Ontogenetic and functional modularity in the rodent mandible.

TL;DR: The research presented here highlights the importance of more naturalistic models of mammalian feeding, and underscores the need for a better understanding of the processes of both morphological and behavioral maturation that follow weaning.

About: This article is published in Zoology.The article was published on 2017-10-01 and is currently open access. It has received 20 citations till now.

Summary

1. Introduction

• The fundamental goal of functional morphology is to understand the diversity of morphological forms in light of their environmental and behavioral roles.
• Phenotypic plasticity refers to the ontogenetic modulation of a phenotype across an environmental gradient (Stearns, 1989; West-Eberhard, 1993 , 2005) and can function as a mechanism for the fine-tuning of form-function relationships across an individual's lifespan (Grant and Grant, 1989; Galis, 1996) .
• Indeed, the extent to which the growth of various morphological components within the mandible is correlated was addressed previously by Atchley et al. (1992) .
• In addition to two treatment groups representing the static homogenous ("annual") diets found in many previous studies, this work also included two variable diet cohorts that experienced a shift in dietary composition during their post-weaning growth period.

2.1. Experimental sample

• All procedures for this project were conducted in accordance with an IACUC-approved protocol.
• Body mass for all animals was measured at least twice weekly to monitor intra-and inter-cohort variation in growth and feeding performance.
• At the end of the experimental period, all animals were euthanized via inhalation of 100% CO2 from a compressed tank using a CO2 chamber.
• Animals were randomly sorted into four dietary treatment cohorts for the duration of the experimental period (Table 1 ).

2.2. Material properties of experimental foods

• The elastic, or Young's, modulus (E) is the stress/strain ratio at small deformations, characterizing the stiffness or resistance to elastic deformation (pellet E = 13.61 MPa).
• Due to the specifications of the food tester, it was possible only to measure the material properties of the whole pellets.
• The meal diet, comprised of ground pellets, primarily differs from whole pellets in the scale of the food particles.

2.3. 3D imaging and landmark digitization

• Between the ages of 4 and 16 weeks, all animals were imaged weekly using microcomputed tomography (μCT) to produce a longitudinal series of three-dimensional (3D) images of the craniofacial skeleton.
• The Siemens Micro-SPECT/CT unit was operated at 80 kV and 500 mA, with reconstruction using 0.126 mm 3 voxels.
• During imaging, animals were anesthetized via inhalation anesthesia using an isoflurane nonrebreathing anesthetic system at 3.0% per minute induction rate, and maintained at the 2.5-3.0% level for the duration of the scan.
• These weeks represent the beginning, middle, and end of the experimental period, respectively.
• 3D landmarks for the right hemimandible (Fig. 1 and Table 2 ) were collected for each ontogenetic point using the landmark placement plugin for eTDIPS (Mullick et al., 1999) .

2.4. Morphometric and statistical analyses

• A generalized Procrustes analysis (GPA) was performed on the right hemimandible landmark sets from each ontogenetic point using the Morphologika v2.5 software package (O'Higgins and Jones, 1998) .
• For all statistical tests involving multiple pairwise (inter-cohort) comparisons, relaxed Bonferroni-adjusted p-values were used (α = 0.017 or 0.05/3 pairwise comparisons per cohort) (Milne and O'Higgins, 2002) .
• CVA combines multiple shape variables to produce a small number of canonical variates (CVs) that maximize the differences among cohorts (Albrecht, 1980) .
• Visualizations of CVA results in MorphoJ use a reference shape representing a Procrustes distance value of zero (CV score of 0.0).
• The observations made from these visualizations were then quantitatively confirmed by the statistical comparison of Euclidean distances between the landmarks of interest (Mann-Whitney U-test, α = 0.05; pairwise comparisons using relaxed Bonferroni-adjusted α = 0.017) (Table 3 ).

3.1. Weaning (week 4)

• Preexisting variation in the size and shape of the right hemimandible was observed during week 4.
• The Procrustes distances between cohorts 1 and 2, and cohorts 1 and 4, were also significant, indicating significant differences in overall shape (Fig. 3 and Table S3 ).
• A CVA revealed that two axes best maximized the distance among the cohorts.
• Thus, at the time of weaning, cohort 1 was characterized by a relatively short mandible, short diastema, and a relatively long mandibular condyle.

3.2. Adolescence (week 10)

• Thus, there were two cohorts (1 and 2) that had been raised on a diet of solid pellets and two cohorts (3 and 4) that had been raised on a diet of powdered pellets/meal.
• A CVA revealed that a single CV axis best separated the cohorts by their dietary treatments.
• Those cohorts raised on pellets (1 and 2) tended to have wider articular processes, greater subcondylar angles formed by the intersection of the articular and angular processes, and shorter rows of cheek teeth.
• These characters were no longer observed to be significantly different among the cohorts in week 10.
• One character (ramus width) did distinguish cohort 1 from the remaining cohorts.

3.3. Adulthood (week 16)

• At adulthood, significant differences in hemimandibular centroid sizes were observed between the two seasonal cohorts.
• Furthermore, Procrustes distances revealed significant overall shape differences between the two annual cohorts (1 and 3), and between the two seasonal cohorts (2 and 4) (Fig. 3 and Table S3 ).
• Axis CV1 accounted for 57.3% of total variance and grouped the cohorts by their early diet, distinguishing cohorts 1 and 2 from cohorts 3 and 4.
• CV1 described differences in mandibular length and coronoid process morphology (Fig. 8 ).
• Individuals who consumed pellets late in life tended to have dorsoventrally taller (as measured by ramus height, RH) and rostrocaudally broader (as measured by coronoid process width, CrW, and ramus width, RW) mandibular rami.

4. Discussion

• The mammalian mandible can be broadly divided into two functional regions: the ramus, consisting of the articular process and multiple attachment sites for the major masticatory muscles, and the corpus, which supports the teeth (Atchley et al., 1992; Klingenberg et al., 2003a) .
• Significantly, the present study found that the osteogenic response to dietary variation differed between mandibular regions, but also among the modules within these regions.
• Here the authors show that the nature of morphological plasticity within these regions and modules is related to function, as well as to the ontogenetic timing of that function and the age of the experimental sample.

4.1. Regional differences in morphological plasticity

• Results from the present study suggest that the muscle hypertrophy and tooth growth models of mandibular morphogenesis (Atchley et al., 1992) in fact need not be exclusive, but that the relative importance of these models may be determined by the ontogenetic stage of the experimental sample.
• This schedule was confirmed for the Sprague Dawley rats included in the present study using μCT scans.
• Early life stages (weaning and adolescence) are thus characterized by tooth eruption, root growth, and immature periodontal sensory input.
• In individuals who have attained skeletal maturity, the muscle hypertrophy model explains the majority of morphological variation among the cohorts.

4.2. Modular differences in morphological plasticity

• As with prior studies of the rodent mandible, results from the present research are consistent with the existence of multiple modules within the ramus and corpus regions (Atchley et al., 1992; Klingenberg et al., 2003a; Anderson et al., 2014) .
• Mandibular size, as measured by ln-adjusted centroid size and mandibular length, tended to group individuals by their early diet, regardless of whether they were in the stable or variable diet cohorts.
• Similarly, the mandibular notch angle, which describes the orientation of the coronoid process relative to the articular process, was found to separate the cohorts on the basis of early diet.
• The delayed ability of these muscles to function in an adult-like manner may thus contribute to the extended growth period of their attachment sites.
• An increased ontogenetic resolution of the relative maturation rates of the masticatory muscles and their impact on masticatory kinematics is needed in order to better understand the nature of juvenile feeding behaviors and morphological plasticity.

4.3. Pre-existing morphological variation

• It is common procedure in experimental studies of phenotypic plasticity to use samples drawn from a similar genetic background, such as littermates, which are then randomly sorted into treatment groups.
• The assumption that pre-existing variation has thus been minimized or eliminated is not always validated in experimental studies.
• The present study found pre-existing morphological variation at the start of the experiment.
• Weanling animals in the annual over-use cohort were found to have shorter mandibles, shorter diastemata, and longer condyles than individuals in the remaining cohorts.
• S1 for cohort means and sample sizes, and Table S2 for pairwise p-values.

Fig. 4.

• CV1 maximizes the distance among cohorts using morphological variation pre-existing to the study, and describes differences in mandibular length, diastema length, and mandibular condyle length.
• Solid red lines indicate an increasing linear or angular dimension; dashed blue lines represent a decreasing linear or angular dimension.
• Cohorts with different superscript numbers are separated by the morphometric variable in CV space but are not significantly different (0.050 < p < 0.017).
• See Table 3 for variable abbreviations and 3 for variable abbreviations.

Figures

Table 1. Experimental design, including dietary treatment groups and the timing of dietary shifts.

Table 2. Landmarks of the right hemimandible used in the morphometric analyses. Landmarks are illustrated in Fig. 1.

Fig. 1. Landmarks of the right hemimandible. Top, 3D landmarks on a surface model of the rat mandible. Middle, landmarks connected to form a wireframe. Bottom, wireframe of the rat hemimandible used for morphometric analyses. See Table 2 for landmark details.

Table 3. Morphometric variables discussed in Section 3, calculated as the angle or Euclidian distance between landmarks.

Frequently asked questions

Q1. What have the authors contributed in "Ontogenetic and functional modularity in the rodent mandible" ?

Furthermore, the mandible is composed of multiple developmental and functional subunits, and the extent to which growth and plasticity among these modules is correlated may be misestimated by studies that examine non-variable masticatory function in adults only. To address these gaps in their current knowledge, this study raised Sprague Dawley rats ( n = 42 ) in four dietary cohorts from weaning to skeletal maturity. The other two cohorts were fed a variable ( “ seasonal ” ) diet consisting of solid/powdered pellets for the first half of the study, followed by a shift to the opposite diet. The research presented here highlights the importance of more naturalistic models of mammalian feeding, and underscores the need for a better understanding of the processes of both morphological and behavioral maturation that follow weaning. Furthermore, adult morphology is influenced by both masticatory function and the ontogenetic timing of this function, e. g., the consumption of a mechanically resistant diet. The morphology of the coronoid process was found to separate cohorts on the basis of their early weanling diet, suggesting that the coronoid process/temporalis muscle module may have an early plasticity window related to high growth rates during this life stage. 

Q2. What was the common method used to assess variation in hemimandible size?

In order to assess variation in hemimandible size, Kruskal–Wallis tests (α = 0.05) were used to statistically compare ln-transformed centroid sizes among cohorts for each longitudinal point. 

Q3. Why was weaning chosen as the starting point for the experimental period?

Weaning was chosen as the starting point for the experimental period because this approximates a shift in masticatory function in the wild and to minimize the confounding influences of postweaning diets other than those included in the present study. 

Q4. What is the goal of functional morphology?

The fundamental goal of functional morphology is to understand the diversity of morphological forms in light of their environmental and behavioral roles. 

Q5. What was used to assess the material properties of pellets?

A portable food tester (Darvell et al., 1996; Lucas et al., 2001) was used to assess the material properties of pellets (Wainwright et al., 1976; Vincent, 1992; Lucas, 1994; Currey, 2002). 

Q6. How was the euthanization of the animals?

At the end of the experimental period, all animals were euthanized via inhalation of 100% CO2 from a compressed tank using a CO2 chamber. 

Q7. What is the effect of the weanling animals on the morphological stability of the jaw?

Weanling animals in the annual over-use cohort were found to have shorter mandibles, shorter diastemata, and longer condyles than individuals in the remaining cohorts. 

Q8. What is the common procedure in experimental studies of phenotypic plasticity to use samples?

It is common procedure in experimental studies of phenotypic plasticity to use samples drawn from a similar genetic background, such as littermates, which are then randomly sorted into treatment groups. 

Q9. What is the morphological relationship between the mandible and the teeth?

The muscle hypertrophy model posits that muscle–bone interactions occurring in the mandibular ramus could drive morphological variation, while the tooth growth model suggests that variation is related to interactions between the teeth and the mandibular corpus. 

Q10. What is the morphology of the masticatory apparatus?

The skeletal morphology of the masticatory apparatus is the product of interactions between genetics, development, and multiple functional pressures (Atchley et al., 1992; Atchley, 1993). 

Q11. What was the morphological feature of the articular and angular processes?

At the adolescent stage, morphological features of the articular and angular processes were found to separate the cohorts on the basis of diet. 

Q12. What is the common morphological variation among the cohorts?

In individuals who have attained skeletal maturity, the muscle hypertrophy model explains the majority of morphological variation among the cohorts. 

Q13. What is the effect of the delayed ability of these muscles to function in an adult-like manner?

The delayed ability of these muscles to function in an adult-like manner may thus contribute to the extended growth period of their attachment sites. 

Q14. What is the importance of the ontogenetic resolution of the masticatory muscles?

An increased ontogenetic resolution of the relative maturation rates of the masticatory muscles and their impact on masticatory kinematics is needed in order to better understand the nature of juvenile feeding behaviors and morphological plasticity. 

Q15. What is the risk of a longitudinal approach to masticatory muscle development?

Without a longitudinal approach that characterizes pre-existing variation, there is a risk of incorrectly correlating this pre-existing variation with the experimental treatment(s) if said variation is observed only at later time points. 

Q16. What were the morphological variables described by the canonical variates?

The morphological variables described by the canonical variates were visually assessed using wireframe deformations and lollipop graphs. 

Q17. What is the nature of phenotypic plasticity in the masticatory apparatus?

the nature of phenotypic plasticity in the masticatory apparatus has important ramifications for feeding function and performance in mammalian taxa that experience ontogenetic changes in feeding behavior and/or inhabit variable environments. 

Q18. What is the morphological difference between the mandible and the corpus?

In sum, results from the present study are consistent with previous work which has suggested that the mandibular ramus, particularly the features related to muscle insertion sites and joint structures, is more plastic with respect to variation in feeding behavior than the mandibular corpus, which may be influenced by early growth processes and spatial factors (McFadden et al., 1986; Daegling, 1996; Taylor, 2002; Terhune, 2013). 

Q19. What is the purpose of phenotypic plasticity?

In recent years, phenotypic plasticity has been highlighted in the biological sciences for its potential to shed light on these form–function relationships.