HyAlx, an aristaless-related gene, is involved in tentacle formation in hydra

Developmental gradients are known to play important roles in axial patterning in hydra. Current efforts are directed toward elucidating the molecular basis of these gradients. We report the isolation and characterization of HyAlx, an aristaless-related gene in hydra. The expression patterns of the gene in adult hydra, as well as during bud formation, head regeneration and the formation of ectopic head structures along the body column, indicate the gene plays a role in the specification of tissue for tentacle formation. The use of RNAi provides more direct evidence for this conclusion. The different patterns of HyAlx expression during head regeneration and bud formation also provide support for a recent version of a reaction-diffusion model for axial patterning in hydra.     

HyBMP5-8b, a BMP5-8 orthologue, acts during axial patterning and tentacle formation in hydra

Developmental gradients play a central role in axial patterning in hydra. As part of the effort towards elucidating the molecular basis of these gradients as well as investigating the evolution of the mechanisms underlying axial patterning, genes encoding signaling molecules are under investigation. We report the isolation and characterization of HyBMP5-8b, a BMP5-8 orthologue, from hydra. Processes governing axial patterning are continuously active in adult hydra. Expression patterns of HyBMP5-8b in normal animals and during bud formation, hydra's asexual form of reproduction, were examined. These patterns, coupled with changes in patterns of expression in manipulated tissues during head regeneration, foot regeneration as well as under conditions that alter the positional value gradient indicate that the gene is active in two different processes. The gene plays a role in tentacle formation and in patterning the lower end of the body axis.     

Large-scale biophysics: ion flows and regeneration

Regeneration requires exquisite orchestration of growth and morphogenesis. A powerful but still largely mysterious system of biophysical signals functions during regeneration, embryonic development and neoplasm. Ion transporters generate pH and voltage gradients, as well as ion fluxes, regulating proliferation, differentiation and migration. Endogenous bioelectrical signals are implicated in the control of wound healing, limb development, left-right patterning and spinal cord regeneration. Recent advances in molecular biology and imaging technology have allowed unprecedented insight into the sources and downstream consequences of ion flows. In complement to the current focus on molecular genetics and stem cell biology, artificial modulation of bioelectrical signals in somatic tissues is a powerful modality that might result in profound advances in understanding and augmentation of regenerative capacity.     

Bioelectric signaling in regeneration: Mechanisms of ionic controls of growth and form

The ability to control pattern formation is critical for the both the embryonic development of complex structures as well as for the regeneration/repair of damaged or missing tissues and organs. In addition to chemical gradients and gene regulatory networks, endogenous ion flows are key regulators of cell behavior. Not only do bioelectric cues provide information needed for the initial development of structures, they also enable the robust restoration of normal pattern after injury. In order to expand our basic understanding of morphogenetic processes responsible for the repair of complex anatomy, we need to identify the roles of endogenous voltage gradients, ion flows, and electric fields. In complement to the current focus on molecular genetics, decoding the information transduced by bioelectric cues enhances our knowledge of the dynamic control of growth and pattern formation. Recent advances in science and technology place us in an exciting time to elucidate the interplay between molecular-genetic inputs and important biophysical cues that direct the creation of tissues and organs. Moving forward, these new insights enable additional approaches to direct cell behavior and may result in profound advances in augmentation of regenerative capacity.     

Pentagone: patrolling BMP morphogen signaling

Orchestration of spatial organization by signaling gradients - morphogen gradients - is a fundamental principle in animal development. Despite their importance in tissue patterning and growth, the exact mechanisms underlying the establishment and maintenance of morphogen gradients are poorly understood. Our recent work on BMP (bone morphogenetic protein) morphogen signaling during wing development identified a novel protein, Pentagone (Pent), as a critical regulator of morphogen activity. In the following, we discuss the properties of Pent and its role as a feed-back loop in morphogen gradient formation.     

H+ pump-dependent changes in membrane voltage are an early mechanism necessary and sufficient to induce Xenopus tail regeneration

In many systems, ion flows and long-term endogenous voltage gradients regulate patterning events, but molecular details remain mysterious. To establish a mechanistic link between biophysical events and regeneration, we investigated the role of ion transport during Xenopus tail regeneration. We show that activity of the V-ATPase H(+) pump is required for regeneration but not wound healing or tail development. The V-ATPase is specifically upregulated in existing wound cells by 6 hours post-amputation. Pharmacological or molecular genetic loss of V-ATPase function and the consequent strong depolarization abrogates regeneration without inducing apoptosis. Uncut tails are normally mostly polarized, with discrete populations of depolarized cells throughout. After amputation, the normal regeneration bud is depolarized, but by 24 hours post-amputation becomes rapidly repolarized by the activity of the V-ATPase, and an island of depolarized cells appears just anterior to the regeneration bud. Tail buds in a non-regenerative ;refractory' state instead remain highly depolarized relative to uncut or regenerating tails. Depolarization caused by V-ATPase loss-of-function results in a drastic reduction of cell proliferation in the bud, a profound mispatterning of neural components, and a failure to regenerate. Crucially, induction of H(+) flux is sufficient to rescue axonal patterning and tail outgrowth in otherwise non-regenerative conditions. These data provide the first detailed mechanistic synthesis of bioelectrical, molecular and cell-biological events underlying the regeneration of a complex vertebrate structure that includes spinal cord, and suggest a model of the biophysical and molecular steps underlying tail regeneration. Control of H(+) flows represents a very important new modality that, together with traditional biochemical approaches, may eventually allow augmentation of regeneration for therapeutic applications.     

Hedgehog signaling controls dorsoventral patterning, blastema cell proliferation and cartilage induction during axolotl tail regeneration

Tail regeneration in urodeles requires the coordinated growth and patterning of the regenerating tissues types, including the spinal cord, cartilage and muscle. The dorsoventral (DV) orientation of the spinal cord at the amputation plane determines the DV patterning of the regenerating spinal cord as well as the patterning of surrounding tissues such as cartilage. We investigated this phenomenon on a molecular level. Both the mature and regenerating axolotl spinal cord express molecular markers of DV progenitor cell domains found during embryonic neural tube development, including Pax6, Pax7 and Msx1. Furthermore, the expression of Sonic hedgehog (Shh) is localized to the ventral floor plate domain in both mature and regenerating spinal cord. Patched1 receptor expression indicated that hedgehog signaling occurs not only within the spinal cord but is also transmitted to the surrounding blastema. Cyclopamine treatment revealed that hedgehog signaling is not only required for DV patterning of the regenerating spinal cord but also had profound effects on the regeneration of surrounding, mesodermal tissues. Proliferation of tail blastema cells was severely impaired, resulting in an overall cessation of tail regeneration, and blastema cells no longer expressed the early cartilage marker Sox9. Spinal cord removal experiments revealed that hedgehog signaling, while required for blastema growth is not sufficient for tail regeneration in the absence of the spinal cord. By contrast to the cyclopamine effect on tail regeneration, cyclopamine-treated regenerating limbs achieve a normal length and contain cartilage. This study represents the first molecular localization of DV patterning information in mature tissue that controls regeneration. Interestingly, although tail regeneration does not occur through the formation of somites, the Shh-dependent pathways that control embryonic somite patterning and proliferation may be utilized within the blastema, albeit with a different topography to mediate growth and patterning of tail tissues during regeneration.     

Pentagone: patrolling BMP morphogen signaling

Orchestration of spatial organization by signaling gradients--morphogen gradients--is a fundamental principle in animal development. Despite their importance in tissue patterning and growth, the exact mechanisms underlying the establishment and maintenance of morphogen gradients are poorly understood. Our recent work on BMP (bone morphogenetic protein) morphogen signaling during wing development identified a novel protein, Pentagone (Pent), as a critical regulator of morphogen activity. In the following, we discuss the properties of Pent and its role as a feed-back loop in morphogen gradient formation.     

Variegated desert vegetation: Covariation of edaphic and fire variables provides a framework for understanding mulga‐spinifex coexistence

Abstract Mulga (Acacia aneura Mimosaceae) and spinifex (Triodia spp. Poaceae) habitats together characterize a large part of arid central Australia. Often very abrupt boundaries form between these two habitats, giving rise to a mosaic pattern of contrasting shrub‐grass alterations across the landscape. Reasons for such patterning remain poorly understood though current niche‐based views relate species' distributions to spatial resource gradients or to fire effects. Field survey work was conducted on central Australian mountain ranges to further quantify floristic, regeneration traits, and structural patterning across mulga‐spinifex transitions and to test resource‐ and disturbance‐models that explain these patterns. Compositional analysis demonstrated variability in transition type – in certain cases boundaries denoted true floristic discontinuity and in others, somewhat more of a structural shift. Moreover, it was shown that minimal between‐habitat floristic overlap coincided with the occurrence of distinct edaphic changes, while greater compositional commonality occurred when soil gradients were more diffuse. This indicated that floristic patterning cannot be ascribed to any one single process. In the case of strong soil gradients, between‐habitat segregation most likely resulted from resource‐based niche differentiation; for weaker gradients, fire‐frequency assumed greatest importance. Disturbance theory most readily accounted for the distribution of woody species' post‐fire regeneration traits across habitat boundaries. The results also suggested that biotic factors –viz competition, facilitation and animal‐mediated dispersal – may be of additional consequence for mulga‐spinifex coexistence. Overall, the study served to emphasize the importance of multi‐factor explanation for within‐ and between‐habitat patterning in these mosaics. It also highlighted the need for experimentation to facilitate distinction between cause and correlation.     

miR-196 is an essential early-stage regulator of tail regeneration, upstream of key spinal cord patterning events

Salamanders have the remarkable ability to regenerate many body parts following catastrophic injuries, including a fully functional spinal cord following a tail amputation. The molecular basis for how this process is so exquisitely well-regulated, assuring a faithful replication of missing structures every time, remains poorly understood. Therefore a study of microRNA expression and function during regeneration in the axolotl, Ambystoma mexicanum, was undertaken. Using microarray-based profiling, it was found that 78 highly conserved microRNAs display significant changes in expression levels during the early stages of tail regeneration, as compared to mature tissue. The role of miR-196, which was highly upregulated in the early tail blastema and spinal cord, was then further analyzed. Inhibition of miR-196 expression in this context resulted in a defect in regeneration, yielding abnormally shortened tails with spinal cord defects in formation of the terminal vesicle. A more detailed characterization of this phenotype revealed downstream components of the miR-196 pathway to include key effectors/regulators of tissue patterning within the spinal cord, including BMP4 and Pax7. As such, our dataset establishes miR-196 as an essential regulator of tail regeneration, acting upstream of key BMP4 and Pax7-based patterning events within the spinal cord.     

Patterning of the head in hydra as visualized by a monoclonal antibody: III. The dynamics of head regeneration

The dynamics of the early patterning processes leading to the regeneration of a head in tissue excised from the body column of Hydra oligactis were examined by using a monoclonal antibody, CP8. This antibody displays position-specific binding, labeling the head ectodermal epithelial cells. During regeneration of a head, antibody labeling is present well before morphological signs of the head, at a time correlated with the determination of the tissue (Javois et al., Dev. Biol., 117:607-618, '86). By quantifying antibody labeling during regeneration of three different pieces of tissue excised from the body column, it was found that the dynamics of the early patterning processes as visualized by CP8 labeling varied. The pattern of labeling observed as well as the spread of labeled tissue suggested that the amount and geometry of apical tissue in the regenerate played a critical role in the patterning processes. Contrary to the labeling pattern observed in heads which formed during bud development or which regenerated following decapitation (Javois et al., '86), not all the CP8+ tissue was confined to the head structures in these regenerates. Several alternative explanations for this surprising result are presented. The usefulness of these data in refining pattern formation models by more explicitly constraining their parameters is discussed.     

Hox genes: Downstream “effectors” of retinoic acid signaling in vertebrate embryogenesis

One of the major regulatory challenges of animal development is to precisely coordinate in space and time the formation, specification, and patterning of cells that underlie elaboration of the basic body plan. How does the vertebrate plan for the nervous and hematopoietic systems, heart, limbs, digestive, and reproductive organs derive from seemingly similar population of cells? These systems are initially established and patterned along the anteroposterior axis (AP) by opposing signaling gradients that lead to the activation of gene regulatory networks involved in axial specification, including the Hox genes. The retinoid signaling pathway is one of the key signaling gradients coupled to the establishment of axial patterning. The nested domains of Hox gene expression, which provide a combinatorial code for axial patterning, arise in part through a differential response to retinoic acid (RA) diffusing from anabolic centers established within the embryo during development. Hence, Hox genes are important direct effectors of retinoid signaling in embryogenesis. This review focuses on describing current knowledge on the complex mechanisms and regulatory processes, which govern the response of Hox genes to RA in several tissue contexts including the nervous system during vertebrate development.     

Wnt signaling is required for antero-posterior patterning of the planarian brain

Wnt signaling functions in axis formation and morphogenesis in various animals and organs. Here we report that Wnt signaling is required for proper brain patterning during planarian brain regeneration. We showed here that one of the Wnt homologues in the planarian Dugesia japonica, DjwntA, was expressed in the posterior region of the brain. When DjwntA-knockdown planarians were produced by RNAi, they could regenerate their heads at the anterior ends of the fragments, but formed ectopic eyes with irregular posterior lateral branches and brain expansion. This suggests that the Wnt signal may be involved in antero-posterior (A-P) patterning of the planarian brain, as in vertebrates. We also investigated the relationship between the DjwntA and nou-darake/FGFR signal systems, as knockdown planarians of these genes showed similar phenotypes. Double-knockdown planarians of these genes did not show any synergistic effects, suggesting that the two signal systems function independently in the process of brain regeneration, which accords with the fact that nou-darake was expressed earlier than DjwntA during brain regeneration. These observations suggest that the nou-darake/FGFR signal may be involved in brain rudiment formation during the early stage of head regeneration, and subsequently the DjwntA signal may function in A-P patterning of the brain rudiment.     

Morphogen-based simulation model of ray growth and joint patterning during fin development and regeneration

The fact that some organisms are able to regenerate organs of the correct shape and size following amputation is particularly fascinating, but the mechanism by which this occurs remains poorly understood. The zebrafish (Danio rerio) caudal fin has emerged as a model system for the study of bone development and regeneration. The fin comprises 16 to 18 bony rays, each containing multiple joints along its proximodistal axis that give rise to segments. Experimental observations on fin ray growth, regeneration and joint formation have been described, but no unified theory has yet been put forward to explain how growth and joint patterns are controlled. We present a model for the control of fin ray growth during development and regeneration, integrated with a model for joint pattern formation, which is in agreement with published, as well as new, experimental data. We propose that fin ray growth and joint patterning are coordinated through the interaction of three morphogens. When the model is extended to incorporate multiple rays across the fin, it also accounts for how the caudal fin acquires its shape during development, and regains its correct size and shape following amputation.     

Advances in pattern formation: Signaling and transcription

The 49(th) Annual Drosophila Research Conference was held in the sunny confines of San Diego. As usual, large numbers of Drosophila scientists working in fields as different as immunology and evolution descended on the venue. The meeting showed that the fly community is still vibrant and diverse even with the funding crunch at the NIH and the renewed rumors that Drosophila may have outlived its usefulness. This short review will focus on one session of platform presentations detailing the recent advances in the field of pattern formation. This session offered a variety of topics reviewing the formation of pattern in various tissues through diverse mechanisms. I will focus on early embryonic patterning through pair-rule genes, specificity of FGF signaling, and tissue regeneration.     

The Kinase Regulator Mob1 Acts as a Patterning Protein for Stentor Morphogenesis

Morphogenesis and pattern formation are vital processes in any organism, whether unicellular or multicellular. But in contrast to the developmental biology of plants and animals, the principles of morphogenesis and pattern formation in single cells remain largely unknown. Although all cells develop patterns, they are most obvious in ciliates; hence, we have turned to a classical unicellular model system, the giant ciliate Stentor coeruleus. Here we show that the RNA interference (RNAi) machinery is conserved in Stentor. Using RNAi, we identify the kinase coactivator Mob1—with conserved functions in cell division and morphogenesis from plants to humans—as an asymmetrically localized patterning protein required for global patterning during development and regeneration in Stentor. Our studies reopen the door for Stentor as a model regeneration system.     

Recognition molecules and neural repair

Neural recognition molecules were discovered and characterized initially for their functional roles in cell adhesion as regulators of affinity between cells and the extracellular matrix in vitro. They were then recognized as mediators or co-receptors which trigger signal transduction mechanisms affecting cell adhesion and de-adhesion. Their involvement in contact attraction and repulsion relies on cell-intrinsic properties that are modulated by the spatial contexts of their expression at particular stages of ontogenetic development, in synaptic plasticity and during regeneration after injury. The functional roles of recognition molecules in cell proliferation and migration, determination of developmental fate, growth cone guidance, and synapse formation, stabilization and modulation have been well documented not only by in vitro, but also by in vivo studies that have been greatly aided by generation of genetically altered mice. More recently, the functions of recognition molecules have been investigated under conditions of neural repair and manipulated using a broad range of genetic and pharmacological approaches to achieve a beneficial outcome. The principal aim of most therapeutically oriented approaches has been to neutralize inhibitory factors. However, less attention has been paid to enhancing repair by stimulating the stimulatory factors. When considering potential therapeutic strategies, it is worth considering that a single recognition molecule can possess domains that are conducive or repellent and that the spatial distribution of recognition molecules can determine the overall function: Recognition molecules may be repellent for neurite outgrowth when presented as barriers or steep-concentration gradients and conducive when presented as uniform substrates. The focus of this review will be on the more recent attempts to study the conducive mechanisms with the expectation that they may be able to tip the balance from a regeneration inhospitable to a hospitable environment. It is likely that a combination of the two principles, as multifactorial as each principle may be in itself, will be of therapeutic value in humans.     

Plant development: auxin in loops

Concentration gradients of the hormone auxin are associated with various patterning events in plants. Recent work has refined our picture of the complex and dynamic system of auxin transport underlying the formation of these gradients.     

Specifying positional information in the embryo: looking beyond morphogens

Concentration gradients of small diffusible molecules called morphogens are key regulators of development, specifying position during pattern formation in the embryo. It is now becoming clear that additional or alternative mechanisms involving interactions among cells are also crucial for positional specification.