Small molecule developmental screens reveal the logic and timing of vertebrate development

Much has been learned about vertebrate development by random mutagenesis followed by phenotypic screening and by targeted gene disruption followed by phenotypic analysis in model organisms. Because the timing of many developmental events is critical, it would be useful to have temporal control over modulation of gene function, a luxury frequently not possible with genetic mutants. Here, we demonstrate that small molecules capable of conditional gene product modulation can be identified through developmental screens in zebrafish. We have identified several small molecules that specifically modulate various aspects of vertebrate ontogeny, including development of the central nervous system, the cardiovascular system, the neural crest, and the ear. Several of the small molecules identified allowed us to dissect the logic of melanocyte and otolith development and to identify critical periods for these events. Small molecules identified in this way offer potential to dissect further these and other developmental processes and to identify novel genes involved in vertebrate development.

Regulation of dorsal fate in the neuraxis by Wnt-1 and Wnt-3a

Members of the Wnt family of signaling molecules are expressed differentially along the dorsal–ventral axis of the developing neural tube. Thus we asked whether Wnt factors are involved in patterning of the nervous system along this axis. We show that Wnt-1 and Wnt-3a, both of which are expressed in the dorsal portion of the neural tube, could synergize with the neural inducers noggin and chordin in Xenopus animal explants to generate the most dorsal neural structure, the neural crest, as determined by the expression of Krox-20, AP-2, and slug. Overexpression of Wnt-1 or Wnt-3a in the neuroectoderm of whole embryos led to a dramatic increase of slug and Krox-20-expressing cells, but the hindbrain expression of Krox-20 remained unaffected. Enlargement in the neural crest population could occur even when cell proliferation was inhibited. Wnt-5A and Wnt-8, neither of which is expressed in the dorsal neuroectoderm, failed to induce neural crest markers. Overexpression of glycogen synthase kinase 3, known to antagonize Wnt signaling, blocked the neural-crest-inducing activity of Wnt-3a in animal explants and inhibited neural crest formation in whole embryos. We suggest that Wnt-1 and Wnt-3a have a role in patterning the neural tube along its dorsoventral axis and function in the differentiation of the neural crest.

Lamprey Dlx genes and early vertebrate evolution

Gnathostome vertebrates have multiple members of the Dlx family of transcription factors that are expressed during the development of several tissues considered to be vertebrate synapomorphies, including the forebrain, cranial neural crest, placodes, and pharyngeal arches. The Dlx gene family thus presents an ideal system in which to examine the relationship between gene duplication and morphological innovation during vertebrate evolution. Toward this end, we have cloned Dlx genes from the lamprey Petromyzon marinus, an agnathan vertebrate that occupies a critical phylogenetic position between cephalochordates and gnathostomes. We have identified four Dlx genes in P. marinus, whose orthology with gnathostome Dlx genes provides a model for how this gene family evolved in the vertebrate lineage. Differential expression of these lamprey Dlx genes in the forebrain, cranial neural crest, pharyngeal arches, and sensory placodes of lamprey embryos provides insight into the developmental evolution of these structures as well as a model of regulatory evolution after Dlx gene duplication events.

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

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

Vertebrate innovations

Vertebrate innovations include neural crest cells and their derivatives, neurogenic placodes, an elaborate segmented brain, endoskeleton, and an increase in the number of genes in the genome. Comparative molecular and developmental data give new insights into the evolutionary origins of these characteristics and the complexity of the vertebrate body.

Neuregulins with an Ig-like domain are essential for mouse myocardial and neuronal development.

Neuregulins are ligands for the erbB family of receptor tyrosine kinases and mediate growth and differentiation of neural crest, muscle, breast cancer, and Schwann cells. Neuregulins contain an epidermal growth factor-like domain located C-terminally to either an Ig-like domain or a cysteine-rich domain specific to the sensory and motor neuron-derived isoform. Here it is shown that elimination of the Ig-like domain-containing neuregulins by homologous recombination results in embryonic lethality associated with a deficiency of ventricular myocardial trabeculation and impairment of cranial ganglion development. The erbB receptors are expressed in myocardial cells and presumably mediate the neuregulin signal originating from endocardial cells. The trigeminal ganglion is reduced in size and lacks projections toward the brain stem and mandible. We conclude that IgL-domain-containing neuregulins play a major role in cardiac and neuronal development.

BCL2 regulates neural differentiation.

A main function attributed to the BCL2 protein is its ability to confer resistance against apoptosis. In addition to the constitutively high expression of BCL2, caused by gene rearrangement in follicular lymphomas, elevated expression of the BCL2 gene has been found in differentiating hematopoietic, neural, and epithelial tissues. To address the question of whether the expression of BCL2 is a cause or consequence of cell differentiation, we used a human neural-crest-derived tumor cell line, Paju, that undergoes spontaneous neural differentiation in vitro. The Paju cell line displays moderate expression of BCL2, the level of which increases in parallel with further neural differentiation induced by treatment with phorbol 12-myristate 13-acetate. Transfection of normal human BCL2 cDNA in sense and antisense orientations had a dramatic impact on the differentiation of the Paju cells. Overexpression of BCL2 cDNA induced extensive neurite outgrowth, even in low serum concentrations, together with an increased expression of neuron-specific enolase. Paju cells expressing the anti-sense BCL2 cDNA construct, which reduced the endogenous levels of BCL2, did not undergo spontaneous neural differentiation. These cells acquired an epithelioid morphology and up-regulated the intermediate filament protein nestin, typically present in primitive neuroectodermal cells. The manipulated levels of BCL2 did not have appreciable impact on cell survival in normal culture. Our findings demonstrate that the BCL2 gene product participates in the regulation of neural differentiation.

Null mutation of endothelin receptor type B gene in spotting lethal rats causes aganglionic megacolon and white coat color.

Mutations in the gene encoding the endothelin receptor type B (EDNRB) produce congenital aganglionic megacolon and pigment abnormalities in mice and humans. Here we report a naturally occurring null mutation of the EDNRB gene in spotting lethal (sl) rats, which exhibit aganglionic megacolon associated with white coat color. We found a 301-bp deletion spanning the exon 1-intron 1 junction of the EDNRB gene in sl rats. A restriction fragment length polymorphism caused by this deletion perfectly cosegregates with the sl phenotype. The deletion leads to production of an aberrantly spliced EDNRB mRNA that lacks the coding sequence for the first and second putative transmembrane domains of the G-protein-coupled receptor. Radioligand binding assays revealed undetectable levels of functional EDNRB in tissues from homozygous sl/sl rats. We conclude that EDNRB plays an essential role in the normal development of two neural crest-derived cell lineages, epidermal melanocytes and enteric neurons, in three mammalian species--humans, mice, and rats. The EDNRB-deficient rat may also prove valuable in defining the postnatal physiologic role of this receptor.

Phylogenetic relationships of the hoatzin, an enigmatic South American bird.

The hoatzin (Opisthocomus hoazin) lives in the humid lowlands of northern and central South America, often in riparian habitats. It is a slender bird approximately 65 cm in length, brownish with lighter streaks and buffy tips to the long tail feathers. The small head has a ragged, bristly crest of reddish-brown feathers, and the bare skin of the face is bright blue. It resembles a chachalaca (Ortalis, Cracidae) in size and shape, but its plumage and markings are similar to those of the smaller guira cuckoo (Guira guira). The hoatzin (pronounced Watson) has been a taxonomic puzzle since it was described in 1776. It usually has been viewed as related to the gallinaceous birds, but alliances to other groups have been suggested, including the cuckoos. We present DNA sequence evidence from the 12S and 16S rRNA mitochondrial genes, and from the nuclear gene that codes for the eye lens protein, alpha A-crystallin. The results indicate that the hoatzin is most closely related to the typical cuckoos and that the divergence occurred at or near the base of the cuculiform phylogenetic tree.

Telomere length and replicative aging in human vascular tissues.

Because repeated injury of the endothelium and subsequent turnover of intimal and medial cells have been implicated in atherosclerosis, we examined telomere length, a marker of somatic cell turnover, in cells from these tissues. Telomere lengths were assessed by Southern analysis of terminal restriction fragments (TRFs) generated by HinfI/Rsa I digestion of human genomic DNA. Mean TRF length decreased as a function of population doublings in human endothelial cell cultures from umbilical veins, iliac arteries, and iliac veins. When endothelial cells were examined for mean TRF length as a function of donor age, there was a significantly greater rate of decrease for cells from iliac arteries than from iliac veins (102 bp/yr vs. 47 bp/yr, respectively, P < 0.05), consistent with higher hemodynamic stress and increased cell turnover in arteries. Moreover, the rate of telomere loss as a function of donor age was greater in the intimal DNA of iliac arteries compared to that of the internal thoracic arteries (147 bp/yr vs. 87 bp/yr, respectively, P < 0.05), a region of the arterial tree subject to less hemodynamic stress. This indicates that the effect is not tissue specific. DNA from the medial tissue of the iliac and internal thoracic arteries showed no significant difference in the rates of decrease, suggesting that chronic stress leading to cellular senescence is more pronounced in the intima than in the media. These observations extend the use of telomere size as a marker for the replicative history of cells and are consistent with a role for focal replicative senescence in cardiovascular diseases.

Ultrassonografia vascular comparada à intravascular no diagnóstico das obstruções venosas ilíacas em portadores de insuficiência venosa crônica

Introdução: O tratamento da Insuficiência Venosa Crônica (IVC) é baseado na correção dos refluxos e obstruções ao fluxo sanguíneo venoso. A detecção, a gravidade e o tratamento dessas obstruções venosas, responsáveis pelos sinais e sintomas da IVC, têm sido recentemente estudados e melhor compreendidos. Estes estudos não definem qual o grau de obstrução significativa nem os critérios ultrassonográficos para sua detecção. O objetivo deste estudo foi determinar critérios ultrassonográficos para o diagnóstico das obstruções venosas ilíacas, avaliando a concordância deste método com o ultrassom intravascular (UI) em pacientes portadores de IVC avançada. Métodos: Foram avaliados 15 pacientes (30 membros; 49,4 ± 10,7 anos; 1 homem) com IVC inicial (Classificação Clínica-Etiológica-Anatômica-Physiopatológica - CEAP C1-2) no grupo I (GI) e 51 pacientes (102 membros; 50,53 ± 14,5 anos; 6 homens) com IVC avançada (CEAP C3-6) no grupo II (GII) pareados por sexo, idade e etnia. Todos pacientes foram submetidos à entrevista clínica e à ultrassonografia vascular com Doppler (UV-D), sendo obtidas as medidas de fasicidade de fluxo, os índices de fluxo e velocidades venosas femorais, e as relações de velocidade e de diâmetro da obstrução ilíaca. Foi analisado o escore de refluxo multisegmentar. Os indivíduos do GI foram avaliados por 3 examinadores independentes. Os pacientes do GII foram submetidos ao UI, sendo obtidos a área dos segmentos venosos comprometidos e comparados com os resultados obtidos pelo UV-D, agrupados em 3 categorias: obstruções < 50%; obstruções entre 50-79% e obstruções >= 80%. Resultados: A classe de severidade clinica CEAP predominante no GI foi C1 em 24/30 (80%) membros, e C3 em 54/102 (52,9%) membros no GII. O refluxo foi severo (escore de refluxo multisegmentar >= 3) em 3/30 (10%) membros no grupo I, e em 45/102 (44,1%) membros no grupo II (p<0,001). Houve uma concordância moderadamente elevada entre o UV-D e o UI, quando agrupadas em 3 categorias (K=0,598; p<0,001), e uma concordância elevada quando agrupadas em 2 categorias (obstruções <50% e >= 50%) (K= 0,784; p<0,001). Os melhores pontos de corte e sua correlação com o UI foram: índice de velocidade (0,9; r=-0,634; p<0,001); índice de fluxo (0,7; r=-0,623; p<0,001); relação de obstrução (0,5; r=0,750; p<0,001); relação de velocidade (2,5; r= 0,790; p<0,001); A ausência de fasicidade de fluxo esteve presente em 88,2% dos pacientes com obstrução >=80% ao UV-D. Foi construído um algoritmo ultrassonográfico vascular, utilizando as medidas e os pontos de corte descritos obtendo-se uma acurácia de 79,6% para 3 categorias (K=0,655; p<0,001) e de 86,7% para 2 categorias (k=0,730; p<0,001). Conclusões: O UV-D apresentou uma concordância elevada com o UI na detecção de obstruções >= 50%. A relação de velocidade na obstrução >= 2,5 é o melhor critério para detecção de obstruções venosas significativas em veias ilíacas.

Análise da correlação entre índices radiomorfométricos de densidade óssea, perfil de qualidade óssea em software de implante, quociente de estabilidade e torque de inserção em implantes

A qualidade óssea, bem como a estabilidade inicial dos implantes, está diretamente relacionada com o sucesso das reabilitações na implantodontia. O presente estudo teve como objetivo analisar a correlação entre índices radiomorfométricos de densidade óssea por meio de radiografias panorâmicas, perfil de qualidade óssea com o auxílio de Tomografia Computadorizada de Feixe Cônico (TCFC) com o uso do software de imagens OsiriX, Análise da Frequência de Ressonância (RFA) e Torque de Inserção do implante. Foram avaliados 160 implantes de 72 indivíduos, com média etária de 55,5 (±10,5) anos. Nas radiografias panorâmicas foram obtidos os índices IM, IPM e ICM, e nas tomografias computadorizadas de feixe cônico, os valores de pixels e a espessura da cortical da crista óssea alveolar, além da estabilidade primária por meio do torque de inserção e análise da frequência de ressonância. Os resultados foram analisados pelo coeficiente de correlação de Spearman, para p<= 0,01 foi obtido entre o torque de inserção e valores de pixels (0.330), o torque de inserção e a espessura da cortical da crista alveolar (0.339), o torque de inserção e o ISQ vestibulo-lingual (0.193), os valores de pixels e espessura da cortical da crista alveolar (0.377), as duas direções vestíbulo-lingual e mesio-distal do ISQ (0.674), o ISQ vestíbulo-lingual e a espessura da cortical da crista alveolar (0.270); os índices radiomorfométricos foram correlacionados entre eles e para p<= 0,05 foi obtido entre torque de inserção e ISQ mesio-distal (0.131), entre o ISQ vestibulo-lingual e os valores de pixels (0.156) e ISQ mesio-distal e IPMI esquerdo (0.149) e ISQ mesio-distal e IPMS esquerdo (0.145). Existe correlação entre a TCFC, o torque de inserção e a RFA na avaliação da qualidade óssea. É possível utilizar, pré-cirurgicamente, os exames de TCFC para avaliar a qualidade e quantidade óssea, tendo em vista as correlações obtidas neste estudo.

Análisis comparativo de morbimortalidad y supervivencia a 12 años del tratamiento de la estenosis carotidea mediante endarterectomía, stent transfemoral y stent transcervical en 481 pacientes. Análisis de coste efectividad

Los avances en la era endovascular han causado una revolución en el abordaje de la estenosis carotidea. La irrupción del abordaje transfemoral y transcervical ha hecho que la comunidad científica nos replanteemos las indicaciones terapeúticas en pacientes con estenosis carotidea sintomática o asintomática. Aunque los resultados del abordaje transfemoral mejoraron con la introducción de los sistemas de neuroprotección, un abundante número de estudios randomizados han sido dirigidos para comparar stent transfemoral frente a la endarterectomia. Endarterectomia frente a angioplastia en pacientes con estenosis severa sintomatica(EVA-3S), angioplastia con stent y neuroprotección frente a endarterectomia (SPACE), Angioplastia carotidea y vertebral (CAVATAS), revascularización carotidea mediante stent versus endarterectomia (CREST) estudio internacional carotideo (ICSS) todos fallaron en demostrar una ventaja global del stent transfemoral frente a la endarterectomia. El abordaje transcervical surge por tanto como opción segura de tratamiento, con buenos resultados clínicos iniciales, eliminando así algunas de las complicaciones del acceso transfemoral. Existe hasta la fecha una falta de estudios comparativos de morbimortalidad entre las tres opciones terapeúticas. Además, dado el elevado número de pacientes candidatos a ellos, es necesario una análisis de resultados y supervivencia de los mismos, asi como un análisis de costes relativos y coste-efectividad que podría tener importantes implicaciones en las políticas de salud y en las guias de tratamiento...

Chicago and vicinity, Illinois, 1953 (Image 3 of 3) (Raster Image)

This layer is a georeferenced raster image of the historic, topographic paper map entitled: Chicago and vicinity, Ill.-Ind. : sheet no. 3 of 3 (Blue Island), 1953, mapped, edited, and published by the Geological Survey. It was published in 1957. Scale 1:24,000. The source map was compiled from 1:24,000 scale maps of Calumet Lake, Blue Island, Palos Park, Sag Bridge, Mokena, Tinley Park, Harvey, and Calumet City 1953 7.5 minute quadrangles. Hydrography from U.S. Lake Survey Chart 755 (1:15,000). This layer is image 3 of 3 total images of the three sheet source map. The image inside the map neatline is georeferenced to the surface of the earth and fit to the Illinois East State Plane Coordinate System NAD27 (in Feet) (Fipszone 1201). All map collar and inset information is also available as part of the raster image, including any inset maps, profiles, statistical tables, directories, text, illustrations, index maps, legends, or other information associated with the principal map. This is a typical topographic map portraying both natural and manmade features. It shows and names works of nature, such as mountains, valleys, lakes, rivers, vegetation, etc. It also identify the principal works of humans, such as roads, railroads, boundaries, transmission lines, major buildings, etc. Relief is shown with standard contour intervals of 5 feet. Depths shown by isolines and soundings. This layer is part of a selection of digitally scanned and georeferenced historic maps from The Harvard Map Collection as part of the Imaging the Urban Environment project. Maps selected for this project represent major urban areas and cities of the world, at various time periods. These maps typically portray both natural and manmade features at a large scale. The selection represents a range of regions, originators, ground condition dates, scales, and purposes.

The Ets domain transcription factor Erm distinguishes rat satellite glia from Schwann cells and is regulated in satellite cells by neuregulin signaling

Distinct glial cell types of the vertebrate peripheral nervous system (PNS) are derived from the neural crest. Here we show that the expression of the Ets domain transcription factor Erm distinguishes satellite glia from Schwann cells beginning early in rat PNS development. In developing dorsal root ganglia (DRG), Erm is present both in presumptive satellite glia and in neurons. In contrast, Erm is not detectable at any developmental stage in Schwann cells in peripheral nerves. In addition, Erm is downregulated in DRG-derived glia adopting Schwann cell traits in culture. Thus, Erm is the first described transcription factor expressed in satellite glia but not in Schwann cells. In culture, the Neuregulin1 (NRG1) isoform GGF2 maintains Erm expression in presumptive satellite cells and reinduces Erm expression in DRG-derived glia but not in Schwann cells from sciatic nerve. These data demonstrate that there are intrinsic differences between these glial subtypes in their response to NRG1 signaling. In neural crest cultures, Erm-positive progenitor cells give rise to two distinct glial subtypes: Erm-positive, Oct-6-negative satellite glia in response to GGF2, and Erm-negative, Oct-6-positive Schwann cells in the presence of serum and the adenylate cyclase activator forskolin. Thus, Erm-positive neural crest-derived progenitor cells and presumptive satellite glia are able to acquire Schwann cell features. Given the in vivo expression of Erm in peripheral ganglia, we suggest that ganglionic Erm-positive cells may be precursors of Schwann cells.