Tmod2 Is a Regulator of Cocaine Responses through Control of Striatal and Cortical Excitability and Drug-Induced Plasticity.

Drugs of abuse induce neuroadaptations, including synaptic plasticity, that are critical for transition to addiction, and genes and pathways that regulate these neuroadaptations are potential therapeutic targets. Tropomodulin 2 (Tmod2) is an actin-regulating gene that plays an important role in synapse maturation and dendritic arborization and has been implicated in substance abuse and intellectual disability in humans. Here, we mine the KOMP2 data and find that Tmod2 knock-out mice show emotionality phenotypes that are predictive of addiction vulnerability. Detailed addiction phenotyping shows that Tmod2 deletion does not affect the acute locomotor response to cocaine administration. However, sensitized locomotor responses are highly attenuated in these knock-outs, indicating perturbed drug-induced plasticity. In addition, Tmod2 mutant animals do not self-administer cocaine indicating lack of hedonic responses to cocaine. Whole-brain MR imaging shows differences in brain volume across multiple regions, although transcriptomic experiments did not reveal perturbations in gene coexpression networks. Detailed electrophysiological characterization of Tmod2 KO neurons showed increased spontaneous firing rate of early postnatal and adult cortical and striatal neurons. Cocaine-induced synaptic plasticity that is critical for sensitization is either missing or reciprocal in Tmod2 KO nucleus accumbens shell medium spiny neurons, providing a mechanistic explanation of the cocaine response phenotypes. Combined, these data, collected from both males and females, provide compelling evidence that Tmod2 is a major regulator of plasticity in the mesolimbic system and regulates the reinforcing and addictive properties of cocaine.

LAMA: automated image analysis for the developmental phenotyping of mouse embryos.

Advanced 3D imaging modalities, such as micro-computed tomography (micro-CT), have been incorporated into the high-throughput embryo pipeline of the International Mouse Phenotyping Consortium (IMPC). This project generates large volumes of raw data that cannot be immediately exploited without significant resources of personnel and expertise. Thus, rapid automated annotation is crucial to ensure that 3D imaging data can be integrated with other multi-dimensional phenotyping data. We present an automated computational mouse embryo phenotyping pipeline that harnesses the large amount of wild-type control data available in the IMPC embryo pipeline in order to address issues of low mutant sample number as well as incomplete penetrance and variable expressivity. We also investigate the effect of developmental substage on automated phenotyping results. Designed primarily for developmental biologists, our software performs image pre-processing, registration, statistical analysis and segmentation of embryo images. We also present a novel anatomical E14.5 embryo atlas average and, using it with LAMA, show that we can uncover known and novel dysmorphology from two IMPC knockout lines.

IRX3/5 regulate mitotic chromatid segregation and limb bud shape.

Pattern formation is influenced by transcriptional regulation as well as by morphogenetic mechanisms that shape organ primordia, although factors that link these processes remain under-appreciated. Here we show that, apart from their established transcriptional roles in pattern formation, IRX3/5 help to shape the limb bud primordium by promoting the separation and intercalation of dividing mesodermal cells. Surprisingly, IRX3/5 are required for appropriate cell cycle progression and chromatid segregation during mitosis, possibly in a nontranscriptional manner. IRX3/5 associate with, promote the abundance of, and share overlapping functions with co-regulators of cell division such as the cohesin subunits SMC1, SMC3, NIPBL and CUX1. The findings imply that IRX3/5 coordinate early limb bud morphogenesis with skeletal pattern formation.

Structural Variant in Mitochondrial-Associated Gene (MRPL3) Induces Adult-Onset Neurodegeneration with Memory Impairment in the Mouse.

An impediment to the development of effective therapies for neurodegenerative disease is that available animal models do not reproduce important clinical features such as adult-onset and stereotypical patterns of progression. Using in vivo magnetic resonance imaging and behavioral testing to study male and female decrepit mice, we found a stereotypical neuroanatomical pattern of progression of the lesion along the limbic system network and an associated memory impairment. Using structural variant analysis, we identified an intronic mutation in a mitochondrial-associated gene (Mrpl3) that is responsible for the decrepit phenotype. While the function of this gene is unknown, embryonic lethality in Mrpl3 knock-out mice suggests it is critical for early development. The observation that a mutation linked to energy metabolism precipitates a pattern of neurodegeneration via cell death across disparate but linked brain regions may explain how stereotyped patterns of neurodegeneration arise in humans or define a not yet identified human disease.SIGNIFICANCE STATEMENT The development of novel therapies for adult-onset neurodegenerative disease has been impeded by the limitations of available animal models in reproducing many of the clinical features. Here, we present a novel spontaneous mutation in a mitochondrial-associated gene in a mouse (termed decrepit) that results in adult-onset neurodegeneration with a stereotypical neuroanatomical pattern of progression and an associated memory impairment. The decrepit mouse model may represent a heretofore undiagnosed human disease and could serve as a new animal model to study neurodegenerative disease.

Deficiency of Natriuretic Peptide Receptor 2 Promotes Bicuspid Aortic Valves, Aortic Valve Disease, Left Ventricular Dysfunction, and Ascending Aortic Dilatations in Mice.

RATIONALE: Aortic valve disease is a cell-mediated process without effective pharmacotherapy. CNP (C-type natriuretic peptide) inhibits myofibrogenesis and osteogenesis of cultured valve interstitial cells and is downregulated in stenotic aortic valves. However, it is unknown whether CNP signaling regulates aortic valve health in vivo. OBJECTIVE: The aim of this study is to determine whether a deficient CNP signaling axis in mice causes accelerated progression of aortic valve disease. METHODS AND RESULTS: In cultured porcine valve interstitial cells, CNP inhibited pathological differentiation via the guanylate cyclase NPR2 (natriuretic peptide receptor 2) and not the G-protein-coupled clearance receptor NPR3 (natriuretic peptide receptor 3). We used Npr2+/- and Npr2+/-;Ldlr-/- mice and wild-type littermate controls to examine the valvular effects of deficient CNP/NPR2 signaling in vivo, in the context of both moderate and advanced aortic valve disease. Myofibrogenesis in cultured Npr2+/- fibroblasts was insensitive to CNP treatment, whereas aged Npr2+/- and Npr2+/-;Ldlr-/- mice developed cardiac dysfunction and ventricular fibrosis. Aortic valve function was significantly impaired in Npr2+/- and Npr2+/-;Ldlr-/- mice versus wild-type littermates, with increased valve thickening, myofibrogenesis, osteogenesis, proteoglycan synthesis, collagen accumulation, and calcification. 9.4% of mice heterozygous for Npr2 had congenital bicuspid aortic valves, with worse aortic valve function, fibrosis, and calcification than those Npr2+/- with typical tricuspid aortic valves or all wild-type littermate controls. Moreover, cGK (cGMP-dependent protein kinase) activity was downregulated in Npr2+/- valves, and CNP triggered synthesis of cGMP and activation of cGK1 (cGMP-dependent protein kinase 1) in cultured porcine valve interstitial cells. Finally, aged Npr2+/-;Ldlr-/- mice developed dilatation of the ascending aortic, with greater aneurysmal progression in Npr2+/- mice with bicuspid aortic valves than those with tricuspid valves. CONCLUSIONS: Our data establish CNP/NPR2 signaling as a novel regulator of aortic valve development and disease and elucidate the therapeutic potential of targeting this pathway to arrest disease progression.

Mouse models of 17q21.31 microdeletion and microduplication syndromes highlight the importance of Kansl1 for cognition.

Koolen-de Vries syndrome (KdVS) is a multi-system disorder characterized by intellectual disability, friendly behavior, and congenital malformations. The syndrome is caused either by microdeletions in the 17q21.31 chromosomal region or by variants in the KANSL1 gene. The reciprocal 17q21.31 microduplication syndrome is associated with psychomotor delay, and reduced social interaction. To investigate the pathophysiology of 17q21.31 microdeletion and microduplication syndromes, we generated three mouse models: 1) the deletion (Del/+); or 2) the reciprocal duplication (Dup/+) of the 17q21.31 syntenic region; and 3) a heterozygous Kansl1 (Kans1+/-) model. We found altered weight, general activity, social behaviors, object recognition, and fear conditioning memory associated with craniofacial and brain structural changes observed in both Del/+ and Dup/+ animals. By investigating hippocampus function, we showed synaptic transmission defects in Del/+ and Dup/+ mice. Mutant mice with a heterozygous loss-of-function mutation in Kansl1 displayed similar behavioral and anatomical phenotypes compared to Del/+ mice with the exception of sociability phenotypes. Genes controlling chromatin organization, synaptic transmission and neurogenesis were upregulated in the hippocampus of Del/+ and Kansl1+/- animals. Our results demonstrate the implication of KANSL1 in the manifestation of KdVS phenotypes and extend substantially our knowledge about biological processes affected by these mutations. Clear differences in social behavior and gene expression profiles between Del/+ and Kansl1+/- mice suggested potential roles of other genes affected by the 17q21.31 deletion. Together, these novel mouse models provide new genetic tools valuable for the development of therapeutic approaches.

Progressive neuroanatomical changes caused by Grin1 loss-of-function mutation.

NMDA receptor dysfunction is central to the encephalopathies caused by missense mutations in the NMDA receptor subunit genes. Missense variants of GRIN1, GRIN2A, and GRIN2B cause similar syndromes with varying severity of intellectual impairment, autism, epilepsy, and motor dysfunction. To gain insight into possible biomarkers of NMDAR hypofunction, we asked whether a loss-of-function variant in the Grin1 gene would cause structural changes in the brain that could be detected by MRI. We also studied the developmental trajectory of these changes to determine whether structural changes coincided with reported cognitive impairments in the mice. We performed magnetic resonance imaging in male Grin1-/- knockdown mice (GluN1KD) that were three, six, or twelve weeks old. Deformation-based morphometry was used to assess neuroanatomical differences. Volumetric reductions were detected in substantia nigra and striatum of GluN1KD mice at all ages. Changes in limbic structures were only evident at six weeks of age. Reductions in white matter volumes were first evident at three weeks, and additional deficits were detected at six and twelve weeks. FluoroJade immunofluorescence revealed degenerating neurons in twelve-week old GluN1KD mice. We conclude that Grin1 loss-of-function mutations cause volume reductions in dopaminergic structures early in development, while changes to limbic and white matter structures are delayed and are more pronounced in post-adolescent ages. The evidence of degenerating neurons in the mature brain indicates an ongoing process of cell loss as a consequence of NMDAR hypofunction.

The effect of automated landmark identification on morphometric analyses.

Morphometric analysis of anatomical landmarks allows researchers to identify specific morphological differences between natural populations or experimental groups, but manually identifying landmarks is time-consuming. We compare manually and automatically generated adult mouse skull landmarks and subsequent morphometric analyses to elucidate how switching from manual to automated landmarking will impact morphometric analysis results for large mouse (Mus musculus) samples (n = 1205) that represent a wide range of 'normal' phenotypic variation (62 genotypes). Other studies have suggested that the use of automated landmarking methods is feasible, but this study is the first to compare the utility of current automated approaches to manual landmarking for a large dataset that allows the quantification of intra- and inter-strain variation. With this unique sample, we investigated how switching to a non-linear image registration-based automated landmarking method impacts estimated differences in genotype mean shape and shape variance-covariance structure. In addition, we tested whether an initial registration of specimen images to genotype-specific averages improves automatic landmark identification accuracy. Our results indicated that automated landmark placement was significantly different than manual landmark placement but that estimated skull shape covariation was correlated across methods. The addition of a preliminary genotype-specific registration step as part of a two-level procedure did not substantially improve on the accuracy of one-level automatic landmark placement. The landmarks with the lowest automatic landmark accuracy are found in locations with poor image registration alignment. The most serious outliers within morphometric analysis of automated landmarks displayed instances of stochastic image registration error that are likely representative of errors common when applying image registration methods to micro-computed tomography datasets that were initially collected with manual landmarking in mind. Additional efforts during specimen preparation and image acquisition can help reduce the number of registration errors and improve registration results. A reduction in skull shape variance estimates were noted for automated landmarking methods compared with manual landmarking. This partially reflects an underestimation of more extreme genotype shapes and loss of biological signal, but largely represents the fact that automated methods do not suffer from intra-observer landmarking error. For appropriate samples and research questions, our image registration-based automated landmarking method can eliminate the time required for manual landmarking and have a similar power to identify shape differences between inbred mouse genotypes.

Activated Hedgehog-GLI Signaling Causes Congenital Ureteropelvic Junction Obstruction.

Intrinsic ureteropelvic junction obstruction is the most common cause of congenital hydronephrosis, yet the underlying pathogenesis is undefined. Hedgehog proteins control morphogenesis by promoting GLI-dependent transcriptional activation and inhibiting the formation of the GLI3 transcriptional repressor. Hedgehog regulates differentiation and proliferation of ureteric smooth muscle progenitor cells during murine kidney-ureter development. Histopathologic findings of smooth muscle cell hypertrophy and stroma-like cells, consistently observed in obstructing tissue at the time of surgical correction, suggest that Hedgehog signaling is abnormally regulated during the genesis of congenital intrinsic ureteropelvic junction obstruction. Here, we demonstrate that constitutively active Hedgehog signaling in murine intermediate mesoderm-derived renal progenitors results in hydronephrosis and failure to develop a patent pelvic-ureteric junction. Tissue obstructing the ureteropelvic junction was marked as early as E13.5 by an ectopic population of cells expressing Ptch2, a Hedgehog signaling target. Constitutive expression of GLI3 repressor in Ptch1-deficient mice rescued ectopic Ptch2 expression and obstructive hydronephrosis. Whole transcriptome analysis of isolated Ptch2+ cells revealed coexpression of genes characteristic of stromal progenitor cells. Genetic lineage tracing indicated that stromal cells blocking the ureteropelvic junction were derived from intermediate mesoderm-derived renal progenitors and were distinct from the smooth muscle or epithelial lineages. Analysis of obstructive ureteric tissue resected from children with congenital intrinsic ureteropelvic junction obstruction revealed a molecular signature similar to that observed in Ptch1-deficient mice. Together, these results demonstrate a Hedgehog-dependent mechanism underlying mammalian intrinsic ureteropelvic junction obstruction.

P63 expression plays a role in developmental rate, embryo size, and local morphogenesis.

BACKGROUND: The p63 gene is integral to the development of many body parts including limb, palate, teeth, and urogenital tract. Loss of p63 expression may alter developmental rate, which is crucial to normal morphogenesis. To validate a novel, unbiased embryo phenotyping software tool, we tested whether delayed development contributes to the pathological phenotype of a p63 mouse mutant (p63-/- ). We quantified dysmorphology in p63-/- embryos and tested for universal growth delay relative to wild-type (WT) embryos. Fixed embryos (n = 6; p63-/- ) aged day (E) 15.5 were micro-CT scanned and quantitatively analyzed using a digital WT atlas that defined volumetric differences between p63-/- and WT embryos. RESULTS: p63-/- embryos showed a growth delay of approximately 22 hr (0.9 days). Among the E15.5 mutants, overall size was closest to WT E14.6 mice but shape was closest to WT E14.0. The atlas clearly identified in p63-/- embryos malformations of epithelial derivatives including limbs, tail, urogenital structures, brain, face, and tooth. CONCLUSIONS: The software atlas technique described the p63-/- phenotype as a combination of developmental delay (i.e., heterochrony) and malformation (i.e., pathological shape; failed organogenesis). This study identifies for the first time global and local roles for p63 in prenatal growth and development. Developmental Dynamics 247:779-787, 2018. © 2018 Wiley Periodicals, Inc.

4D atlas of the mouse embryo for precise morphological staging.

After more than a century of research, the mouse remains the gold-standard model system, for it recapitulates human development and disease and is quickly and highly tractable to genetic manipulations. Fundamental to the power and success of using a mouse model is the ability to stage embryonic mouse development accurately. Past staging systems were limited by the technologies of the day, such that only surface features, visible with a light microscope, could be recognized and used to define stages. With the advent of high-throughput 3D imaging tools that capture embryo morphology in microscopic detail, we now present the first 4D atlas staging system for mouse embryonic development using optical projection tomography and image registration methods. By tracking 3D trajectories of every anatomical point in the mouse embryo from E11.5 to E14.0, we established the first 4D atlas compiled from ex vivo 3D mouse embryo reference images. The resulting 4D atlas comprises 51 interpolated 3D images in this gestational range, resulting in a temporal resolution of 72 min. From this 4D atlas, any mouse embryo image can be subsequently compared and staged at the global, voxel and/or structural level. Assigning an embryonic stage to each point in anatomy allows for unprecedented quantitative analysis of developmental asynchrony among different anatomical structures in the same mouse embryo. This comprehensive developmental data set offers developmental biologists a new, powerful staging system that can identify and compare differences in developmental timing in wild-type embryos and shows promise for localizing deviations in mutant development.

Automated pipeline for anatomical phenotyping of mouse embryos using micro-CT.

The International Mouse Phenotyping Consortium (IMPC) plans to phenotype 20,000 single-gene knockout mice to gain an insight into gene function. Approximately 30% of these knockout mouse lines will be embryonic or perinatal lethal. The IMPC has selected three-dimensional (3D) imaging to phenotype these mouse lines at relevant stages of embryonic development in an attempt to discover the cause of lethality using detailed anatomical information. Rate of throughput is paramount as IMPC production centers have been given the ambitious task of completing this phenotyping project by 2021. Sifting through the wealth of data within high-resolution 3D mouse embryo data sets by trained human experts is infeasible at this scale. Here, we present a phenotyping pipeline that identifies statistically significant anatomical differences in the knockout, in comparison with the wild type, through a computer-automated image registration algorithm. This phenotyping pipeline consists of three analyses (intensity, deformation, and atlas based) that can detect missing anatomical structures and differences in volume of whole organs as well as on the voxel level. This phenotyping pipeline was applied to micro-CT images of two perinatal lethal mouse lines: a hypomorphic mutation of the Tcf21 gene (Tcf21-hypo) and a knockout of the Satb2 gene. With the proposed pipeline we were able to identify the majority of morphological phenotypes previously published for both the Tcf21-hypo and Satb2 mutant mouse embryos in addition to novel phenotypes. This phenotyping pipeline is an unbiased, automated method that highlights only those structural abnormalities that survive statistical scrutiny and illustrates them in a straightforward fashion.

Partitioning k-space for cylindrical three-dimensional rapid acquisition with relaxation enhancement imaging in the mouse brain.

Three-dimensional rapid acquisition with relaxation enhancement (RARE) scans require the assignment of each phase encode step in two dimensions to an echo in the echo train. Although this assignment is frequently made across the entire Cartesian grid, collection of only the central cylinder of k-space by eliminating the corners in each phase encode dimension reduces the scan time by ~22% with negligible impact on image quality. The recipe for the assignment of echoes to grid points for such an acquisition is less straightforward than for the simple full Cartesian acquisition case, and has important implications for image quality. We explored several methods of partitioning k-space-exploiting angular symmetry in one extreme or emulating a cropped Cartesian acquisition in the other-and acquired three-dimensional RARE magnetic resonance imaging (MRI) scans of the ex vivo mouse brain. We evaluated each partitioning method for sensitivity to artifacts and then further considered strategies to minimize these through averaging or interleaving of echoes and by empirical phase correction. All scans were collected 16 at a time with multiple-mouse MRI. Although all schemes considered could be used to generate images, the results indicate that the emulation of a standard Cartesian echo assignment, by partitioning preferentially along one dimension within the cylinder, is more robust to artifacts. Samples at the periphery of the bore showed larger phase deviations and higher sensitivity to artifacts, but images of good quality could still be obtained with an optimized acquisition protocol. A protocol for high-resolution (40 µm) ex vivo images using this approach is presented, and has been used routinely with a success rate of 99% in over 1000 images.

A coming of age: advanced imaging technologies for characterising the developing mouse.

The immense challenge of annotating the entire mouse genome has stimulated the development of cutting-edge imaging technologies in a drive for novel information. These techniques promise to improve understanding of the genes involved in embryo development, at least one third of which have been shown to be essential. Aligning advanced imaging technologies with biological needs will be fundamental to maximising the number of phenotypes discovered in the coming years. International efforts are underway to meet this challenge through an integrated and sophisticated approach to embryo phenotyping. We review rapid advances made in the imaging field over the past decade and provide a comprehensive examination of the relative merits of current and emerging techniques. The aim of this review is to provide a guide to state-of-the-art embryo imaging that will enable informed decisions as to which technology to use and fuel conversations between expert imaging laboratories, researchers, and core mouse production facilities.

Diffusible iodine-based contrast-enhanced computed tomography (diceCT): an emerging tool for rapid, high-resolution, 3-D imaging of metazoan soft tissues.

Morphologists have historically had to rely on destructive procedures to visualize the three-dimensional (3-D) anatomy of animals. More recently, however, non-destructive techniques have come to the forefront. These include X-ray computed tomography (CT), which has been used most commonly to examine the mineralized, hard-tissue anatomy of living and fossil metazoans. One relatively new and potentially transformative aspect of current CT-based research is the use of chemical agents to render visible, and differentiate between, soft-tissue structures in X-ray images. Specifically, iodine has emerged as one of the most widely used of these contrast agents among animal morphologists due to its ease of handling, cost effectiveness, and differential affinities for major types of soft tissues. The rapid adoption of iodine-based contrast agents has resulted in a proliferation of distinct specimen preparations and scanning parameter choices, as well as an increasing variety of imaging hardware and software preferences. Here we provide a critical review of the recent contributions to iodine-based, contrast-enhanced CT research to enable researchers just beginning to employ contrast enhancement to make sense of this complex new landscape of methodologies. We provide a detailed summary of recent case studies, assess factors that govern success at each step of the specimen storage, preparation, and imaging processes, and make recommendations for standardizing both techniques and reporting practices. Finally, we discuss potential cutting-edge applications of diffusible iodine-based contrast-enhanced computed tomography (diceCT) and the issues that must still be overcome to facilitate the broader adoption of diceCT going forward.

Micro-CT imaging: Developing criteria for examining fetal skeletons in regulatory developmental toxicology studies - A workshop report.

During the past two decades the use and refinements of imaging modalities have markedly increased making it possible to image embryos and fetuses used in pivotal nonclinical studies submitted to regulatory agencies. Implementing these technologies into the Good Laboratory Practice environment requires rigorous testing, validation, and documentation to ensure the reproducibility of data. A workshop on current practices and regulatory requirements was held with the goal of defining minimal criteria for the proper implementation of these technologies and subsequent submission to regulatory agencies. Micro-computed tomography (micro-CT) is especially well suited for high-throughput evaluations, and is gaining popularity to evaluate fetal skeletons to assess the potential developmental toxicity of test agents. This workshop was convened to help scientists in the developmental toxicology field understand and apply micro-CT technology to nonclinical toxicology studies and facilitate the regulatory acceptance of imaging data. Presentations and workshop discussions covered: (1) principles of micro-CT fetal imaging; (2) concordance of findings with conventional skeletal evaluations; and (3) regulatory requirements for validating the system. Establishing these requirements for micro-CT examination can provide a path forward for laboratories considering implementing this technology and provide regulatory agencies with a basis to consider the acceptability of data generated via this technology.

Delayed myelination in a mouse model of fragile X syndrome.

Fragile X Syndrome is the most common inherited cause of autism. Fragile X mental retardation protein (FMRP), which is absent in fragile X, is an mRNA binding protein that regulates the translation of hundreds of different mRNA transcripts. In the adult brain, FMRP is expressed primarily in the neurons; however, it is also expressed in developing glial cells, where its function is not well understood. Here, we show that fragile X (Fmr1) knockout mice display abnormalities in the myelination of cerebellar axons as early as the first postnatal week, corresponding roughly to the equivalent time in human brain development when symptoms of the syndrome first become apparent (1-3 years of age). At postnatal day (PND) 7, diffusion tensor magnetic resonance imaging showed reduced volume of the Fmr1 cerebellum compared with wild-type mice, concomitant with an 80-85% reduction in the expression of myelin basic protein, fewer myelinated axons and reduced thickness of myelin sheaths, as measured by electron microscopy. Both the expression of the proteoglycan NG2 and the number of PDGFRα+/NG2+ oligodendrocyte precursor cells were reduced in the Fmr1 cerebellum at PND 7. Although myelin proteins were still depressed at PND 15, they regained wild-type levels by PND 30. These findings suggest that impaired maturation or function of oligodendrocyte precursor cells induces delayed myelination in the Fmr1 mouse brain. Our results bolster an emerging recognition that white matter abnormalities in early postnatal brain development represent an underlying neurological deficit in Fragile X syndrome.

A novel 3D mouse embryo atlas based on micro-CT.

The goal of the International Mouse Phenotyping Consortium (IMPC) is to phenotype targeted knockout mouse strains throughout the whole mouse genome (23,000 genes) by 2021. A significant percentage of the generated mice will be embryonic lethal; therefore, phenotyping methods tuned to the mouse embryo are needed. Methods that are robust, quantitative, automated and high-throughput are attractive owing to the numbers of mice involved. Three-dimensional (3D) imaging is a useful method for characterizing morphological phenotypes. However, tools to automatically quantify morphological information of mouse embryos from 3D imaging have not been fully developed. We present a representative mouse embryo average 3D atlas comprising micro-CT images of 35 individual C57BL/6J mouse embryos at 15.5 days post-coitum. The 35 micro-CT images were registered into a consensus average image with our automated image registration software and 48 anatomical structures were segmented manually. We report the mean and variation in volumes for each of the 48 segmented structures. Mouse organ volumes vary by 2.6-4.2% on a linear scale when normalized to whole body volume. A power analysis of the volume data reports that a 9-14% volume difference can be detected between two classes of mice with sample sizes of eight. This resource will be crucial in establishing baseline anatomical phenotypic measurements for the assessment of mutant mouse phenotypes, as any future mutant embryo image can be registered to the atlas and subsequent organ volumes calculated automatically.

Reptilian heart development and the molecular basis of cardiac chamber evolution.

The emergence of terrestrial life witnessed the need for more sophisticated circulatory systems. This has evolved in birds, mammals and crocodilians into complete septation of the heart into left and right sides, allowing separate pulmonary and systemic circulatory systems, a key requirement for the evolution of endothermy. However, the evolution of the amniote heart is poorly understood. Reptilian hearts have been the subject of debate in the context of the evolution of cardiac septation: do they possess a single ventricular chamber or two incompletely septated ventricles? Here we examine heart development in the red-eared slider turtle, Trachemys scripta elegans (a chelonian), and the green anole, Anolis carolinensis (a squamate), focusing on gene expression in the developing ventricles. Both reptiles initially form a ventricular chamber that homogenously expresses the T-box transcription factor gene Tbx5. In contrast, in birds and mammals, Tbx5 is restricted to left ventricle precursors. In later stages, Tbx5 expression in the turtle (but not anole) heart is gradually restricted to a distinct left ventricle, forming a left-right gradient. This suggests that Tbx5 expression was refined during evolution to pattern the ventricles. In support of this hypothesis, we show that loss of Tbx5 in the mouse ventricle results in a single chamber lacking distinct identity, indicating a requirement for Tbx5 in septation. Importantly, misexpression of Tbx5 throughout the developing myocardium to mimic the reptilian expression pattern also results in a single mispatterned ventricular chamber lacking septation. Thus ventricular septation is established by a steep and correctly positioned Tbx5 gradient. Our findings provide a molecular mechanism for the evolution of the amniote ventricle, and support the concept that altered expression of developmental regulators is a key mechanism of vertebrate evolution.

Vertebrate intersectin1 is repurposed to facilitate cortical midline connectivity and higher order cognition.

Invertebrate studies have highlighted a role for EH and SH3 domain Intersectin (Itsn) proteins in synaptic vesicle recycling and morphology. Mammals have two Itsn genes (Itsn1 and Itsn2), both of which can undergo alternative splicing to include DBL/PH and C2 domains not present in invertebrate Itsn proteins. To probe for specific and redundant functions of vertebrate Itsn genes, we generated Itsn1, Itsn2, and double mutant mice. While invertebrate mutants showed severe synaptic abnormalities, basal synaptic transmission and plasticity were unaffected at Schaffer CA1 synapses in mutant mice. Surprisingly, intercortical tracts-corpus callosum, ventral hippocampal, and anterior commissures-failed to cross the midline in mice lacking Itsn1, but not Itsn2. In contrast, tracts extending within hemispheres and those that decussate to more caudal brain segments appeared normal. Itsn1 mutant mice showed severe deficits in Morris water maze and contextual fear memory tasks, whereas mice lacking Itsn2 showed normal learning and memory. Thus, coincident with the acquisition of additional signaling domains, vertebrate Itsn1 has been functionally repurposed to also facilitate interhemispheric connectivity essential for high order cognitive functions.