Anatomical comparison across heads, fore- and hindlimbs in mammals using network models.

Animal body parts evolve with variable degrees of integration that nonetheless yield functional adult phenotypes: but, how? The analysis of modularity with Anatomical Network Analysis (AnNA) is used to quantitatively determine phenotypic modules based on the physical connection among anatomical elements, an approach that is valuable to understand developmental and evolutionary constraints. We created anatomical network models of the head, forelimb, and hindlimb of two taxa considered to represent a 'generalized' eutherian (placental: mouse) and metatherian (marsupial: opossum) anatomical configuration and compared them with our species, which has a derived eutherian configuration. In these models, nodes represent anatomical units and links represent their physical connection. Here, we aimed to identify: (1) the commonalities and differences in modularity between species, (2) whether modules present a potential phylogenetic character, and (3) whether modules preferentially reflect either developmental or functional aspects of anatomy, or a mix of both. We predicted differences between networks of metatherian and eutherian mammals that would best be explained by functional constraints, versus by constraints of development and/or phylogeny. The topology of contacts between bones, muscles, and bones + muscles showed that, among all three species, skeletal networks were more similar than musculoskeletal networks. There was no clear indication that humans and mice are more alike when compared to the opossum overall, even though their musculoskeletal and skeletal networks of fore- and hindlimbs are slightly more similar. Differences were greatest among musculoskeletal networks of heads and next of forelimbs, which showed more variation than hindlimbs, supporting previous anatomical studies indicating that in general the configuration of the hindlimbs changes less across evolutionary history. Most observations regarding the anatomical networks seem to be best explained by function, but an exception is the adult opossum ear ossicles. These ear bones might form an independent module because the incus and malleus are involved in forming a functional primary jaw that enables the neonate to attach to the teat, where this newborn will complete its development. Additionally, the human data show a specialized digit 1 module (thumb/big toe) in both limb types, likely the result of functional and evolutionary pressures, as our ape ancestors had highly movable big toes and thumbs.

Unexpected variation of human molar size patterns.

A tenet of mammalian, including primate dental evolution, is the Inhibitory Cascade Model, where first molar (M1) size predicts in a linear cline the size and onset time of the second (M2) and third (M3) molars: a larger M1 portends a progressively smaller and later-developing M2 and M3. In contemporary modern Homo sapiens, later-developing M3s are less likely to erupt properly. The Inhibitory Cascade Model is also used to predict molar sizes of extinct taxa, including fossil Homo. The extent to which Inhibitory Cascade Model predictions hold in contemporary H. sapiens molars is unclear, including whether this tenet informs about molar initiation, development, and eruption. We tested these questions here. In our radiographic sample of 323 oral quadrants and molar rows from contemporary humans based on mesiodistal crown lengths, we observed the distribution of molar proportions with a central tendency around parity (M1 = M2 = M3) that parsed into 13 distinct molar size ratio patterns. These patterns presented at different frequencies (e.g., M1 > M2 > M3 in about one-third of cases) that reflected whether the molar row was located in the maxilla or mandible and included both linear (e.g., M1 < M2 < M3) and nonlinear molar size ratio progressions (e.g., M1 > M2 < M3). Up to four patterns were found in the same subject's mouth. Lastly, M1 size alone does not predict M3 size, developmental timing, or eruption; rather, M2 size is integral to predicting M3 size. Our study indicates that human molar size is genetically 'softwired' and sensitive to factors local to the human upper jaw vs. lower jaw. The lack of a single stereotypical molar size ratio for contemporary H. sapiens suggests that predictions of fossil H. sapiens molar sizes using the Inhibitory Cascade Model must be made with caution.

Biological clocks and incremental growth line formation in dentine.

Dentine- and enamel-forming cells secrete matrix in consistent rhythmic phases, resulting in the formation of successive microscopic growth lines inside tooth crowns and roots. Experimental studies of various mammals have proven that these lines are laid down in subdaily, daily (circadian), and multidaily rhythms, but it is less clear how these rhythms are initiated and maintained. In 2001, researchers reported that lesioning the so-called master biological clock, the suprachiasmatic nucleus (SCN), halted daily line formation in rat dentine, whereas subdaily lines persisted. More recently, a key clock gene (Bmal1) expressed in the SCN in a circadian manner was also found to be active in dentine- and enamel- secretory cells. To probe these potential neurological and local mechanisms for the production of rhythmic lines in teeth, we reexamined the role of the SCN in growth line formation in Wistar rats and investigated the presence of daily lines in Bmal1 knockout mice (Bmal1-/- ). In contrast to the results of the 2001 study, we found that both daily and subdaily growth lines persisted in rat dentine after complete or partial SCN lesion in the majority of individuals. In mice, after transfer into constant darkness, daily rhythms continued to manifest as incremental lines in the dentine of each Bmal1 genotype (wild-type, Bmal+/- , and Bmal1-/- ). These results affirm that the manifestation of biological rhythms in teeth is a robust phenomenon, imply a more autonomous role of local biological clocks in tooth growth than previously suggested, and underscore the need further to elucidate tissue-specific circadian biology and its role in incremental line formation. Investigations of this nature will strengthen an invaluable system for determining growth rates and calendar ages from mammalian hard tissues, as well as documenting the early lives of fossil hominins and other primates.

Expression of new genes in vertebrate tooth development and p63 signaling.

BACKGROUND: p63 is an evolutionarily ancient transcription factor essential to vertebrate tooth development. Our recent gene expression screen comparing wild-type and "toothless" p63-/- mouse embryos implicated in tooth development several new genes that we hypothesized act downstream of p63 in dental epithelium, where p63 is also expressed. RESULTS: Via in situ hybridization and immunohistochemistry, we probed mouse embryos (embryonic days 10.5-14.5) and spotted gar fish embryos (14 days postfertilization) for these newly linked genes, Cbln1, Cldn23, Fermt1, Krt15, Pltp and Prss8, which were expressed in mouse and gar dental epithelium. Loss of p63 altered expression levels but not domains. Expression was comparable between murine upper and lower tooth organs, implying conserved gene functions in maxillary and mandibular dentitions. Our meta-analysis of gene expression databases supported that these genes act within a p63-driven gene regulatory network important to tooth development in mammals and more evolutionary ancient vertebrates (fish, amphibians). CONCLUSIONS: Cbln1, Cldn23, Fermt1, Krt15, Pltp, and Prss8 were expressed in mouse and fish dental epithelium at placode, bud, and/or cap stages. We theorize that these genes participate in cell-cell adhesion, cell polarity, and extracellular matrix signaling to support dental epithelium integrity, folding, and epithelial-mesenchymal cross talk during tooth development.

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.

Upper jaw development in the absence of teeth: New insights for craniodental evo-devo integration.

In p63-null mice (p63-/- ), teeth fail to form but the mandible forms normally; conversely, the upper jaw skeleton is malformed. Here we explored whether lack of dental tissues contributed to midfacial dysmorphologies in p63-/- mice by testing if facial prominence defects appeared before odontogenesis failed. We also investigated gene dose effects by testing if one wild type (WT) p63 allele (p63+/- ) was sufficient for normal upper jaw skeleton formation. We micro-CT scanned PFA-fixed p63-/- , p63+/- , and WT (p63+/+ ) adult and embryonic mice aged E10-E14. Next, we landmarked mandibular (MdP), maxillary (MxP) and nasal prominences (NPs), and facial bones. 3D landmark data were assessed using Principal Component, Canonical Variate, Partial Least Squares, and other statistical analyses. The p63-/- embryos showed MxP and NP malformations by E12, despite the presence of dental tissues. MdP shape was comparable among p63-/- , p63+/- , and p63+/+ embryos. Upper jaw shape was comparable between p63+/+ and p63+/- adults. The upper jaw and its dentition both require p63 signaling, but not each other's presence, to form properly. One WT p63 allele enables normal midfacial morphogenesis; gene dose may be a target for jaw macroevolution. Jaw-specific genetic mechanisms likely integrate the evo-devo of dentitions with upper versus lower jaws.

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.

Implications of Vertebrate Craniodental Evo-Devo for Human Oral Health.

Highly processed diets eaten by postindustrial modern human populations coincide with higher frequencies of third molar impaction, malocclusion, and temporomandibular joint disorders that affect millions of people worldwide each year. Current treatments address symptoms, not causes, because the multifactorial etiologies of these three concerns mask which factors incline certain people to malocclusion, impaction, and/or joint issues. Deep scientific curiosity about the origins of jaws and dentitions continues to yield rich insights about the developmental genetic mechanisms that underpin healthy craniodental morphogenesis and integration. Mounting evidence from evolution and development (Evo-Devo) studies suggests that function is another mechanism important to healthy craniodental integration and fit. Starting as early as weaning, softer diets and thus lower bite forces appear to relax or disrupt integration of oral tissues, alter development and growth, and catalyze impaction, malocclusion, and jaw joint disorders. How developing oral tissues respond to bite forces remains poorly understood, but biomechanical feedback seems to alter balances of local bone resorption and deposition at the tooth-bone interface as well as affect tempos and amounts of facial outgrowth. Also, behavioral changes in jaw function and parafunction contribute to degeneration and pain in joint articular cartilages and masticatory muscles. The developmental genetic contribution to craniodental misfits and disorders is undeniable but still unclear; however, at present, human diet and jaw function remain important and much more actionable clinical targets. New Evo-Devo studies are needed to explain how function interfaces with craniodental phenotypic plasticity, variation, and evolvability to yield a spectrum of healthy and mismatched dentitions and jaws.

Detangling the evolutionary developmental integration of dentate jaws: evidence that a p63 gene network regulates odontogenesis exclusive of mandible morphogenesis.

Vertebrate jaws and dentitions fit and function together, yet the genetic processes that coordinate cranial and dental morphogenesis and evolution remain poorly understood. Teeth but not jaws fail to form in the edentate p63-/- mouse mutant, which we used here to identify genes important to odontogenesis, but not jaw morphogenesis, and that may allow dentitions to change during development and evolution without necessarily affecting the jaw skeleton. With the working hypothesis that tooth and jaw development are autonomously controlled by discreet gene regulatory networks, using gene expression microarray assays validated by quantitative reverse-transcription PCR we contrasted expression in mandibular prominences at embryonic days (E) 10-13 of mice with normal lower jaw development but either normal (p63+/- , p63+/+ ) or arrested (p63-/- ) tooth development. The p63-/- mice showed significantly different expression (fold change ≥2, ≤-2; P ≤ 0.05) of several genes. Some of these are known to help regulate odontogenesis (e.g., p63, Osr2, Cldn3/4) and/or to be targets of p63 (e.g., Jag1/2, Fgfr2); other genes have no previously reported roles in odontogenesis or the p63 pathway (e.g., Fermt1, Cbln1, Pltp, Krt8). As expected, from E10 to E13, few genes known to regulate mandible morphogenesis differed in expression between mouse strains. This study newly links several genes to odontogenesis and/or to the p63 signaling network. We propose that these genes act in a novel odontogenic network that is exclusive of lower jaw morphogenesis, and posit that this network evolved in oral, not pharyngeal, teeth.

Third-molar mineralization as a function of available retromolar space.

OBJECTIVE: To test in the maxilla and mandible for an association between stage of third-molar (M3) mineralization and space in the jaws for M3 eruption. Mineralization is hypothesized to be delayed not only for impacted M3s but also for M3s with eruption space less than their mesiodistal crown diameter. MATERIAL AND METHODS: Retrospective cone beam computed tomography (CBCT) scans of 37 females and 32 males aged 17-24 years, for a total sample of 197 upper and lower M3s, were used to assess the status of M3 eruption and measure the M3 crown diameter (CD) relative to the length of the retromolar space (RS). Stage of M3 mineralization was then compared between impacted and erupting M3s as well as between two conditions of relative eruption space (RS/CD ≥ 1 versus RS/CD < 1) using Mann-Whitney U tests. RESULTS: Impacted M3s were at significantly earlier (delayed) stages of mineralization compared to erupting M3s. Mineralization was also delayed for M3s with eruption space less than their mesiodistal crown diameter (e.g. RS/CD < 1). A moderate positive correlation between stage of M3 mineralization and space was seen in both jaws, and was stronger in the mandible. CONCLUSION: Our study shows for the first time that stage of M3 mineralization is associated not only with impaction but also with amount of retromolar space, and that these associations are consistent in upper and lower jaws. Present findings underscore that M3 mineralization stage may be a clinically useful predictor of M3 impaction that thus merits further investigation.

A multivariate approach to assess variation in tooth mineralization using free-lived and captive-raised chimpanzees (P. troglodytes).

OBJECTIVES: Understanding variation in dental development among primates is important to accurately characterize species-specific sequences and times of tooth formation. Conventional approaches that summarize dental development data (i.e., dental maturity score, DMS) inherently omit information about the full range of variation in raw scores; thus, classic bivariate analyses are limited for exploring patterns of variation in detail. Here we report a new multivariate approach to simultaneously assess all raw dental scores, for all teeth, among all individuals for all groups, thus retaining much greater detail about population-specific patterns of variation. METHODS: We scored (0-12) permanent tooth mineralization using radiographs of mandibles of captive-raised known-age chimpanzees (Pan troglodytes; n = 114) and free-lived age-unknown conspecifics (n = 54). As a test of our method we also scored free-lived baboons (Papio anubis, n = 50) because of well-described contrasting patterns of permanent molar initiation between Papio and Pan. Using principal component analysis (PCA), we investigated how crypt, crown and root formation scores covaried with each other in all three genera, and with chronological age in captive chimpanzees. RESULTS: PCA successfully captured additional detail about variation among raw scores. Also, compared to DMS, PC1 scores correlated equivalently well with known ages but had lower prediction error. CONCLUSIONS: We found different patterns of variation in scores between younger juvenile free and captive chimpanzees but saw no wholesale differences between groups. Pan and Papio showed different patterns of variation, further validating this multivariate approach to visualize, quantify and compare raw dental score datasets among primate species.

Food texture and vitamin D influence mouse mandible form and molar roots.

Industrialization influenced several facets of lifestyle, including softer nutrient-poor diets that contributed to vitamin D deficiency in post-industrzialized populations, with concomitantly increased dental problems. Here we simulated a post-industrialized diet in a mouse model to test the effects of diet texture and vitamin D level on mandible and third molar (M3) forms. Mice were raised on a soft diet with vitamin D (VitD) or without it (NoD), or on a hard diet with vitamin D. We hypothesized that a VitD/hard diet is optimal for normal mandible and tooth root form, as well as for timely M3 initiation. Subsets of adult NoD/soft and VitD/soft groups were bred to produce embryos that were micro-computed tomography (µCT) scanned to stage M3 development. M3 stage did not differ between embryos from mothers fed VitD and NoD diets, indicating that vitamin D does not affect timing of M3 onset. Sacrificed adult mice were µCT-scanned, their mandibles 3D-landmarked and M3 roots were measured. Principal component (PC) analysis described the largest proportion of mandible shape variance (PC1, 30.1%) related to diet texture, and nominal shape variance (PC2, 13.8%) related to vitamin D. Mice fed a soft diet had shorter, relatively narrower, and somewhat differently shaped mandibles that recapitulated findings in human populations. ANOVA and other multivariate tests found significantly wider M3 roots and larger root canals in mice fed a soft diet, with vitamin D having little effect. Altogether our experiments using a mouse model contribute new insights about how a post-industrial diet may influence human craniodental variation.

Jaw growth in the absence of teeth: the developmental morphology of edentulous mandibles using the p63 mouse mutant.

Mammalian tooth and jaw development must be coordinated well enough that these systems continue to function together properly throughout growth, thus optimizing an animal's survival and fitness, as well as a species' success. The persistent question is how teeth and jaws remain developmentally and functionally viable despite sometimes monumental evolutionary changes to tooth and jaw shape and size. Here we used the p63 mouse mutant to test the effect of tooth development - or the lack thereof - on normal mandible developmental morphology. Using 3D geometric morphometrics, we compared for the first time mandible shape among mice with normal tooth and jaw development against p63 double knock-out mice, with failed tooth development but apparently normal jaw development. Mandible shape differed statistically between toothless (p63(-/-) ) and toothed (p63(+/-) , p63(+/+) ) mice as early as embryonic day (E) 18. As expected, most of the shape difference in the p63(-/-) mandibles was due to underdeveloped alveolar bone related to arrested odontogenesis in these E18-aged mice. Mandible shape did not differ statistically between p63(+/-) and p63(+/+) adult mice, which showed normal tooth development. Our results support the idea of a gene regulatory network that is exclusive to the mandible and independent of the dentition. This study also underscores the biomechanical impact of the teeth on the developing alveolar bone. Importantly, this work shows quantitatively that the p63 mutant is an apt model with which to study mandible morphogenesis in isolation of odontogenesis to clarify developmental relationships between the teeth and jaws.

Quantifying the relationship between cell proliferation and morphology during development of the face.

Morphogenesis requires highly coordinated, complex interactions between cellular processes: proliferation, migration, and apoptosis, along with physical tissue interactions. How these cellular and tissue dynamics drive morphogenesis remains elusive. Three dimensional (3D) microscopic imaging poses great promise, and generates elegant images. However, generating even moderate through-put quantified images is challenging for many reasons. As a result, the association between morphogenesis and cellular processes in 3D developing tissues has not been fully explored. To address this critical gap, we have developed an imaging and image analysis pipeline to enable 3D quantification of cellular dynamics along with 3D morphology for the same individual embryo. Specifically, we focus on how 3D distribution of proliferation relates to morphogenesis during mouse facial development. Our method involves imaging with light-sheet microscopy, automated segmentation of cells and tissues using machine learning-based tools, and quantification of external morphology via geometric morphometrics. Applying this framework, we show that changes in proliferation are tightly correlated to changes in morphology over the course of facial morphogenesis. These analyses illustrate the potential of this pipeline to investigate mechanistic relationships between cellular dynamics and morphogenesis during embryonic development.

Permanent tooth mineralization in bonobos (Pan paniscus) and chimpanzees (P. troglodytes).

The timing of tooth mineralization in bonobos (Pan paniscus) is virtually uncharacterized. Analysis of these developmental features in bonobos and the possible differences with its sister species, the chimpanzee (P. troglodytes), is important to properly quantify the normal ranges of dental growth variation in closely related primate species. Understanding this variation among bonobo, chimpanzee and modern human dental development is necessary to better contextualize the life histories of extinct hominins. This study tests whether bonobos and chimpanzees are distinguished from each other by covariance among the relative timing and sequences of tooth crown initiation, mineralization, root extension, and completion. Using multivariate statistical analyses, we compared the relative timing of permanent tooth crypt formation, crown mineralization, and root extension between 34 P. paniscus and 80 P. troglodytes mandibles radiographed in lateral and occlusal views. Covariance among our 12 assigned dental scores failed to statistically distinguish between bonobos and chimpanzees. Rather than clustering by species, individuals clustered by age group (infant, younger or older juvenile, and adult). Dental scores covaried similarly between the incisors, as well as between both premolars. Conversely, covariance among dental scores distinguished the canine and each of the three molars not only from each other, but also from the rest of the anterior teeth. Our study showed no significant differences in the relative timing of permanent tooth crown and root formation between bonobos and chimpanzees.

Epigenetic integration of the developing brain and face.

The integration of the brain and face and to what extent this relationship constrains or enables evolutionary change in the craniofacial complex is an issue of long-standing interest in vertebrate evolution. To investigate brain-face integration, we studied the covariation between the forebrain and midface at gestational days 10-10.5 in four strains of laboratory mice. We found that phenotypic variation in the forebrain is highly correlated with that of the face during face formation such that variation in the size of the forebrain correlates with the degree of prognathism and orientation of the facial prominences. This suggests strongly that the integration of the brain and face is relevant to the etiology of midfacial malformations such as orofacial clefts. This axis of integration also has important implications for the evolutionary developmental biology of the mammalian craniofacial complex.

Timing of Mouse Molar Formation Is Independent of Jaw Length Including Retromolar Space.

For humans and other mammals to eat effectively, teeth must develop properly inside the jaw. Deciphering craniodental integration is central to explaining the timely formation of permanent molars, including third molars which are often impacted in humans, and to clarifying how teeth and jaws fit, function and evolve together. A factor long-posited to influence molar onset time is the jaw space available for each molar organ to form within. Here, we tested whether each successive molar initiates only after a minimum threshold of space is created via jaw growth. We used synchrotron-based micro-CT scanning to assess developing molars in situ within jaws of C57BL/6J mice aged E10 to P32, encompassing molar onset to emergence. We compared total jaw, retromolar and molar lengths, and molar onset times, between upper and lower jaws. Initiation time and developmental duration were comparable between molar upper and lower counterparts despite shorter, slower-growing retromolar space in the upper jaw, and despite size differences between upper and lower molars. Timing of molar formation appears unmoved by jaw length including space. Conditions within the dental lamina likely influence molar onset much more than surrounding jaw tissues. We theorize that molar initiation is contingent on sufficient surface area for the physical reorganization of dental epithelium and its invagination of underlying mesenchyme.

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

BACKGROUND: 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 (microCT) 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 microCT 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 microCT-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 microCT 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. RESULTS: 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 microCT 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. CONCLUSIONS: 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 microCT-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.

Fast interactive integration of cross-sectional image datasets and surface data for morphometric analysis.

To investigate external facial morphology and cell proliferation patterns and their relationship with cleft lip malformation in mice, we need to compare samples of mice tissue photographs and surface reconstructions from micro-CT scans obtained from mouse embryos. Tissue samples obtained through digital photography are typically misaligned with respect to each other, which prevents further analysis. We have developed a system for fast interactive alignment of these image stacks for volume reconstruction and data visualization and analysis in 3D. The system is designed to work in multiprocessor environments and can utilize an arbitrary number of processors, cutting down significantly the turnaround time and allowing users to quickly process sets of hundreds of high resolution images using a combination of automated and interactive tools. Additional modules are used to reconstruct the shape of the original subject. Our system is interactive, fully scalable and can be applied to any photographic sliced dataset, regardless of subject and reduces significantly the processing time for stack alignment.