Micro-computed tomography-based phenotypic approaches in embryology: procedural artifacts on assessments of embryonic craniofacial growth and development

<p>Abstract</p> <p>Background</p> <p>Growing demand for three dimensional (3D) digital images of embryos for purposes of phenotypic assessment drives implementation of new histological and imaging techniques. Among these micro-computed tomography (μCT) has recently been utilized as an effective and practical method for generating images at resolutions permitting 3D quantitative analysis of gross morphological attributes of developing tissues and organs in embryonic mice. However, histological processing in preparation for μCT scanning induces changes in organ size and shape. Establishing normative expectations for experimentally induced changes in size and shape will be an important feature of 3D μCT-based phenotypic assessments, especially if quantifying differences in the values of those parameters between comparison sets of developing embryos is a primary aim. Toward that end, we assessed the nature and degree of morphological artifacts attending μCT scanning following use of common fixatives, using a two dimensional (2D) landmark geometric morphometric approach to track the accumulation of distortions affecting the embryonic head from the native, uterine state through to fixation and subsequent scanning.</p> <p>Results</p> <p>Bouin's fixation reduced average centroid sizes of embryonic mouse crania by approximately 30% and substantially altered the morphometric shape, as measured by the shift in Procrustes distance, from the unfixed state, after the data were normalized for naturally occurring shape variation. Subsequent μCT scanning produced negligible changes in size but did appear to reduce or even reverse fixation-induced random shape changes. Mixtures of paraformaldehyde + glutaraldehyde reduced average centroid sizes by 2-3%. Changes in craniofacial shape progressively increased post-fixation.</p> <p>Conclusions</p> <p>The degree to which artifacts are introduced in the generation of random craniofacial shape variation relates to the degree of specimen dehydration during the initial fixation. Fixation methods that better maintain original craniofacial dimensions at reduced levels of dehydration and tissue shrinkage lead to the progressive accumulation of random shape variation during handling and data acquisition. In general, to the degree that embryonic organ size and shape factor into μCT-based phenotypic assessments, procedurally induced artifacts associated with fixation and scanning will influence results. Experimental designs will need to address these significant effects, either by employing alternative methods that minimize artifacts in the region of focus or in the interpretation of statistical patterns.</p

Consequences of Lineage-Specific Gene Loss on Functional Evolution of Surviving Paralogs: ALDH1A and Retinoic Acid Signaling in Vertebrate Genomes

Genome duplications increase genetic diversity and may facilitate the evolution of gene subfunctions. Little attention, however, has focused on the evolutionary impact of lineage-specific gene loss. Here, we show that identifying lineage-specific gene loss after genome duplication is important for understanding the evolution of gene subfunctions in surviving paralogs and for improving functional connectivity among human and model organism genomes. We examine the general principles of gene loss following duplication, coupled with expression analysis of the retinaldehyde dehydrogenase Aldh1a gene family during retinoic acid signaling in eye development as a case study. Humans have three ALDH1A genes, but teleosts have just one or two. We used comparative genomics and conserved syntenies to identify loss of ohnologs (paralogs derived from genome duplication) and to clarify uncertain phylogenies. Analysis showed that Aldh1a1 and Aldh1a2 form a clade that is sister to Aldh1a3-related genes. Genome comparisons showed secondarily loss of aldh1a1 in teleosts, revealing that Aldh1a1 is not a tetrapod innovation and that aldh1a3 was recently lost in medaka, making it the first known vertebrate with a single aldh1a gene. Interestingly, results revealed asymmetric distribution of surviving ohnologs between co-orthologous teleost chromosome segments, suggesting that local genome architecture can influence ohnolog survival. We propose a model that reconstructs the chromosomal history of the Aldh1a family in the ancestral vertebrate genome, coupled with the evolution of gene functions in surviving Aldh1a ohnologs after R1, R2, and R3 genome duplications. Results provide evidence for early subfunctionalization and late subfunction-partitioning and suggest a mechanistic model based on altered regulation leading to heterochronic gene expression to explain the acquisition or modification of subfunctions by surviving ohnologs that preserve unaltered ancestral developmental programs in the face of gene loss

Synaptic incorporation of GluA1-containing AMPA receptors during memory processes

It is generally understood that modifications in synaptic strength are the basis for learning and memory and that the strength of a synapse is largely governed by the abundance and distribution of synaptic receptors, especially alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPA receptors), which mediate most of the fast synaptic transmission in the brain. GluA1-containing AMPARs are incorporated into the synapse following activity and posttranslational modifications to the carboxyl-terminus affect which proteins interact with the receptor and determine whether the receptor is inserted or removed from the synapse. In vitro research has discovered that the phosphorylation of three serine residues contained on the carboxyl-terminus (Ser 818, Ser-831 and Ser-845) regulates GluA1 synaptic incorporation; however, in vivo research investigating the relative importance of these phosphorylation sites on long-term memory formation is currently limited to knock in studies.To block the interactions between these phosphorylation sites and their binding partners in an inducible, temporally sensitive manner, we infused interference peptides containing these residues during consolidation and reconsolidation. We hypothesized that if the synaptic incorporation of GluA1 containing AMPA receptors is required for memory formation, and if this incorporation required the residues contained on the interference peptide, we would see an impairment in long-term memory expression when the peptide was infused at the time of training, or at the time of retrieval.Infusing the interference peptide GluA1-CT, containing Ser-831 and Ser 845, 1 hour before auditory fear conditioning produced no impairment in memory expression 24 hours later. However, infusing the interference peptide GluA1-MPR, containing Ser-818, 1 hour before training did produce an impairment in memory expression 24 hours later. We did not observe an impairment in long-term memory expression when both peptides were infused 1 hour before memory reactivation.Il est généralement accepté que les modifications de la force synaptique sont à la base de l'apprentissage et la mémoire et que la force d'une synapse est largement régie par l'abondance et la distribution de récepteurs synaptiques, en particulier de récepteurs alpha-amino-3-hydroxy-5-méthyl-4- isoxazole propionate (récepteurs AMPA), qui interviennent dans la plupart des transmissions synaptiques rapides dans le cerveau. Les récepteurs AMPA contenant la sous-unité GluA1 sont incorporés dans la synapse suite à son activation et des modifications post-traductionnelles de l'extrémité carboxy-terminale influencent quelles protéines interagissent avec le récepteur et détermine si le récepteur est inséré ou retiré de la synapse. Des recherches in vitro ont découvert que la phosphorylation de trois résidus sérine contenus sur l'extrémité carboxy-terminale (Ser-818, Ser-831 et Ser-845) régie l'incorporation synaptique de GluA1; cependant, les recherches in vivo étudiant l'importance de ces sites de phosphorylation sur la formation de la mémoire à long terme est actuellement limitée à des études utilisant des « knock in ». Pour bloquer les interactions entre ces sites de phosphorylation et de leurs partenaires de liaison de manière inductible et temporellement sensibles, nous avons infusé des peptides d'interférence contenant ces résidus lors de la consolidation et la reconsolidation. Nous émettons l'hypothèse que si l'incorporation synaptique des récepteurs AMPA contenant GluA1 est nécessaire à la formation de la mémoire, et si cette incorporation exige les résidus contenus dans le peptide d'interférence, nous verrions une déficience dans l'expression de mémoire à long terme lorsque le peptide a été infusé au moment du conditionnement ou du rappel du souvenir.L'infusion du peptide d'interférence GluA1-CT, contenant les sérines Ser 831 et Ser-845, 1 heure avant le conditionnement de peur auditive n'a produit aucune altération dans l'expression de mémoire 24 heures plus tard. Cependant, l'infusion du peptide d'interférence GluA1-MPR, contenant la sérine Ser-818, 1 heure avant le conditionnement a produit une déficience dans l'expression de mémoire 24 heures plus tard. Nous n'avons pas observé d'altération dans l'expression de mémoire à long terme lorsque les deux peptides ont été infusés 1 heure avant la réactivation