Cichlids, one of the most species-rich family of vertebrates!
Fig. 1 Skeletal image of the jaw of a Cichlid fish at 21 days post-fertilization (Albertson et. al 2005).
Q1. Why study the morphogenesis of jaw development in Cichlids?
In respect to studying the jaw and other factors that influence craniofacial diversity, Cichlids serve as an excellent model organism as its feeding apparatus has undergone extensive changes in evolution (Kocher 2006). The study is significant in regards to the craniofacial abnormalities that affect several human syndromes, such as TMJ and clefts. The specialized craniofacial shape of cichlids are most apparent during its early embryonic development stages. Furthermore, there is not much known about the mechanisms through which the jaw is shaped and through studies, we are able to observe factors involved in regulating variation in the particular jaw shape.
Introduction to Vertebrate Jaw Development:
Cichlids’ jaw development has evolved in response to strong, divergent selection (Kocher 2006). Early in their developmental stage, adaptive variation has been observed in the jaw shape and is found to be closely associated with bone morphogenetic protein 4 (Bmp4) expression. Bmp4, along with calmodulin (CaM) play a rather essential role in craniofacial development and diversity.
Additionally, the cranial neural crest cells (CNCs) contribute significantly to the development of the craniofacial skeleton. Unlike sensory cells and nerves, CNCs are able to form skeletogenic tissues derived from the forebrain, midbrain, and hindbrain. CNCs consists of two regions: anterior and posterior domains. In the anterior region, anterior cells are progenitors for the face and are distinguished from posterior CNCs by their lack of Hox gene expression. In particular, work on cichlids has been focused on various morphological aspects, such as the jaw and teeth, both derived from the neural crest (Kocher 2006).
Fig. 2 The differences in the Cranial Neural Crest cells (CNCs) reveal possible differences in adult feeding morphology (Kocher 2006).
Q2. How was the experiment conducted?
In a particular study carried out by Parsons and Albertson, they utilized a Quantitative trait locus (QTL) analysis, which is a statistical search for loci that is associated with the variation of a quantitative trait. Essentially, this method reveals the genetic basis of variation in complex traits by examining both phenotypic and genetic aspects through traits and molecular markers, respectively (Miles and Wayne 2008). They examined two different strains, Labeotropheus fuelleborni (LF) and the Metriaclima zebra (MZ), as the analysis requires two or more strains of organisms that differ genetically with regard to the trait of interest in order to examine significant effects.
By utilizing in situ hybridization, they then assessed the extent of bmp4 in regulating functional shape differences by manipulating levels of bmp4 mRNA during embryonic development in a cross between the robust-jawed LF and the relatively slender-jawed MZ. These two particular species differ greatly in their oral jaw morphology, which in turn, will reflect their alternate modes of feeding. In situ hybridization was an invaluable tool in examining the particular gene expression and the involvement of bmp4 in craniofacial development.
Parsons and Albertson continued to apply both whole mount in situ hybridization and QTL mapping to the same LF x MZ cross to analyze the role of CaM in craniofacial development. Specifically, to examine CaM’s expression during jaw morphogenesis, they cloned cichlid CaM and designed in situ hybridization probes (Parsons and Albertson 2009).
Q3. What were the Results Gathered from the Observation?
Functional Roles of Bmp4 and CaM in the Expression of Jaw Development:
The growth factor, Bmp4, is known to regulate embryonic cartilage development and shows restricted patterns of expression during vertebrate (in this case, Cichlid) jaw morphogenesis.
- Member of the TGF-β superfamily whose proteins are involved in embryogenesis and cell differentiation.
- Essential in mesoderm formation, tooth formation, limb morphogenesis, and the initial induction during neuralation of CNCs (Parsons and Albertson 2009).
- Soluble, local signaling proteins that bind to specific receptors on the cell surface. These receptors transduce the signal via Smad proteins which activate particular genes (Parsons and Albertson 2009).
- In extracellular cases, Bmps can either stimulate cell proliferation, promote cell differentiation, or initiate apoptosis. The type of target cell type, Bmp4 concentration, or timing of the signal determine the various functions of Bmps. (Parsons and Albertson 2009).
- Bmps at low concentrations might stimulate cell proliferation, whereas higher concentrations might promote differentiation.
- Bmp4 may upregulate inhibitors inside (Smads) or outside (Chordin or Noggin, both of which are Bmp extracellular antagonists) the cell suggesting a negative feedback loop (Parsons and Albertson 2009).
- Antagonists that dampen the effects of Bmp4 may have particularly important effects on craniofacial phenotypes.
- To examine further details in regards to the Bmp signaling pathway, please view this video.
Fig. 3 This image shows the result of Albertson’s experiment, where they observed a difference in Bmp4 expression in the lower jaw of LF x MZ cross by examination through in situ hybridization (Albertson et. al 2005).
Fig. 4 Effects of higher vs. lower levels of bmp4 concentration on jaw morphology in Cichlids (Albertson’s Lab)
In figure 4, higher levels of bmp4 develop short robust jaws (since increased levels of bmp4 were associated with biting morphologies), whereas Cichlids with low levels of bmp4 develop thin elongate jaws.
- Calmodulin (CaM) is a highly conserved, intracellular molecule involved in mediating calcium (Ca2+) signaling by binding to calcium.
- Ca2+ is critical for signaling transduction as the binding of Ca2+ leads to intra-and extracellular interactions.
- CaM is ubiquitous to all eukaryotes and is involved in processes including inflammation, apoptosis, muscle contraction, intracellular movement, memory, and the immune response (Parsons and Albertson 2009).
- CaM is highly versatile as it has flexible binding and has an array of diverse target enzymes involved in the intracellular signaling pathway. Check out this interactive site to observe its targets. Or, for more specifics in regards to its signaling pathway, please observe this video.
- CaM expression leads to the activation of the cyclin protein expression, which allows the acceleration of osteoblast cell proliferation. This will eventually increase localized bone tissue (Parsons and Albertson 2009).
- Genes having antagonistic interactions with Bmp4 may have important spatial/ temporal regulators of craniofacial development by binding and releasing gene products (Parsons and Albertson 2009).
Fig 5 QTL analysis revealing the linkage between jaw width and CaM1. In figure a, the species LF has a wide jaw that crops algae from rocks. In figure b, MZ has a lower and narrower jaw that suction feeds in water (Parsons and Albertson 2009).
While using the same cross, CaM1 is linked to a QTL for jaw width. By designing a cross-species experiment between LF and MZ, they were able to identify a QTL for jaw width on linkage group 19, centered over CaM1 (refer to figure 5, graph). They assessed the phenotype for the recombinant lines and the genotypic markers that vary between the parental strains. The QTL accounted for 10% of phenotypic variance in the jaw width trait and was found to be significant at the 95% genome-wide confidence level (Parsons and Albertson 2009). The results of this analysis is presented as a plot of the test statistic against the chromosomal map position, in recombination units (cM). Markers that are genetically linked to a QTL influencing the trait of interest will segregate more frequently. Unlinked markers will not show significant association with the jaw width (Miles and Wayne 2008). However, more extensive mapping is needed to fully support this conclusion.
In the results stemming from the QTL analysis, mapping of the appropriate QTL revealed that small number of genes, such as pleiotropic genes, were responsible for differences in species. More specifically, they found a QTL that segregated with allelic variation at the bmp4 locus and thus affected the shape of the lower jaw (Parsons and Albertson 2009). The QTL approach enabled the researchers to determine that alternate expression patterns of Bmp4 and CaM1 in this tissue are implicated in the development of species-specific jaw shapes.
Fig. 5 This picture retrieved from Albertson’s lab demonstrates how QTL co-segregates with bmp4 to regulate jaw morphogenesis (Albertson’s Lab).
The overexpression of bmp4 elicited the growth of the jaw along the dorsal-ventral axis, increasing the depth of the jaw. More specifically, the differences in jaw shape are mapped on a linkage group (19) containing bmp4.
Fig 6 Expression of CaM1 is associated with skeletogenesis and varies among cichlid species. There is a difference in expression levels between the robust LF (refer to a, b) and another less robust species, Tropheops (e, f) (Parsons and Albertson 2009).
In figure 6, it was found that CaM1 expression is associated with bone development in cichlids and the differential pattern expressions vary in species with differently shaped jaws. By observing figure 6a and 6e, there is localized expression observed around the developing jaws. Eight days after post fertilization, the craniofacial skeleton begins to ossify, or turn into a rigid bone. This particular pattern of expression is consistent for CaM1 in controlling the proliferation or differentiation of osteogenic (~ossification) precursor cells. Because LF and TRC (less robust species, see figure) show different expression levels, the data suggests that CaM signaling is critical in formation of the craniofacial skeleton.
-Interaction between CaM1 and Bmp-
- Both may interact through their respective signal transduction pathways.
- CaM may stimulate Bmp signaling by increasing Smad1 activity (Parsons and Albertson 2009).
- CaM signaling may indirectly act as a negative regulator of Bmp signaling by recruiting the CREB binding protein away from Bmp (Parsons and Albertson 2009).
- In relation to jaw development, high levels of expression for CaM1 in the developing jaw may in turn locally reduce and/or spatially modulate levels of Bmp4 expression (Parsons and Albertson 2009)
Q4: Are there any relations of CaM and Bmp4 to humans? Any interesting facts, in particular?
- In other in vitro studies, CaM signaling has been shown to modulate osteoblast differentiation and proliferation
- CaM1 may increase susceptibility of hip osteroarthritis in humans!
- The resorption of bone results in an increase in extracellular concentrations of Ca2+. As a results, levels of osteoblast proliferation and differentiation increases through a CaM-mediated response (Parsons and Albertson 2009).
This particular study highlighted the significance of Bmp4 and CaM in craniofacial development, specifically in embryonic jaw development, and how they may play critical roles in the maintenance of adaptive phenotypes. By employing QTL and whole mount in-situ hybridization in studying Bmp4 and CaM’s involvement in their corresponding pathways, we can also explore future studies in examining jaw morphology and observing responses in gene expression patterning. This may allow us to address why induced changes in gene expression resemble differences in other regulatory networks found between species (Parsons and Albertson 2009).
The roles for Bmp and CaM signaling involvement in CNCs and skeletogenesis signify that CNCs, Bmp4, and CaM are implicated in the diverse jaw morphological development for specific species. The interaction between Bmp4 and CaM permits a possible mechanism that could reveal different jaw dimensions (Parsons and Albertson 2009).
Overall, this experiment provides a basic understanding of processes underlying craniofacial development. that are important to the study of human diseases. Induced mutations in these cichlid models can reveal essential studies of monogenetic human diseases.
Q5: What were the strengths and weaknesses of the main and subset studies?
The study conducted by Dr. Albertson in his papers, Parsons and Albertson and Albertson et. al, thoroughly provided a clear layout concerning the methods involved and results acquired. The images provided in the particular study were interesting and well laid out. The authors also addressed the importance of these pathways in future implications, such as their possible involvement in developmental plasticity and their significance in studies that tie in both evolution and developmental biology. Furthermore, they provided essential background information that facilitated the understanding of the study. Additionally, the paper written by Miles and Wayne demonstrated how to properly interpret and analyze a quantitative trait locus.
The authors could have addressed some of the caveats or limitations to the particular chosen methods, especially with the QTL analysis. The sample size was not clarified or implied very thoroughly in the paper, and the sample size is important, according to Miles and Wayne 2008), and could have been acknowledged.
Additionally, while the study by Albertson et. al mentions in-depth details and functions of Bmp4 and Cam1, there lacks sufficient images to provide substantial evidence. They could have included supplemental information in regards to their findings.
Albertson, R.C., Streelman, J.T., Kocher, T.D, and Yelick, P.C. “Integration and evolution of the cichlid mandible: The molecular basis of alternate feeding strategies.” Proceedings of the National Academy of Sciences: Biological Sciences- Evolution. 102.45 (2005): 16287-16292.
Kocher, T.D., Baroiller, J., Fernard, R., Hey, J., Hofmann, H., Meyer, A., Okada, N., Penman, D., Seehausen, O., and Streelman, T. “Genetic Basis of Vertebrate Diversity: the Cichlid Fish Model.” The International Cichlid Genome Consortium (2006).
Images & HyperLinks Citations: Fig. 1. Skeletal image of Cichlid Fish. http://www.pnas.org/content/102/45.cover-expansion.
 Cichlids. http://en.wikipedia.org/wiki/Cichlid.
 TMJ. http://www.webmd.com/oral-health/guide/temporomandibular-disorders.
 Cranial Neural Crest Cells. http://www.nature.com/hdy/journal/v97/n3/fig_tab/6800864f7.html.
 Quantitative Trait Locus. http://www.nature.com/scitable/topicpage/quantitative-trait-locus-qtl-analysis-53904.
 In situ hybridization. http://en.wikipedia.org/wiki/In_situ_hybridization
 Fig. 3. Bmp4 expression. http://www.genome.gov/Pages/Research/Sequencing/SeqProposals/CichlidGenomeSeq.pdf.
 Albertson’s Lab. http://albertsonlab.syr.edu/CNC.html.
 Calmodulin Video. http://www.youtube.com/watch?v=sufhrXpuoF8.
 Bmp4 Signaling Pathway Video. http://www.youtube.com/watch?v=QoJBIfM0bmU.