Polarity Gene Research Articles

The impact of function perturbations in Boolean networks

A network is said to be robust relative to a certain network characteristic if a small change in network structure does not significantly affect the characteristic. From the perspective of network stability, robustness is desirable; however, from the perspective of intervention to exert influence on network behavior, it is undesirable. For Boolean networks, there are two fundamental types of robustness. One type pertains to perturbing the state of the network and the other to perturbing the rule-based structure. This article explores the impact of function perturbations in Boolean networks from two aspects: (1) analysis: predict the impact on network state transitions and attractors via analytical approaches or identify a perturbation by observing its consequences; (2) synthesis: preserve or modify the network characteristics, especially attractors, by introducing a judicious change to the functions. The results are applied to achieve intervention that structurally alters the network to achieve a more favorable steady-state distribution and to identify the function perturbation that has led to altered observed behavior. The intervention procedure is applied to a WNT5A network to reduce the risk of metastasis in melanoma, and the identification procedure is applied to a Drosophila melanogaster segmentation polarity gene network to identify regulatory function perturbation.

Identification of the C. elegansanaphase promoting complex subunit Cdc26 by phenotypic profiling and functional rescue in yeast

BackgroundRNA interference coupled with videorecording of C. elegans embryos is a powerful method for identifying genes involved in cell division processes. Here we present a functional analysis of the gene B0511.9, previously identified as a candidate cell polarity gene in an RNAi videorecording screen of chromosome I embryonic lethal genes.ResultsWhereas weak RNAi inhibition of B0511.9 causes embryonic cell polarity defects, strong inhibition causes embryos to arrest in metaphase of meiosis I. The range of defects induced by RNAi of B0511.9 is strikingly similar to those displayed by mutants of anaphase-promoting complex/cyclosome (APC/C) components. Although similarity searches did not reveal any obvious homologue of B0511.9 in the non-redundant protein database, we found that the N-terminus shares a conserved sequence pattern with the N-terminus of the small budding yeast APC/C subunit Cdc26 and its orthologues from a variety of other organisms. Furthermore, we show that B0511.9 robustly complements the temperature-sensitive growth defect of a yeast cdc26Δ mutant.ConclusionThese data demonstrate that B0511.9 encodes the C. elegans APC/C subunit CDC-26.

Gene expression patterns in primary neuronal clusters of the Drosophila embryonic brain

The brain of Drosophila is formed by approximately 100 lineages, each lineage being derived from a stem cell-like neuroblast that segregates from the procephalic neurectoderm of the early embryo. A neuroblast map has been established in great detail for the early embryo, and a suite of molecular markers has been defined for all neuroblasts included in this map [Urbach, R., Technau, G.M. (2003a) Molecular markers for identified neuroblasts in the developing brain of Drosophila. Development 130, 3621–3637]. However, the expression of these markers was not followed into later embryonic or larval stages, mainly due to the fact that anatomical landmarks to which expression patterns could be related had not been defined. Such markers, in the form of stereotyped clusters of neurons whose axons project along cohesive bundles (“primary axon bundles” or “PABs”) are now available [Younossi-Hartenstein, A., Nguyen, B., Shy, D., Hartenstein, V. 2006. Embryonic origin of the Drosophila brain neuropile. J. Comp. Neurol. 497, 981–998]. In the present study we have mapped the expression of molecular markers in relationship to primary neuronal clusters and their PABs. The markers we analyzed include many of the genes involved in patterning of the brain along the anteroposterior axis (cephalic gap genes, segment polarity genes) and dorso-ventral axis (columnar patterning genes), as well as genes expressed in the dorsal protocerebrum and visual system (early eye genes). Our analysis represents an important step along the way to identify neuronal lineages of the mature brain with genes expressed in the early embryo in discrete neuroblasts. Furthermore, the analysis helped us to reconstruct the morphogenetic movements that transform the two-dimensional neuroblast layer of the early embryo into the three-dimensional larval brain and provides the basis for deeper understanding of how the embryonic brain develops.

Polarité planaire chez les Mammifères : similitudes et divergences avec la Drosophile

Planar cell polarity (PCP) genes were originally identified in invertebrates (Drosophila Melanogaster) for their role in the uniform orientation of a structure within the plane of the epithelium (hair, group of cells). During the last five years, numerous studies have shown that vertebrate, but more importantly, mammalian homologues of some of these genes are involved in various developmental processes such as neural tube closure, polycystic kidney disease, inner ear functions (hearing, balance) or Bardet Biedl syndrome. These processes rely on a set of genes whose PCP function is conserved in mammals and Drosophila Melanogaster for some, or only present in mammals for others. In 2003, the inner ear was identified as a model to study PP in mammals and allowed the identification of the first important genes. These genes encode a variety of cell surface molecules as well as intracellular adapters whose molecular mechanisms are still poorly understood. It is clear that the identification of the PP pathways in mammals will come from a comparison with the genes in Drosophila, but also from the identification of genes specific to mammals.

Mouse naked cuticle 2 (mNkd2) as a direct transcriptional target of Hoxc8 in vivo

Mouse naked cuticle 2 (mNkd2), the mammalian homolog of the Drosophila segment polarity gene naked cuticle (nkd), encodes an EF hand protein that regulates early Wg activity by acting as an inducible antagonist. The transcription factor, Hoxc8, a member of the homeobox gene family, is vital for growth and differentiation. Chromatin immunoprecipitation (ChIP) assay, an electrophoretic mobility shift assay (EMSA), and a reporter assay demonstrated that endogenous Hoxc8 protein binds directly to the enhancer region of the mNkd2 gene, implying a Hoxc8-dependent transcriptional activity. Introduction of exogenous Hoxc8 into NIH3T3 cell lines lacking wild-type Hoxc8 dramatically reduced expression of mNkd2 mRNA. If, as the results suggest, mNkd2 is a direct target of Hoxc8, it represents a novel mechanism by which Hoxc8 might cross-talk with the Wnt signaling pathway by regulating mNkd2.

Tracking down the “head blob”: Comparative analysis of wingless expression in the developing insect procephalon reveals progressive reduction of embryonic visual system patterning in higher insects

The evolution of larval head morphology in holometabolous insects is characterized by reduction of antennal appendages and the visual system components. Little insight has been gained into molecular developmental changes underlying this morphological diversification. Here we compare the expression of the segment polarity gene wingless ( wg) in the pregnathal head of fruit fly, flour beetle and grasshopper embryos. We provide evidence that wg activity contributes to segment border formation, and, subsequently, the separation of the visual system and protocerebrum anlagen in the anterior procephalon. In directly developing insects like grasshopper, seven expression domains are formed during this process. The activation of four of these, which correspond to polar expression pairs in the optic lobe anlagen and the protocerebral ectoderm, has shifted to postembryonic stages in flour beetle and Drosophila. The remaining three domains map to the protocerebral neuroectoderm, and form by disintegration of a large precursor domain in flour beetle and grasshopper. In Drosophila, the precursor domain remains intact, constituting the previously described “head blob”. These data document major changes in the expression of an early patterning gene correlated with the dramatic evolution of embryonic visual system development in the Holometabola.

Even-skipped has gap-like, pair-rule-like, and segmental functions in the cricket Gryllus bimaculatus, a basal, intermediate germ insect (Orthoptera)

Developmental mechanisms of segmentation appear to be varied among insects in spite of their conserved body plan. Although the expression patterns of the segment polarity genes in all insects examined imply well conserved function of this class of genes, expression patterns and function of the pair-rule genes tend to exhibit diversity. To gain further insights into the evolution of the segmentation process and the role of pair-rule genes, we have examined expression and function of an ortholog of the Drosophila pair-rule gene even-skipped ( eve) in a phylogenetically basal insect, Gryllus bimaculatus (Orthoptera, intermediate germ cricket). We find that Gryllus eve ( Gb'eve) is expressed as stripes in each of the prospective gnathal, thoracic, and abdominal segments and as a broad domain in the posterior growth zone. Dynamics of stripe formation vary among Gb' eve stripes, representing one of the three modes, the segmental, incomplete pair-rule, and complete pair-rule mode. Furthermore, we find that RNAi suppression of Gb'eve results in segmentation defects in both anterior and posterior regions of the embryo. Mild depletion of Gb'eve shows a pair-rule-like defect in anterior segments, while stronger depletion causes a gap-like defect showing deletion of anterior and posterior segments. These results suggest that Gb'eve acts as a pair-rule gene at least during anterior segmentation and also has segmental and gap-like functions. Additionally, Gb'eve may be involved in the regulation of hunchback and Krüppel expression. Comparisons with eve functions in other species suggest that the Gb'eve function may represent an intermediate state of the evolution of pair-rule patterning by eve in insects.

JNK- and Fos-regulated Mmp1 expression cooperates with Ras to induce invasive tumors in Drosophila

Loss of the epithelial polarity gene scribble in clones of Drosophila imaginal disc cells can cooperate with Ras signaling to induce malignant tumors. Such mutant tissue overproliferates, resists apoptosis, leaves its place of origin and invades other organs, ultimately causing lethality. We show that increased Jun N-terminal kinase (JNK) signaling resulting from the loss of scribble promotes the movement of transformed cells to secondary sites. This effect requires Fos-dependent transcriptional activation of a matrix metalloprotease gene mmp1 downstream of JNK. Expression of the Mmp inhibitor Timp or Mmp RNAi knockdown suppresses cell invasiveness. The proinvasive function of the JNK pathway is revealed in a tumor context when active Ras signaling prevents the apoptotic response to JNK activity as it occurs in nontransformed cells. Based on these results, we present a model that explains the oncogenic cooperation between JNK and Ras, and describes how aberrant regulation of cell survival, proliferation and mobilization cooperate to incite malignant tumor formation.

Evolutionary flexibility of pair-rule patterning revealed by functional analysis of secondary pair-rule genes, paired and sloppy-paired in the short-germ insect, Tribolium castaneum

In the Drosophila segmentation hierarchy, periodic expression of pair-rule genes translates gradients of regional information from maternal and gap genes into the segmental expression of segment polarity genes. In Tribolium, homologs of almost all the eight canonical Drosophila pair-rule genes are expressed in pair-rule domains, but only five have pair-rule functions. even-skipped, runt and odd-skipped act as primary pair-rule genes, while the functions of paired (prd) and sloppy-paired (slp) are secondary. Since secondary pair-rule genes directly regulate segment polarity genes in Drosophila, we analyzed Tc-prd and Tc-slp to determine the extent to which this paradigm is conserved in Tribolium. We found that the role of prd is conserved between Drosophila and Tribolium; it is required in both insects to activate engrailed in odd-numbered parasegments and wingless (wg) in even-numbered parasegments. Similarly, slp is required to activate wg in alternate parasegments and to maintain the remaining wg stripes in both insects. However, the parasegmental register for Tc-slp is opposite that of Drosophila slp1. Thus, while prd is functionally conserved, the fact that the register of slp function has evolved differently in the lineages leading to Drosophila and Tribolium reveals an unprecedented flexibility in pair-rule patterning.

Differential activities of the core planar polarity proteins during Drosophila wing patterning

During planar polarity patterning of the Drosophila wing, a “core” group of planar polarity genes has been identified which acts downstream of global polarity cues to locally coordinate cell polarity and specify trichome production at distal cell edges. These genes encode protein products that assemble into asymmetric apicolateral complexes that straddle the proximodistal junctional region between adjacent cells. We have carried out detailed genetic analysis experiments, analysing the requirements of each complex component for planar polarity patterning. We find that the three transmembrane proteins at the core of the complex, Frizzled, Strabismus and Flamingo, are required earliest in development and are the only components needed for intercellular polarity signalling. Notably, cells that lack both Frizzled and Strabismus are unable to signal, revealing an absolute requirement for both proteins in cell–cell communication. In contrast the cytoplasmic components Dishevelled, Prickle and Diego are not needed for intercellular communication. These factors contribute to the cell–cell propagation of polarity, most likely by promotion of intracellular asymmetry. Interestingly, both local polarity propagation and trichome placement occur normally in mutant backgrounds where asymmetry of polarity protein distribution is undetectable, suggesting such asymmetry is not an absolute requirement for any of the functions of the core complex.

Wheat TaAS2, a member of LOB family, affects the adaxial–abaxial polarity of leaves in transgenic Arabidopsis

Establishment of adaxial–abaxial polarity is very important to the pattern formation of leaves. Previous studies indicated that ASYMMETRIC LEAVES2 ( AS2) in Arabidopsis is a critical gene that functions on the determination of adaxial–abaxial polarity. To understand the molecular mechanism of polarity establishment in wheat, a gene that shares higher homology with AS2 was isolated from wheat ( Triticum aestivum L.) and it was designated as TaAS2. TaAS2 encoded a protein with a LOB-domain containing a C-motif and a leucine-zipper-like sequence and it was expressed throughout leaf primordia and prevascular tissues and vascular bundles. Ectopic expression of TaAS2 in Arabidopsis mainly resulted in the adaxialization of abaxial mesophyll tissues and the alteration of the vascular pattern in leaves and petioles. Further study showed that the expression of TaAS2 affected the transcript levels of several polarity genes and KNOX genes in Arabidopsis. Our results suggest that TaAS2 may play important roles in regulating plant development.

SERRATE: a new player on the plant microRNA scene

MicroRNAs (miRNAs) function as sequence-specific guides that control gene expression by post-transcriptional gene silencing. Many miRNAs influence plant development by regulating the accumulation of transcripts that encode transcription factors. Mutants defective in miRNA accumulation, such as dcl1, hen1, hyl1 and ago1, have pleiotropic developmental phenotypes. The serrate-1 (se-1) mutant of Arabidopsis also shows a highly pleiotropic phenotype, which overlaps with the phenotypes of mutants defective in miRNA accumulation. Although it has been proposed that SERRATE (SE) functions specifically in miRNA-mediated repression of the leaf polarity genes PHABULOSA and PHAVOLUTA, microarray analysis shows upregulation of many genes known to be the targets of miRNAs in se-1. We show that SE is a general regulator of miRNA levels affecting the processing of primary miRNA to miRNA.

The ten Hox genes of the millipede Glomeris marginata

We have isolated the ten Hox genes from the pill millipede Glomeris marginata (Myriapoda:Diplopoda). All ten genes are expressed in characteristic Hox-gene-like expression patterns. The register of Hox gene expression borders is conserved and the expression profiles show that the anterior-most limb-bearing segment in arthropods (antennal/cheliceral segment) does not express any Hox gene, while the next segment (intercalary/second-antennal/premandibular/pedipalpal segment) does express Hox genes. The Hox expression patterns in this millipede thus support the conclusion that all arthropods possess a deuterocerebral segment. We find that there is an apparent posterior shift of Hox gene expression domains dorsally relative to their ventral patterns, indicating that the decoupling of dorsal and ventral segmentation is not restricted to the level of segment polarity genes but apparently includes the Hox genes. Although the mechanism for the decoupling of dorsal and ventral segmentation remains unsolved, the decoupling must be at a level higher in the hierarchy than that of the segment polarity and Hox genes. The expression patterns of Ultrabithorax and abdominal-A suggest a correlation between the function of these genes and the delayed outgrowth of posterior trunk appendages. This delay may be caused by an assumed repressor function of Ultrabithorax, which might partially repress the activation of the Distal-less gene. The Glomeris fushi tarazu gene is expressed in a Hox-like domain and in the developing central nervous system, but not in segmental stripes such as has been reported in another myriapod species, the centipede Lithobius. In contrast to the Lithobius fushi tarazu gene, there is no indication for a role in segment formation for the millipede fushi tarazu gene, suggesting that fushi tarazu first acquired its segmentation function in the lineage of the insects.

Loss of Cell Polarity Drives Tumor Growth and Invasion through JNK Activation in Drosophila

Apparent defects in cell polarity are often seen in human cancer. However, the underlying mechanisms of how cell polarity disruption contributes to tumor progression are unknown. Here, using a Drosophila genetic model for Ras-induced tumor progression, we show a molecular link between loss of cell polarity and tumor malignancy. Mutation of different apicobasal polarity genes activates c-Jun N-terminal kinase (JNK) signaling and downregulates the E-cadherin/beta-catenin adhesion complex, both of which are necessary and sufficient to cause oncogenic Ras(V12)-induced benign tumors in the developing eye to exhibit metastatic behavior. Furthermore, activated JNK and Ras signaling cooperate in promoting tumor growth cell autonomously, as JNK signaling switches its proapoptotic role to a progrowth effect in the presence of oncogenic Ras. Our finding that such context-dependent alterations promote both tumor growth and metastatic behavior suggests that metastasis-promoting mutations may be selected for based primarily on their growth-promoting capabilities. Similar oncogenic cooperation mediated through these evolutionarily conserved signaling pathways could contribute to human cancer progression.

AINTEGUMENTA Contributes to Organ Polarity and Regulates Growth of Lateral Organs in Combination with YABBY Genes

Lateral organs in flowering plants display polarity along their adaxial-abaxial axis with distinct cell types forming at different positions along this axis. Members of three classes of transcription factors in Arabidopsis (Arabidopsis thaliana; the Class III homeodomain/leucine zipper [HD-ZIP] proteins, KANADI proteins, and YABBY proteins) are expressed in either the adaxial or abaxial domain of organ primordia where they confer these respective identities. Little is known about the factors that act upstream of these polarity-determining genes to regulate their expression. We have investigated the relationship between AINTEGUMENTA (ANT), a gene that promotes initiation and growth of lateral organ primordia, and polarity genes. Although ant single mutants do not display any obvious defects in organ polarity, loss of ANT activity in combination with mutations in one or more YABBY genes results in polarity defects greater than those observed in the yabby mutants alone. Our results suggest that ANT acts in combination with the YABBY gene FILAMENTOUS FLOWER (FIL) to promote organ polarity by up-regulating the expression of the adaxial-specifying HD-ZIP gene PHABULOSA. Furthermore, we show that ANT acts with FIL to up-regulate expression of the floral homeotic gene APETALA3. Our work defines new roles for ANT in the development of lateral organs.

Maintenance of segment and appendage primordia by the Tribolium gene knödel

For homeotic and segment-polarity genes in Drosophila, a switch in gene regulation has been described that distinguishes patterning and maintenance phases. Maintenance of segment and organ primordia involves secondary patterning and differentiation steps, as well as survival factors regulating proliferation and organ size. In a screen for embryonic lethal mutations in the flour beetle Tribolium castaneum, we have recovered two alleles of the knödel gene, which result in short, bag-like embryos. These embryos have severely reduced appendages and differentiate a cuticle that lacks most overt signs of segmentation. In addition, they lack bristles and display defects in the nervous system. Early patterning in knödel mutant embryos is normal up to the extended germ band stage, as indicated by the formation of regular even-skipped ( Tc'eve) and wingless ( Tc'wg) stripes. Afterwards, however, these patterns degenerate. Similarly, proximo-distal growth and patterning of limbs are nearly normal initially, but limb primordia shrink, and proximo-distal patterns degenerate, during subsequent stages. knödel could be a segment polarity gene required for segment border maintenance in both trunk and appendages. Alternatively, it may have a more general role in tissue or organ maintenance.

A pair-rule gene circuit defines segments sequentially in the short-germ insect Tribolium castaneum

In Drosophila, a hierarchy of maternal, gap, pair-rule, and segment polarity gene interactions regulates virtually simultaneous blastoderm segmentation. For the last decade, studies have focused on revealing the extent to which Drosophila segmentation mechanisms are conserved in other arthropods where segments are added sequentially from anterior to posterior in a cellular environment. Despite our increased knowledge of individual segmentation genes, details of their interactions in non-Drosophilid insects are not well understood. We analyzed the Tribolium orthologs of Drosophila pair-rule genes, which display pair-rule expression patterns. Tribolium castaneum paired (Tc-prd) and sloppy-paired (Tc-slp) genes produced pair-rule phenotypes when their transcripts were severely reduced by RNA interference. In contrast, similar analysis of T. castaneum even-skipped (Tc-eve), runt (Tc-run), or odd-skipped (Tc-odd) genes produced severely truncated, almost completely asegmental phenotypes. Analysis of interactions between pair-rule components revealed that Tc-eve, Tc-run, and Tc-odd form a three-gene circuit to regulate one another as well as their downstream targets, Tc-prd and Tc-slp. The complement of primary pair-rule genes in Tribolium differs from Drosophila in that it includes Tc-odd but not Tc-hairy. This gene circuit defines segments sequentially in double segment periodicity. Furthermore, this single mechanism functions in the early blastoderm stage and subsequently during germ-band elongation. The periodicity of the Tribolium pair-rule gene interactions reveals components of the genetic hierarchy that are regulated in a repetitive circuit or clock-like mechanism. This pair-rule gene circuit provides insight into short-germ segmentation in Tribolium that may be more generally applicable to segmentation in other arthropods.

SEUSS and LEUNIG regulate cell proliferation, vascular development and organ polarity in Arabidopsis petals

Unlike in animals where cell migrations and programmed cell death play key roles in organ shape determination, in plants organ shape is largely a result of coordinated cellular growth (cell divisions and cell elongations). We have investigated the role of the SEUSS and LEUNIG genes in Arabidopsis thaliana (L.) Heynh. petal development to better understand the molecular mechanisms through which cellular growth and organ shape are coordinated in plants. SEUSS and LEUNIG encode components of a putative transcriptional regulatory complex that controls organ identity specification through the repression of the floral organ identity gene AGAMOUS. SEUSS and LEUNIG also regulate petal shape through AGAMOUS-independent mechanisms; however, the molecular and cellular actions of SEUSS and LEUNIG during petal development are unknown. Here we show that SEUSS and LEUNIG control blade cell number and vasculature development within the petal. Furthermore, SEUSS and LEUNIG regulate petal polarity along the adaxial/abaxial axis. We present a model where SEUSS and LEUNIG are required to potentiate the key polarity genes PHABULOSA and FILAMENTOUS FLOWER/YABBY1 and thus influence cellular growth within the developing petal blade.

Functions of the segment polarity genes midline and H15 in Drosophila melanogaster neurogenesis

The Drosophila melanogaster ventral nerve cord derives from neural progenitor cells called neuroblasts. Individual neuroblasts have unique gene expression profiles and give rise to distinct clones of neurons and glia. The specification of neuroblast identity provides a cell intrinsic mechanism which ultimately results in the generation of progeny which are different from each other. Segment polarity genes have a dual function in early neurogenesis: within distinct regions of the neuroectoderm, they are required both for neuroblast formation and for the specification of neuroblast identity. Previous studies of segment polarity gene function largely focused on neuroblasts that arise within the posterior part of the segment. Here we show that the segment polarity gene midline is required for neuroblast formation in the anterior-most part of the segment. Moreover, midline contributes to the specification of anterior neuroblast identity by negatively regulating the expression of Wingless and positively regulating the expression of Mirror. In the posterior-most part of the segment, midline and its paralog, H15, have partially redundant functions in the regulation of the NB marker Eagle. Hence, the segment polarity genes midline and H15 play an important role in the development of the ventral nerve cord in the anterior- and posterior-most part of the segment.

Palmitoylation of the EGFR Ligand Spitz by Rasp Increases Spitz Activity by Restricting Its Diffusion

Lipid modifications such as palmitoylation or myristoylation target intracellular proteins to cell membranes. Secreted ligands of the Hedgehog and Wnt families are also palmitoylated; this modification, which requires the related transmembrane acyltransferases Rasp and Porcupine, can enhance their secretion, transport, or activity. We show here that rasp is also essential for the developmental functions of Spitz, a ligand for the Drosophila epidermal growth factor receptor (EGFR). In cultured cells, Rasp promotes palmitate addition to the N-terminal cysteine residue of Spitz, and this cysteine is required for Spitz activity in vivo. Palmitoylation reduces Spitz secretion and enhances its plasma membrane association, but does not alter its ability to activate the EGFR in vitro. In vivo, overexpressed unpalmitoylated Spitz has an increased range of action but reduced activity. These data suggest a role for palmitoylation in restricting Spitz diffusion, allowing its local concentration to reach the threshold required for biological function.