Neogenin May Functionally Substitute for Dcc in Chicken

Dcc is the key receptor that mediates attractive responses of axonal growth cones to netrins, a family of axon guidance cues used throughout evolution. However, a Dcc homolog has not yet been identified in the chicken genome, raising the possibility that Dcc is not present in avians. Here we show that the closely related family member neogenin may functionally substitute for Dcc in the developing chicken spinal cord. The expression pattern of chicken neogenin in the developing spinal cord is a composite of the distribution patterns of both rodent Dcc and neogenin. Moreover, whereas the loss of mouse neogenin has no effect on the trajectory of commissural axons, removing chicken neogenin by RNA interference results in a phenotype similar to the functional inactivation of Dcc in mouse. Taken together, these data suggest that the chick neogenin is functionally equivalent to rodent Dcc

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Sequence effects of single base loops in intramolecular quadruplex DNA

We have examined the properties of intramolecular G-quadruplexes in which the G3 tracts are separated by single base loops. The most stable complex contained 1?,2?-dideoxyribose in all three loops, while loops containing T and C were slightly less stable (by about 2°C). Quadruplexes containing loops with single A residues were less stable by 8°C for each T to A substitution. These folded sequences display similar CD spectra, which are consistent with the formation of parallel stranded complexes with double-chain reversal loops. These results demonstrate that loop sequence, and not just length, affects quadruplex stability

Comparison of extent of amino acid similarity between Dcc and neogenin in different species.

<p>Comparison of extent of amino acid similarity between Dcc and neogenin in different species.</p

Commissural axons stall in the absence of neogenin in chicken embryos.

<p>(A–L) HH stage 14/15 chicken embryos were <i>in ovo</i> electroporated with either short hairpin micro RNAs directed against neogenin under the control of the U6 promoter (U6::shRNA(neo)-GFP, A–D, I–L) or the empty vector (E–H). This RNA interference (RNAi) vector includes GFP sequences that permit the efficacy of vector delivery to be monitored (A, C). Embryos were taken for analysis at HH stage 22. (A, B) Electroporation of the U6::shRNA(neo)-GFP construct (green) results in an observable reduction in the levels of neogenin protein (red, arrowhead, B). (C, D) The loss of neogenin (green) results in fewer axonin1⁺ axons (red) extending towards the floor plate (FP); compare thickness of the commissural axon bundle on the electroporated side (open arrow, D) to that on the non-electroporated side (arrows, D). Note that, at this stage in spinal cord development, axonin1 antibodies also label the motor axons. (E–L) To monitor the extent of axon outgrowth towards the floor plate (FP), Lhx2/9⁺ commissural neurons (red, G, H, K, L) were expressing fGFP under the control of the Math1 enhancer (Math1::fGFP, green E–L). (E–H) Math1⁺ axons project normally to the FP (arrowhead, F, H) after electroporation of the empty RNAi vector. (I–J) In contrast, the outgrowth of Math1⁺ axons is severely compromised after electroporation with U6::shRNA(neo) (arrowheads, J, H). (M) The level of neogenin protein was reduced by 38±5% (n = 12 sections from 4 embryos) on the electroporated side of the embryo compared to the non-electroporated side. This reduction in neogenin intensity is significantly different (p<2.6×10⁻⁵, student's t-test) from the effect of the empty vector on neogenin levels (n = 16 sections from 2 embryos). (N) The extent of commissural axon outgrowth was quantified by determining whether Math1⁺ axons had crossed one of four arbitrary lines in the spinal cord: mid-dorsal (MD), intermediate (INT), mid-ventral (MV) or the FP. (O) By HH stage 22, 80% of the control Lhx2/9⁺ neurons have extended GFP⁺ axons, of which 57% have extended into the intermediate spinal cord and 12% have reached the FP (n = 30 sections from 2 embryos). In contrast, after reducing the levels of neogenin, only 67% of the Lhx2/9⁺ neurons have extended GFP⁺ axons (p<0.013, similar to control, n = 52 sections from 4 embryos). Of these axons, 42% have projected into the intermediate spinal cord (p<0.00014, similar to control) but only 1.7% have reached the FP (p<2.9×10⁻⁵, similar to control), suggesting that although the experimental Math1⁺ axons largely projected to the intermediate spinal cord, they did not proceed further. (P) This defect did not result from a loss of Lhx2/9⁺ neurons. The number of Lhx2/9⁺ neurons on the U6::shRNA(neo)-GFP electroporated side was statistically indistinguishable from either the non-electroporated side (p>0.65, similar to control) or control electroporations (p>0.07, similar to control. Scale bar: 30 µm.</p

Mouse commissural axons stall in the absence of Dcc, but not neogenin.

<p>(A–J) Transverse sections taken from brachial levels of the spinal cord from E11.5 <i>Neo1^Gt/Gt</i> (C, D) and <i>Dcc^−/−</i> (H–I) embryos and their respective wild-type littermates (A, B, E–G) labeled with antibodies against Lhx2/9 (red, A, C, E, G, H, J) and Tag1 (green, A–F, H, I) which decorate commissural nuclei and axons, respectively. Tag1 also transiently labels motor neurons (m). (A, B, E–G) By E11.5, Tag1⁺ commissural axons in both wild-type littermate controls have extended robustly to the floor plate (FP, arrowhead, B, F). Their Lhx2/9⁺ cell bodies have also started to migrate ventrally to a deeper layer of the dorsal spinal cord (arrowhead, G). (C, D) In the <i>Neo1^Gt/Gt</i> mutant, the extent of Tag1⁺ commissural axon outgrowth (arrowhead, D) and the migration pattern of Lhx2/9⁺ neurons is indistinguishable from control littermates. (H–J) However, in <i>Dcc</i> mutants, Tag1⁺ axons show severe defects in their extent of outgrowth: they stall in the intermediate spinal cord (arrowheads, I) with very few axons reaching the FP. There also appears to be a delay/stall in Lhx2/9⁺ cell migration (arrowhead, J) Scale bar: 40 µm.</p

Summary of structure and sequence similarities between chicken, mouse and human neogenin and Dcc proteins.

<p>(A) Chicken neogenin is structurally similar to both mouse neogenin1 and Dcc, containing four immunoglobulin (Ig) domains and six fibronectin (FN) type III repeats on the extracellular side and three intracellular P domains. The individual domains of chicken neogenin show highest similarity at the amino acid level to mouse neogenin, although there is also considerable similarity between chicken neogenin and mouse Dcc. (B) Amino acid alignment of chicken, mouse and human neogenin with mouse and human Dcc. Identical amino acids are highlighted in grey. Additionally highlighted in FN domains 1 (red), 2 (blue), 3 (green) and 5 (yellow) are non-conservative amino acid changes that result in chicken neogenin being more similar to human/mouse Dcc than human/mouse neogenin1.</p

Chick <i>neogenin</i> is expressed in a similar manner to mouse <i>Dcc</i>.

<p>(A–H) <i>In situ</i> hybridization experiments for <i>neogenin</i> (A–C, G–I) and <i>Dcc</i> (D–F) on transverse sections of the spinal cord of E10.5 (A, D) and E11.5 (B, C, E, F) mouse embryos and HH stage 20 (G), 23 (E) and 26 (I) chicken embryos. (A–C) In mouse, <i>neogenin</i> is expressed first in the intermediate spinal cord (bracket, A). By E11.5, neogenin is present at high levels in the ventral ventricular zone as well as broadly in motor neurons (brackets, B, C) (D–F) Mouse <i>Dcc</i> is expressed at highest levels in the dorsal-most neural progenitors (arrowhead, D, E) and in post-mitotic neurons (brackets, D–F) throughout the dorsal spinal cord as well as at lower levels in a broad population of motor neurons. (G–I) The distribution of chicken <i>neogenin</i> is a composite of the expression patterns of both mouse <i>Dcc</i> and <i>neogenin</i>. At all stages, the highest levels of <i>neogenin</i> expression is in the dorsal-most spinal cord, in dorsal neural progenitors (arrowheads, G, I) and in a population of post-mitotic dorsal neurons whose position is consistent with their being commissural neurons (arrowhead, H). Neogenin is also present at lower levels in both the ventral ventricular zone and in motor neurons (brackets, H, I). Scale bar: A, D, G: 30 µm, B, C, E, F, H, I: 40 µm.</p

Brief behavioural surveys in routine HIV sentinel surveillance: a new tool for monitoring the HIV epidemic in Viet Nam

In this report we describe a new approach in HIV sentinel surveillance that was piloted in Viet Nam in 2009 and is currently being rolled out in all provinces. It comprises a brief behavioural questionnaire added to the HIV sentinel surveillance surveys conducted routinely among people who inject drugs, female sex workers and men who have sex with men. Timely reporting of data from this system has resulted in improvements to HIV prevention efforts for most at-risk populations