Primers to highly conserved elements optimized for qPCR‐based telomere length measurement in vertebrates
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Stephanie F. Hudon↗
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Amphioxus and lamprey AP-2 genes: implications for neural crest evolution and migration patterns
The neural crest is a uniquely vertebrate cell type present in the most basal vertebrates, but not in cephalochordates. We have studied differences in regulation of the neural crest marker AP-2 across two evolutionary transitions: invertebrate to vertebrate, and agnathan to gnathostome. Isolation and comparison of amphioxus, lamprey and axolotl AP-2 reveals its extensive expansion in the vertebrate dorsal neural tube and pharyngeal arches, implying co-option of AP-2 genes by neural crest cells early in vertebrate evolution. Expression in non-neural ectoderm is a conserved feature in amphioxus and vertebrates, suggesting an ancient role for AP-2 genes in this tissue. There is also common expression in subsets of ventrolateral neurons in the anterior neural tube, consistent with a primitive role in brain development. Comparison of AP-2 expression in axolotl and lamprey suggests an elaboration of cranial neural crest patterning in gnathostomes. However, migration of AP-2-expressing neural crest cells medial to the pharyngeal arch mesoderm appears to be a primitive feature retained in all vertebrates. Because AP-2 has essential roles in cranial neural crest differentiation and proliferation, the co-option of AP-2 by neural crest cells in the vertebrate lineage was a potentially crucial event in vertebrate evolution.
NASA Discipline Evolutionary Biology↗
Id expression in amphioxus and lamprey highlights the role of gene cooption during neural crest evolution
Neural crest cells are unique to vertebrates and generate many of the adult structures that differentiate them from their closest invertebrate relatives, the cephalochordates. Id genes are robust markers of neural crest cells at all stages of development. We compared Id gene expression in amphioxus and lamprey to ask if cephalochordates deploy Id genes at the neural plate border and dorsal neural tube in a manner similar to vertebrates. Furthermore, we examined whether Id expression in these cells is a basal vertebrate trait or a derived feature of gnathostomes. We found that while expression of Id genes in the mesoderm and endoderm is conserved between amphioxus and vertebrates, expression in the lateral neural plate border and dorsal neural tube is a vertebrate novelty. Furthermore, expression of lamprey Id implies that recruitment of Id genes to these cells occurred very early in the vertebrate lineage. Based on expression in amphioxus we postulate that Id cooption conferred sensory cell progenitor-like properties upon the lateral neurectoderm, and pharyngeal mesoderm-like properties upon cranial neural crest. Amphioxus Id expression is also consistent with homology between the anterior neurectoderm of amphioxus and the presumptive placodal ectoderm of vertebrates. These observations support the idea that neural crest evolution was driven in large part by cooption of multipurpose transcriptional regulators from other tissues and cell types.
NASA Program Fundamental Space Biology↗
Evolutionary conservation of the presumptive neural plate markers AmphiSox1/2/3 and AmphiNeurogenin in the invertebrate chordate amphioxus
Amphioxus, as the closest living invertebrate relative of the vertebrates, can give insights into the evolutionary origin of the vertebrate body plan. Therefore, to investigate the evolution of genetic mechanisms for establishing and patterning the neuroectoderm, we cloned and determined the embryonic expression of two amphioxus transcription factors, AmphiSox1/2/3 and AmphiNeurogenin. These genes are the earliest known markers for presumptive neuroectoderm in amphioxus. By the early neurula stage, AmphiNeurogenin expression becomes restricted to two bilateral columns of segmentally arranged neural plate cells, which probably include precursors of motor neurons. This is the earliest indication of segmentation in the amphioxus nerve cord. Later, expression extends to dorsal cells in the nerve cord, which may include precursors of sensory neurons. By the midneurula, AmphiSox1/2/3 expression becomes limited to the dorsal part of the forming neural tube. These patterns resemble those of their vertebrate and Drosophila homologs. Taken together with the evolutionarily conserved expression of the dorsoventral patterning genes, BMP2/4 and chordin, in nonneural and neural ectoderm, respectively, of chordates and Drosophila, our results are consistent with the evolution of the chordate dorsal nerve cord and the insect ventral nerve cord from a longitudinal nerve cord in a common bilaterian ancestor. However, AmphiSox1/2/3 differs from its vertebrate homologs in not being expressed outside the CNS, suggesting that additional roles for this gene have evolved in connection with gene duplication in the vertebrate lineage. In contrast, expression in the midgut of AmphiNeurogenin together with the gene encoding the insulin-like peptide suggests that amphioxus may have homologs of vertebrate pancreatic islet cells, which express neurogenin3. In addition, AmphiNeurogenin, like its vertebrate and Drosophila homologs, is expressed in apparent precursors of epidermal chemosensory and possibly mechanosensory cells, suggesting a common origin for protostome and deuterostome epidermal sensory cells in the ancestral bilaterian. Copyright 2000 Academic Press.
NASA Discipline Evolutionary Biology↗
Forest and wildlife habitat analysis using remote sensing and geographic information systems
Forest and wildlife habitat analyses were conducted at the H.J. Andrews Experimental Forest in the Central Cascade Mountains of Oregon using remotely sensed data and a geographic information system (GIS). Landsat Thematic Mapper (TM) data were used to determine forest successional stages, and to analyze the structure of both old and young conifer forests. Two successional stage maps were developed. One was developed from six TM spectral bands alone, and the second was developed from six TM spectral bands and a relative sun incidence band. Including the sun incidence band in the classification improved the mapping accuracy in the two youngest successional stages, but did not improve overall accuracy or accuracy of the two oldest successional stages. Mean spectral values for old-growth and mature stands were compared in seven TM bands and seven band transformations. Differences between mature and old-growth successional stages were greatest for the band ratio of TM 4/5 (P = 0.00005) and the multiband transformation of wetness (P = 0.00003). The age of young conifer stands had the highest correlation to TM 4/5 values (r = 0.9559) of any of the TM band or band transformations used. TM 4/5 ratio values of poorly regenerated conifer stands were significantly different from well regenerated conifer stands after age 15 (P = 0.0000). TM 4/5 was named a 'Successional Stage Index' (SSI) because of its ability to distinguish forest successional stages. The forest successional stage map was used as input into a vertebrate richness model using GIS. The three variables of (1) successional stage, (2) elevation, and (3) site moisture were used in the GIS to predict the spatial occurrence of small mammal, amphibian, and reptile species based on primary and secondary habitat requirements. These occurrence or habitat maps were overlayed to tally the predicted number of vertebrate at any given point in the study area. Overall, sixty-three and sixty-seven percent of the model predictions for vertebrate occurrence matched the vertebrates that were trapped in the field in eight forested stands. Of the three model variables, site moisture appeared to have the greatest influence on the pattern of high vertebrate richness in all vertebrate classes.
Fiorella, Maria↗
Basic Gravitational Reflexes in the Larval Frog
This investigation was designed to determine how a primitive vertebrate, the bullfrog tadpole, is able to sense and process gravitational stimuli. Because of the phylogenetic similarities of the vestibular systems in all vertebrates, the understanding of the gravitational reflexes in this relatively simple vertebrate should elucidate a skeletal framework on a elementary level, upon which the more elaborate reflexes of higher vertebrates may be constructed. The purpose of this study was to understand how the nervous system of the larval amphibian processes gravitational information. This study involved predominantly electrophysiological investigations of the isolated, alert (forebrain removed) bullfrog tadpole head. The focus of these experiments is threefold: (1) to understand from whole extraocular nerve recordings the signals sent to the eye following static gravitational tilt of the head; (2) to localize neuronal centers responsible for generating these signals through reversible pharmacological ablation of these centers; and (3) to record intracellularly from neurons within these centers in order to determine the single neuron's role in the overall processing of the center. This study has provided information on the mechanisms by which a primitive vertebrate processes gravitational reflexes.
Cochran, Stephen L.↗
A single-cell atlas of the bobtail squid visual and nervous system highlights molecular principles of convergent evolution
Abstract The cephalopod and vertebrate visual systems are a textbook example of convergent evolution with unknown molecular underpinnings. Here we characterize 98,537 single-cell transcriptomes in the bobtail squidEuprymna berryito understand how the cephalopod retina and optic lobes relate to the vertebrate retina. We confirm the overall relative simplicity of the cephalopod retina but identify two related photoreceptor cell subtypes expressing distinct r-opsins. By contrast, the adult optic lobe contains a diverse repertoire of neuronal and glial cell types, with a predominance of dopaminergic neurons. We show that cephalopod-specific gene duplicates probably contributed to this cell type diversification. Comparing neuronal cell population in the optic lobes of hatchlings and adults, we reveal a switch towards dopaminergic neurotransmitter usage with age, indicative of a maturation process. We further identify an FMRF-amide-based retrograde signal from the optic lobe towards the retina that supports the functional analogy of the cephalopod optic lobe cortex and the vertebrate inner retina in visual signal processing from a molecular standpoint. Finally, comparative analyses with vertebrate and arthropod cells suggest a scenario in which two photoreceptor types and two neuronal populations may have already been present in the eye of the bilaterian ancestor.
Environmental Sciences & Ecology↗
Neural crest contributions to the lamprey head
The neural crest is a vertebrate-specific cell population that contributes to the facial skeleton and other derivatives. We have performed focal DiI injection into the cranial neural tube of the developing lamprey in order to follow the migratory pathways of discrete groups of cells from origin to destination and to compare neural crest migratory pathways in a basal vertebrate to those of gnathostomes. The results show that the general pathways of cranial neural crest migration are conserved throughout the vertebrates, with cells migrating in streams analogous to the mandibular and hyoid streams. Caudal branchial neural crest cells migrate ventrally as a sheet of cells from the hindbrain and super-pharyngeal region of the neural tube and form a cylinder surrounding a core of mesoderm in each pharyngeal arch, similar to that seen in zebrafish and axolotl. In addition to these similarities, we also uncovered important differences. Migration into the presumptive caudal branchial arches of the lamprey involves both rostral and caudal movements of neural crest cells that have not been described in gnathostomes, suggesting that barriers that constrain rostrocaudal movement of cranial neural crest cells may have arisen after the agnathan/gnathostome split. Accordingly, neural crest cells from a single axial level contributed to multiple arches and there was extensive mixing between populations. There was no apparent filling of neural crest derivatives in a ventral-to-dorsal order, as has been observed in higher vertebrates, nor did we find evidence of a neural crest contribution to cranial sensory ganglia. These results suggest that migratory constraints and additional neural crest derivatives arose later in gnathostome evolution.
Non-NASA Center↗
Evolution of hematopoiesis: Three members of the PU.1 transcription factor family in a cartilaginous fish, Raja eglanteria
T lymphocytes and B lymphocytes are present in jawed vertebrates, including cartilaginous fishes, but not in jawless vertebrates or invertebrates. The origins of these lineages may be understood in terms of evolutionary changes in the structure and regulation of transcription factors that control lymphocyte development, such as PU.1. The identification and characterization of three members of the PU.1 family of transcription factors in a cartilaginous fish, Raja eglanteria, are described here. Two of these genes are orthologs of mammalian PU.1 and Spi-C, respectively, whereas the third gene, Spi-D, is a different family member. In addition, a PU.1-like gene has been identified in a jawless vertebrate, Petromyzon marinus (sea lamprey). Both DNA-binding and transactivation domains are highly conserved between mammalian and skate PU.1, in marked contrast to lamprey Spi, in which similarity is evident only in the DNA-binding domain. Phylogenetic analysis of sequence data suggests that the appearance of Spi-C may predate the divergence of the jawed and jawless vertebrates and that Spi-D arose before the divergence of the cartilaginous fish from the lineage leading to the mammals. The tissue-specific expression patterns of skate PU.1 and Spi-C suggest that these genes share regulatory as well as structural properties with their mammalian orthologs.
NASA Discipline Evolutionary Biology↗
Estimates of volumetric bone density from projectional measurements improve the discriminatory capability of dual X-ray absorptiometry
To determine whether estimates of volumetric bone density from projectional scans of the lumbar spine have weaker associations with height and weight and stronger associations with prevalent vertebral fractures than standard projectional bone mineral density (BMD) and bone mineral content (BMC), we obtained posteroanterior (PA) dual X-ray absorptiometry (DXA), lateral supine DXA (Hologic QDR 2000), and quantitative computed tomography (QCT, GE 9800 scanner) in 260 postmenopausal women enrolled in two trials of treatment for osteoporosis. In 223 women, all vertebral levels, i.e., L2-L4 in the DXA scan and L1-L3 in the QCT scan, could be evaluated. Fifty-five women were diagnosed as having at least one mild fracture (age 67.9 +/- 6.5 years) and 168 women did not have any fractures (age 62.3 +/- 6.9 years). We derived three estimates of "volumetric bone density" from PA DXA (BMAD, BMAD*, and BMD*) and three from paired PA and lateral DXA (WA BMD, WA BMDHol, and eVBMD). While PA BMC and PA BMD were significantly correlated with height (r = 0.49 and r = 0.28) or weight (r = 0.38 and r = 0.37), QCT and the volumetric bone density estimates from paired PA and lateral scans were not (r = -0.083 to r = 0.050). BMAD, BMAD*, and BMD* correlated with weight but not height. The associations with vertebral fracture were stronger for QCT (odds ratio [QR] = 3.17; 95% confidence interval [CI] = 1.90-5.27), eVBMD (OR = 2.87; CI 1.80-4.57), WA BMDHol (OR = 2.86; CI 1.80-4.55) and WA-BMD (OR = 2.77; CI 1.75-4.39) than for BMAD*/BMD* (OR = 2.03; CI 1.32-3.12), BMAD (OR = 1.68; CI 1.14-2.48), lateral BMD (OR = 1.88; CI 1.28-2.77), standard PA BMD (OR = 1.47; CI 1.02-2.13) or PA BMC (OR = 1.22; CI 0.86-1.74). The areas under the receiver operating characteristic (ROC) curves for QCT and all estimates of volumetric BMD were significantly higher compared with standard PA BMD and PA BMC. We conclude that, like QCT, estimates of volumetric bone density from paired PA and lateral scans are unaffected by height and weight and are more strongly associated with vertebral fracture than standard PA BMD or BMC, or estimates of volumetric density that are solely based on PA DXA scans.
Non-NASA Center↗
Plasticity of Neurovestibular Systems Following Micro- and Hyper-Gravity Exposure and Readaptation to Earth's 1G
The gravity-sensing organs sense the sum of inertial force due to head translation and head orientation relative to gravity. Normally gravity is constant, and yet the neural sensors show remarkable plasticity. When the force of gravity changes, such as in spaceflight or during centrifugation, the neurovestibular system responds by regulating its neural output, and this response is similar for the vertebrate utricular nerve afferents and for the statocyst hair cell in invertebrates. First, we examine the response of utricular afferents in toadfish following exposure to G on two orbital missions (STS-90 and 95). Within the first day after landing, magnitude of neural response to an applied acceleration was significantly elevated, and re-adaptation back to control values occurred within approximately 30 hours. Time course of return to normal approximately parallels the decrease in vestibular disorientation in astronauts following return. Next, we use well-controlled hyper-G experiments in the vertebrate model to address: If G leads to adaptation and subsequent re-adaptation neural processes, does the transfer from 1G to hyper-G impart the opposite effects and do the effects accompanying transfer from the hyper-G back to the 1G conditions resemble as an analog the transfer from 1G to the microG Results show a biphasic pattern in reaction to 3G exposures: an initial sensitivity up-regulation (3- and 4-day) followed by a significant decrease after longer exposure. Return to control values is on the order of 4-8 days. Utricular sensitivity is strongly regulated up or down by gravity load and the duration of exposure. Interestingly, we found no correlation of response and hair cell synaptic body counts despite the large gain difference between 4- and 16-Day subjects. Lastly, we examine responses of statocyst receptors in land snail following exposure to G on two unmanned Russian Orbital missions (Foton M-2 and -3). Here, we have the ability to measure the output directly from the hair cells. Similar to afferents in vertebrates the hair cells increased their response sensitivity to vestibular stimulation. Two major pieces of information are needed: the precise vertebrate hair cell response to altered gravity and the impact of longer duration exposures on sensory plasticity.
Boyle, Richard D.↗
Neural Mechanisms to the Space Environment
Highly conserved neural systems have evolved to sense the inertial forces due to head translation and head tilt relative to gravitational vertical. These structures consist of ciliated mechanosensitive receptor cells inserted into a neuroepithelium surmounted by biomineral grains of calcium carbonate (CaCO3) called oto- (vertebrates) or stato-conia (invertebrates). Detection of these forces by receptor cells relies on the CaCO3 mass being weighted in Earths 1G. A change in gravity or orientation with respect to gravity has a profound effect on how an organism interacts with its environment, and it is evident that the nervous system responds to the new gravity state. This response might involve the peripheral receptors, the CaCO3 mass, the brain or any combination of these mechanisms based on the intensity and duration of the gravity change. Here, we examine the arguments supporting the different mechanisms of adaptation to the space environment. First, a pre- or post-synaptic alteration in the strength of synaptic transmission between the receptor cell and nerve afferent can adjust the system output. The number of synaptic ribbons in certain type II hair cells in rodent is labile, increasing following exposure to microgravity. An increase in number of synaptic ribbons in toadfish otolith hair cells following exposure to microgravity could potentially explain the observed afferent hypersensitivity to acceleration postflight. The physiological findings in the isolated statocyst in snails are in line with the vertebrate data, and conform to the proposition that G exposure leads to changes in gravireceptor function. At the same time this similarity in neural response to G exposure between the vertebrates and invertebrates is intriguing: the increased neural sensitivity in the vertebrate was detected in the nerve afferents, one synapse away from the receptor cell, whereas the increased neural sensitivity observed in the snail was detected directly at the receptor level. Second, the CaCO3 mass provides mechanical loading of receptor cell cilia, and their density alters sensitivity. A widely considered mechanism by which the animal responds to a chronic change in amplitude of gravity is a change in weight-lending CaCO3 mass. In G, it is argued, the organism counters the loss of gravity by increasing CaCO3 production, thereby increasing its mass, as a means to increase system gain. In hypergravity, the converse is argued. Earlier evidence in mollusks and recent results in mice suggest a remodeling might occur, especially after long-term space exposure. Lastly, we have to distinguish at least two kinds of neural feedbacks. One is connected with local mechanisms of self-regulation and specific for initial period of organ development when the neural connections are still absent. And the other feedback is related to neural self-regulation and specific for later stages of the organ development, and includes an efferent vestibular feedback. Complexity of the problem is enhanced by incompleteness of experiments, and consequently the experimental results have not led to a clear interpretation despite the numerous studies.
Boyle, Richard D.↗
Functional Responses in Otolith Structures from Micro- to Hyper-Gravity
Vertebrates and invertebrates sense gravito-inertial acceleration by mechanoreceptors in the otolith and statolith organs, respectively. These structures consist of ciliated sensory hair cells surmounted by biomineral grains of calcium carbonate (CaCO3) called oto-orstato-conia. The grains provide mechanical loading of hair cell cilia, and their high density increases sensitivity to acceleration. A widely considered mechanism by which the animal responds to a chronic change in amplitude of gravity is a change in weight lending otoconia. In G, it is argued, the organism counters the loss of gravity by increasing CaCO3 production, thereby increasing otolith mass, as a means to increase system gain. In hypergravity (HG), the converse is argued. Here, we present the results obtained in 3 species exposed both to G and HG. Adult toadfish, Opsanus tau, were exposed to G in 2 short-duration shuttle missions and to 1.24 1.73G centrifugation for 1-32 days; re-adaptation was studied following 1-8 days of 1G. Results show a biphasic pattern in response to 1.73G: initial hypersensitivity, similar to that observed after G exposure, followed by transition to a significant decrease at 16-32 days. Recovery from HG exposure is 4-8 days. Next, we examined directly the responses of statocyst receptors in the land snail after exposure to G on two unmanned Russian Orbital missions and at 1.24G. Similar to vertebrate afferents snail receptors increased their sensitivity to tilt after G exposure, and decrease it after 16-32 days of HG. Two major pieces of information are still needed: vertebrate hair cell response to altered gravity and impact of longer duration exposures on sensory plasticity. To address the latter, we applied electron microscopic techniques to image otoconia mass obtained from 1) mice subjected to 91-days of weightlessness in the Mouse Drawer System (MDS) flown on International Space Station, 2) mice subjected to 91-days of 1.24G centrifugation on ground, and 3) mice flown on 2 short-duration orbital missions. Images indicate a clear restructuring of individual otoconia, suggesting deposition to the outer shell. Images from their HG counterparts indicate the converse - an ablation of the otoconia mass. For shorter duration exposures to weightlessness on 13-day shuttle missions, mice otoconia appear normal. Despite the permanence of 1G in evolution, the animal senses exposure to a novel, non-1G, environment and adaptive mechanisms are initiated - in the short term, compensation is likely confined to the peripheral sensory receptors, the brain or both. For longer exposures structural modifications of the endorgan may also result. Support Contributed By: NASA 03-OBPR-04 and 11-11_Omni_2-0002
Vertebrates↗
The crystal structure of Acinetobacter baumannii bacterioferritin reveals a heteropolymer of bacterioferritin and ferritin subunits
Iron storage proteins, e.g., vertebrate ferritin, and the ferritin-like bacterioferritin (Bfr) and bacterial ferritin (Ftn), are spherical, hollow proteins that catalyze the oxidation of Fe 2+ at binuclear iron ferroxidase centers (FOC) and store the Fe 3+ in their interior, thus protecting cells from unwanted Fe 3+ /Fe 2+ redox cycling and storing iron at concentrations far above the solubility of Fe 3+ . Vertebrate ferritins are heteropolymers of H and L subunits with only the H subunits having FOC. Bfr and Ftn were thought to coexist in bacteria as homopolymers, but recent evidence indicates these molecules are heteropolymers assembled from Bfr and Ftn subunits. Despite the heteropolymeric nature of vertebrate and bacterial ferritins, structures have been determined only for recombinant proteins constituted by a single subunit type. Herein we report the structure of Acinetobacter baumannii bacterioferritin, the first structural example of a heteropolymeric ferritin or ferritin-like molecule, assembled from completely overlapping Ftn homodimers harboring FOC and Bfr homodimers devoid of FOC but binding heme. The Ftn homodimers function by catalyzing the oxidation of Fe 2+ to Fe 3+ , while the Bfr homodimers bind a cognate ferredoxin (Bfd) which reduces the stored Fe 3+ by transferring electrons via the heme, enabling Fe 2+ mobilization to the cytosol for incorporation in metabolism.
59 BASIC BIOLOGICAL SCIENCES↗
Rocks, resolution, and the record at the terrestrial K/T boundary, eastern Montana and western North Dakota
Reconstructions of mass extinction events are based upon faunal patterns, reconstructed from numerical and diversity data ultimately derived from rocks. It follows that geological complexity must not be subsumed in the desire to establish patterns. This is exemplified at the Terrestrial Cretaceous-Tertiary (K/T) boundary in eastern Montana and western North Dakota, where there are represented all of the major indicators of the terrestrial K/T transition: dinosaurian and non-dinosaurian vertebrate faunas, pollen, a megaflora, iridium, and shocked quartz. It is the patterns of these indicators that shape ideas about the terrestrial K/T transition. In eastern Montana and western North Dakota, the K/T transition is represented lithostratigraphically by the Cretaceous Hell Creek Formation, and the Tertiary Tullock Formation. Both of these are the result of aggrading, meandering, fluvial systems, a fact that has important consequences for interpretations of fossils they contain. Direct consequences of the fluvial depositional environments are: facies are lenticular, interfingering, and laterally discontinuous; the occurrence of fossils in the Hell Creek and Tullock formations is facies-dependent; and the K/T sequence in eastern Montana and western North Dakota is incomplete, as indicated by repetitive erosional contacts and soil successions. The significance for faunal patterns of lenticular facies, facies-dependent preservation, and incompleteness is discussed. A project attempting to reconstruct vertebrate evolution in a reproducible manner in Hell Creek-type sediments must be based upon a reliable scale of correlations, given the lenticular nature of the deposits, and a recognition of the fact that disparate facies are not comparable in terms of either numbers of preserved vertebrates or depositional rates.
Fastovsky, D. E.↗
Functional Characterization of G12, a Gene Required for Mitotic Progression during Gastrulation in Zebrafish
In a differential RNA display screen we have isolated a zebrafish gene, G12, for which homologs can only be found in DNA databases for vertebrates, but not invertebrates. This suggests that this is a gene required specifically in vertebrates. G12 expression is upregulated at mid-blastula transition (MBT). Morpholino inactivation of this gene by injection into 1-cell embryos results in mitotic defects and apoptosis shortly after MBT. Nuclei in morpholino treated embryos also display segregation defects. We have characterized the localization of this gene as a GFP fusion in live and fixed embryos. Overexpression of G12-GFP is non-toxic. Animals retain GFP expression for at least 7 days with no developmental defects, Interestingly in these animals G12-GFP is never detectable in blood cells though blood is present. In the deep cells of early embryos, G 12GFP is localized to nuclei and cytoskeletal elements in interphase and to the centrosome and spindle apparatus during mitosis. In the EVL, G12-GFP shows additional localization to the cell periphery, especially in mitosis. In the yolk syncytium, G12-GFP again localizes to nuclei and strongly to cytoplasmic microtubules of migrating nuclei at the YSL margin. Morpholinc, injection specifically into the YSL after cellularization blocks epiboly and nuclei of the YSL show mitotic defects while deep cells show no mitotic defects and continue to divide. Rescue experiments in which morpholino and G12-GFP RNA are co-injected indicate partial rescue by the G12-GFP. The rescue is cell autonomous; that is, regions of the embryo with higher G12-GFP expression show fewer mitotic defects. Spot 14, the human bomolog of G12, has been shown to be amplified in aggressive breast tumors. This finding, along with our functional and morphological data suggest that G12 and spot 14 are vertebrate-specific and may function either as mitotic checkpoints or as structural components of the spindle apparatus.
Reinsch, Sigrid↗
The retinoic acid signaling pathway regulates anterior/posterior patterning in the nerve cord and pharynx of amphioxus, a chordate lacking neural crest
Amphioxus, the closest living invertebrate relative of the vertebrates, has a notochord, segmental axial musculature, pharyngeal gill slits and dorsal hollow nerve cord, but lacks neural crest. In amphioxus, as in vertebrates, exogenous retinoic acid (RA) posteriorizes the embryo. The mouth and gill slits never form, AmphiPax1, which is normally downregulated where gill slits form, remains upregulated and AmphiHox1 expression shifts anteriorly in the nerve cord. To dissect the role of RA signaling in patterning chordate embryos, we have cloned the single retinoic acid receptor (AmphiRAR), retinoid X receptor (AmphiRXR) and an orphan receptor (AmphiTR2/4) from amphioxus. AmphiTR2/4 inhibits AmphiRAR-AmphiRXR-mediated transactivation in the presence of RA by competing for DR5 or IR7 retinoic acid response elements (RAREs). The 5' untranslated region of AmphiTR2/4 contains an IR7 element, suggesting possible auto- and RA-regulation. The patterns of AmphiTR2/4 and AmphiRAR expression during embryogenesis are largely complementary: AmphiTR2/4 is strongly expressed in the cerebral vesicle (homologous to the diencephalon plus anterior midbrain), while AmphiRAR expression is high in the equivalent of the hindbrain and spinal cord. Similarly, while AmphiTR2/4 is expressed most strongly in the anterior and posterior thirds of the endoderm, the highest AmphiRAR expression is in the middle third. Expression of AmphiRAR is upregulated by exogenous RA and completely downregulated by the RA antagonist BMS009. Moreover, BMS009 expands the pharynx posteriorly; the first three gill slit primordia are elongated and shifted posteriorly, but do not penetrate, and additional, non-penetrating gill slit primordia are induced. Thus, in an organism without neural crest, initiation and penetration of gill slits appear to be separate events mediated by distinct levels of RA signaling in the pharyngeal endoderm. Although these compounds have little effect on levels of AmphiTR2/4 expression, RA shifts pharyngeal expression of AmphiTR2/4 anteriorly, while BMS009 extends it posteriorly. Collectively, our results suggest a model for anteroposterior patterning of the amphioxus nerve cord and pharynx, which is probably applicable to vertebrates as well, in which a low anterior level of AmphiRAR (caused, at least in part, by competitive inhibition by AmphiTR2/4) is necessary for patterning the forebrain and formation of gill slits, the posterior extent of both being set by a sharp increase in the level of AmphiRAR. Supplemental data available on-line.
NASA Discipline Evolutionary Biology↗
Conservation of Pax gene expression in ectodermal placodes of the lamprey
Ectodermal placodes contribute to the cranial ganglia and sense organs of the head and, together with neural crest cells, represent defining features of the vertebrate embryo. The identity of different placodes appears to be specified in part by the expression of different Pax genes, with Pax-3/7 class genes being expressed in the trigeminal placode of mice, chick, frogs and fish, and Pax-2/5/8 class genes expressed in the otic placode. Here, we present the cloning and expression pattern of lamprey Pax-7 and Pax-2, which mark the trigeminal and otic placodes, respectively, as well as other structures characteristic of vertebrate Pax genes. These results suggest conservation of Pax genes and placodal structures in basal and derived vertebrates.
NASA Discipline Evolutionary Biology↗