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Much remains to be learned about how cis-regulatory elements such as enhancers function, especially during vertebrate organ development. To increase knowledge in this area, we have examined the cis-regulation of the transcription factor dlx2b during zebrafish larval tooth formation. We have created a GFP knock-in line that replicates dlx2b expression during tooth development and have also isolated a minimal enhancer of dlx2b (MTE1) sufficient for activating most of the tooth germ expression pattern. We have found that four evolutionarily conserved predicted transcription factor binding sites are required for the function of this minimal enhancer in both contexts. When the conserved sequences are mutated in a transgene it eliminates the activity of the enhancer and when they are deleted at the dlx2b locus it causes a dramatic alteration in the expression pattern. We hypothesize that disabling this enhancer at the dlx2b locus may be enabling other nearby cis-regulatory elements to take control of the promoter. These experiments reveal details of how cis-regulatory elements are working to control gene expression during organogenesis and highlight how much remains to be learned by empirical studies of gene regulation.Graphical Abstract

Explaining how the extensive diversity in form of vertebrate teeth arose in evolution and the mechanisms by which teeth are made during embryogenesis are intertwined questions that can merit from a better understanding of the roles of retinoic acid (RA) in tooth development. Pioneering studies in rodents showed that dietary vitamin A (VA), and eventually RA (one of the major active metabolites of VA), are required for proper tooth formation and that dentin-forming odontoblast cells seem to be especially sensitive to changes in RA levels. Later, rodent studies further indicated that RA signaling interactions with other cell-signaling pathways are an important part of RA's actions in odontogenesis. Recent investigations employing zebrafish and other teleost fish continued this work in an evolutionary context, and specifically demonstrated that RA is required for the initiation of tooth development. RA is also sufficient in certain circumstances to induce de novo tooth formation. Both effects appear to involve cranial-neural crest cells, again suggesting that RA signaling has a particular influence on odontoblast development. These teleost studies have also highlighted both evolutionary conservation and change in how RA is employed during odontogenesis in different vertebrate lineages, and thus raises the possibility that developmental changes to RA signaling has led to some of the diversity of form seen across vertebrate dentitions. Future progress in this area will come at least in part from expanding the species examined to get a better picture of how often RA signaling has changed in evolution and how this relates to the evolution of dental form.

Explaining how the extensive diversity in form of vertebrate teeth arose in evolution and the mechanisms by which teeth are made during embryogenesis are intertwined questions that can merit from a better understanding of the roles of retinoic acid (RA) in tooth development. Pioneering studies in rodents showed that dietary vitamin A (VA), and eventually RA (one of the major active metabolites of VA), are required for proper tooth formation and that dentin-forming odontoblast cells seem to be especially sensitive to changes in RA levels. Later, rodent studies further indicated that RA signaling interactions with other cell-signaling pathways are an important part of RA's actions in odontogenesis. Recent investigations employing zebrafish and other teleost fish continued this work in an evolutionary context, and specifically demonstrated that RA is required for the initiation of tooth development. RA is also sufficient in certain circumstances to induce de novo tooth formation. Both effects appear to involve cranial-neural crest cells, again suggesting that RA signaling has a particular influence on odontoblast development. These teleost studies have also highlighted both evolutionary conservation and change in how RA is employed during odontogenesis in different vertebrate lineages, and thus raises the possibility that developmental changes to RA signaling has led to some of the diversity of form seen across vertebrate dentitions. Future progress in this area will come at least in part from expanding the species examined to get a better picture of how often RA signaling has changed in evolution and how this relates to the evolution of dental form.

Teeth are one of the most fascinating innovations of vertebrates. Their diversity of shape, size, location, and number in vertebrates is astonishing. If the molecular mechanisms underlying the morphogenesis of individual teeth are now relatively well understood, thanks to the detailed experimental work that has been performed in model organisms (mainly mouse and zebrafish), the mechanisms that control the organization of the dentition are still a mystery. Mammals display simplified dentitions when compared to other vertebrates with only a single tooth row positioned in the anterior part of the mouth, whereas other vertebrates exhibit tooth rows in many locations. As proposed 60 years ago, tooth rows can be formed sequentially from an initiator tooth. Recent results in zebrafish have now largely confirmed this hypothesis. Here this observation is generalized upon and it is suggested that in most vertebrates tooth rows could form sequentially from a single initiator tooth.

Background: Intercellular communication by the hedgehog cell signaling pathway is necessary for tooth development throughout the vertebrates, but it remains unclear which specific developmental signals control cell behavior at different stages of odontogenesis. To address this issue, we have manipulated hedgehog activity during zebrafish tooth development and visualized the results using confocal microscopy. Results: We first established that reporter lines for dlx2b, fli1, NF-κB, and prdm1a are markers for specific subsets of tooth germ tissues. We then blocked hedgehog signaling with cyclopamine and observed a reduction or elimination of the cranial neural crest derived dental papilla, which normally contains the cells that later give rise to dentin-producing odontoblasts. Upon further investigation, we observed that the dental papilla begins to form and then regresses in the absence of hedgehog signaling, through a mechanism unrelated to cell proliferation or apoptosis. We also found evidence of an isometric reduction in tooth size that correlates with the time of earliest hedgehog inhibition. Conclusions: We hypothesize that these results reveal a previously uncharacterized function of hedgehog signaling during tooth morphogenesis, regulating the number of cells in the dental papilla and thereby controlling tooth size. Developmental Dynamics 244:577–590, 2015. © 2015 Wiley Periodicals, Inc.

SUMMARY Teeth with two or more cusps have arisen independently from an ancestral unicuspid condition in a variety of vertebrate lineages, including sharks, teleost fishes, amphibians, lizards, and mammals. One potential explanation for the repeated origins of multicuspid teeth is the existence of multiple adaptive pathways leading to them, as suggested by their different uses in these lineages. Another is that the addition of cusps required only minor changes in genetic pathways regulating tooth development. Here we provide support for the latter hypothesis by demonstrating that manipulation of the levels of Fibroblast growth factor (Fgf) or Bone morphogenetic protein (Bmp) signaling produces bicuspid teeth in the zebrafish (Danio rerio), a species lacking multicuspid teeth in its ancestry. The generality of these results for teleosts is suggested by the conversion of unicuspid pharyngeal teeth into bicuspid teeth by similar manipulations of the Mexican Tetra (Astyanax mexicanus). That these manipulations also produced supernumerary teeth in both species supports previous suggestions of similarities in the molecular control of tooth and cusp number. We conclude that despite their apparent complexity, the evolutionary origin of multicuspid teeth is positively constrained, likely requiring only slight modifications of a pre-existing mechanism for patterning the number and spacing of individual teeth.

Zebrafish lost anterior teeth during evolution but retain a posterior pharyngeal dentition that requires retinoic acid (RA) cell-cell signaling for its development. The purposes of this study were to test the sufficiency of RA to induce tooth development and to assess its role in evolution. We found that exposure of embryos to exogenous RA induces a dramatic anterior expansion of the number of pharyngeal teeth that later form and shifts anteriorly the expression patterns of genes normally expressed in the posterior tooth-forming region, such as pitx2 and dlx2b. After RA exposure, we also observed a correlation between cartilage malformations and ectopic tooth induction, as well as abnormal cranial neural crest marker gene expression. Additionally, we observed that the RA-induced zebrafish anterior teeth resemble in pattern and number the dentition of fish species that retain anterior pharyngeal teeth such as medaka but that medaka do not express the aldh1a2 RA-synthesizing enzyme in tooth-forming regions. We conclude that RA is sufficient to induce anterior ectopic tooth development in zebrafish where teeth were lost in evolution, potentially by altering neural crest cell development, and that changes in the location of RA synthesis correlate with evolutionary changes in vertebrate dentitions.-Seritrakul, P., Samarut, E., Lama, T. T. S., Gibert, Y., Laudet, V., Jackman, W. R. Retinoic acid expands the evolutionarily reduced dentition of zebrafish. FASEB J. 26, 5014-5024 (2012). www.fasebj.org

It has been considered a “law” that a lost structure cannot reappear in evolution. The common explanation, that genes required for the development of the lost structure degrade by mutation, remains largely theoretical, however. Additionally, the extent to which this mechanism applies to systems of repeated parts, where individual modules are likely to exhibit few unique aspects of genetic control, is unclear. We investigated reversibility of evolution in one such system, the vertebrate dentition, using as a model loss of oral teeth in cypriniform fishes, which include the zebrafish. This evolutionary event, which occurred >50 million years ago, has not been reversed despite subsequent diversification of feeding modes and retention of pharyngeal teeth. We asked whether the cis-regulatory region of a gene whose expression loss parallels cypriniform tooth loss, Dlx2b, retains the capacity for expression in oral teeth. We first created a zebrafish reporter transgenic line that recapitulates endogenous dlx2b expression. We then showed that this zebrafish construct drives reporter expression in oral teeth of the related characiform Astyanax mexicanus . This result, along with our finding that Dlx genes are required for normal tooth development, suggests that changes in trans-acting regulators of these genes were responsible for loss of cypriniform oral teeth. Preservation of oral enhancer function unused for >50 million years could be the result of pleiotropic function in the pharyngeal dentition. If enhancers of other genes in the tooth developmental pathway are similarly preserved, teeth lost from specific regions may be relatively easy to reacquire in evolution.

The club-shaped gland (csg) is a prominent organ during the development of amphioxus. However, the evolutionary significance of this pharyngeal structure has been a mystery because of the lack of an obvious corollary in vertebrates or other close relatives. To address the homology of the csg by molecular means, we report the cloning and expression patterns of two amphioxus genes expressed during its development, crabp and maf. Amphioxus maf is a bzip transcription factor expressed early in csg formation in the forming of the ventral duct of the gland. crabp encodes a cellular retinoic acid binding protein and is expressed widely in the csg later in its development. We compare these genes to the expression of AmphiKrox, a zinc-finger transcription factor previously reported to be expressed during csg development. Together these genes mark different spatial and temporal aspects of csg formation. However, we find little evidence to suggest homology of the csg with other organs in amphioxus or other chordates. We therefore propose that the csg can be viewed as an evolutionary novelty that probably arose within the amphioxus lineage.

The segmentation of the vertebrate hindbrain into rhombomeres is highly conserved, but how early hindbrain patterning is established is not well understood. We show that rhombomere 4 (r4) functions as an early-differentiating signaling center in the zebrafish hindbrain. Time-lapse analyses of zebrafish hindbrain development show that r4 forms first and hindbrain neuronal differentiation occurs first in r4. Two signaling molecules, FGF3 and FGF8, which are both expressed early in r4, are together required for the development of rhombomeres adjacent to r4, particularly r5 and r6. Transplantation of r4 cells can induce expression of r5/r6 markers, as can misexpression of either FGF3 or FGF8. Genetic mosaic analyses also support a role for FGF signaling acting from r4. Taken together, our findings demonstrate a crucial role for FGF-mediated inter-rhombomere signaling in promoting early hindbrain patterning and underscore the significance of organizing centers in patterning the vertebrate neural plate.

The vertebrate embryonic hindbrain is segmented into rhombomeres. Gene expression studies suggest that amphioxus, the closest invertebrate relative of vertebrates, has a hindbrain homolog. However, this region is not overtly segmented in amphioxus, raising the question of how hindbrain segmentation arose in chordate evolution. Vertebrate hindbrain segmentation includes the patterning of cranial motor neurons, which can be identified by their expression of the LIM-homeodomain transcription factor islet1. To learn if the amphioxus hindbrain homolog is cryptically segmented, we cloned an amphioxus gene closely related to islet1, which we named simply islet. We report that amphioxus islet expression includes a domain of segmentally arranged cells in the ventral hindbrain homolog. We hypothesize that these cells are developing motor neurons and reveal a form of hindbrain segmentation in amphioxus. Hence, vertebrate rhombomeres may derive from a cryptically segmented brain present in the amphioxus/vertebrate ancestor. Other islet expression domains provide evidence for amphioxus homologs of the pineal gland, adenohypophysis, and endocrine pancreas. Surprisingly, homologs of vertebrate islet1-expressing spinal motor neurons and Rohon-Beard sensory neurons appear to be absent.

Homologs of the Drosophila snail gene have been characterized in several vertebrates. In addition to being expressed in mesoderm during gastrulation, vertebrate snail genes are also expressed in presumptive neural crest and/or its derivatives. Given that neural crest is unique to vertebrates and is considered to be of fundamental importance in their evolution, we have cloned and characterized the expression of a snail gene from amphioxus, a cephalochordate widely accepted as the sister group of the vertebrates. We show that, at the amino acid sequence level, the amphioxus snail gene is a clear phylogenetic outgroup to all the characterized vertebrate snail genes. During embryogenesis snail expression initially becomes restricted to the paraxial or presomitic mesoderm of amphioxus. Later, snail is expressed at high levels in the lateral neural plate, where it persists during neurulation. Our results indicate that an ancestral function of snail genes in the lineage leading to vertebrates is to define the paraxial mesoderm. Furthermore, our results indicate that a cell population homologous to the vertebrate neural crest may be present in amphioxus, thus providing an important link in the evolution of this key vertebrate tissue.

The fossil record indicates that cypriniform fishes, a group including the zebrafish, lost oral teeth over 50 million years ago. Despite subsequent diversification of feeding modes, no cypriniform has regained oral teeth,suggesting the zebrafish as a model for studying the developmental genetic basis of evolutionary constraint. To investigate the mechanism of cypriniform tooth loss, we compared the oral expression of seven genes whose mammalian orthologs are involved in tooth initiation in the zebrafish and the Mexican tetra, Astyanax mexicanus, a related species retaining oral teeth. The most significant difference we found was an absence in zebrafish oral epithelium of expression of dlx2a and dlx2b, transcription factors that are expressed in early Astyanax odontogenic epithelium. Analysis of orthologous genes in the Japanese medaka (Oryzias latipes) and a catfish (Synodontis multipunctatus) suggests that expression was lost in cypriniforms, rather than gained in Astyanax. Treatment of Astyanax with an inhibitor of Fibroblast growth factor(Fgf) signaling produced a partial phenocopy of the zebrafish oral region, in that oral teeth, and expression of dlx2a and dlx2b, were lost, whereas shh and pitx2, genes whose expression is present in zebrafish oral epithelium, were unaffected. We hypothesize that a loss of Fgf signaling to oral epithelium was associated with cypriniform tooth loss.

The segmental patterning of the vertebrate hindbrain has been intensely investigated, yet the evolutionary origin of hindbrain segmentation remains unclear. In the vertebrate sister group, amphioxus (Cephalochordata), the embryonic neural tube lacks obvious morphological segmentation, but comparative Hox gene expression analysis has suggested the presence of a region homologous to the vertebrate hindbrain. Does this region contain ancient segmental features shared with the vertebrate hindbrain? To help address this question we cloned the paired-like amphioxus homeodomain gene shox and found that its expression is segmental in the amphioxus neural tube. We also uncovered a previously uncharacterized iterated neural tube expression pattern of the zinc-finger gene AmphiKrox. We propose that these genes, along with amphioxus islet and AmphiMnx, share a one-somite width periodicity of expression in the neural tube, the coincidence of which may reflect an underlying segmental organization. We hypothesize that the segmental patterning of neurons in the neural tube was present in the amphioxus/vertebrate ancestor, but the establishment of a bona fide segmented hindbrain may indeed have arisen in the vertebrate lineage.

Use of scat analysis to determine food habits of carnivores is dependent on accurate species identification of scat. Thin-layer chromatography (TLC) of fecal bile acids has been used to identify carnivore scat but relies on an untested assumption that diet does not affect the profile (incidence and concentration) of fecal bile acids in ways that obscure species-specific patterns. We conducted a feeding experiment to evaluate the effect of diet on the reliability of TLC as a means of uniquely identifying coyote (Canis latrans) scats. We detected deoxycholic and chenodeoxycholic acids in all scats from coyotes fed a control diet composed of commercial feed