Preliminary Skull Characterization and Comparison
of C57BL/6J, C3H/HeSnJ, BALB/cByJ and
DBA/2J Inbred Mice.
Animal Core
The Jackson Laboratory
J.D. Bauschatz, V.E. Guido, C.C. Marden, M.T. Davisson, L.R. Donahue
Skulls were collected from six male and six female eight week old C57BL/6J, C3H/HeSnJ, BALB/cByJ and DBA/2J mice in order to establish craniofacial characteristics in these four common inbred strains. These strains were chosen based on the prevalence of craniofacial mutations on their backgrounds. During the collection process, ear pinnae were measured with digital calipers measuring in millimeters (Stoelting, Wood Dale, Ill), and a bone mineral density scan (PIXImus, LUNAR, Madison, WI) was obtained of the skull and body of each mouse. The skulls were then prepared by incomplete maceration in potassium hydroxide, stained with alizarin red, and stored in undiluted glycerin.
Seven measurements of the prepared skulls were then taken using the digital caliper. The measurements were as follows; total skull length, (tip of the nasal bone to back of the occipital bone) nose length, (nasal bone to coronal suture) skull height, (lowest point of the angular process of lower jaw to frontal bone) skull width, (measured at the widest points of the parietal bone) inner canthal distance, and length of both jaws. These measurements were selected from those used by Dr. Joan Richtsmeier to characterize craniofacial differences in mouse models of Down Syndrome (Richtsmeier et. al., 2000 Developmental Dynamics 217 (2):140.)
Table 1a: Digital Caliper Measurements of Eight Week Old Male and Female Skulls of Four Inbred Strains Stained with Alizarin Red
(Mean± SEM; n=12; 6 Male, 6 Female).
| C57BL/6J | C3H/HeSnJ | BALB/cByJ | DBA2/J | |
| Skull Length (mm) | 22.273±0.125bd | 22.103±0.193e | 21.733±0.097f | 21.152±0.185 |
| Nose Length (mm) | 14.804±0.093abd | 14.407±0.123c | 14.018±0.058 | 14.285±0.133 |
| Skull Height (mm) | 9.873±0.90bd | 9.855±0.145ce | 9.468±0.124f | 9.053±0.119 |
| Skull Width (mm) | 10.644±0.053abd | 10.876±0.061ce | 10.374±0.076f | 10.079±0.105 |
| Inner Canthal Distance (mm) | 4.182±0.039b | 4.170±0.027c | 3.962±0.033f | 4.123±0.096 |
| Lower Jaw Length (mm) | 11.014±0.165d | 10.843±0.13e | 10.992±0.148f | 10.222±0.126 |
| Upper Jaw Length (mm) | 15.161±0.173bd | 15.079±0.154e | 14.632±0.116 | 14.482±0.203 |
| Ear Pinna Length (mm) | 12.359±0.244ab | 13.123±0.179ce | 13.577±0.288* | 13.826±0.235 |
b: PIXImus Densitometric Measurements of Exvivo Skulls.
| Head BMD (g/cm2) | 0.099±0.01ad | 0.108±0.00e | 0.098±.0010f | 0.091±0.002 |
| Head BMC (g/cm2) | 0.198±0.003ad | 0.224±0.006ce | 0.186±0.003 | 0.173±0.006 |
a:p? 0.05 B6 vs. C3H. b:p?0.05 B6 vs. BALB. c:p?0.05 C3H vs. BALB. d:p<0.05 B6 vs. DBA e:p<0.05 C3H vs. DBA f:p<0.05 BALB vs. DBA *n=9 (6 females, 3 males)
The preceding tables show the results of eight hand caliper measurements along with the bone mineral density (BMD) and bone mineral content (BMC) of the skulls by strain. Skull width was the only measurement found to be significantly different between all four strains. The skulls of the C57BL/6J and C3H/HeSnJ mice were the most alike, a significant difference between the two strains being found in only five of ten measurements. The skulls of both C57BL/6J and C3H/HeSnJ were found to be the most different from DBA/2J skulls, being significantly different in eight of ten measurements. The C57BL/6J and C3H/HeSnJ inbred strains were both found to be significantly different from BALB/cByJ in seven of ten measurements. And BALB/cByJ was found to be significantly different from DBA/2J in six of ten measurements.
Two of our measurements, skull height and skull BMD showed significant difference in all intrastrain comparisons except one. The following graphs show those comparisons.
Table 2a: Digital Caliper Measurements of Eight Week Old Alizerin Red Stained Skulls of Four Inbred Strains Split by Sex (Mean± SEM; n=6)
| Measurement | Sex | C57BL/6J | C3H/HeSnJ | BALB/cByJ | DBA/2J |
| Skull Length (mm) | M | 22.447±0.084bd | 22.650±0.130ceg | 21.877±0.136 | 21.567±0.130g |
| F | 22.100±0.222d | 21.557±0.166e | 21.588±0.122f | 20.738±0.256 | |
| Nose Length (mm) | M | 14.863±0.129bd | 14.582±0.161c | 14.077±0.091f | 14.492±0.058 |
| F | 14.745±0.142abd | 14.232±0.168 | 13.960±0.071 | 14.078±0.241 | |
| Skull Height (mm) | M | 9.867±0.126abd | 10.253±0.128ceg | 9.495±0.094 | 9.357±0.116g |
| F | 9.880±0.140d | 9.457±0.111e | 9.442±0.243f | 8.750±0.107 | |
| Skull Width (mm) | M | 10.630±0.077ad | 11.042±0.050ceg | 10.460±0.109 | 10.293±0.108g |
| F | 10.658±0.080bd | 10.708±0.055ce | 10.288±0.104f | 9.865±0.134 | |
| Inner Canthal Distance (mm) | M | 4.198±0.058 | 4.210±0.037 | 4.008±0.060f | 4.312±0.130g |
| F | 4.165±0.058bd | 4.130±0.034ce | 3.917±0.017 | 3.935±0.095 | |
| Lower Jaw Length (mm) | M | 11.040±0.214d | 10.987±0.229 | 10.995±0.100 | 10.492±0.150g |
| F | 10.988±0.273d | 10.700±0.122e | 10.988±0.294f | 9.952±0.133 | |
| Upper Jaw Length (mm) | M | 15.243±0.238b | 15.497±0.110gce | 14.640±0.206 | 14.782±0.214 |
| F | 15.078±0.270d | 14.662±0.152 | 14.623±0.129 | 14.182±0.315 | |
| Ear Pinna Length (mm) | M | 12.957±0.211dg | 12.937±0.312e | 13.473±0.494* | 14.328±0.167g |
| F | 11.762±0.272abd | 13.308±0.171 | 13.628±0.386 | 13.323±0.337 |
b. PIXImus Densitometric Measurements of Exvivo Skulls
| Skull BMD (g/cm2) | M | 0.098±0.001ad | 0.110±0.001gce | 0.097±0.001 | 0.094±0.001 |
| F | 0.100±0.001ad | 0.106±0.001ce | 0.098±0.002f | 0.088±0.002 | |
| Skull BMC (g/cm2) | M | 0.195±0.003a | 0.238±0.007ceg | 0.183±0.004 | 0.187±0.005g |
| F | 0.202±0.006d | 0.210±0.007ce | 0.189±0.003f | 0.160±0.008 |
a:p?0.05 B6 vs. C3H within gender. b:p?0.05 B6 vs. BALB within gender. c:p?0.05 C3H vs. BALB within gender. d:p?0.05 B6 vs. DBA within gender. e:p?0.05 C3H vs. DBA within gender. f:p?0.05 BALB vs. DBA within gender. g:p?0.05 male vs. female within strain. *n=3.
Tables 2a&b indicate that no significant differences were found between males and females of the BALB/cByJ strain in any of ten measurements. The C57BL/6J strain showed a significant difference when split by sex only in ear pinna length. These results are quite different from C3H/HeSnJ and DBA/2J strains which showed significant differences between males and females in six and seven of ten measurements respectively. C57BL/6J and DBA/2J female skulls were found to be significantly different from each other in all ten measurements, whereas BALB/cByJ and DBA/2J male skulls only differed significantly from each other in two measurements.
The measurement found to be most variable when split by sex was male skull height, which was significantly different in five comparisons as shown below.
The measurements with the least difference between the strains when split by sex were female inner canthal distance, lower jaw length, and upper jaw length, each of which demonstrated significant difference in only one comparison. Nose length was the only measurement in which no significant difference was found between the sexes in any strain.
