Development beyond the embryo.
Forward genetic mutagenesis screens in zebrafish have revealed critical insights into the molecular signaling pathways that regulate early craniofacial patterning and development. The efficiency of this methodology, however, is also one of its greatest limitations. By targeting early embryonic phenotypes, these screens typically identify mutants characterized by gross, qualitative defects that are lethal soon after embryonic development. Most zebrafish craniofacial mutants, for example, die around 5 days post-fertilization (dpf), well before most of the bones of the pharyngeal skeleton begin to ossify.
The craniofacial skeleton of the adult zebrafish (right panel) is substantially more complex than that of the 5 day larvae (left panel, images not to scale). In stark contrast to the growing body of literature aimed at understanding development of the larval skeleton, significantly less is known about the molecular mechanisms that regulate adult form.
We are interested in development beyond embryonic stages. Specifically, we seek to understand the molecular signaling pathways that contribute to the coordinated formation, growth and remodeling of the skeletal system. We suggest that to better understand the mechanisms that regulate postembryonic skeletal development, traditional techniques used to study early zebrafish development should be bolstered by the application of new methodologies, including: (1) genetic screens targeting post larval phenotypes (i.e., mineralized tissues); (2) a shift from qualitative to quantitative characterizations of mutant phenotypes; and (3) a transition away from assessment of defects at isolated developmental stages, to the continuous monitoring of mutant phenotypes throughout development.
Morphometric shape analysis of craniofacial mutants.
Geometric morphometrics is a powerful tool used to define and describe variation in shape. A geometric approach to shape analysis is based on landmark data – homologous anatomical points recorded as a Cartesian coordinate (x,y) system. Instead of describing shape as a series of lengths, widths, or angles, landmark data emphasize the geometry of a structure, allowing shape variation to be described relative to other structures. Furthermore, results of geometric morphometric shape analyses can be reported as a pictorial representation of the organism or structure under investigation, providing an intuitive representation of biologically meaningful variation in shape.
We believe that the application of quantitative shape analyses to the study of zebrafish craniofacial mutants will facilitate an understanding of clinically relevant defects in craniofacial shape.
Landmarks used to define the craniofacial skeleton in the adult zebrafish (right panel), and biomedically relevant deformation in shape of the cranium in mutant zebrafish, characterized by and expanded frontal region of the skull (blue triangle, right panel), relative to wild type skull geometry (middle panel).
For example, craniosynostosis is a medical condition that involves the premature closure of the cranial sutures and occurs at a frquencey of 1 in 2500 live births. Affected individuals are characterized by various skull deformities including a bulging and malformed cranial vault. Mutations have been identified in both syndromic and non-syndromic cases of craniosynostosis, the majority of which occur in fibroblast growth factor receptors (Fgfrs). We find that zebrafish deficient in fgf8 exhibit skull deformities similar in presentation to individuals with craniosynostosis, including an expanded frontal region of the skull (above).
The identification of zebrafish mutants with defects that are consistent with human craniofacial syndromes will facilitate an understanding of the pathophysiology of these disorders.
Cytochemical analysis of craniofacial development.
Skeletal development in the zebrafish is regulated in much the same way as in other vertebrates, by balanced activities of bone forming cells (osteoblasts) and bone resorbing cells (osteoclasts). While descriptions of skeletal development have been reported for zebrafish (Bird and Mabee, 2003; Cubbage and Mabee, 1996), the molecular basis of this process is virtually unknown.
Images of TRAP and AP staining in the adult zebrafish. From left to right: TRAP activity was observed in osteoclasts cells (first panel from the left), as well as in bony structures subjected to continuous growth and remodeling including the scales (second panel), and fin rays (middle). AP staining of osteoblasts was observed at sites undergoing endochondral ossification (fin radials, forth panel) and intramembranous ossification (lateral line canal, last panel).
As a first step toward a mechanistic understanding of bone growth, we are assaying the activities of osteogenic cells over extended periods of development. Osteoclast and osteoblast cells each have a distinct chemical signature - tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (AP), respectfully. Whole-mount staining for TRAP and AP provides a generalized view of where and when osteoclast and osteoblast cells are active.
We are using this approach in conjuction with morphometric shape analyses (described above) to characterize patterns of bone growth and remodeling in wild type zebrafish. Once this reference is established, we'll be able to compare skeletal development in wild type and mutant zebrafish.
Specific areas of research:
If you are interested in participating in one or more of these projects please contact me: rcalbert@syr.edu
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