bluuebunny/crossref_metadata_2025 huggingface dataset:
defective proventriculusis required for pattern formation along the proximodistal axis, cell proliferation and formation of veins in theDrosophilawing
Authors: Kölzer Stefan , Fuss Bernhard , Hoch Michael , Klein Thomas
Published: July 2003

Many genes have been identified that are required for the establishment of the dorsoventral (DV) and anteroposterior (AP) axes of the Drosophilawing. By contrast, little is known about the genes and mechanisms that pattern the proximodistal (PD) axis. Vestigial (Vg) is instrumental in patterning this axis, but the genes that mediate its effects and the mechanisms that operate during PD patterning are not known. We show that the gene defective proventriculus (dve) is required for a region of the PD axis encompassing the distal region of the proximal wing (PW) and a small part of the adjacent wing pouch. Loss-of-function of dve results in the deletion of this region and, consequently, shortening of the PD axis. dve expression is activated by Vg in a non-autonomous manner, and is repressed at the DV boundary through the combined activity of Nubbin and Wg. Besides its role in the establishment of the distal part of the PW, dve is also required for the formation of the wing veins 2 and 5, and the proliferation of wing pouch cells, especially in regions anterior to wing vein 3 and posterior to wing vein 4. The study of the regulation of dve expression provides information about the strategies employed to subdivide and pattern the PD axis, and reveals the importance of vgduring this process.
Msx1is required for dorsal diencephalon patterning
Authors: Bach Antoine , Lallemand Yvan , Nicola Marie-Anne , Ramos Casto , Mathis Luc , Maufras Mathilde , Robert Benoît
Published: July 2003

The dorsal midline of the neural tube has recently emerged as a major signaling center for dorsoventral patterning. Msx genes are expressed at the dorsal midline, although their function at this site remains unknown. Using Msx1nlacZ mutant mice, we show that the normal expression domain of Msx1 is interrupted in the pretectum of mutant embryos. Morphological and gene expression data further indicate that a functional midline is not maintained along the whole prosomere 1 in Msx1 mutant mice. This results in the downregulation of genes expressed laterally to the midline in prosomere 1, confirming the importance of the midline as a signaling center. Wnt1 is essential for dorsoventral patterning of the neural tube. In the Msx1 mutant, Wnt1 is downregulated before the midline disappears, suggesting that its expression depends on Msx1. Furthermore, electroporation in the chick embryo demonstrates that Msx1 can induce Wnt1 expression in the diencephalon neuroepithelium and in the lateral ectoderm. In double Msx1/Msx2 mutants, Wnt1 expression is completely abolished at the dorsal midline of the diencephalon and rostral mesencephalon. This indicates that Msx genes may regulate Wnt1 expression at the dorsal midline of the neural tube. Based on these results, we propose a model in which Msx genes are intermediary between Bmp and Wnt at this site.
Conservation ofEndo16expression in sea urchins despite evolutionary divergence in both cis and trans-acting components of transcriptional regulation
Authors: Romano Laura A. , Wray Gregory A.
Published: July 2003

Evolutionary changes in transcriptional regulation undoubtedly play an important role in creating morphological diversity. However, there is little information about the evolutionary dynamics of cis-regulatory sequences. This study examines the functional consequence of evolutionary changes in the Endo16 promoter of sea urchins. The Endo16 gene encodes a large extracellular protein that is expressed in the endoderm and may play a role in cell adhesion. Its promoter has been characterized in exceptional detail in the purple sea urchin, Strongylocentrotus purpuratus. We have characterized the structure and function of the Endo16 promoter from a second sea urchin species, Lytechinus variegatus. The Endo16 promoter sequences have evolved in a strongly mosaic manner since these species diverged ∼35 million years ago: the most proximal region (module A) is conserved, but the remaining modules (B-G) are unalignable. Despite extensive divergence in promoter sequences, the pattern of Endo16 transcription is largely conserved during embryonic and larval development. Transient expression assays demonstrate that 2.2 kb of upstream sequence in either species is sufficient to drive GFP reporter expression that correctly mimics this pattern of Endo16transcription. Reciprocal cross-species transient expression assays imply that changes have also evolved in the set of transcription factors that interact with the Endo16 promoter. Taken together, these results suggest that stabilizing selection on the transcriptional output may have operated to maintain a similar pattern of Endo16 expression in S. purpuratus and L. variegatus, despite dramatic divergence in promoter sequence and mechanisms of transcriptional regulation.
Nitric oxide and cyclic nucleotides are regulators of neuronal migration in an insect embryo
Authors: Haase Annely , Bicker Gerd
Published: July 2003

The dynamic regulation of nitric oxide synthase (NOS) activity and cGMP levels suggests a functional role in the development of nervous systems. We report evidence for a key role of the NO/cGMP signalling cascade on migration of postmitotic neurons in the enteric nervous system of the embryonic grasshopper. During embryonic development, a population of enteric neurons migrates several hundred micrometers on the surface of the midgut. These midgut neurons (MG neurons) exhibit nitric oxide-induced cGMP-immunoreactivity coinciding with the migratory phase. Using a histochemical marker for NOS, we identified potential sources of NO in subsets of the midgut cells below the migrating MG neurons. Pharmacological inhibition of endogenous NOS, soluble guanylyl cyclase (sGC) and protein kinase G (PKG) activity in whole embryo culture significantly blocks MG neuron migration. This pharmacological inhibition can be rescued by supplementing with protoporphyrin IX free acid,an activator of sGC, and membrane-permeant cGMP, indicating that NO/cGMP signalling is essential for MG neuron migration. Conversely, the stimulation of the cAMP/protein kinase A signalling cascade results in an inhibition of cell migration. Activation of either the cGMP or the cAMP cascade influences the cellular distribution of F-actin in neuronal somata in a complementary fashion. The cytochemical stainings and experimental manipulations of cyclic nucleotide levels provide clear evidence that NO/cGMP/PKG signalling is permissive for MG neuron migration, whereas the cAMP/PKA cascade may be a negative regulator. These findings reveal an accessible invertebrate model in which the role of the NO and cyclic nucleotide signalling in neuronal migration can be analyzed in a natural setting.
Interplay between the tumor suppressor p53 and TGFβ signaling shapes embryonic body axes inXenopus
Authors: Takebayashi-Suzuki Kimiko , Funami Jun , Tokumori Daisuke , Saito Akira , Watabe Tetsuro , Miyazono Kohei , Kanda Akifumi , Suzuki Atsushi
Published: July 2003

The transcription factor p53 has been shown to mediate cellular responses to diverse stresses such as DNA damage. However, the function of p53 in cellular differentiation in response to growth factor stimulations has remained obscure. We present evidence that p53 regulates cellular differentiation by modulating signaling of the TGFβ family of growth factors during early Xenopus embryogenesis. We show that p53 functionally and physically interacts with the activin and bone morphogenetic protein pathways to directly induce the expression of the homeobox genes Xhox3 and Mix.1/2. Furthermore, functional knockdown of p53 in embryos by an antisense morpholino oligonucleotide reveals that p53 is required for the development of dorsal and ventral mesoderm. Our data illustrate a pivotal role of interplay between the p53 and TGFβ pathways in cell fate determination during early vertebrate embryogenesis.
The role of PAR-1 in regulating the polarised microtubule cytoskeleton in theDrosophilafollicular epithelium
Authors: Doerflinger Hélène , Benton Richard , Shulman Joshua M. , Johnston Daniel St
Published: July 2003

The PAR-1 kinase plays a conserved role in cell polarity in C. elegans,Drosophila and mammals. We have investigated the role of PAR-1 in epithelial polarity by generating null mutant clones in the Drosophila follicular epithelium. Large clones show defects in apicobasal membrane polarity, but small clones induced later in development usually have a normal membrane polarity. However, all cells that lack PAR-1 accumulate spectrin and F-actin laterally, and show a strong increase in the density of microtubules. This is consistent with the observation that the mammalian PAR-1 homologues, the MARKs, dramatically reduce the number of microtubules, when overexpressed in tissue culture cells. The MARKs have been proposed to destabilize microtubules by inhibiting the stabilizing activity of the Tau family of microtubule-associated proteins. This is not the case in Drosophila, however, as null mutations in the single taufamily member in the genome have no effect on the microtubule organisation in the follicle cells. Furthermore, PAR-1 activity stabilises microtubules, as microtubules in mutant cells depolymerise much more rapidly after cold or colcemid treatments. Loss of PAR-1 also disrupts the basal localisation of the microtubule plus ends, which are mislocalised to the centre of mutant cells. Thus, Drosophila PAR-1 regulates the density, stability and apicobasal organisation of microtubules. Although the direct targets of PAR-1 are unknown, we suggest that it functions by regulating the plus ends,possibly by capping them at the basal cortex.
SCD1 is required for cell cytokinesis and polarized cell expansion inArabidopsis thaliana
Authors: Falbel Tanya G. , Koch Lisa M. , Nadeau Jeanette A. , Segui-Simarro Jose M. , Sack Fred D. , Bednarek Sebastian Y.
Published: July 2003

In the leaf epidermis, guard mother cells undergo a stereotyped symmetric division to form the guard cells of stomata. We have identified a temperature-sensitive Arabidopsis mutant, stomatal cytokinesis-defective 1-1 (scd1-1), which affects this specialized division. At the non-permissive temperature, 22°C, defective scd1-1 guard cells are binucleate, and the formation of their ventral cell walls is incomplete. Cytokinesis was also disrupted in other types of epidermal cells such as pavement cells. Further phenotypic analysis of scd1-1 indicated a role for SCD1 in seedling growth, root elongation and flower morphogenesis. More severe scd1 T-DNA insertion alleles (scd1-2 and scd1-3) markedly affect polar cell expansion, most notably in trichomes and root hairs. SCD1 is a unique gene in Arabidopsis that encodes a protein related to animal proteins that regulate intracellular protein transport and/or mitogen-activated protein kinase signaling pathways. Consistent with a role for SCD1 in membrane trafficking, secretory vesicles were found to accumulate in cytokinesis-defective scd1 cells. In addition the scd1 mutant phenotype was enhanced by low doses of inhibitors of cell plate consolidation and vesicle secretion. We propose that SCD1 functions in polarized vesicle trafficking during plant cytokinesis and cell expansion.
Phyllotactic pattern and stem cell fate are determined by theArabidopsishomeobox geneBELLRINGER
Authors: Byrne Mary E. , Groover Andrew T. , Fontana Joseph R. , Martienssen Robert A.
Published: July 2003

Lateral organs in plants arise from the meristem in a stereotypical pattern known as phyllotaxy. Spiral patterns result from initiation of successive organs at a fixed angle of divergence but variable patterns of physical contact. Such patterns ultimately give rise to individual leaves and flowers at positions related to each other by consecutive terms in the mathematical series first described by Leonardo Fibonacci. We demonstrate that a BELL1 related homeodomain protein in Arabidopsis, BELLRINGER, maintains the spiral phyllotactic pattern. In the absence of BELLRINGER, the regular pattern of organ initiation is disturbed and lateral organs are initiated more frequently. BELLRINGER is also required for maintenance of stem cell fate in the absence of the regulatory genes SHOOT MERISTEMLESS and ASYMMETRIC LEAVES1. We propose a model whereby BELLRINGER coordinates the maintenance of stem cells with differentiation of daughter cells in stem cell lineages.
Selective degradation of excess Ldb1 by Rnf12/RLIM confers proper Ldb1 expression levels and Xlim-1/Ldb1 stoichiometry inXenopusorganizer functions
Authors: Hiratani Ichiro , Yamamoto Naoko , Mochizuki Toshiaki , Ohmori Shin-ya , Taira Masanori
Published: July 2003

The Xenopus LIM homeodomain (LIM-HD) protein, Xlim-1, is expressed in the Spemann organizer and cooperates with its positive regulator, Ldb1, to activate organizer gene expression. While this activation is presumably mediated through Xlim-1/Ldb1 tetramer formation, the mechanisms regulating proper Xlim-1/Ldb1 stoichiometry remains largely unknown. We isolated the Xenopus ortholog (XRnf12) of the RING finger protein Rnf12/RLIM and explored its functional interactions with Xlim-1 and Ldb1. Although XRnf12 functions as a E3 ubiquitin ligase for Ldb1 and causes proteasome-dependent degradation of Ldb1, we found that co-expression of a high level of Xlim-1 suppresses Ldb1 degradation by XRnf12. This suppression requires both the LIM domains of Xlim-1 and the LIM interaction domain of Ldb1, suggesting that Ldb1, when bound to Xlim-1, escapes degradation by XRnf12. We further show that a high level of Ldb1 suppresses the organizer activity of Xlim-1/Ldb1,suggesting that excess Ldb1 molecules disturb Xlim-1/Ldb1 stoichiometry. Consistent with this, Ldb1 overexpression in the dorsal marginal zone suppresses expression of several organizer genes including postulated Xlim-1 targets, and importantly, this suppression is rescued by co-expression of XRnf12. These data suggest that XRnf12 confers proper Ldb1 protein levels and Xlim-1/Ldb1 stoichiometry for their functions in the organizer. Together with the similarity in the expression pattern of Ldb1 and XRnf12throughout early embryogenesis, we propose Rnf12/RLIM as a specific regulator of Ldb1 to ensure its proper interactions with LIM-HD proteins and possibly other Ldb1-interacting proteins in the organizer as well as in other tissues.
Novelas1andas2defects in leaf adaxial-abaxial polarity reveal the requirement forASYMMETRIC LEAVES1and2andERECTAfunctions in specifying leaf adaxial identity
Authors: Xu Lin , Xu Yi , Dong Aiwu , Sun Yue , Pi Limin , Xu Yuquan , Huang Hai
Published: July 2003

The shoot apical meristem (SAM) of seed plants is the site at which lateral organs are formed. Once organ primordia initiate from the SAM, they establish polarity along the adaxial-abaxial, proximodistal and mediolateral axes. Among these three axes, the adaxial-abaxial polarity is of primary importance in leaf patterning. In leaf development, once the adaxial-abaxial axis is established within leaf primordia, it provides cues for proper lamina growth and asymmetric development. It was reported previously that the Arabidopsis ASYMMETRIC LEAVES1 (AS1) and ASYMMETRIC LEAVES2 (AS2) genes are two key regulators of leaf polarity. In this work, we demonstrate a new function of the AS1 and AS2genes in the establishment of adaxial-abaxial polarity by analyzing as1 and as2 alleles in the Landsberg erecta(Ler) genetic background. We provide genetic evidence that the Arabidopsis ERECTA (ER) gene is involved in the AS1-AS2 pathway to promote leaf adaxial fate. In addition, we show that AS1 and AS2 bind to each other, suggesting that AS1 and AS2 may form a complex that regulates the establishment of leaf polarity. We also report the effects on leaf polarity of overexpression of the AS1 or AS2genes under the control of the cauliflower mosaic virus (CAMV) 35S promoter. Although plants with as1 and as2 mutations have very similar phenotypes, 35S::AS1/Ler and 35S::AS2/Lertransgenic plants showed dramatically different morphologies. A possible model of the AS1, AS2 and ER action in leaf polarity formation is discussed.
EDL/MAE regulates EGF-mediated induction by antagonizing Ets transcription factor Pointed
Authors: Yamada Takuma , Okabe Masataka , Hiromi Yasushi
Published: July 2003

Inductive patterning mechanisms often use negative regulators to coordinate the effects and efficiency of induction. During Spitz EGF-mediated neuronal induction in the Drosophila compound eye and chordotonal organs,Spitz causes activation of Ras signaling in the induced cells, resulting in the activation of Ets transcription factor Pointed P2. We describe developmental roles of a novel negative regulator of Ras signaling, EDL/MAE, a protein with an Ets-specific Pointed domain but not an ETS DNA-binding domain. The loss of EDL/MAE function results in reduced number of photoreceptor neurons and chordotonal organs, suggesting a positive role in the induction by Spitz EGF. However, EDL/MAE functions as an antagonist of Pointed P2, by binding to its Pointed domain and abolishing its transcriptional activation function. Furthermore, edl/mae appears to be specifically expressed in cells with inducing ability. This suggests that inducing cells,which can respond to Spitz they themselves produce, must somehow prevent activation of Pointed P2. Indeed hyperactivation of Pointed P2 in inducing cells interferes with their inducing ability, resulting in the reduction in inducing ability. We propose that EDL/MAE blocks autocrine activation of Pointed P2 so that inducing cells remain induction-competent. Inhibition of inducing ability by Pointed probably represents a novel negative feedback system that can prevent uncontrolled spread of induction of similar cell fates.
Targeted expression of the dominant-negative FGFR4a in the eye usingXrx1Aregulatory sequences interferes with normal retinal development
Authors: Zhang Li , El-Hodiri Heithem M. , Ma Hai-Fei , Zhang Xue , Servetnick Marc , Wensel Theodore G. , Jamrich Milan
Published: July 2003

Molecular analysis of vertebrate eye development has been hampered by the availability of sequences that can selectively direct gene expression in the developing eye. We report the characterization of the regulatory sequences of the Xenopus laevis Rx1A gene that can direct gene expression in the retinal progenitor cells. We have used these sequences to investigate the role of Fibroblast Growth Factor (FGF) signaling in the development of retinal cell types. FGFs are signaling molecules that are crucial for correct patterning of the embryo and that play important roles in the development of several embryonic tissues. FGFs and their receptors are expressed in the developing retina, and FGF receptor-mediated signaling has been implicated to have a role in the specification and survival of retinal cell types. We investigated the role of FGF signaling mediated by FGF receptor 4a in the development of retinal cell types in Xenopus laevis. For this purpose, we have made transgenic Xenopus tadpoles in which the dominant-negative FGFR4a(ΔFGFR4a) coding region was linked to the newly characterized regulatory sequences of the Xrx1A gene. We found that the expression ofΔFGFR4a in retinal progenitor cells results in abnormal retinal development. The retinas of transgenic animals expressing ΔFGFR4a show disorganized cell layering and specifically lack photoreceptor cells. These experiments show that FGFR4a-mediated FGF signaling is necessary for the correct specification of retinal cell types. Furthermore, they demonstrate that constructs using Xrx1A regulatory sequences are excellent tools with which to study the developmental processes involved in retinal formation.
The role ofSix1in mammalian auditory system development
Authors: Zheng Weiming , Huang Li , Wei Zhu-Bo , Silvius Derek , Tang Bihui , Xu Pin-Xian
Published: July 2003

The homeobox Six genes, homologues to Drosophila sine oculis(so) gene, are expressed in multiple organs during mammalian development. However, their roles during auditory system development have not been studied. We report that Six1 is required for mouse auditory system development. During inner ear development, Six1 expression was first detected in the ventral region of the otic pit and later is restricted to the middle and ventral otic vesicle within which, respectively, the vestibular and auditory epithelia form. By contrast, Six1 expression is excluded from the dorsal otic vesicle within which the semicircular canals form. Six1 is also expressed in the vestibuloacoustic ganglion. At E15.5, Six1 is expressed in all sensory epithelia of the inner ear. Using recently generated Six1 mutant mice, we found that all Six1+/- mice showed some degree of hearing loss because of a failure of sound transmission in the middle ear. By contrast, Six1-/- mice displayed malformations of the auditory system involving the outer, middle and inner ears. The inner ear development in Six1-/- embryos arrested at the otic vesicle stage and all components of the inner ear failed to form due to increased cell death and reduced cell proliferation in the otic epithelium. Because we previously reported that Six1 expression in the otic vesicle is Eya1dependent, we first clarified that Eya1 expression was unaffected in Six1-/- otic vesicle, further demonstrating that the Drosophila Eya-Six regulatory cassette is evolutionarily conserved during mammalian inner ear development. We also analyzed several other otic markers and found that the expression of Pax2 and Pax8 was unaffected in Six1-/- otic vesicle. By contrast, Six1 is required for the activation of Fgf3 expression and the maintenance of Fgf10 and Bmp4 expression in the otic vesicle. Furthermore, loss of Six1 function alters the expression pattern of Nkx5.1 and Gata3, indicating that Six1is required for regional specification of the otic vesicle. Finally, our data suggest that the interaction between Eya1 and Six1 is crucial for the morphogenesis of the cochlea and the posterior ampulla during inner ear development. These analyses establish a role for Six1 in early growth and patterning of the otic vesicle.
The identification of novel genes required forDrosophilaanteroposterior axis formation in a germline clone screen using GFP-Staufen
Authors: Martin Sophie G. , Leclerc Vincent , Smith-Litière Katie , Johnston Daniel St
Published: July 2003

The anteroposterior axis of Drosophila is defined during oogenesis, when the polarisation of the oocyte microtubule cytoskeleton directs the localisation of bicoid and oskar mRNAs to the anterior and posterior poles, respectively. Although maternal-effect lethal and female-sterile screens have identified many mutants that disrupt these processes, these screens could not recover mutations in essential genes. Here we describe a genetic screen in germline clones for mutants that disrupt the localisation of GFP-Staufen in living oocytes, which overcomes this limitation. As Staufen localises to the posterior with oskar mRNA and to the anterior with bicoid mRNA, it acts as a marker for both poles of the oocyte, allowing the identification of mutants that affect the localisation of either mRNA, as well as mutants that disrupt oocyte polarity. Using this approach, we have identified 23 novel complementation groups on chromosome 3R that disrupt anteroposterior axis formation. Analyses of new alleles of spn-E and orb show that both SPN-E and ORB proteins are required to organise the microtubule cytoskeleton at stage 9, and to prevent premature cytoplasmic streaming. Furthermore, yps mutants partially suppress the premature cytoplasmic streaming of orbmutants. As orb, yps and spn-E encode RNA-binding proteins,they may regulate the translation of unidentified RNAs necessary for the polarisation of the microtubule cytoskeleton.
The pro-BMP activity of Twisted gastrulation is independent of BMP binding
Authors: Oelgeschläger Michael , Reversade Bruno , Larraín Juan , Little Shawn , Mullins Mary C. , De Robertis E. M.
Published: July 2003

The determination of the vertebrate dorsoventral body axis is regulated in the extracellular space by a system of interacting secreted molecules consisting of BMP, Chordin, Tolloid and Twisted Gastrulation (Tsg). Tsg is a BMP-binding protein that forms ternary complexes with BMP and Chordin. We investigated the function of Tsg in embryonic patterning by generating point mutations in its two conserved cysteine-rich domains. Surprisingly, Tsg proteins with mutations in the N-terminal domain were unable to bind BMP, yet ventralized the embryo very effectively, indicating strong pro-BMP activity. This hyperventralizing Tsg activity required an intact C-terminal domain and could block the anti-BMP activity of isolated BMP-binding modules of Chordin(CRs) in embryonic assays. This activity was specific for CR-containing proteins as it did not affect the dorsalizing effects of Noggin or dominant-negative BMP receptor. The ventralizing effects of the xTsg mutants were stronger than the effect of Chordin loss-of-function in Xenopusor zebrafish. The results suggest that xTsg interacts with additional CR-containing proteins that regulate dorsoventral development in embryos.
Complementary roles for Nkx6 and Nkx2 class proteins in the establishment of motoneuron identity in the hindbrain
Authors: Pattyn Alexandre , Vallstedt Anna , Dias Jose M. , Sander Maike , Ericson Johan
Published: July 2003

The genetic program that underlies the generation of visceral motoneurons in the developing hindbrain remains poorly defined. We have examined the role of Nkx6 and Nkx2 class homeodomain proteins in this process, and provide evidence that these proteins mediate complementary roles in the specification of visceral motoneuron fate. The expression of Nkx2.2 in hindbrain progenitor cells is sufficient to mediate the activation of Phox2b, a homeodomain protein required for the generation of hindbrain visceral motoneurons. The redundant activities of Nkx6.1 and Nkx6.2, in turn, are dispensable for visceral motoneuron generation but are necessary to prevent these cells from adopting a parallel program of interneuron differentiation. The expression of Nkx6.1 and Nkx6.2 is further maintained in differentiating visceral motoneurons, and consistent with this the migration and axonal projection properties of visceral motoneurons are impaired in mice lacking Nkx6.1 and/or Nkx6.2 function. Our analysis provides insight also into the role of Nkx6 proteins in the generation of somatic motoneurons. Studies in the spinal cord have shown that Nkx6.1 and Nkx6.2 are required for the generation of somatic motoneurons,and that the loss of motoneurons at this level correlates with the extinguished expression of the motoneuron determinant Olig2. Unexpectedly, we find that the initial expression of Olig2 is left intact in the caudal hindbrain of Nkx6.1/Nkx6.2 compound mutants, and despite this, all somatic motoneurons are missing. These data argue against models in which Nkx6 proteins and Olig2 operate in a linear pathway, and instead indicate a parallel requirement for these proteins in the progression of somatic motoneuron differentiation. Thus, both visceraland somatic motoneuron differentiation appear to rely on the combined activity of cell intrinsic determinants, rather than on a single key determinant of neuronal cell fate.
The double contact phase in walking children
Authors: Bastien G. J. , Heglund N. C. , Schepens B.
Published: July 2003

During walking, when both feet are on the ground (the double contact phase), the legs push against each other, and both positive and negative work are done simultaneously. The work done by one leg on the other(Wint,dc) is not counted in the classic measurements of the positive muscular work done during walking. Using force platforms, we studied the effect of speed and age (size) on Wint,dc. In adults and in 3-12-year-old children, Wint,dc (J kg-1 m-1) as a function of speed shows an inverted U-shaped curve, attaining a maximum value that is independent of size but that occurs at higher speeds in larger subjects. Normalising the speed with the Froude number shows that Wint,dc is maximal at about 0.3 in both children and adults. Differences due to size disappear for the most part when normalised with the Froude number, indicating that these speed-dependent changes are primarily a result of body size changes. At its maximum, Wint,dc represents more than 40% of Wext (the positive work done to move the centre of mass of the body relative to the surroundings) in both children and adults.
Into thin air: contributions of aerodynamic and inertial-elastic forces to wing bending in the hawkmothManduca sexta
Authors: Combes S. A. , Daniel T. L.
Published: July 2003

During flapping flight, insect wings must withstand not only fluid-dynamic forces, but also inertial-elastic forces generated by the rapid acceleration and deceleration of their own mass. Estimates of overall aerodynamic and inertial forces vary widely, and the relative importance of these forces in determining passive wing deformations remains unknown. If aeroelastic interactions between a wing and the fluid-dynamic forces it generates are minor compared to the effects of wing inertia, models of insect flight that account for passive wing flexibility would be far simpler to develop. We used an experimental approach to examine the contributions of aerodynamic and inertial-elastic forces to wing bending in the hawkmoth Manduca sexta. We attached fresh Manduca wings to a motor and flapped them at a realistic wing-beat frequency and stroke amplitude. We compared wing bending in normal air versus helium (approx. 15% air density), in which the contribution of fluid-dynamic forces to wing deformations is significantly reduced. This 85% reduction in air density produced only slight changes in the pattern of Manduca wing deformations, suggesting that fluid-dynamic forces have a minimal effect on wing bending. We used a simplified finite element model of a wing to show that the differences observed between wings flapped in air versus helium are most likely due to fluid damping, rather than to aerodynamic forces. This suggests that damped finite element models of insect wings (with no fluid-dynamic forces included) may be able to predict overall patterns of wing deformation prior to calculations of aerodynamic force production, facilitating integrative models of insect flight.
Muscle force-length dynamics during levelversusincline locomotion: a comparison ofin vivoperformance of two guinea fowl ankle extensors
Authors: Daley Monica A. , Biewener Andrew A.
Published: July 2003

For a terrestrial animal to move in the complex natural environment, the limb muscles must modulate force and work performance to meet changing mechanical requirements; however, it is not clear whether this is accomplished via a collective shift in function by all limb muscles, or a division of labor among limb muscles. Do muscles differ in their ability to modulate force-length contractile function to meet the mechanical demands of different locomotor tasks? We explore this question by examining the in vivoforce-length performance of the guinea fowl Numida meleagris lateral gastrocnemius (LG) and digital flexor-IV (DF-IV), during level and incline locomotion. During level locomotion, the LG and DF-IV exhibit differing muscle fascicle strain patterns: the LG shortens by 10-15% while developing force,whereas the DF-IV undergoes a stretch-shorten cycle with large strain amplitudes and small net strains of 1-8%. Furthermore, the DF-IV operates at higher muscle stresses (92-130 kPa, compared to 23-39 kPa for LG) and possesses a longer tendon, which allows the DF-IV tendon to recover greater elastic energy than the LG tendon. During incline locomotion, these muscles contribute only one-third of the energy expected for their mass, with the DF-IV exhibiting high stride-to-stride variability in work output. While the stretch-shorten cycle of the DF-IV muscle may allow more economic force production, it also leads to large changes in work output with small changes in the relative timing of force and strain. Thus, while the primary determinants of LG work are net strain and mean force, the primary determinant of DF-IV work is the phase relationship between force and strain. Our results suggest that, in addition to influencing a muscle's mechanical performance during steady level locomotion, morphology also affects its capacity and mechanism for altering work output for different locomotor tasks.
Seasonal acclimatization in water flux rate, urine osmolality and kidney water channels in free-living degus: molecular mechanisms, physiological processes and ecological implications
Authors: Bozinovic Francisco , Gallardo Pedro A. , Visser G. Henk , Cortés Arturo
Published: July 2003

The environmental modification of an organism's physiology in the field is often hypothesized to be responsible for allowing an organism to adjust to changing biotic and abiotic environmental conditions through increases in biological performance. Here, we examine the phenotypic flexibility of water flux rate, urine osmolality and the expression of kidney aquaporins (AQP; or water channels) in free-ranging Octodon degus, a South American desert-dwelling rodent, through an integrative study at cellular, systemic and organismal levels. Water flux rates varied seasonally and were significantly lower in austral summer than in winter, while urine osmolality was higher in summer than during winter. The observed water influx rate during summer was 10.3±2.3 ml day-1 and during winter was 40.4±9.1 ml day-1. Mean urine osmolality was 3137±472 mosmol kg-1 during summer and 1123±472 mosmol kg-1during winter. AQP-2 medullary immunolabeling was more abundant in the kidneys of degus captured during summer than those captured during winter. This immunoreactivity was higher in apical cell membranes of medullary collecting ducts of degus in summer. AQP-1 immunostaining did not differ between seasons. Consistently, AQP-2 protein levels were increased in medulla from the summer individuals, as judged by the size of the 29 kDa band in the immunoblot. Here,we reveal how the integration of flexible mechanisms acting at cellular,systemic and organismal levels allows a small desert-dwelling mammal to cope with seasonal water scarcity in its semi-arid habitat.
The osmotic response of the Asian freshwater stingray (Himantura signifer) to increased salinity: a comparison with marine (Taeniura lymma) and Amazonian freshwater (Potamotrygon motoro)stingrays
Authors: Tam Wai L. , Wong Wai P. , Loong Ai M. , Hiong Kum C. , Chew Shit F. , Ballantyne James S. , Ip Yuen K.
Published: July 2003

The white-edge freshwater whip ray Himantura signifer can survive in freshwater (0.7‰) indefinitely or in brackish water (20‰) for at least two weeks in the laboratory. In freshwater, the blood plasma was maintained hyperosmotic to that of the external medium. There was approximately 44 mmol l-1 of urea in the plasma, with the rest of the osmolality made up mainly by Na+ and Cl-. In freshwater, it was not completely ureotelic, excreting up to 45% of its nitrogenous waste as urea. Unlike the South American freshwater stingray Potamotrygon motoro, H. signifer has a functional ornithine-urea cycle (OUC) in the liver, with hepatic carbamoylphosphate synthetase III (CPS III) and glutamine synthetase (GS) activities lower than those of the marine blue-spotted fan tail ray Taeniura lymma. More importantly, the stomach of H. signifer also possesses a functional OUC, the capacity(based on CPS III activity) of which was approximately 70% that in the liver. When H. signifer was exposed to a progressive increase in salinity through an 8-day period, there was a continuous decrease in the rate of ammonia excretion. In 20‰ water, urea levels in the muscle, brain and plasma increased significantly. In the plasma, osmolality increased to 571 mosmol kg-1, in which urea contributed 83 mmol l-1. Approximately 59% of the excess urea accumulated in the tissues of the specimens exposed to 20‰ water was equivalent to the deficit in ammonia excretion through the 8-day period, indicating that an increase in the rate of urea synthesis de novo at higher salinities would have occurred. Indeed, there was an induction in the activity of CPS III in both the liver and stomach, and activities of GS, ornithine transcarbamoylase and arginase in the liver. Furthermore, there was a significant decrease in the rate of urea excretion during passage through 5‰, 10‰ and 15‰ water. Although the local T. lymma in full-strength sea water (30‰)had a much greater plasma urea concentration (380 mmol l-1), its urea excretion rate (4.7 μmol day-1 g-1) was comparable with that of H. signifier in 20‰ water. Therefore, H. signifer appears to have reduced its capacity to retain urea in order to survive in the freshwater environment and, consequently, it could not survive well in full-strength seawater.
Postnatal suppression of myomesin, muscle creatine kinase and the M-line in rat extraocular muscle
Authors: Porter John D. , Merriam Anita P. , Gong Bendi , Kasturi Sriram , Zhou Xiaohua , Hauser Kurt F. , Andrade Francisco H. , Cheng Georgiana
Published: July 2003

The M-line and its associated creatine kinase (CK) M-isoform (CK-M) are ubiquitous features of skeletal and cardiac muscle. The M-line maintains myosin myofilaments in register, links the contractile apparatus to the cytoskeleton for external force transfer and localizes CK-based energy storage and transfer to the site of highest ATP demand. We establish here that the muscle group responsible for movements of the eye, extraocular muscle (EOM),is divergent from other striated muscles in lacking both an M-line and its associated CK-M. Although an M-line forms during myogenesis, both in vivo and in vitro, it is actively repressed after birth. Transcripts of the major M-line structural proteins, myomesin 1 and myomesin 2, follow the same pattern of postnatal downregulation, while the embryonic heart-specific EH-myomesin 1 transcript is expressed early and retained in adult eye muscle. By immunocytochemistry, myomesin protein is absent from adult EOM sarcomeres. M-line suppression does not occur in organotypic co-culture with oculomotor motoneurons, suggesting that the mechanism for suppression may lie in muscle group-specific activation or workload patterns experienced only in vivo. The M-line is, however, still lost in dark-reared rats, despite the developmental delay this paradigm produces in the visuomotor system and EOMs. EOM was low in all CK isoform transcripts except for the sarcomeric mitochondrial (Ckmt2) isoform. Total CK enzyme activity of EOM was one-third that of hindlimb muscle. These findings are singularly unique among fast-twitch skeletal muscles. Since EOM exhibits isoform diversity for other sarcomeric proteins, the M-line/CK-M divergence probably represents a key physiological adaptation for the unique energetics and functional demands placed on this muscle group in voluntary and reflexive eye movements.
Recovery of C-starts, equilibrium and targeted feeding after whole spinal cord crush in the adult goldfishCarassius auratus
Authors: Zottoli S. J. , Freemer M. M.
Published: July 2003

Central nervous system neurons of many adult teleost fish are capable of regrowth across spinal cord lesions, which may result in behavioral recovery of swimming. Since there have been few, if any, studies that examine the return of behaviors other than swimming, we provide a quantitative analysis of the recovery of C-starts that occur in adult goldfish after spinal cord injury. In addition, we include a qualitative analysis of the return of targeted feeding and equilibrium. Whole spinal cord crushes near the junction of the brain and spinal cord [spinomedullary level (SML)] were made in 45 experimental fish. Eight sham-operated goldfish served as controls for the effects of the surgery procedures alone. After spinal cord crush and recovery from the anesthetic, experimental fish lay on their sides with no movement caudal to the wound. The fish were monitored for the return of behaviors for up to 190 days postoperatively. Twenty-five fish survived the course of this study. Of these fish, 12 regained equilibrium and C-starts, two regained equilibrium but not C-starts, and 11 did not regain equilibrium (one of these did display a C-start). Twenty-two of the 25 experimental fish that survived the 190 days were able to target food from the water surface. Quantitative analysis of recovered C-starts in this study revealed that the probability of eliciting the response is reduced, that latencies from stimulus to response are longer and that movement parameters (i.e. angles, distance and velocity)are reduced compared with those of sham-operated control animals for up to 190 days postoperatively. The recovery of C-starts, equilibrium and targeted feeding was due to re-growth across the wound site, since re-crushing the spinal cord at the SML resulted in the loss of these behaviors. Mauthner cells are known to initiate C-starts in goldfish. Since the majority of M-axons that regrow across a crush wound associate with an inappropriate pathway (i.e. the first ventral root), it is unlikely that these cells play a major role in the return of C-starts. We propose that regeneration of Mauthner cell homologues across the wound site is responsible for the recovery of most C-starts. The identifiability of the M-cell and its homologues provides a unique opportunity to analyze the mechanisms underlying behavioral recovery at the cellular level.
Stimulatory effect of sulphide on thiotaurine synthesis in three hydrothermal-vent species from the East Pacific Rise
Authors: Pruski Audrey M. , Fiala-Médioni Aline
Published: July 2003

Symbiotic associations between marine invertebrates and sulphur-oxidising bacteria are a common feature in communities from sulphide-rich environments,such as those flourishing in the vicinity of hydrothermal vents. While the bacterial endosymbionts provide the host with an undoubted nutritional advantage, their presence also requires specific adaptations for the transport and storage of sulphide, which is a potent toxin of aerobic respiration. Although different mechanisms such as the reversible binding of sulphide to serum binding proteins or its oxidation to less toxic forms have been described, many questions still remained unanswered. In the last decade, large amounts of thiotaurine, an unusual sulphur-amino acid, have been reported in sulphur-based symbioses from hydrothermal vents and cold seeps. Compounds such as thiotaurine are known to take part in trans-sulphuration reactions, so the involvement of thiotaurine in sulphide metabolism has been suggested. We present here an experimental study on thiotaurine biosynthesis in three sulphur-oxidising symbiont-bearing species from the East Pacific Rise: the vesicomyid Calyptogena magnifica, the mytilid Bathymodiolus thermophilus and the vestimentiferan Riftia pachyptila. In all three species, thiotaurine synthesis is stimulated in vitro by an input of sulphide, as well as by thiosulphate in B. thermophilus. Several distinct metabolic pathways seem to occur, however, since hypotaurine is the only precursor in the bivalves C. magnifica and B. thermophilus, whereas thiotaurine is also produced from taurine in R. pachyptila. Hypotaurine(NH2-CH2-CH2-SO2H) and thiotaurine(NH2-CH2-CH2-SO2SH) are two free sulphur amino acids whose chemical formulae differ by only one atom of sulphur. It appears that the extent of thiotaurine synthesis is strongly dependent on the initial equilibrium between these two amino acids, since the strongest thiotaurine synthesis rates are found in tissues with the lowest initial thiotaurine concentration. Moreover, the lack of any effect of sulphide in symbiont-free tissues and in gills of the methanotrophic mussel Bathymodiolus childressi reinforces the assumption that thiotaurine synthesis is a specific adaptation to the thiotrophic mode of life. While the precise function (i.e. transport and/or storage of sulphide) of hypotaurine and thiotaurine has yet to be established, our results strongly support a general role for these free amino acids in the metabolism of sulphide in hydrothermal-vent thiotrophic symbioses.
Environment temperature affects cell proliferation in the spinal cord and brain of juvenile turtles
Authors: Radmilovich Milka , Fernández Anabel , Trujillo-Cenóz Omar
Published: July 2003

The spinal cords and brains - comprising dorsal cortex (DC), medial cortex(MC) and diencephalon (Dien) - of juvenile turtles acclimated to warm temperature [27-30°C; warm-acclimated turtles (WATs)] revealed higher density values of bromodeoxyuridine-labeled cells (BrdU-LCs) than those acclimated to a cooler environment [5-14°C; cold-acclimated turtles(CATs)]. Both populations were under the influence of the seasonal daily light-dark rhythms. Pronounced differences between WATs and CATs (independent t-test; confidence level, P<0.01) were found in the central area of the spinal gray matter and in the ependymal epithelium lining the brain ventricles. Forebrain regions (DC, MC and Dien) also revealed significant differences between WATs and CATs (independent t-test;confidence level, P<0.01-0.05). Unexplored biological clocks that may be affecting cell proliferation were equalized by performing paired experiments involving one WAT and one CAT. Both animals were injected on the same day at the same time and both were sacrificed 24 h later. These experiments confirmed that a warm environment increased cell proliferation in the CNS of turtles. Double- and triple-labeling experiments involving anti-BrdU antibody together with anti-glial protein antibodies revealed that temperature modulates not only cell populations expressing glial markers but also other cells that do not express them. As expected, in the case of short post-injection (BrdU) surviving time points, no cells were found colabeling for BrdU and NeuN (neuronal marker). The probable direct effect of temperature on the cell division rate should be analyzed together with potential indirect effects involving increased motor activity and increased food intake. The fate of the increased BrdU-LCs (death, permanence as progenitor cells or differentiation following neuronal or glial lines) remains a matter for further investigation. Results are discussed in the light of current opinions concerned with post-natal neurogenesis in vertebrates.
Evolution of the coordinate regulation of glycolytic enzyme genes by hypoxia
Authors: Webster Keith A.
Published: July 2003

Two billion years of aerobic evolution have resulted in mammalian cells and tissues that are extremely oxygen-dependent. Exposure to oxygen tensions outside the relatively narrow physiological range results in cellular stress and toxicity. Consequently, hypoxia features prominently in many human diseases, particularly those associated with blood and vascular disorders,including all forms of anemia and ischemia. Bioenergetic enzymes have evolved both acute and chronic oxygen sensing mechanisms to buffer changes of oxygen tension; at normal PO oxidative phosphorylation is the principal energy supply for eukaryotic cells, but when the PO falls below a critical mark metabolic switches turn off mitochondrial electron transport and activate anaerobic glycolysis. Without this switch cells would suffer an immediate energy deficit and death at low PO. An intriguing feature of the switching is that the same conditions that regulate energy metabolism also regulate bioenergetic genes, so that enzyme activity and transcription are regulated simultaneously,albeit with different time courses and signaling pathways. In this review we explore the pathways mediating hypoxia-regulated glycolytic enzyme gene expression, focusing on their atavistic traits and evolution.