An integrated genomics approach to identify genetic regions associated with neonatal growth trait in mice.

Neonatal growth during the early post-partum period is closely associated with lactation performance. Neonatal growth reflects milk output and is a complex variable trait among inbred mouse strains, but few studies have compared this trait systematically across more than a few strains. In the present study, 11 inbred strains of mice were measured for a neonatal growth phenotype during the first eight days of lactation. Significant differences in neonatal growth trait were observed with QSi5 (3.71±0.05 g) and DBA/1J (2.67±0.06 g) strains defining the two extremes of the phenotype. In silico association analysis was performed for trait variability using the high density SNP information on inbred strains of mice. We found strong evidence to refine a previously identified large neonatal growth QTL on mouse chromosome 9, Neogq1. When an integrated strategy that combined fine mapping and analysis of mammary transcriptome expression profiles of lactating mice with divergent phenotypes was applied, we identified neogenin (Neo1), a gene important for mammary gland morphogenesis, as a likely quantitative trait gene (QTG) underlying the Neogq1 QTL in mice.

Enhancing the value of psychiatric mouse models; differential expression of developmental behavioral and cognitive profiles in four inbred strains of mice.

The behavioral characterization of animal models of psychiatric disorders is often based upon independent traits measured at adult age. To model the neurodevelopmental aspects of psychiatric pathogenesis, we introduce a novel approach for a developmental behavioral analysis in mice. C57BL/6J (C57) mice were used as a reference strain and compared with 129S1/SvImJ (129Sv), BTBR T+tf/J (BTBR) and A/J (AJ) strains as marker strains for aberrant development. Mice were assessed at pre-adolescence (4 weeks), adolescence (6 weeks), early adulthood (8 weeks) and in adulthood (10-12 weeks) on a series of behavioral tasks measuring general health, neurological reflexes, locomotor activity, anxiety, short- and long-term memory and cognitive flexibility. Developmental delays in short-term object memory were associated with either a hypo-reactive profile in 129Sv mice or a hyper-reactive profile in BTBR mice. Furthermore, BTBR mice showed persistent high levels of repetitive grooming behavior during all developmental stages that was associated with the adult expression of cognitive rigidity. In addition, strain differences in development were observed in puberty onset, touch escape, and body position. These data showed that this longitudinal testing battery provides sufficient behavioral and cognitive resolution during different development stages and offers the opportunity to address the behavioral developmental trajectory in genetic mouse models for neurodevelopmental disorders. Furthermore, the data revealed that the assessment of multiple behavioral and cognitive domains at different developmental stages is critical to determine confounding factors (e.g., impaired motor behavior) that may interfere with the behavioral testing performance in mouse models for brain disorders.

Milk ejection in mice LG/J x SM/J.

In mammals, milk provision is crucial to offspring survival and growth from birth to weaning. Milk deficiency early in life may cause death or changes in the progeny metabolism that later may lead to obesity and metabolic disorders. This study investigates milk ejection (ME) the first day after birth (D1) in F(2) females from the intercross of LG/J and SM/J inbred mice strains. The absence of milk in F(3) pups' stomach at D1 is directly associated with their survival (p < 0.001) and growth pattern (p < 0.001) in the early stages of life. Furthermore, late growth pattern is also affected by this lack of nutrients at D1 because pups that survive this absence, mostly males, are heavier at weaning (p < 0.001) which, after necropsy, is shown to be due to significant higher total fat deposition (p < 0.01). We performed QTL analysis for ME at D1 in these F(2) females. Maternal performance of ME revealed a complex genetic architecture which even though it contains only a single QTL (accounting for 8 % of the variation in ME), it is totally context-dependent on the genetic background. We discovered many regions involved in epistatic interactions that together with the single QTL explain 19 % of the genetic variation for this trait. Milk ejection is an important component of maternal care, and understanding the mechanisms modulating its variation, along with other maternal features, may help to disentangle the complexity that is the mother/offspring relationship.

Developmental and genetic origins of murine long bone length variation.

If we wish to understand whether development influences the rate or direction of morphological evolution, we must first understand the developmental bases of morphological variation within species. However, quantitative variation in adult morphology is the product of molecular and cellular processes unfolding from embryonic development through juvenile growth to maturity. The Atchley-Hall model provides a useful framework for dissecting complex morphologies into their component parts as a way of determining which developmental processes contribute to variation in adult form. We have examined differences in postnatal allometry and the patterns of genetic correlation between age-specific traits for ten recombinant inbred strains of mice generated from an intercross of LG/J and SM/J. Long bone length is closely tied to body size, but variation in adult morphology is more closely tied to differences in growth rate between 3 and 5 weeks of age. These analyses show that variation generated during early development is overridden by variation generated later in life. To more precisely determine the cellular processes generating this variation we then examined the cellular dynamics of long bone growth plates at the time of maximum elongation rate differences in the parent strains. Our analyses revealed that variation in long bone length is the result of faster elongation rates of the LG/J stain. The developmental bases for these differences in growth rate involve the rate of cell division and chondrocyte hypertrophy in the growth plate.

Phenotypic integration of skeletal traits during growth buffers genetic variants affecting the slenderness of femora in inbred mouse strains.

Compensatory interactions among adult skeletal traits are critical for establishing strength but complicate the search for fracture susceptibility genes by allowing many genetic variants to exist in a population without loss of function. A better understanding of how these interactions arise during growth will provide new insight into genotype-phenotype relationships and the biological controls that establish skeletal strength. We tested the hypothesis that genetic variants affecting growth in width relative to growth in length (slenderness) are coordinated with movement of the inner bone surface and matrix mineralization to match stiffness with weight-bearing loads during postnatal growth. Midshaft femoral morphology and tissue-mineral density were quantified at ages of 1 day and at 4, 8, and 16 weeks for a panel of 20 female AXB/BXA recombinant inbred mouse strains. Path Analyses revealed significant compensatory interactions among outer-surface expansion rate, inner-surface expansion rate, and tissue-mineral density during postnatal growth, indicating that genetic variants affecting bone slenderness were buffered mechanically by the precise regulation of bone surface movements and matrix mineralization. Importantly, the covariation between morphology and mineralization resulted from a heritable constraint limiting the amount of tissue that could be used to construct a functional femur. The functional interactions during growth explained 56-99% of the variability in adult traits and mechanical properties. These functional interactions provide quantitative expectations of how genetic or environmental variants affecting one trait should be compensated by changes in other traits. Variants that impair this process or that cannot be fully compensated are expected to alter skeletal growth leading to underdesigned (weak) or overdesigned (bulky) structures.

Variations of eye size parameters among different strains of mice.

In the mouse, only a few genes have been definitively associated with a small-eye phenotype; the paired-box gene Pax6 and the gene coding for the microphthalmia-associated transcription factor (Mitf). Mutant alleles were recovered by crude phenotype screens and their effects on eye size are relatively large. This feature points to a bias during screening for eye-size mutants, selecting preferentially more severe phenotypes. An unbiased method determining eye-size parameters in an observer-independent, quantitative manner is expected to pick up variations in other genes, which will be confirmed as pathologic mutations in confirmation crosses. The present study used optical low coherent interferometry (OLCI) to compare the axial eye length, the cornea and lens thicknesses, and the anterior chamber depth in four common wild-type, laboratory inbred strains (C57BL/6J, C3HeB/FeJ, 129S2/SvPasCrl, and BALB/cByJ) between 4 and 15 weeks of age. There were no differences between left and right eyes; differences between the size parameters of males and females have been observed only in a few cases. An optimal screening age for OLCI measurements was defined as 11 weeks of age. At this age, we checked two other inbred strains (AKR/J and DBA/2NCrl) as well as CD-1 outbred mice. CD-1 mice have the largest axial length. The most impressive differences among inbred strains were, first, the anterior chamber depth, where the DBA mice have significantly lower values than the other strains. Second, the cornea in C3H mice is approximately 20% thicker than in the other inbred strains. Finally, wild-type intervals (mean +/- 3 SD) for axial length, anterior chamber depth, and cornea and lens thicknesses were calculated allowing a quick identification of pathologic outliers.

Growth and hepatic in vitro metabolism of ergotamine in mice divergently selected for response to endophyte toxicity.

This study investigated if genetic differences exhibited in endophyte-resistant and -susceptible mouse lines had persisted after 13 generations in which the integrity of lines was maintained yet selection ceased. Experimental groups were mouse lines fed an endophyte-free (E-) or -infected (E+) diet. The in vitro metabolism of the ergot alkaloid ergotamine in mouse liver microsomes was characterized by LC-MS/MS and compared between both lines before and after exposure to E+ feed. No difference in the average daily weight gain of pups between resistant and susceptible mice was observed on the E+ diet. Thus, for the weight gain selection criterion under study, the difference established between the two lines appears not to have persisted over the extended period of relaxed selection. Microsomal incubations produced nine predominate peaks in the HPLC assay. The peaks were confirmed by LC-MS/MS to be ergotamine, ergotamine epimer, monohydroxylated metabolites (M1, M2, M1e, M2e) and dihydroxylated metabolites (M3--5). A gender difference for metabolite formation was observed on the E- diet, in that females produced a greater amount of M1, M1e and M3--5 than males. When challenged with the E+ diet, mice showed differences in concentration of M3 for line (resistant > susceptible) and gender (female > male) and of M4 and M5 for gender (female > male). Gender differences in the metabolism of ergotamine have not been shown before in these lines of mice or other species used to study ergot alkaloid metabolism. This adds a potential source of variation in the susceptibility to fescue toxicity not explored previously and would be of value to investigate further.

Genetic variation and correlation of dietary response in an advanced intercross mouse line produced from two divergent growth lines.

Levels of human obesity have increased over the past 20 years worldwide, primarily due to changes in diet and activity levels. Although environmental changes are clearly responsible for the increasing prevalence of obesity, individuals may show genetic variation in their response to an obesogenic environment. Here, we measure genetic variation in response to a high-fat diet in a mouse model, an F16 Advanced Intercross Line derived from the cross of SM/J and LG/J inbred mouse strains. The experimental population was separated by sex and fed either a high-fat (42% of energy from fat) or low-fat (15% of energy from fat) diet. A number of phenotypic traits related to obesity and diabetes such as growth rate, glucose tolerance traits, organ weights and fat pad weights were collected and analysed in addition to serum levels of insulin, free fatty acids, cholesterol and triglycerides. Most traits are different between the sexes and between dietary treatments and for a few traits, including adult growth, fat pad weights, insulin and glucose tolerance, the dietary effect is stronger in one sex than the other. We find that fat pad weights, liver weight, serum insulin levels and adult growth rates are all phenotypically and genetically correlated with one another in both dietary treatments. Critically, these traits have relatively low genetic correlations across environments (average r =0.38). Dietary responses are also genetically correlated across these traits. We found substantial genetic variation in dietary response and low cross environment genetic correlations for traits aligned with adiposity. Therefore, genetic effects for these traits are different depending on the environment an animal is exposed to.

Regulatory variation at glypican-3 underlies a major growth QTL in mice.

The genetic basis of variation in complex traits remains poorly understood, and few genes underlying variation have been identified. Previous work identified a quantitative trait locus (QTL) responsible for much of the response to selection on growth in mice, effecting a change in body mass of approximately 20%. By fine-mapping, we have resolved the location of this QTL to a 660-kb region containing only two genes of known function, Gpc3 and Gpc4, and two other putative genes of unknown function. There are no non-synonymous polymorphisms in any of these genes, indicating that the QTL affects gene regulation. Mice carrying the high-growth QTL allele have approximately 15% lower Gpc3 mRNA expression in kidney and liver, whereas expression differences at Gpc4 are non-significant. Expression profiles of the two other genes within the region are inconsistent with a factor responsible for a general effect on growth. Polymorphisms in the 3' untranslated region of Gpc3 are strong candidates for the causal sequence variation. Gpc3 loss-of-function mutations in humans and mice cause overgrowth and developmental abnormalities. However, no deleterious side-effects were detected in our mice, indicating that genes involved in Mendelian diseases also contribute to complex trait variation. Furthermore, these findings show that small changes in gene expression can have substantial phenotypic effects.

Performance of mouse lines divergently selected for heat loss when exposed to different environmental temperatures. I. Reproductive performance, pup survival, and metabolic hormones.

Mouse populations differing in metabolic rate have been developed through selection for high (MH) and low (ML) heat loss, along with the unselected controls (MC). Objectives of the study were to compare the MH, ML, and MC lines for reproductive performance, pup survival, and metabolic hormones when reared at 12, 22, and 31 degrees C, and to search for line x environment interactions. Conception and litter size were recorded on the parent generation mice introduced to the environments at 11 wk of age and bred after a 3-wk acclimatization period. Survival of pups (preweaning to 3 wk; postweaning from 3 to 9 wk of age) was measured with continuous exposure in the designated environment from birth to the time of measurement. Corticosterone, triiodothyronine (T3), and thyroxine (T4) serum concentrations were measured on the parent generation after producing litters and on the pup generation at 9 wk. No line x environment interaction was detected for conception rate, preweaning mortality, postweaning survival, pup weaning weight, or body temperature. There were no differences in conception rate among lines and environments. Environments affected survival of pups, but there were no line differences. Rectal body temperatures were greater for MH than ML mice, and MC mice were intermediate; body temperature of mice did not differ among the environments. Lines differed significantly in litter size only in the 22 degrees C environment. No significant line differences were found for serum corticosterone or serum T3 or T4. Line x environment interaction was detected only for litter size and for serum corticosterone concentration in dams. Contrary to the other two lines, ML dam performance relative to MH and MC was not affected negatively by either of the thermal environments. Results from this study do not raise concern that selection to decrease maintenance requirements will produce livestock with any greater liability to cope and perform under an array of environmental temperatures.

Performance of mouse lines divergently selected for heat loss when exposed to different environmental temperatures. II. Feed intake, growth, fatness, and body organs.

Mouse populations differing in metabolic rate have been developed through selection for high (MH) and low (ML) heat loss, along with the unselected controls (MC). Objectives of the study were to compare the MH, ML, and MC lines for feed intake, growth, body fatness, and organ weights when reared at 12, 22, and 31 degrees C, and investigate potential line x environment interactions. Feed intake was recorded weekly from 3 to 9 wk of age, and BW at 3, 6, and 9 wk of age. Body fat percent and organ weights were measured at 9 wk of age. No line x environment interactions were detected for any of the traits measured. The MH mice consumed more feed than ML mice from 5 to 9 wk of age. Between 8 and 9 wk of age, MH mice consumed 13% more feed than the ML mice, but they were relatively leaner (14.45 vs. 16.32% body fat); MC mice were intermediate for both traits. Mice in the cold environment consumed the greatest amount of feed, and those in the hot environment consumed the least. Males consumed more feed than females, and the difference was greater in the cold than in the hot environment. No differences in BW were found between the lines. Mice in the 22 degrees C environment were heavier than their age-matched counterparts in the other two environments, and males were heavier than females at all ages. Relative to BW, the three lines had similar tail length, body length, and liver weight. Mice in the cold environment had heavier spleens and livers than those in the hot environment but relatively shorter bodies and tails; the normal environment was intermediate for these traits. Results from this study indicate that selection to decrease maintenance requirements did not produce mice with any less ability to grow and perform under an array of environmental temperatures.

A large-sample QTL study in mice: I. Growth.

By use of long-term selection lines for high and low growth, a large-sample (n = approximately 1,000 F2) experiment was conducted in mice to further understand the genetic architecture of complex polygenic traits. In combination with previous work, we conclude that QTL analysis has reinforced classic polygenic paradigms put in place prior to molecular analysis. Composite interval mapping revealed large numbers of QTL for growth traits with an exponential distribution of magnitudes of effects and validated theoretical expectations regarding gene action. Of particular significance, large effects were detected on Chromosome (Chr) 2. Regions on Chrs 1, 3, 6, 10, 11, and 17 also harbor loci with significant contributions to phenotypic variation for growth. Despite the large sample size, average confidence intervals of approximately 20 cM exhibit the poor resolution for initial estimates of QTL location. Analysis with genome-wide and chromosomal polygenic models revealed that, under certain assumptions, large fractions of the genome may contribute little to phenotypic variation for growth. Only a few epistatic interactions among detected QTL, little statistical support for gender-specific QTL, and significant age effects on genetic architecture were other primary observations from this study.

A large-sample QTL study in mice: II. Body composition.

Using lines of mice having undergone long-term selection for high and low growth, a large-sample (n = approximately 1,000 F2) experiment was conducted to gain further understanding of the genetic architecture of complex polygenic traits. Composite interval mapping on data from male F2 mice (n = 552) detected 50 QTL on 15 chromosomes impacting weights of various organ and adipose subcomponents of growth, including heart, liver, kidney, spleen, testis, and subcutaneous and epididymal fat depots. Nearly all aggregate growth QTL could be interpreted in terms of the organ and fat subcomponents measured. More than 25% of QTL detected map to MMU2, accentuating the relevance of this chromosome to growth and fatness in the context of this cross. Regions of MMU7, 15, and 17 also emerged as important obesity "hot-spots." Average degrees of directional dominance are close to additivity, matching expectations for body composition traits. A strong QTL congruency is evident among heart, liver, kidney, and spleen weights. Liver and testis are organs whose genetic architectures are, respectively, most and least aligned with that for aggregate body weight. In this study, growth and body weight are interpreted in terms of organ subcomponents underlying the macro aggregate traits, and anchored on the corresponding genomic locations.

Phenotype of cloned mice: development, behavior, and physiology.

Cloning technology has potential to be a valuable tool in basic research, clinical medicine, and agriculture. However, it is critical to determine the consequences of this technique in resulting offspring before widespread use of the technology. Mammalian cloning using somatic cells was first demonstrated in sheep in 1997 and since then has been extended to a number of other species. We examined development, behavior, physiology, and longevity in B6C3F1 female mice cloned from adult cumulus cells. Control mice were naturally fertilized embryos subjected to the same in vitro manipulation and culture conditions as clone embryos. Clones attained developmental milestones similar to controls. Activity level, motor ability and coordination, and learning and memory skills of cloned mice were comparable with controls. Interestingly, clones gained more body weight than controls during adulthood. Increased body weight was attributable to higher body fat and was associated with hyperleptinemia and hyperinsulinemia indicating that cloned mice are obese. Cloned mice were not hyperphagic as adults and had hypersensitive leptin and melanocortin signaling systems. Longevity of cloned mice was comparable with that reported by the National Institute on Aging and the causes of death were typical for this strain of mouse. These studies represent the first comprehensive set of data to characterize cloned mice and provide critical information about the long-term effects of somatic cell cloning.

BMP mRNA and protein expression in the developing mouse olfactory system.

The bone morphogenetic proteins (BMPs) play fundamental roles during the organization of the central nervous system. The presence of these proteins has also been demonstrated in regions of the adult brain that are characterized by neural plasticity. In this study, we examined the expression of BMP4, 6, and 7 mRNAs and proteins in the murine olfactory system. The olfactory system is a useful model for studying cell proliferation and neural differentiation because both of these processes persist throughout life in the olfactory epithelium (OE) and olfactory bulb (OB). Our results demonstrate a differential expression of BMP4, 6, and 7 in the embryonic, postnatal, and adult olfactory system. In particular, BMP4 and BMP7 showed similar immunostaining patterns, being expressed in the olfactory region from the earliest stages studied (embryonic day 15.5) to adulthood. During development BMPs were expressed in the OE, olfactory bulb nerve layer, glomerular layer (GL), mitral cell layer (MCL), and subventricular zone. During the first postnatal week of life, BMP4 and 7 immunoreactivity (-ir) was particularly evident in the GL, MCL, and in the subependymal layer (SEL), which originates postnatally from the subventricular zone. In adults, BMP4 and 7 immunostaining was present in the GL and SEL. Within the SEL, BMP4 and 7 proteins were expressed primarily in association with the astrocytic glial compartment. BMP6-ir was always found in mature olfactory receptor neurons and their axonal projections to the OB. In summary, these data support the hypothesis that BMPs play a role in the morphogenesis of the olfactory system during development and in its plasticity during adulthood.

Facial nerve lesion response; strain differences but no involvement of IFN-gamma, STAT4 or STAT6.

Facial nerve lesions lead to a retrograde response characterized by activation of glia surrounding axotomized motoneurons and up-regulation of immunological cell surface molecules such as major histocompatibility complex (MHC) antigens. Cytokines, in particular interferon-gamma, are potent inducers of MHC expression and glial activation. We have here tested whether axotomy-induced activation is changed in transgenic mouse strains lacking components of the IFN-gamma signaling pathway, STAT4 or STAT6. No differences regarding astrocyte activation, ss2-microglobulin or MHC class I expression were discernible as compared to wild type controls. In contrast, there were conspicuous differences in the reaction between the examined wild type strains (C57BL/6J, BALB/c and 129/SvJ), suggesting considerable polymorphisms in the genetic regulation of these events, however, not involving IFN-gamma, STAT4 or STAT6.

Contribution of maternal effect QTL to genetic architecture of early growth in mice.

Existing approaches to characterizing quantitative trait loci (QTL) utilize a paradigm explicitly focused on the direct effects of genes, where phenotypic variation among individuals is mapped onto genetic variation of those individuals. For many characters, however, the genotype of the mother via its maternal effect accounts for a considerable portion of the genetically based variation in progeny phenotypes. Thus the focus on direct effect QTL may result in an insufficient or misleading characterization of genetic architecture due to the omission of the potentially important source of genetic variance contributed by maternal effects. We analyze the relative contribution of direct and maternal effect (ME) QTL to early growth in mice using a three-generation intercross of the Small (SM/J) and Large (LG/J) inbred mouse lineages. Using interval mapping and composite interval mapping, direct effect (DE) QTL for early growth (change in body mass during the interval from week 1 to 2) were detected in the F(2) generation of the intercross (n = 510), where no maternal genetic effect variance is present (all individuals are progeny of genetically identical F(1) mothers). ME QTL were detected by treating the phenotypes of cross-fostered F(3) pups as a characteristic of their nurse-dam (n = 168 dams with cross-fostered progeny). Five DE QTL, significant at a chromosome wide level (alpha = 0.05), were detected, with two significant at a genome wide level. FourME QTL significant at the chromosome wide level were detected, with three significant at the genome wide level. A model containing only DE QTL accounted for 11.8% of phenotypic variance, while a model containing only ME QTL accounted for 31.5% of the among litter variance in growth. There was no evidence for pleiotropy of DE and ME loci since there was no overlap between loci detected in these two analyses. Epistasis between all pairs of loci was analyzed for both DEs and MEs. Ten pairs of loci showed significant epistasis for MEs (alpha = 0.05 corrected for multiple comparisons) while four pairs showed significant epistasis for DEs on early growth.

Genetic variability in forced and voluntary endurance exercise performance in seven inbred mouse strains.

The goal of this study was to characterize the genetic contribution to both forced and voluntary exercise performance and to determine whether performance in these two paradigms is controlled by similar genetic influences. There were marked strain differences in treadmill exercise performance, with Swiss Webster (SW) and FVB/NJ mice showing elevated performance and C57BL/6J animals showing decreased performance compared with all other strains. There was no apparent relationship between treadmill performance and voluntary wheel performance, with the exception of SW mice, which demonstrated high performances on both the treadmill and the voluntary wheel. Numerous properties were measured to attempt to understand the basis for these differences in exercise performance. DBA/1J and SW mice exhibited significantly greater cardiac contractility than all other analyzed strains. Conversely, BALB/cByJ mice exhibited significantly reduced cardiac contractility compared with all other strains. Expression of molecular indicators of hypertrophy (atrial natriuretic factor and beta-myosin heavy chain) was significantly elevated in DBA/2J myocardium compared with all other analyzed strains.

Inbred lines of mice derived from long-term growth selected lines: unique resources for mapping growth genes.

Lines of mice selected for many generations for high or low growth in several laboratories around the world have been collected, and from these, inbred lines are being developed by recurrent full-sib mating in Edinburgh. There are seven high selected lines and four low lines (each low line is from the same base population as one of the high lines), and the histories of each are summarized. Mean body weight of males at 70 days of age in the Edinburgh laboratory in the heaviest inbred line (77 g) is 4.8-fold higher than in the lightest line (16 g), and 1.9-fold higher than in the least extreme high line (41 g). Litter size, food intake, and fat content also differ substantially. These inbred extreme selected lines are a uniquely valuable resource for QTL or gene mapping, candidate gene identification, and elucidation of epistatic effects.

Altered timing of the extracellular-matrix-mediated epithelial-mesenchymal interaction that initiates mandibular skeletogenesis in three inbred strains of mice: development, heterochrony, and evolutionary change in morphology.

Subtle changes in embryonic development are a source of significant morphological alterations during evolution. The mammalian mandibular skeleton, which originates from the cranial neural crest, is a complex structure comprising several components that interact late in embryogenesis to produce a single functional unit. It provides a model system in which individual developmental events at the basis of population-level evolutionary change can be investigated experimentally. Inbred mouse strains exhibit obvious morphological differences despite the relatively short time since their divergence from one another. Some of these differences can be traced to small changes in the timing of early developmental events such as the formation of the cellular condensations that initiate skeletogenesis. This paper examines an even earlier event for changes in timing, the epithelial-mesenchymal interaction(s) required to initiate chondrogenesis of Meckel's cartilage and osteogenesis of the dentary bone. Using three inbred strains of mice (CBA, C3H and C57) we found that, within each strain, cartilage and bone are induced at the same time and by the same (mandibular) epithelium, that chondrogenesis and osteogenesis are initiated by a matrix-mediated epithelial-mesenchymal interaction, and that timing of the interactions differs among the three inbred strains. These results are discussed with respect to the possible molecular basis of such temporal shifts in inductive interactions and how such studies can be used to shed light on heterochrony as a mechanism of evolutionary change in morphology.