Institute of Neuroscience Faculty

 

What do skeleton-forming cells do, specifically, when they make cartilages and bones of specific shapes and sizes? What do the cells do differently when they make cartilages and bones of different shapes and sizes?

During development a skeletal element reshapes as it develops and grows to larger size (Fig. 1). And vertebrate morphological evolution is fundamentally based on reprogramming development to yield elements of novel shapes and sizes. Defects in development, such as may be due to perturbations in the genome or to environmental insults, yield malformed cartilages and bones, as in cleft palate.

The underlying cellular and molecular-genetic bases of developmental, evolutionary, and disruptive change in the shapes and sizes of cartilages and bones are poorly understood, and to learn more about developmental cell behaviors underlying morphological change, our lab uses a model-system approach.

Fig. 1. Live confocal imaging of the opercle bone (OP, matrix red, green cells expressing GPF) in 3 and 4 dpf zebrafish larvae.

We take a genetic focus to investigate the neural crest-derived head skeleton development in two species of teleost fishes, the zebrafish Danio rerio, and the threespine stickleback Gasterosteus aculeatus. In both, the first craniofacial elements arise from small populations of mesenchymal cells, some of these populations positioned where we can observe them directly in the living and intact organism during early morphogenesis (Fig. 1).

A general hypothesis that guides much of our work is that the key cellular determinant of skeletal shaping is the patterning of arrangements of skeleton forming cells, chondroblasts and osteoblasts. Reshaping involves cellular rearrangement. By this hypothesis, how genes control cell arrangement becomes a central issue in our understanding of skeletal morphology.

The Endothelin-1 (Edn1) pathway in pharyngeal patterning

With zebrafish we use forward genetics, mutagenesis and mutant screens, to initially identify important genes in skeletal morphogenesis and to lead us toward learning the roles of these genes. This approach led us to investigation of an upstream genetic pathway regulated by a secreted signaling peptide, Edn1, that is a critical molecular regulator dorsal-ventral patterning and sizing of pharyngeal skeletal elements of the jaw and opercular (gill cover) regions. (refs [1,5,6,12,14]).

Edn1 might act as an upstream morphogen, specifying identity of ventral elements and is particularly important in the control of an “intermediate” domain along the dorsal-ventral axis. This intermediate domain is where articulating joints are made (e.g. between the upper and lower jaws), and is currently is being studied in the lab by Jared Talbot and Mary Swartz.

For example, in the End1 pathway mutant “hoover” joints are lost, effectively turning two hyoid arch cartilages into one larger cartilage, and two bones developing near the joint into one larger bone (Fig. 2). Joint loss might involve ectopic cartilage formation where the joint would normally be made, and bone fusion might involve ectopic bone formation between where the bones are normally made, as we are investigating.

The hoover gene functions in neural-crest derived cells and we hypothesize that hoover is negatively regulating the intermediate domain specification of chrondroblasts and osteoblasts. The function of hoover in the intermediate domain is itself negatively regulated by edn1. We are currently using time-lapse analyses to learn how the skeletal defects arise.

Fig. 2. Cartilages (blue) and bone (red) are reconfigured in a zebrafish mutant (6 dpf, arrow: joint region, *: bone-free region). Images by Mary Swartz.

Hedgehog signaling in palatogenesis

The intercellular signaling protein sonic hedgehog (Shh) regulates formation of the neural crest-derived palatal skeleton that forms between the oral cavity and the forebrain in the late embryo. Johann Eberhart discovered that Shh control is surprisingly indirect: Secreted by the ventral brain long before migration of neural crest from the dorsal brain, Shh acts not on the crest, but on oral ectoderm, toward which then neural crest eventually migrates [11]. Then, when neural crest migrates into the neighborhood of the oral ectoderm a signaling dialog is set up between these two tissues.

Aspects of the reciprocal signaling depends on the early Shh ‘priming’ event, and on function of “space case” a gene we identified in our mutant screens, and which mosaic analyses show to function in the neural crest. The space case mutant is missing midline palatal cartilage, yielding a zebrafish version of cleft palate(Fig. 3).

Fig. 3. A zebrafish model of cleft palate, 6 dpf. Images by Johann Eberhart.

 

The replacement of cartilage with bone

Upstream signaling molecules such as Edn1 and Shh act to pattern the skeleton in part through regulation of factors that control the histogenesis of cartilage and bone, including Sox9 and Runx2, transcription factors critical for chondroblast and osteoblast identity.

To learn more about such specifications of skeletal identity, Brian Eames, in collaboration with investigators in John Postlethwait’s lab, has begun a study of the developmental program of so-called “endochondral ossification” in which the early head cartilages are eventually all replaced with bone. This replacement begins in the young larva (evident in Fig. 1A and B as patches of red staining over both dorsal and ventral cartilages).

Our genetic skeletal screens, using staining as in Fig. 1, have yielded a set of mutants in which the replacement of cartilage by bone occurs precociously. Brian’s genetic studies, including mapping and complementation, show that these mutants represent at least 4 genes. Discovery and characterizations of the pathway(s) along which these genes function provides for exciting future studies.

Evo devo of facial bone morphology

Our zebrafish studies provide candidate genes important for evolution of skeletal morphologies [2,13].
In collaboration with Bill Cresko’s lab (Center of Ecology and Evolutionary Biology) we are investigating morphological changes in some of these same bones when Alaskan threespine sticklebacks evolve from oceanic (ancestral) forms to derived freshwater residents in isolated lakes.

A wonderful advantage for studying the evolution of development with sticklebacks is that the ancestral (oceanic) populations can be gathered, grown in the lab, and studied as well as the evolutionarily derived populations (lacustrine). Furthermore, we can cross the two forms and obtain fully fertile offspring – hence genetic analyses are possible.

Notably, the opercle bone undergoes prominent reshaping in evolving sticklebacks (Fig. 4), and developmental study shows that the two morphs are distinguishable in newly hatched larva, about a week after fertilization, when bone formation is just beginning to get underway [9]. In this system, therefore, we can study changes along the entire developmental trajectory of OP development. The variation is inherited as a quantitative trait, and we have begun genetic mapping to begin to parse out loci that control phases of the developmental trajectory, and to molecularly identify the genes

 

Lab citations

1.Miller, C.T., T.F. Schilling, K.-H. Lee, J. Parker, and C.B. Kimmel (2000) sucker encodes a zebrafish Endothelin-1 required for ventral pharyngeal arch development. Development 127, 3815-3828.

2.Kimmel, C.B., C.T. Miller, and R.D. Keynes (2001) Neural crest patterning and the evolution of the jaw. J. Anat. 199, 105-120.

3.Kimmel, C.B., C.T. Miller, and C.B. Moens (2001) Specification and morphogenesis of the zebrafish larval head skeleton. Dev. Biol. 233, 239-257.

4.Glickman N.S., Kimmel C.B., Jones M.A., and R.J. Adams.(2003) Shaping the zebrafish notochord. Development 130, 873-887.

5.Kimmel, C.B., B. Ullmann, M. Walker, C.T. Miller, and J.G. Crump (2003) Endothelin 1-mediated regulation of pharyngeal bone development in zebrafish. Development 130, 1339-1351.

6.Miller, C.T., D. Yelon, D.Y.R. Stainier, and C.B. Kimmel (2003) Two endothelin1 effectors, hand2 and bapx1, pattern ventral pharyngeal cartilage and the jaw joint. Development 130, 1353-1365.

7.Crump J.G., M.E. Swartz and C.B. Kimmel (2004) An integrin-dependent role of pouch endoderm in hyoid cartilage development. PLoS Biol. 2:244.

8.Crump, J. G., L. Maves, N. Lawson, B. Weinstein and C.B. Kimmel (2004) An essential role for Fgfs in endodermal pouch formation influences later craniofacial skeletal patterning. Development 131: 5703-5716.

9.Kimmel, C.B., B. Ullmann, C. Walker, C. Wilson, M. Currey, P.C. Phillips, M.A. Bell, J.H. Postlethwait and W.A. Cresko (2005) Evolution and development of facial bone morphology in threespine sticklebacks. PNAS. 102, 579-586.

10.Crump, J., Swartz, M., Eberhart, J. K. and Kimmel, C.B. (2006). moz-dependent hox expression controls segment-specific fate maps of skeletal precursors in the face. Development 133: 2661-2669

11.Eberhart, J. K., M.E. Swartz, J.G. Crump and C.B Kimmel (2006). Early Hedgehog signaling from neural to oral epithelia organizes anterior craniofacial development. Development 133, 1069-1077.

12.Walker, M.B., Miller, C.T., Talbot, J.C., Stock, D.W., Kimmel, C.B. (2006) Zebrafish furin mutants reveal intricacies in regulating Endothelin1 signaling in craniofacial patterning. Dev. Biology 295, 194-205.

13.Kimmel, C.B., Walker, M.B., and Miller, C.T. (2007) Morphing the Hyomandibular Skeleton in development and Evolution. J. Exp. Zoology Part B: Mol. & Dev. Evo. in press.

14.Walker, M.B., Miller, C.T., Kimmel, C.B. (2007) phospholipase C, beta 3 is required for Endothelin1 regulation of pharyngeal patterning in zebrafish. Dev. Biol. in press.

Full Publications List

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• Department of Biology
• Department of Psychology
• Department of Human Physiology