assembled monolayer of alkanethiolates on gold that could be switched from a state that prevents the attachment of cells to a state that promotes the integrin-mediated attachment of cells. Monolayers were patterned into regions having this electroactive monolayer and a second set of regions that were adhesive. After Swiss 3T3 fibroblasts had attached to the adhesive regions of this substrate, the second set of regions was activated electrically to permit the attachment of a second population of fibroblast cells. This method provides a general strategy for patterning the attachment of multiple cell types and will be important for studying heterotypic cell-cell interactions.

Sonic hedgehog protein signals not as a hydrolytic enzyme but as an apparent ligand for Patched

The amino-terminal signaling domain of the Sonic hedgehog secreted protein (Shh-N), which derives from the Shh precursor through an autoprocessing reaction mediated by the carboxyl-terminal domain, executes multiple functions in embryonic tissue patterning, including induction of ventral and suppression of dorsal cell types in the developing neural tube. An apparent catalytic site within Shh-N is suggested by structural homology to a bacterial carboxypeptidase. We demonstrate here that alteration of residues presumed to be critical for a hydrolytic activity does not cause a loss of inductive activity, thus ruling out catalysis by Shh-N as a requirement for signaling. We favor the alternative, that Shh-N functions primarily as a ligand for the putative receptor Patched (Ptc). This possibility is supported by new evidence for direct binding of Shh-N to Ptc and by a strong correlation between the affinity of Ptc-binding and the signaling potency of Shh-N protein variants carrying alterations of conserved residues in a particular region of the protein surface. These results together suggest that direct Shh-N binding to Ptc is a critical event in transduction of the Shh-N signal.

Molecular genetics of pattern formation in the inner ear: Do compartment boundaries play a role?

The membranous labyrinth of the inner ear establishes a precise geometrical topology so that it may subserve the functions of hearing and balance. How this geometry arises from a simple ectodermal placode is under active investigation. The placode invaginates to form the otic cup, which deepens before pinching off to form the otic vesicle. By the vesicle stage many genes expressed in the developing ear have assumed broad, asymmetrical expression domains. We have been exploring the possibility that these domains may reflect developmental compartments that are instrumental in specifying the location and identity of different parts of the ear. The boundaries between compartments are proposed to be the site of inductive interactions required for this specification. Our work has shown that sensory organs and the endolymphatic duct each arise near the boundaries of broader gene expression domains, lending support to this idea. A further prediction of the model, that the compartment boundaries will also represent lineage-restriction compartments, is supported in part by fate mapping the otic cup. Our data suggest that two lineage-restriction boundaries intersect at the dorsal pole of the otocyst, a convergence that may be critical for the specification of endolymphatic duct outgrowth. We speculate that the patterning information necessary to establish these two orthogonal boundaries may emanate, in part, from the hindbrain. The compartment boundary model of ear development now needs to be tested through a variety of experimental perturbations, such as the removal of boundaries, the generation of ectopic boundaries, and/or changes in compartment identity.

A modular misexpression screen in Drosophila detecting tissue-specific phenotypes.

Genetic screens in Drosophila have lead to the discovery of many genes important for patterning and signal transduction in diverse organisms. Traditionally, the phenotypic effects of loss-of-function mutations are analyzed. As an alternative way to link genes and function, I have developed a versatile misexpression screen in Drosophila, the first such screen in higher eukaryotes. The screen identifies genes that, when over- or misexpressed in a pattern of interest, give a specific phenotype or modulate an existing mutant phenotype. It is based on Gal4 transactivation of a mobile enhancer and promoter that "targets" random endogenous genes for expression. The modular design of the screen allows directed expression in any temporal or spatial pattern. When activated in the developing eye, 4% of target inserts gave dominant phenotypes. One insertion was in the gene encoding Ras GTPase-activating protein; its overexpression phenotype was strongly enhanced by a mutation in Ras1. Thus, biologically relevant phenotypes and genetic interactions are identified using this method. The screen is a powerful new tool for developmental genetics; similar approaches can also be developed for other organisms.