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Rapid brain enlargement requires a hydraulic mechanism in the chick embryo. Such a mechanism involves a closed, fluid-filled system that generates positive pressure. For the chick embryo this study determined when rapid brain enlargement begins, assessed the relative contributions of cavity expansion and tissue growth to overall brain enlargement, and evaluated mathematical models of overall brain enlargement and expansion and growth of the component parts. Three to five embryos were collected at each Hamburger and Hamilton state (11, 12, 14, 16, and 18) and processed for paraffin serial sectioning. Brain growth was determined over a 24-hr period (stages 11-18) by calculating volumes from area measurements of sections of brains from individual embryos by using a computerized image-analysis system. Statistical analysis indicated that a linear model adequately described cavity expansion, and a linear model was rejected for the description of tissue growth and total brain enlargement. At the onset of brain enlargement, the cavity expands faster than the tissue grows; but after 12 hr the reverse is true. Initially (i.e., at stage 11), the cavity accounts for 60% of the total brain volume and tissue for 40%. At stages 12-16, cavity and tissue contribute 50% each. Finally at stage 18, cavity accounts for 55% and tissue for 45%. In order to better distinguish changes in cavity expansion and tissue growth over the 24-hr period studied, this period was divided into four intervals (I-IV). The rates of both cavity expansion and tissue growth increase between intervals I and II, decrease between intervals II and III, and increase between intervals III and IV.(ABSTRACT TRUNCATED AT 250 WORDS)

SUMMARYThe genetic load associated with the method of sex determination in honey bees has been a great deterrent to selective breeding programmes, primarily because of a lack of understanding of the population genetics of the system. In this paper we examine the distribution of diploid brood viabilities in closed, random mating populations, as a function both of the system of sex determination and of multiple mating in queens. Analytically we show that for a given number of sex alleles, an increase in the numbers of matings of queens, reduces the variance in brood viability, but does not affect the mean. The results of a computer simulation demonstrate that the equilibrium number of sex alleles is approached very slowly from above, so that small populations that start with large numbers of sex alleles will maintain high brood viability for a long time. The practical significance of this is discussed.

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Previous workers (McKenzie and Parsons, 1972, 1974; McKenzie, 1974; Briscoe et al., 1975) have found anomalous distributions of species of Drosophila, of sexes of D. melanogaster, and of Adh alleles in and around wineries in Australia and Spain. Field studies in California's Sonoma Valley provide evidence that the explanations advanced for these distributions may incorrect. The anomalous distribution of species was attributed to alcohol, either as a selective agent or as a behavioral stimulus. We find a virtually identical species distribution in the absence of environmental alcohol. The anomalous sex ratio was attributedd to differential survivall of the sexes when raised on alcohol. We present crude evidence thatehe difference may simply be a behavioral response to some product of fermentation, which need not be alcohol. Finally, the allele frequency difference reported from Spain was attributed to differential adult mortality on alcohol. We do not find an allele frequency difference even when alcohol is exposed, and therefore suggest that selection is occurring in pre-adult stages.

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BackgroundClustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided adaptive immune systems are found in prokaryotes to defend cells from foreign DNA. CRISPR Cas9 systems have been modified and employed as genome editing tools in wide ranging organisms. Here, we provide a detailed protocol to truncate genes in mammalian cells using CRISPR Cas9 editing. We describe custom donor vector construction using Gibson assembly with the commonly utilized pcDNA3 vector as the backbone.ResultsWe describe a step-by-step method to truncate genes of interest in mammalian cell lines using custom-made donor vectors. Our method employs 2 guide RNAs, mutant Cas9D10A nickase (Cas9 = CRISPR associated sequence 9), and a custom-made donor vector for homologous recombination to precisely truncate a gene of interest with a selectable neomycin resistance cassette (NPTII: Neomycin Phosphotransferase II). We provide a detailed protocol on how to design and construct a custom donor vector using Gibson assembly (and the commonly utilized pcDNA3 vector as the backbone) allowing researchers to obtain specific gene modifications of interest (gene truncation, gene deletion, epitope tagging or knock-in mutation). Selection of mutants in mammalian cell lines with G418 (Geneticin) combined with several screening methods: western blot analysis, polymerase chain reaction, and Sanger sequencing resulted in streamlined mutant isolation. Proof of principle experiments were done in several mammalian cell lines.ConclusionsHere we describe a detailed protocol to employ CRISPR Cas9 genome editing to truncate genes of interest using the commonly employed expression vector pcDNA3 as the backbone for the donor vector. Providing a detailed protocol for custom donor vector design and construction will enable researchers to develop unique genome editing tools. To date, detailed protocols for CRISPR Cas9 custom donor vector construction are limited (Lee et al. in Sci Rep 5:8572, 2015; Ma et al. in Sci Rep 4:4489, 2014). Custom donor vectors are commercially available, but can be expensive. Our goal is to share this protocol to aid researchers in performing genetic investigations that require custom donor vectors for specialized applications (specific gene truncations, knock-in mutations, and epitope tagging applications).Electronic supplementary materialThe online version of this article (10.1186/s12867-018-0105-8) contains supplementary material, which is available to authorized users.

Simple evolutionary processes can regularly generate events that appear hig~ly unlikely when compared to the universe of all possIble events. Models incorporating this evolutionary dynamic we call "constructionist" and we contrast them with traditional models which focus on static, equilibrium aspects. In popUlation genetics, construc~ionist models have provided a different perspective on several long-standing problems. We review three examples of the results from our own work. These results are: (1) simple evolutionary processes can result in multiple-allele polymorphisms maintained by selection that are highly unlikely to occur if fitnesses are chosen simultaneously and at random; (2) the frequency at which viabilities (e.g., heter?-zygote advantage or recessive lethality) occur III mutations is likely to be very different from that observed in established polymorphisms; and (3) the power of the Ewens-Watterson test of neutrality is low. Several extensions of the models are discussed, together with the sorts of questions they may answer. We conclude that a complete understanding of the evolutionary forces responsible for molecular polymorphism requires consideration of its temporal development as well as examination of its current form. B93016