Quantifying three‐dimensional morphology and RNA from individual embryos

Green, Rebecca M.; Leach, Courtney L.; Hoehn, Natasha; Marcucio, Ralph; Hallgrímsson, Benedikt

doi:10.1002/dvdy.24490

Cited by 8 publications

(12 citation statements)

References 17 publications

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“…Embryonic specimens from A/WySnJ (AWS), C57BL/6J (C57), and WSB/EiJ (WSB) inbred backgrounds were collected at the University of Calgary at embryonic day (E) 11.5 and processed as recently described [ 30 ]. Briefly, embryos were fixed overnight in PaxGene tissue fix solution (Qiagen, PreAnalytics, cat #765312) then stored at −20 °C in the PaxGene tissue stabilizer solution prepared to manufacturer specification (Qiagen, PreAnalytics, cat #765512).…”

Section: Methodsmentioning

confidence: 99%

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Developmental constraint through negative pleiotropy in the zygomatic arch

Percival

Green

Roseman

et al. 2018

EvoDevo

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Background Previous analysis suggested that the relative contribution of individual bones to regional skull lengths differ between inbred mouse strains. If the negative correlation of adjacent bone lengths is associated with genetic variation in a heterogeneous population, it would be an example of negative pleiotropy, which occurs when a genetic factor leads to opposite effects in two phenotypes. Confirming negative pleiotropy and determining its basis may reveal important information about the maintenance of overall skull integration and developmental constraint on skull morphology.ResultsWe identified negative correlations between the lengths of the frontal and parietal bones in the midline cranial vault as well as the zygomatic bone and zygomatic process of the maxilla, which contribute to the zygomatic arch. Through gene association mapping of a large heterogeneous population of Diversity Outbred (DO) mice, we identified a quantitative trait locus on chromosome 17 driving the antagonistic contribution of these two zygomatic arch bones to total zygomatic arch length. Candidate genes in this region were identified and real-time PCR of the maxillary processes of DO founder strain embryos indicated differences in the RNA expression levels for two of the candidate genes, Camkmt and Six2.ConclusionsA genomic region underlying negative pleiotropy of two zygomatic arch bones was identified, which provides a mechanism for antagonism in component bone lengths while constraining overall zygomatic arch length. This type of mechanism may have led to variation in the contribution of individual bones to the zygomatic arch noted across mammals. Given that similar genetic and developmental mechanisms may underlie negative correlations in other parts of the skull, these results provide an important step toward understanding the developmental basis of evolutionary variation and constraint in skull morphology.Electronic supplementary materialThe online version of this article (10.1186/s13227-018-0092-3) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

“…Following a recently published analysis [ 30 ], RNA was extracted using the PaxGene RNA extraction kit (Qiagen, PreAnalytics cat #766134), which includes a DNA removal step. RNA was analyzed using a NanoDrop 1000 (ThermoFisher).…”

Section: Methodsmentioning

confidence: 99%

Developmental constraint through negative pleiotropy in the zygomatic arch

Percival

Green

Roseman

et al. 2018

EvoDevo

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“…Embryos were fixed and set in the PAXgene tissue system as outlined in Green et al The embryos were microCT scanned using a Scanco UCT 35 desktop scanner also according to the protocol outlined in Reference , briefly, embryos contrasted in 1% iodine metal in PAX tissue stabilizer for 1 hour and were scanned in air in a sealed microfuge tube with a small amount of clean PAX tissue stabilizer. They were scanned at 11 μm resolution at 55 kpv.…”

Section: Methodsmentioning

confidence: 99%

“…Recent improvements in fixation and imaging methods now allow us to generate a three‐dimensional (3D) microCT scan, DNA and RNA from the same individual embryo . This is important as it allows us to study directly the relationships between morphology and gene expression at the level of individual embryos.…”

Section: Introductionmentioning

confidence: 99%

“…Recent improvements in fixation and imaging methods now allow us to generate a three-dimensional (3D) microCT scan, DNA and RNA from the same individual embryo. 29 This is important as it allows us to study directly the relationships between morphology and gene expression at the level of individual embryos. Here, we present the first direct correlations between gene expression and morphology among individual mammalian embryos that exhibit differences in incidence and severity of clefting.…”

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Nonlinear gene expression‐phenotype relationships contribute to variation and clefting in the A/WySn mouse

Green

Leach

Diewert

et al. 2019

Developmental Dynamics

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Background Cleft lip and palate is one of the most common human birth defects, but the underlying etiology is poorly understood. The A/WySn mouse is a spontaneously occurring model of multigenic clefting in which 20% to 30% of individuals develop an orofacial cleft. Recent work has shown altered methylation at a specific retrotransposon insertion downstream of the Wnt9b locus in clefting animals, which results in decreased Wnt9b expression. Results Using a newly developed protocol that allows us to measure morphology, gene expression, and DNA methylation in the same embryo, we relate gene expression in an individual embryo directly to its three‐dimensional morphology for the first time. We find that methylation at the retrotransposon relates to Wnt9b expression and morphology. IAP methylation relates to shape of the nasal process in a manner consistent with clefting. Embryos with low IAP methylation exhibit increased among‐individual variance in facial shape. Conclusions Methylation and gene expression relate nonlinearly to nasal process morphology. Individuals at one end of a continuum of phenotypic states display a clinical phenotype and increased phenotypic variation. Variable penetrance and expressivity in this model is likely determined both by among‐individual variation in methylation and changes in phenotypic robustness along the underlying liability distribution for orofacial clefting.

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Phosphotungstic acid‐enhanced microCT: Optimized protocols for embryonic and early postnatal mice

Lesciotto

Perrine

Kawasaki

et al. 2019

Developmental Dynamics

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Background Given the need for descriptive and increasingly mechanistic morphological analyses, contrast‐enhanced microcomputed tomography (microCT) represents perhaps the best method for visualizing 3D biological soft tissues in situ. Although staining protocols using phosphotungstic acid (PTA) have been published with beautiful visualizations of soft tissue structures, these protocols are often aimed at highly specific research questions and are applicable to a limited set of model organisms, specimen ages, or tissue types. We provide detailed protocols for micro‐level visualization of soft tissue structures in mice at several embryonic and early postnatal ages using PTA‐enhanced microCT. Results Our protocols produce microCT scans that enable visualization and quantitative analyses of whole organisms, individual tissues, and organ systems while preserving 3D morphology and relationships with surrounding structures, with minimal soft tissue shrinkage. Of particular note, both internal and external features of the murine heart, lungs, and liver, as well as embryonic cartilage, are captured at high resolution. Conclusion These protocols have broad applicability to mouse models for a variety of diseases and conditions. Minor experimentation in the staining duration can expand this protocol to additional age groups, permitting ontogenetic studies of internal organs and soft tissue structures within their 3D in situ position.

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Quantifying three‐dimensional morphology and RNA from individual embryos

Cited by 8 publications

References 17 publications

Developmental constraint through negative pleiotropy in the zygomatic arch

Developmental constraint through negative pleiotropy in the zygomatic arch

Nonlinear gene expression‐phenotype relationships contribute to variation and clefting in the A/WySn mouse

Phosphotungstic acid‐enhanced microCT: Optimized protocols for embryonic and early postnatal mice

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