Precise coordination of cell-ECM adhesion is essential for efficient melanoblast migration during development.

Melanoblasts disperse throughout the skin and populate hair follicles through long-range cell migration. During migration, cells undergo cycles of coordinated attachment and detachment from the extracellular matrix (ECM). Embryonic migration processes that require cell-ECM attachment are dependent on the integrin family of adhesion receptors. Precise regulation of integrin-mediated adhesion is important for many developmental migration events. However, the mechanisms that regulate integrin-mediated adhesion in vivo in melanoblasts are not well understood. Here, we show that autoinhibitory regulation of the integrin-associated adapter protein talin coordinates cell-ECM adhesion during melanoblast migration in vivo Specifically, an autoinhibition-defective talin mutant strengthens and stabilizes integrin-based adhesions in melanocytes, which impinges on their ability to migrate. Mice with defective talin autoinhibition exhibit delays in melanoblast migration and pigmentation defects. Our results show that coordinated integrin-mediated cell-ECM attachment is essential for melanoblast migration and that talin autoinhibition is an important mechanism for fine-tuning cell-ECM adhesion during cell migration in development.

Cyclic DNA remethylation following active demethylation at euchromatic regions in mouse embryonic stem cells.

DNA methylation is an essential epigenetic mark that regulates normal mammalian embryonic development. DNA methylation profiles are not always static, especially during germline development. In zygotes, DNA is typically highly methylated but, during preimplantation, DNA methylation is erased globally. Then, at the start of post-implantation development in mouse embryos, DNA again becomes dramatically hypermethylated. Chromatin structure regulates the accessibility of DNA-modifying enzymes to target DNA. Beyond that, however, our understanding of the pathway by which chromatin regulation initiates changes in global DNA methylation during mouse embryonic development remains incomplete. To analyse the relationship between global regulation of DNA methylation and chromatin status, we examined 5-methylcytosine (5mC), modified by the DNA methyltransferase DNMT, and the oxidative derivative 5-hydroxymethylation (5hmC), converted from 5mC by TET-family enzymes, by means of immunofluorescence staining of mitotic chromosomes in mouse embryonic stem cells (ESCs). Our comparison of immunostaining patterns for those epigenetic modifications in wild-type, DNMT-deficient, and TET-deficient ESCs allowed us to visualise cell cycle-mediated DNA methylation changes, especially in euchromatic regions. Our findings suggest that DNA methylation patterns in undifferentiated mouse ESCs are stochastically balanced by the opposing effects of two activities: demethylation by TET and subsequent remethylation by DNMT.

Can we build a neuroecology of innovativeness similar to that pioneered by David Sherry for spatial memory?

David Sherry's pioneering work on the neuroecology of spatial memory has three characteristics that could inspire studies on other cognitive processes: it was grounded in a robust prior literature in psychology and neuroscience; it identified several natural history contexts in which repeated independent evolution of spatial memory differences had occurred in different clades; it involved a precise cognitive ability with a precise neural substrate. We discuss the application of these three principles to a more domain-general trait-innovation. We argue that targeting the caudolateral nidopallium and its connected areas, favoring problem-solving over reversal learning as an experimental assay, and focusing on situations that involve environmental change, such as urbanization and invasion, can help the study of innovation progress, like the field of spatial memory has since 1989.

Setdb1 is required for germline development and silencing of H3K9me3-marked endogenous retroviruses in primordial germ cells.

Transcription of endogenous retroviruses (ERVs) is inhibited by de novo DNA methylation during gametogenesis, a process initiated after birth in oocytes and at approximately embryonic day 15.5 (E15.5) in prospermatogonia. Earlier in germline development, the genome, including most retrotransposons, is progressively demethylated. Young ERVK and ERV1 elements, however, retain intermediate methylation levels. As DNA methylation reaches a low point in E13.5 primordial germ cells (PGCs) of both sexes, we determined whether retrotransposons are marked by H3K9me3 and H3K27me3 using a recently developed low-input ChIP-seq (chromatin immunoprecipitation [ChIP] combined with deep sequencing) method. Although these repressive histone modifications are found predominantly on distinct genomic regions in E13.5 PGCs, they concurrently mark partially methylated long terminal repeats (LTRs) and LINE1 elements. Germline-specific conditional knockout of the H3K9 methyltransferase SETDB1 yields a decrease of both marks and DNA methylation at H3K9me3-enriched retrotransposon families. Strikingly, Setdb1 knockout E13.5 PGCs show concomitant derepression of many marked ERVs, including intracisternal A particle (IAP), ETn, and ERVK10C elements, and ERV-proximal genes, a subset in a sex-dependent manner. Furthermore, Setdb1 deficiency is associated with a reduced number of male E13.5 PGCs and postnatal hypogonadism in both sexes. Taken together, these observations reveal that SETDB1 is an essential guardian against proviral expression prior to the onset of de novo DNA methylation in the germline.

Transcription factor ASCL2 is required for development of the glycogen trophoblast cell lineage.

The basic helix-loop-helix (bHLH) transcription factor ASCL2 plays essential roles in diploid multipotent trophoblast progenitors, intestinal stem cells, follicular T-helper cells, as well as during epidermal development and myogenesis. During early development, Ascl2 expression is regulated by genomic imprinting and only the maternally inherited allele is transcriptionally active in trophoblast. The paternal allele-specific silencing of Ascl2 requires expression of the long non-coding RNA Kcnq1ot1 in cis and the deposition of repressive histone marks. Here we show that Del7AI, a 280-kb deletion allele neighboring Ascl2, interferes with this process in cis and leads to a partial loss of silencing at Ascl2. Genetic rescue experiments show that the low level of Ascl2 expression from the paternal Del7AI allele can rescue the embryonic lethality associated with maternally inherited Ascl2 mutations, in a level-dependent manner. Despite their ability to support development to term, the rescued placentae have a pronounced phenotype characterized by severe hypoplasia of the junctional zone, expansion of the parietal trophoblast giant cell layer, and complete absence of invasive glycogen trophoblast cells. Transcriptome analysis of ectoplacental cones at E7.5 and differentiation assays of Ascl2 mutant trophoblast stem cells show that ASCL2 is required for the emergence or early maintenance of glycogen trophoblast cells during development. Our work identifies a new cis-acting mutation interfering with Kcnq1ot1 silencing function and establishes a novel critical developmental role for the transcription factor ASCL2.

Speed-accuracy trade-off, detour reaching and response to PHA in Carib grackles.

Performance on different cognitive tasks varies between individuals within species. Recent evidence suggests that, in some species, this variation reflects the existence of coherent cognitive strategies bringing together positive and negative relationships between tasks. For example, Carib grackles show a speed-accuracy trade-off, where individuals that are fast at solving novel problems make more errors at discrimination learning than individuals that are slow solvers. Pathogens are thought to play a major role in shaping variation in cognition, either because different cognitive strategies lead to differential exposure to pathogens, or because investment in cognitive abilities is costly, limiting the ability to invest in anti-pathogen responses. In both cases, immunocompetence is expected to co-vary with cognition. Here, using wild-caught Carib grackles, we tested whether performance on reversal learning and detour-reaching tasks is associated with the speed-accuracy trade-off found in a previous study. In parallel, we measured the response of individuals to a phytohemagglutinin (PHA) injection, an immunoecological technique that assesses general immunity. Performance on two problem-solving tasks and two learning tasks was characterized by a speed-accuracy trade-off, reversal learning and discrimination learning performance being better in individuals with slower problem-solving performance. Detour-reaching performance was independent from this trade-off. Finally, our results show that PHA response was higher in accurate but slow grackles, and higher in grackles with better detour-reaching performance. Investigating the emergence and maintenance of variation in cognition in a framework integrating variation in physiology and life history is likely a major step towards a better understanding of the evolution of cognition.

Innovativeness and the effects of urbanization on risk-taking behaviors in wild Barbados birds.

The effects of urbanization on avian cognition remain poorly understood. Risk-taking behaviors like boldness, neophobia and flight distance are thought to affect opportunism and innovativeness, and should also vary with urbanization. Here, we investigate variation in risk-taking behaviors in the field in an avian assemblage of nine species that forage together in Barbados and for which innovation rate is known from previous work. We predicted that birds from highly urbanized areas would show more risk-taking behavior than conspecifics from less urbanized parts of the island and that the differences would be strongest in the most innovative of the species. Overall, we found that urban birds are bolder, less neophobic and have shorter flight distances than their less urbanized conspecifics. Additionally, we detected between-species differences in the effect of urbanization on flight distance, more innovative species showing smaller differences in flight distance between areas. Our results suggest that, within successful urban colonizers, species differences in innovativeness may affect the way species change their risk-taking behaviors in response to the urban environment.

Relative Brain Size and Its Relation with the Associative Pallium in Birds.

Despite growing interest in the evolution of enlarged brains, the biological significance of brain size variation remains controversial. Much of the controversy is over the extent to which brain structures have evolved independently of each other (mosaic evolution) or in a coordinated way (concerted evolution). If larger brains have evolved by the increase of different brain regions in different species, it follows that comparisons of the whole brain might be biologically meaningless. Such an argument has been used to criticize comparative attempts to explain the existing variation in whole-brain size among species. Here, we show that pallium areas associated with domain-general cognition represent a large fraction of the entire brain, are disproportionally larger in large-brained birds and accurately predict variation in the whole brain when allometric effects are appropriately accounted for. While this does not question the importance of mosaic evolution, it suggests that examining specialized, small areas of the brain is not very helpful for understanding why some birds have evolved such large brains. Instead, the size of the whole brain reflects consistent variation in associative pallium areas and hence is functionally meaningful for comparative analyses.

Ecological generalism and behavioural innovation in birds: technical intelligence or the simple incorporation of new foods?

Generalist species are more successful than specialists in anthropogenically modified environments or in environments in which they have been introduced, but the nature of the link between generalism and establishment success is unclear. A higher feeding innovation rate has previously been reported in habitat generalist birds from North America. By allowing them to exploit new resources, this higher feeding innovation rate might explain the generalists' advantage. This result might be due to generalists being more likely to find new resources because they are exposed to more diverse environmental conditions. Alternatively, they might differ from specialists in other traits, in particular cognitive skills that might allow them to innovate more complex food searching and handling techniques. To test these hypotheses, we separated avian feeding innovations into a 'technical' (novel searching and handling behaviour) and a 'food type' (incorporation of a new food in a species' diet) category. Technical innovations, but not food type innovations, have previously been shown to correlate with avian brain size, suggesting they reflect cognitive ability. We used a world-wide data base of 2339 feeding innovations recorded in the literature, covering a total of 765 avian species and assessed the correlations between brain size and feeding innovation rates on one side and habitat and diet generalism on the other. Habitat generalism was positively related with food type innovation rate, but not technical innovation rate or brain size. This suggests that habitat generalist species are more likely to incorporate new food types in their diet because of higher chances to find new food resources in their environment, or of a higher opportunism, but not enhanced cognitive skills. In contrast, diet generalist species had higher food type and technical innovation rates, as well as larger brains, suggesting that cognitive skills might help species expand their diet breadth or that an increase in diet breadth might favour the evolution of enhanced cognitive abilities. Our results provide new insights into the nature of the generalists' advantage in the face of environmental changes, and suggest that dietary and habitat generalism are different, but convergent, routes to feeding flexibility and adaptation to changed environments.

Morphological and molecular sexing of the monochromatic Barbados bullfinch, Loxigilla barbadensis.

The bullfinch Loxigilla barbadensis is an endemic passerine on the Caribbean island of Barbados that has only recently been taxonomically split from the Lesser Antillean bullfinch L. noctis. The trait that most clearly distinguishes L. barbadensis from L. noctis is the absence in the male of sexually dimorphic coloration of the body and throat feathers, with L. barbadensis males and females sharing the same dull brown plumage. Here we report, in 64 individuals netted throughout the island, the results of a discriminant analysis on two (wing length and tail length) to four morphological traits showing very high (97%) concordance with sexing via PCR using blood samples. Females also show a paler lower mandible, a trait that yields an 80% concordance with PCR sexing. We found one L. barbadensis male that had a noctis-like reddish throat patch, supporting the idea that sexual dichromatism is the ancestral condition and that male Barbados bullfinches have evolved cryptic coloration that now makes the species monochromatic.

Tissue-specific alternative polyadenylation at the imprinted gene Mest regulates allelic usage at Copg2.

The gene Mest (also known as Peg1) is regulated by genomic imprinting in the mouse and only the paternal allele is active for transcription. MEST is similarly imprinted in humans, where it is a candidate for the growth retardation Silver-Russell syndrome. The MEST protein belongs to an ancient family of hydrolases but its function is still unknown. It is highly conserved in vertebrates although imprinted expression is only observed in marsupials and eutherians, thus a recent evolutionary event. Here we describe the identification of new imprinted RNA products at the Mest locus, longer variants of the RNA, called MestXL, transcribed >10 kb into the downstream antisense gene Copg2. During development MestXL is produced exclusively in the developing central nervous system (CNS) by alternative polyadenylation. Copg2 is biallelically expressed in the embryo except in MestXL-expressing tissues, where we observed preferential expression from the maternal allele. To analyze the function of the MestXL transcripts in Copg2 regulation, we studied the effects of a targeted allele at Mest introducing a truncation in the mRNA. We show that both the formation of the MestXL isoforms and the allelic bias at Copg2 are lost in the CNS of mutants embryos. Our results propose a new mechanism to regulate allelic usage in the mammalian genome, via tissue-specific alternative polyadenylation and transcriptional interference in sense-antisense pairs at imprinted loci.

Roles of endogenous retroviral elements in the establishment and maintenance of imprinted gene expression.

DNA methylation (DNAme) has long been recognized as a host defense mechanism, both in the restriction modification systems of prokaryotes as well as in the transcriptional silencing of repetitive elements in mammals. When DNAme was shown to be implicated as a key epigenetic mechanism in the regulation of imprinted genes in mammals, a parallel with host defense mechanisms was drawn, suggesting perhaps a common evolutionary origin. Here we review recent work related to this hypothesis on two different aspects of the developmental imprinting cycle in mammals that has revealed unexpected roles for long terminal repeat (LTR) retroelements in imprinting, both canonical and noncanonical. These two different forms of genomic imprinting depend on different epigenetic marks inherited from the mature gametes, DNAme and histone H3 lysine 27 trimethylation (H3K27me3), respectively. DNAme establishment in the maternal germline is guided by transcription during oocyte growth. Specific families of LTRs, evading silencing mechanisms, have been implicated in this process for specific imprinted genes. In noncanonical imprinting, maternally inherited histone marks play transient roles in transcriptional silencing during preimplantation development. These marks are ultimately translated into DNAme, notably over LTR elements, for the maintenance of silencing of the maternal alleles in the extraembryonic trophoblast lineage. Therefore, LTR retroelements play important roles in both establishment and maintenance of different epigenetic pathways leading to imprinted expression during development. Because such elements are mobile and highly polymorphic among different species, they can be coopted for the evolution of new species-specific imprinted genes.

Problem-solving skills are predicted by technical innovations in the wild and brain size in passerines.

Behavioural innovations can provide key advantages for animals in the wild, especially when ecological conditions change rapidly and unexpectedly. Innovation rates can be compared across taxa by compiling field reports of novel behaviours. Large-scale analyses have shown that innovativeness reduces extinction risk, increases colonization success and is associated with increased brain size and pallial neuron numbers. However, appropriate laboratory measurements of innovativeness, necessary to conduct targeted experimental studies, have not been clearly established, despite decades of speculation on the most suitable assay. Here we implemented a battery of cognitive tasks on 203 birds of 15 passerine species and tested for relationships at the interspecific and intraspecific levels with ecological metrics of innovation and brain size. We found that species better at solving extractive foraging problems had higher technical innovation rates in the wild and larger brains. By contrast, performance on other cognitive tasks often subsumed under the term behavioural flexibility, namely, associative and reversal learning, as well as self-control, were not related to problem-solving, innovation in the wild or brain size. Our study yields robust support for problem-solving as an accurate experimental proxy of innovation and suggests that novel motor solutions are more important than self-control or learning of modified cues in generating technical innovations in the wild.

Brain size and morphology of the brood-parasitic and cerophagous honeyguides (Aves: Piciformes).

Honeyguides (Indicatoridae, Piciformes) are unique among birds in several respects. All subsist primarily on wax, are obligatory brood parasites and one species engages in 'guiding' behavior in which it leads human honey hunters to bees' nests. This unique life history has likely shaped the evolution of their brain size and morphology. Here, we test that hypothesis using comparative data on relative brain and brain region size of honeyguides and their relatives: woodpeckers, barbets and toucans. Honeyguides have significantly smaller relative brain volumes than all other piciform taxa. Volumetric measurements of the brain indicate that honeyguides have a significantly larger cerebellum and hippocampal formation (HF) than woodpeckers, the sister clade of the honeyguides, although the HF enlargement was not significant across all of our analyses. Cluster analyses also revealed that the overall composition of the brain and telencephalon differs greatly between honeyguides and woodpeckers. The relatively smaller brains of the honeyguides may be a consequence of brood parasitism and cerophagy ('wax eating'), both of which place energetic constraints on brain development and maintenance. The inconclusive results of our analyses of relative HF volume highlight some of the problems associated with comparative studies of the HF that require further study.

Insertion profiles of 4 headless compression screws.

PURPOSE: In practice, the surgeon must rely on screw position (insertion depth) and tactile feedback from the screwdriver (insertion torque) to gauge compression. In this study, we identified the relationship between interfragmentary compression and these 2 factors. METHODS: The Acutrak Standard, Acutrak Mini, Synthes 3.0, and Herbert-Whipple implants were tested using a polyurethane foam scaphoid model. A specialized testing jig simultaneously measured compression force, insertion torque, and insertion depth at half-screw-turn intervals until failure occurred. RESULTS: The peak compression occurs at an insertion depth of -3.1 mm, -2.8 mm, 0.9 mm, and 1.5 mm for the Acutrak Mini, Acutrak Standard, Herbert-Whipple, and Synthes screws respectively (insertion depth is positive when the screw is proud above the bone and negative when buried). The compression and insertion torque at a depth of -2 mm were found to be 113 ± 18 N and 0.348 ± 0.052 Nm for the Acutrak Standard, 104 ± 15 N and 0.175 ± 0.008 Nm for the Acutrak Mini, 78 ± 9 N and 0.245 ± 0.006 Nm for the Herbert-Whipple, and 67 ± 2N, 0.233 ± 0.010 Nm for the Synthes headless compression screws. CONCLUSIONS: All 4 screws generated a sizable amount of compression (> 60 N) over a wide range of insertion depths. The compression at the commonly recommended insertion depth of -2 mm was not significantly different between screws; thus, implant selection should not be based on compression profile alone. Conically shaped screws (Acutrak) generated their peak compression when they were fully buried in the foam whereas the shanked screws (Synthes and Herbert-Whipple) reached peak compression before they were fully inserted. Because insertion torque correlated poorly with compression, surgeons should avoid using tactile judgment of torque as a proxy for compression. CLINICAL RELEVANCE: Knowledge of the insertion profile may improve our understanding of the implants, provide a better basis for comparing screws, and enable the surgeon to optimize compression.

Adding intelligence to mobile asset management in hospitals: the true value of RFID.

RFID (Radio Frequency Identification) technology is expected to play a vital role in the healthcare arena, especially in times when cost containments are at the top of the priorities of healthcare management authorities. Medical equipment represents a significant share of yearly healthcare operational costs; hence, ensuring an effective and efficient management of such key assets is critical to promptly and reliably deliver a diversity of clinical services at the patient bedside. Empirical evidence from a phased-out RFID implementation in one European hospital demonstrates that RFID has the potential to transform asset management by improving inventory management, enhancing asset utilization, increasing staff productivity, improving care services, enhancing maintenance compliance, and increasing information visibility. Most importantly, RFID allows the emergence of intelligent asset management processes, which is, undoubtedly, the most important benefit that could be derived from the RFID system. Results show that the added intelligence can be rather basic (auto-status change) or a bit more advanced (personalized automatic triggers). More importantly, adding intelligence improves planning and decision-making processes.

Parent-of-origin detection and chromosome-scale haplotyping using long-read DNA methylation sequencing and Strand-seq.

Hundreds of loci in human genomes have alleles that are methylated differentially according to their parent of origin. These imprinted loci generally show little variation across tissues, individuals, and populations. We show that such loci can be used to distinguish the maternal and paternal homologs for all human autosomes without the need for the parental DNA. We integrate methylation-detecting nanopore sequencing with the long-range phase information in Strand-seq data to determine the parent of origin of chromosome-length haplotypes for both DNA sequence and DNA methylation in five trios with diverse genetic backgrounds. The parent of origin was correctly inferred for all autosomes with an average mismatch error rate of 0.31% for SNVs and 1.89% for insertions or deletions (indels). Because our method can determine whether an inherited disease allele originated from the mother or the father, we predict that it will improve the diagnosis and management of many genetic diseases.

Partial loss of Ascl2 function affects all three layers of the mature placenta and causes intrauterine growth restriction.

Several imprinted genes have been implicated in the regulation of placental function and embryonic growth. On distal mouse chromosome 7, two clusters of imprinted genes, each regulated by its own imprinting center (IC), are separated by a poorly characterized region of 280kb (the IC1-IC2 interval). We previously generated a mouse line in which this IC1-IC2 interval has been deleted (Del(7AI) allele) and found that maternal inheritance of this allele results in low birth weights in newborns. Here we report that Del(7AI) causes a partial loss of Ascl2, a maternally expressed gene in the IC2 cluster, which when knocked out leads to embryonic lethality at midgestation due to a lack of spongiotrophoblast formation. The hypomorphic Ascl2 allele causes embryonic growth restriction and an associated placental phenotype characterized by a reduction in placental weight, reduced spongiotrophoblast population, absence of glycogen cells, and an expanded trophoblast giant cell layer. We also uncovered severe defects in the labyrinth layer of maternal mutants including increased production of the trilaminar labyrinth trophoblast cell types and a disorganized labyrinthine vasculature. Our results have important implications for our understanding of the role played by the spongiotrophoblast layer during placentation and show that regulation of the dosage of the imprinted gene Ascl2 can affect all three layers of the chorio-allantoic placenta.

The placental imprintome and imprinted gene function in the trophoblast glycogen cell lineage.

Imprinted genes represent a unique class of autosomal genes expressed from only one of the parental alleles during development. The choice of the expressed allele is not random but rather is determined by the parental origin of the allele. Consequently, the mouse genome contains more than 100 genes expressed preferentially or exclusively from the maternally or the paternally inherited allele. Current research efforts are focused on understanding the molecular mechanism of this epigenetic phenomenon as well as the biological functions of the genes under its regulation. Both theoretical considerations and experimental results support a role for genomic imprinting in the regulation of embryonic growth and placental biology. In this review, recent efforts to establish the complete set of genes showing imprinted expression in the mouse placenta are first discussed. Then, the evidence suggesting that imprinted genes might be implicated in the emergence, maintenance and function of trophoblast glycogen cells is presented. Although the origin and functions of this trophoblast cell lineage are currently unknown, the analysis of mutations in imprinted genes in the mouse are providing new insights into these issues. The implications of this work for placental pathologies in human are also discussed.

The Otto Aufranc Award: Demineralized bone matrix around porous implants promotes rapid gap healing and bone ingrowth.

BACKGROUND: Noncemented revision arthroplasty is often complicated by the presence of bone implant gaps that reduce initial stability and biologic fixation. Demineralized bone matrix has osteoinductive properties and therefore the potential to enhance gap healing and porous implant fixation. QUESTIONS/PURPOSES: We determined at what times and to what extent demineralized bone matrix promotes gap healing and bone ingrowth around a porous implant. METHODS: We inserted porous titanium implants into the proximal metaphyses of canine femora and humeri, with an initial 3-mm gap between host cancellous bone and implants. We left the gaps empty (control; n = 12) or filled them with either demineralized bone matrix (n = 6) or devitalized demineralized bone matrix (negative control; n = 6) and left them in situ for 4 or 12 weeks. We quantified volume healing of the gap with new bone using three-dimensional micro-CT scanning and quantified apposition and ingrowth using backscattered scanning electron microscopy. RESULTS: The density of bone inside gaps filled with demineralized bone matrix reached 64% and 93% of surrounding bone density by 4 and 12 weeks, respectively. Compared with empty controls and negative controls at 4 and 12 weeks, gap healing using demineralized bone matrix was two to three times greater and bone ingrowth and apposition were up to 15 times greater. CONCLUSIONS: Demineralized bone matrix promotes rapid bone ingrowth and gap healing around porous implants. CLINICAL RELEVANCE: Demineralized bone matrix has potential for enhancing implant fixation in revision arthroplasty.