Fgf8 haploinsufficency results in craniofacial asymmetries
Mandibular positioning relative to the midline was measured in aceti282a/+ adult zebrafish and their homozygous wild-type siblings. A significant difference was observed in the deviation of the mandibluar symphasis from the cranial midline in aceti282a/+ (n=52) and wild-type (n=45) zebrafish (F-ratio = 8.55, p = 0.0052). On average, deviation in aceti282a/+ zebrafish was twice as much as that observed in wild-type zebrafish (90 and 46 μm, respectively). Sidedness of mandibular asymmetry was determined by comparing heterozygous and homozygous phenotypes that fell outside the 95% confidence interval for each genotype. This comparison was used to control for the effects of extreme phenotypes. The average deviation from the midline was 9.2 μm to the left side (i.e., the left side was smaller than the right) in wild-type zebrafish (n=11), which was significantly different from the heterozygous phenotype where the average deviation of the mandibular symphasis was 128.8 μm to the left side (n=10) (F-ratio = 4.07, p = 0.05). Sidedness of mandibular asymmetry in aceti282a/+ zebrafish was confirmed using a Chi-square Goodness of Fit Test. Of 52 aceti282a/+ animals analyzed, 10 (19%) exhibited mandibular asymmetries outside the 95% confidence interval (). Eight of the 10 (80%) asymmetric aceti282a/+ animals presented mandibles skewed to the left side, which is significantly more than what would be expected by chance (X2 = 3.6, df = 1, p = 0.05). These data are consistent with mandibular asymmetry having a directional (R>L) bias in aceti282a/+ zebrafish.
Opercle asymmetry was measured by comparing left and right opercle centroid sizes (CS). A significant difference was observed in opercle asymmetry in aceti282a/+
(n=50) and homozygous wild-type (n=48) zebrafish (F-ratio = 3.72, p = 0.058). Sidedness of opercle asymmetry was examined by comparing heterozygous and homozygous asymmetries that fell outside the 95% confidence interval for each genotype. The difference between the left and right sides of the opercle was skewed slightly to the right (R>L) in wild-type animals (n=18, L-R = −0.05), which is significantly different from the left-side bias observed in aceti282a/+
animals (n=14, L-R = 0.230, F-ratio = 3.49, p = 0.071). Sidedness of opercle asymmetry in aceti282a/+
zebrafish was confirmed using a Chi-square Goodness of Fit Test. Of 50 aceti282a/+
animals analyzed, 14 (28%) exhibited opercle asymmetries that exceeded at least one 95% confidence interval. Of these, 11 presented cases where the left opercle was larger than the right opercle, which is significantly more than what would be expected by chance (X2
= 4.6, df = 1, p = 0.05 (). The frequency (e.g., 28%) and bias (e.g., L>R) of opercle size asymmetry in aceti282a/+
zebrafish is consistent with observations in 7 day post fertilization (dpf) homozygous recessive aceti282a
mutants (Albertson and Yelick, 2005
), where 28% exhibited pharyngeal bone asymmetries characterized by skeletal elements (typically the opercle) missing from one side of the pharynx. Sixty percent of asymmetric aceti282a
homozygous recessive mutants were missing bones from the right side of the head. Thus, whereas aceti282a
mutants exhibited an asymmetry in the presence/absence
of the opercle, aceti282a
heterozygous zebrafish exhibited an asymmetry in the size
of the opercle. Both asymmetries have a directional (L>R) bias. Notably, the sidedness of opercle and mandibular asymmetries was different in aceti282a/+
animals. Whether this observation speaks to an independent or inversely dependent origin of these defects is a question to be investigated in future studies.
Opercle defects in aceti282a/+
animals mainly affected the relative width of the element (). This defect, however, was only observed in a percentage of aceti282a/+
animals (16/50 = 32%) and was often (but not always) asymmetrically expressed. Irregular opercle shapes observed in aceti282a/+
zebrafish were distinct from those in aceti282a
homozygous recessive mutants (). Hyoid arch dermal bones in aceti282a
larvae were typically either missing, small or generally misshaped (n=194). A fraction (~10%) of mutants, however, exhibited more revealing bone phenotypes including fused dermal elements (), enlarged “opercle-gain” morphologies () (Kimmel et al., 2003
), and homeosis with both the opercle adopting the shape of the branchiostegal ray (), and the branchiostegal ray adopting the shape of the opercle (). These phenotypes are consistent with defects in DV patterning of the hyoid arch (Kimmel et al., 2003
). Furthermore these bone defects were only observed in aceti282a
homozygous recessive larvae - heterozygous larvae exhibited normal dermal bone shapes, suggesting separate roles for fgf8
during pre- and post-larval opercle development.
Aceti282a heterozygous zebrafish exhibit defects in cranial suturing and aberrant craniofacial geometry
Cranial suture patterns were examined in aceti282a/+ and homozygous wild-type animals. Specifically, we measured the deviation of interfrontal and sagittal sutures from the cranial midline. In wild-type zebrafish, neither the interfrontal or sagittal sutures deviated significantly from the cranial midline (n=25). In aceti282a/+ zebrafish, however, these sutures exhibited considerable divergence from the cranial midline (n=52) (). This difference in suture morphology was statically significant for both the interfrontal and sagittal sutures (F-ratio = 20.8, p <0.0001, and F-ratio = 8.36, p = 0.006, respectively). The coronal and lambdoid (transverse) sutures appeared to be unaffected in aceti282a/+ animals.
Aceti282a/+ zebrafish also exhibited aberrant craniofacial shape. Landmark-based GM analysis revealed significant differences in the length, height and overall geometry of the craniofacial skeleton between aceti282a/+ animals and their wild-type siblings. Fourteen landmarks on the oral jaws, suspensorium, pectoral girdle, and skull were used to describe craniofacial architecture (). A relative warp analysis was performed to identify major axes of shape variation. Aceti282a/+ zebrafish were significantly different from wild-type siblings along principal component (PC) axes 1 and 3 (F-ratio = 8.38, p = 0.006; F-ratio = 9.94, p = 0.003, respectfully), which collectively accounted for 37% of the total variation in craniofacial shape (). Heterozygous and wild-type animals did not differ in their loadings on PC 2, which explained 17% of the variance and mainly defined the rotation of the upper jaw relative to the skull. This variation was artificially induced due to varying degrees of jaw adduction among samples, and is therefore not biologically meaningful. Deformation of shape along the axis that discriminates aceti282a/+ and wild-type siblings revealed specific differences in the shape of the craniofacial complex. Of particular interest is the deviation in the shape of the frontal bone in aceti282a/+ zebrafish (purple triangle, ), which is expanded both in length and height relative to the posterior portion of the skull (green line, ). Aceti282a/+ zebrafish also exhibited concomitant changes in the configuration of the upper jaws (blue triangle, ), and in the relative height of the skull (yellow line, ).
Aceti282a heterozygous zebrafish exhibit aberrant bone formation
In addition to specific defects in craniofacial shape, aceti282a/+ zebrafish also exhibited more generalized bone defects, including irregular and ectopic bone formation. These defects seemed to be limited to the mandible (), upper jaws, and anterior region of the skull, but until a careful dissection of the articulated craniofacial skeleton is performed we cannot rule out the possibility that other bones might also be affected. The appearance of ectopic bone in aceti282a/+ animals is consistent with our previous observations of homozygous recessive aceti282a mutants, where ectopic bones and cartilages were observed around the developing mandible in approximately 30% of mutants (Albertson, unpublished data).
Figure 5 TRAP and AP activity is elevated in the jaws of aceti282a/+ zebrafish. A greater proportion of the lower jaw was TRAP positive in aceti282a/+ animals relative to homozygous wild-types siblings (A, B, F, F-ratio = 30.6, p < 0.001). Aceti282a/+ (more ...)
The intricate configuration of the oral jaw apparatus in bony fishes is constrained by the mechanical requirements of respiration and feeding. These actions impose forces on the craniofacial skeleton that serve to continuously shape and remodel the oral jaw apparatus for optimal performance as the animal grows. We therefore hypothesized that the bone defects observed in aceti282a/+ zebrafish might relate to the mechanisms that underlie bone growth and remodeling.
Aceti282a heterozygotes exhibit increased bone remodeling activity
Tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (AP) activities were assessed using a whole-mount procedures to identify global patterns of bone remodeling in adult zebrafish (). We observed increased levels of both TRAP (n=20) and AP (n=20) in the jaws of aceti282a/+ zebrafish relative to their homozygous wild-type siblings (n=20 each TRAP and AP). TRAP levels were ubiquitously elevated in the mandibles of aceti282a/+ animals (), whereas elevated AP levels were restricted to distal regions of the jaw (). Elevated levels of TRAP and AP observed in the jaws aceti282a/+ animals were consistent with the frequent appearance of irregular and ectopic bones (). TRAP and AP activities were, on average, two-fold greater in aceti282a/+ zebrafish than in wild-type zebrafish (-ratio = 30.6, p < 0.001, and G, F-ratio = 7.24, p = 0.01). Similar increases in TRAP and AP levels were noted for the skull (data not shown). These data suggest that both osteoclast and osteoblast activities are elevated in aceti282a/+ animals, consistent with fgf8 functioning as a negative regulator of bone turnover in zebrafish.
Zebrafish development is characterized by extensive allometric growth, making it an excellent model with which to study skeletal remodeling (Witten et al., 2001
). However, this attribute also means that skeletal development is particularly dynamic in zebrafish, characterized by shifts in the appearance and location of osteogenic cells depending on the size of the animal (Witten et al., 2001
; Albertson, unpublished data). It is also clear that Fgfs play dynamic roles during skeletal development (Colvin et al., 1996
; Deng et al., 1996
; Govindarajan and Overbeek, 2006
; Sobue et al., 2005
; Valta et al., 2006
; Yu et al., 2003
). In this study, we present a characterization of bone remodeling activities in adult aceti282a/+
zebrafish, providing insights into Fgf8 functions at this developmental stage. Additional ongoing studies of zebrafish at multiple developmental stages are currently being performed to define the roles of Fgf8 signaling in bone formation and remodeling throughout development.
Fgf8 is expressed in the operculum, oral jaws and cranial sutures
was expressed in discrete anatomical domains of the mature craniofacial skeleton. illustrates fgf8
expression in the head of a 6 month-old zebrafish. Expression was clearly seen in the upper jaw apparatus (black arrow, ), opercular apparatus (white arrow, ), cranial sutures (inset, ), and lower jaw (). Fgf8
expression around the upper jaws was associated with the premaxilla, maxilla, kinethmoid, and the ethmoid region of the skull. This region of the craniofacial skeleton is a functional “hot spot” comprised of an intricate configuration of bones and ligaments that participate in action of upper jaw protrusion (Hernandez, 2000
; Otten, 1983
). Forces associated with this action, and imposed on the upper jaws, are likely to play important roles in the growth and remodeling of this region. Fgf8
expression associated with the opercle was most pronounced at the location where the opercle articulates with the skull and along the ventral/proximal edge of the bone (arrow, ). Fgf8
was also expressed in the sagittal cranial suture (inset, ), and along the ventral surface of the mandible (). These expression patterns are consistent with defects observed in aceti282a/+
zebrafish, including upregulated osteoblast and osteoclast activities, suggesting a causal relationship between fgf8
deficiency and observed skeletal defects.
Figure 6 Fgf8 mRNA is expressed in distinct anatomical regions of the mature craniofacial skeleton. (A) A 6 month wild-type zebrafish head showing fgf8 expression in the upper jaw apparatus (black arrow), operculum (white arrow), and sagittal suture (inset). Scale (more ...)