Convergent Evolution of Cichlid Fish Pharyngeal Jaw Dentitions in Mollusk-Crushing Predators: Comparative X-Ray Computed Tomography of Tooth Sizes, Numbers, and Replacement.

Dental convergence is a hallmark of cichlid fish adaptive radiations. This type of repeated evolution characterizes both the oral jaws of these fishes as well as their pharyngeal jaws that are modified gill arches used to functionally process prey like hard-shelled mollusks. To test several hypotheses regarding the evolution of cichlid crushing pharyngeal dentitions, we used X-ray computed tomography scans to comparatively examine dental evolution in the pharyngeal jaw of a diversity of New World Heroine cichlid lineages. The substantial variation in erupted tooth sizes and numbers as well as replacement teeth found in these fishes showed several general patterns. Larger toothed species tended to have fewer teeth suggesting a potential role of spatial constraints in cichlid dental divergence. Species with larger numbers of erupted pharyngeal teeth also had larger numbers of replacement teeth. Replacement tooth size is almost exactly predicted (r = 0.99) from the size of erupted teeth across all of the species. Mollusk crushing was, therefore, highly associated with not only larger pharyngeal teeth, but also larger replacement teeth. Whether dental divergence arises as a result of environmental induced plasticity or originates via trophic polymorphism as found in the species Herichthys minckleyi, there appear to be general rules that structure interspecific divergence in cichlid pharyngeal erupted and replacement dentitions.

Ecotype differences in aggression, neural activity and behaviorally relevant gene expression in cichlid fish.

In Lake Malawi, two ecologically distinct lineages of cichlid fishes (rock- vs sand-dwelling ecotypes, each comprised of over 200 species) evolved within the last million years. The rock-dwelling species (Mbuna) are aggressively territorial year-round and males court and spawn with females over rocky substrate. In contrast, males of sand-dwelling species are not territorial and instead aggregate on seasonal breeding leks in which males construct courtship "bowers" in the sand. However, little is known about how phenotypic variation in aggression is produced by the genome. In this study, we first quantify and compare behavior in seven cichlid species, demonstrating substantial ecotype and species differences in unconditioned mirror-elicited aggression. Second, we compare neural activity in mirror-elicited aggression in two representative species, Mchenga conophoros (sand-dwelling) and Petrotilapia chitimba (rock-dwelling). Finally, we compare gene expression patterns between these two species, specifically within neurons activated during mirror aggression. We identified a large number of genes showing differential expression in mirror-elicited aggression, as well as many genes that differ between ecotypes. These genes, which may underly species differences in behavior, include several neuropeptides, genes involved in the synthesis of steroid hormones and neurotransmitter activity. This work lays the foundation for future experiments using this emerging genetic model system to investigate the genomic basis of evolved species differences in both brain and behavior.

Developmental plasticity of epithelial stem cells in tooth and taste bud renewal.

In Lake Malawi cichlids, each tooth is replaced in one-for-one fashion every ∼20 to 50 d, and taste buds (TBs) are continuously renewed as in mammals. These structures are colocalized in the fish mouth and throat, from the point of initiation through adulthood. Here, we found that replacement teeth (RT) share a continuous band of epithelium with adjacent TBs and that both organs coexpress stem cell factors in subsets of label-retaining cells. We used RNA-seq to characterize transcriptomes of RT germs and TB-bearing oral epithelium. Analysis revealed differential usage of developmental pathways in RT compared to TB oral epithelia, as well as a repertoire of genome paralogues expressed complimentarily in each organ. Notably, BMP ligands were expressed in RT but excluded from TBs. Morphant fishes bathed in a BMP chemical antagonist exhibited RT with abrogated shh expression in the inner dental epithelium (IDE) and ectopic expression of calb2 (a TB marker) in these very cells. In the mouse, teeth are located on the jaw margin while TBs and other oral papillae are located on the tongue. Previous study reported that tongue intermolar eminence (IE) oral papillae of Follistatin (a BMP antagonist) mouse mutants exhibited dysmorphic invagination. We used these mutants to demonstrate altered transcriptomes and ectopic expression of dental markers in tongue IE. Our results suggest that vertebrate oral epithelium retains inherent plasticity to form tooth and taste-like cell types, mediated by BMP specification of progenitor cells. These findings indicate underappreciated epithelial cell populations with promising potential in bioengineering and dental therapeutics.

Behavior-dependent cis regulation reveals genes and pathways associated with bower building in cichlid fishes.

Many behaviors are associated with heritable genetic variation [Kendler and Greenspan (2006) Am J Psychiatry 163:1683-1694]. Genetic mapping has revealed genomic regions or, in a few cases, specific genes explaining part of this variation [Bendesky and Bargmann (2011) Nat Rev Gen 12:809-820]. However, the genetic basis of behavioral evolution remains unclear. Here we investigate the evolution of an innate extended phenotype, bower building, among cichlid fishes of Lake Malawi. Males build bowers of two types, pits or castles, to attract females for mating. We performed comparative genome-wide analyses of 20 bower-building species and found that these phenotypes have evolved multiple times with thousands of genetic variants strongly associated with this behavior, suggesting a polygenic architecture. Remarkably, F1 hybrids of a pit-digging and a castle-building species perform sequential construction of first a pit and then a castle bower. Analysis of brain gene expression in these hybrids showed that genes near behavior-associated variants display behavior-dependent allele-specific expression with preferential expression of the pit-digging species allele during pit digging and of the castle-building species allele during castle building. These genes are highly enriched for functions related to neurodevelopment and neural plasticity. Our results suggest that natural behaviors are associated with complex genetic architectures that alter behavior via cis-regulatory differences whose effects on gene expression are specific to the behavior itself.

Genome-wide protein phylogenies for four African cichlid species.

BACKGROUND: The thousands of species of closely related cichlid fishes in the great lakes of East Africa are a powerful model for understanding speciation and the genetic basis of trait variation. Recently, the genomes of five species of African cichlids representing five distinct lineages were sequenced and used to predict protein products at a genome-wide level. Here we characterize the evolutionary relationship of each cichlid protein to previously sequenced animal species. RESULTS: We used the Treefam database, a set of preexisting protein phylogenies built using 109 previously sequenced genomes, to identify Treefam families for each protein annotated from four cichlid species: Metriaclima zebra, Astatotilapia burtoni, Pundamilia nyererei and Neolamporologus brichardi. For each of these Treefam families, we built new protein phylogenies containing each of the cichlid protein hits. Using these new phylogenies we identified the evolutionary relationship of each cichlid protein to its nearest human and zebrafish protein. This data is available either through download or through a webserver we have implemented. CONCLUSION: These phylogenies will be useful for any cichlid researchers trying to predict biological and protein function for a given cichlid gene, understanding the evolutionary history of a given cichlid gene, identifying recently duplicated cichlid genes, or performing genome-wide analysis in cichlids that relies on using databases generated from other species.

Applying gene regulatory network logic to the evolution of social behavior.

Animal behavior is ultimately the product of gene regulatory networks (GRNs) for brain development and neural networks for brain function. The GRN approach has advanced the fields of genomics and development, and we identify organizational similarities between networks of genes that build the brain and networks of neurons that encode brain function. In this perspective, we engage the analogy between developmental networks and neural networks, exploring the advantages of using GRN logic to study behavior. Applying the GRN approach to the brain and behavior provides a quantitative and manipulative framework for discovery. We illustrate features of this framework using the example of social behavior and the neural circuitry of aggression.

Developmental Plasticity of Patterned and Regenerating Oral Organs.

In many aquatic vertebrates, including bony and cartilaginous fishes, teeth and taste buds colocalize on jaw elements. In these animals, taste buds are renewed continuously throughout life, whereas teeth undergo cycled whole-organ replacement by various means. Recently, studies of cichlid fishes have yielded new insights into the development and regeneration of these dental and sensory oral organs. Tooth and taste bud densities covary positively across species with different feeding strategies, controlled by common regions of the genome and integrated molecular signals. Developing teeth and taste buds share a bipotent epithelium during early patterning stages, from which dental and taste fields are specified. Moreover, these organs share a common epithelial ribbon that supports label-retaining cells during later stages of regeneration. During both patterning and regeneration stages, dental organs can be converted to taste bud fate by manipulation of BMP signaling. These observations highlight a surprising long-term plasticity between dental and sensory organ types. Here, we review these findings and discuss the implications of developmental plasticity that spans the continuum of craniofacial organ patterning and regeneration.

Coevolutionary patterning of teeth and taste buds.

Teeth and taste buds are iteratively patterned structures that line the oro-pharynx of vertebrates. Biologists do not fully understand how teeth and taste buds develop from undifferentiated epithelium or how variation in organ density is regulated. These organs are typically studied independently because of their separate anatomical location in mammals: teeth on the jaw margin and taste buds on the tongue. However, in many aquatic animals like bony fishes, teeth and taste buds are colocalized one next to the other. Using genetic mapping in cichlid fishes, we identified shared loci controlling a positive correlation between tooth and taste bud densities. Genome intervals contained candidate genes expressed in tooth and taste bud fields. sfrp5 and bmper, notable for roles in Wingless (Wnt) and bone morphogenetic protein (BMP) signaling, were differentially expressed across cichlid species with divergent tooth and taste bud density, and were expressed in the development of both organs in mice. Synexpression analysis and chemical manipulation of Wnt, BMP, and Hedgehog (Hh) pathways suggest that a common cichlid oral lamina is competent to form teeth or taste buds. Wnt signaling couples tooth and taste bud density and BMP and Hh mediate distinct organ identity. Synthesizing data from fish and mouse, we suggest that the Wnt-BMP-Hh regulatory hierarchy that configures teeth and taste buds on mammalian jaws and tongues may be an evolutionary remnant inherited from ancestors wherein these organs were copatterned from common epithelium.

Quantitative multivariate analysis of dynamic multicellular morphogenic trajectories.

Interrogating fundamental cell biology principles that govern tissue morphogenesis is critical to better understanding of developmental biology and engineering novel multicellular systems. Recently, functional micro-tissues derived from pluripotent embryonic stem cell (ESC) aggregates have provided novel platforms for experimental investigation; however elucidating the factors directing emergent spatial phenotypic patterns remains a significant challenge. Computational modelling techniques offer a unique complementary approach to probe mechanisms regulating morphogenic processes and provide a wealth of spatio-temporal data, but quantitative analysis of simulations and comparison to experimental data is extremely difficult. Quantitative descriptions of spatial phenomena across multiple systems and scales would enable unprecedented comparisons of computational simulations with experimental systems, thereby leveraging the inherent power of computational methods to interrogate the mechanisms governing emergent properties of multicellular biology. To address these challenges, we developed a portable pattern recognition pipeline consisting of: the conversion of cellular images into networks, extraction of novel features via network analysis, and generation of morphogenic trajectories. This novel methodology enabled the quantitative description of morphogenic pattern trajectories that could be compared across diverse systems: computational modelling of multicellular structures, differentiation of stem cell aggregates, and gastrulation of cichlid fish. Moreover, this method identified novel spatio-temporal features associated with different stages of embryo gastrulation, and elucidated a complex paracrine mechanism capable of explaining spatiotemporal pattern kinetic differences in ESC aggregates of different sizes.

Common developmental pathways link tooth shape to regeneration.

In many non-mammalian vertebrates, adult dentitions result from cyclical rounds of tooth regeneration wherein simple unicuspid teeth are replaced by more complex forms. Therefore and by contrast to mammalian models, the numerical majority of vertebrate teeth develop shape during the process of replacement. Here, we exploit the dental diversity of Lake Malawi cichlid fishes to ask how vertebrates generally replace their dentition and in turn how this process acts to influence resulting tooth morphologies. First, we used immunohistochemistry to chart organogenesis of continually replacing cichlid teeth and discovered an epithelial down-growth that initiates the replacement cycle via a labial proliferation bias. Next, we identified sets of co-expressed genes from common pathways active during de novo, lifelong tooth replacement and tooth morphogenesis. Of note, we found two distinct epithelial cell populations, expressing markers of dental competence and cell potency, which may be responsible for tooth regeneration. Related gene sets were simultaneously active in putative signaling centers associated with the differentiation of replacement teeth with complex shapes. Finally, we manipulated targeted pathways (BMP, FGF, Hh, Notch, Wnt/ß-catenin) in vivo with small molecules and demonstrated dose-dependent effects on both tooth replacement and tooth shape. Our data suggest that the processes of tooth regeneration and tooth shape morphogenesis are integrated via a common set of molecular signals. This linkage has subsequently been lost or decoupled in mammalian dentitions where complex tooth shapes develop in first generation dentitions that lack the capacity for lifelong replacement. Our dissection of the molecular mechanics of vertebrate tooth replacement coupled to complex shape pinpoints aspects of odontogenesis that might be re-evolved in the lab to solve problems in regenerative dentistry.

Origins of shared genetic variation in African cichlids.

Cichlid fishes have evolved tremendous morphological and behavioral diversity in the waters of East Africa. Within each of the Great Lakes Tanganyika, Malawi, and Victoria, the phenomena of hybridization and retention of ancestral polymorphism explain allele sharing across species. Here, we explore the sharing of single nucleotide polymorphisms (SNPs) between the major East African cichlid assemblages. A set of approximately 200 genic and nongenic SNPs was ascertained in five Lake Malawi species and genotyped in a diverse collection of ~160 species from across Africa. We observed segregating polymorphism outside of the Malawi lineage for more than 50% of these loci; this holds similarly for genic versus nongenic SNPs, as well as for SNPs at putative CpG versus non-CpG sites. Bayesian and principal component analyses of genetic structure in the data demonstrate that the Lake Malawi endemic flock is not monophyletic and that river species have likely contributed significantly to Malawi genomes. Coalescent simulations support the hypothesis that river cichlids have transported polymorphism between lake assemblages. We observed strong genetic differentiation between Malawi lineages for approximately 8% of loci, with contributions from both genic and nongenic SNPs. Notably, more than half of these outlier loci between Malawi groups are polymorphic outside of the lake. Cichlid fishes have evolved diversity in Lake Malawi as new mutations combined with standing genetic variation shared across East Africa.

The genetic basis of a complex functional system.

The relationship between form and function can have profound effects on evolutionary dynamics and such effects may differ for simple versus complex systems. In particular, functions produced by multiple structural configurations (many-to-one mapping, MTOM) may dampen constituent trade-offs and promote diversification. Unfortunately, we lack information about the genetic architecture of MTOM functional systems. The skulls of teleost fishes contain both simple (lower jaw levers) as well as more complex (jaws modeled as 4-bar linkages) functional systems within the same craniofacial unit. We examined the mapping of form to function and the genetic basis of these systems by identifying quantitative trait loci (QTL) in hybrids of two Lake Malawi cichlid species. Hybrid individuals exhibited novelty (transgressive segregation) in morphological components and function of the simple and complex jaw systems. Functional novelty was proportional to the prevalence of extreme morphologies in the simple levers; by contrast, recombination of parental morphologies produced transgression in the MTOM 4-bar linkage. We found multiple loci of moderate effect and epistasis controlling jaw phenotypes in both the simple and complex systems, with less phenotypic variance explained by QTL for the 4-bar. Genetic linkage between components of the simple and complex systems partly explains phenotypic correlations and may constrain functional evolution.

Integrated brain diversification along the early neuraxes.

Brains develop under the influence of signaling centers that link major dorsal/ventral (DV) and anterior/posterior (AP) axes. Over ontogeny, these 'developmental neuraxes' progress from near global signaling gradients into more localized gene expression domains separated by molecular boundaries. Therefore, developmental changes along a neuraxis can have major consequences across the brain, or more precise effects on a specific structure, depending upon the time during ontogeny in which change occurs. It is well known from mammalian systems how evolution has acted later in development, via differential neurogenesis, to reshape the cortex. Recent studies in fishes show how early changes in DV and AP patterning result in divergence of integrated brain regions that ultimately define visual versus olfactory ecotypes. We explore the generality of this trend and suggest that such early developmental differences integrating brain diversification along the neuraxes may be a common theme in vertebrates. Early differences in brain patterning among species imply that adult variation in sensory function and behavior manifests in the embryo.

Divergence in cis-regulatory sequences surrounding the opsin gene arrays of African cichlid fishes.

BACKGROUND: Divergence within cis-regulatory sequences may contribute to the adaptive evolution of gene expression, but functional alleles in these regions are difficult to identify without abundant genomic resources. Among African cichlid fishes, the differential expression of seven opsin genes has produced adaptive differences in visual sensitivity. Quantitative genetic analysis suggests that cis-regulatory alleles near the SWS2-LWS opsins may contribute to this variation. Here, we sequence BACs containing the opsin genes of two cichlids, Oreochromis niloticus and Metriaclima zebra. We use phylogenetic footprinting and shadowing to examine divergence in conserved non-coding elements, promoter sequences, and 3'-UTRs surrounding each opsin in search of candidate cis-regulatory sequences that influence cichlid opsin expression. RESULTS: We identified 20 conserved non-coding elements surrounding the opsins of cichlids and other teleosts, including one known enhancer and a retinal microRNA. Most conserved elements contained computationally-predicted binding sites that correspond to transcription factors that function in vertebrate opsin expression; O. niloticus and M. zebra were significantly divergent in two of these. Similarly, we found a large number of relevant transcription factor binding sites within each opsin's proximal promoter, and identified five opsins that were considerably divergent in both expression and the number of transcription factor binding sites shared between O. niloticus and M. zebra. We also found several microRNA target sites within the 3'-UTR of each opsin, including two 3'-UTRs that differ significantly between O. niloticus and M. zebra. Finally, we examined interspecific divergence among 18 phenotypically diverse cichlids from Lake Malawi for one conserved non-coding element, two 3'-UTRs, and five opsin proximal promoters. We found that all regions were highly conserved with some evidence of CRX transcription factor binding site turnover. We also found three SNPs within two opsin promoters and one non-coding element that had weak association with cichlid opsin expression. CONCLUSIONS: This study is the first to systematically search the opsins of cichlids for putative cis-regulatory sequences. Although many putative regulatory regions are highly conserved across a large number of phenotypically diverse cichlids, we found at least nine divergent sequences that could contribute to opsin expression differences in cis and stand out as candidates for future functional analyses.

The macroecology of rapid evolutionary radiation.

A long-standing debate in ecology addresses whether community composition is the result of stochastic factors or assembly rules. Non-random, over-dispersed patterns of species co-occurrence have commonly been attributed to competition--a particularly important force in adaptive radiation. We thus examined the macroecology of the recently radiated cichlid rock-fish assemblage in Lake Malawi, Africa at a spectrum of increasingly fine spatial scales (entire lake to depth within rock-reef sites). Along this range of spatial scales, we observed a signal of community structure (decreased co-occurrence of species) at the largest and smallest scales, but not in between. Evidence suggests that the lakewide signature of structure is driven by extreme endemism and micro-allopatric speciation, while patterns of reduced co-occurrence with depth are indicative of species interactions. We identified a 'core' set of rock-reef species, found in combination throughout the lake, whose depth profiles exhibited replicated positive and negative correlation. Our results provide insight into how ecological communities may be structured differently at distinct spatial scales, re-emphasize the importance of local species interactions in community assembly, and further elucidate the processes shaping speciation in this model adaptive radiation.

Evolution of microRNAs and the diversification of species.

MicroRNAs (miRNAs) are ancient, short noncoding RNA molecules that regulate the transcriptome through post-transcriptional mechanisms. miRNA riboregulation is involved in a diverse range of biological processes, and misregulation is implicated in disease. It is generally thought that miRNAs function to canalize cellular outputs, for instance as "fail-safe" repressors of gene misexpression. Genomic surveys in humans have revealed reduced genetic polymorphism and the signature of negative selection for both miRNAs themselves and the target sequences to which they are predicted to bind. We investigated the evolution of miRNAs and their binding sites across cichlid fishes from Lake Malawi (East Africa), where hundreds of diverse species have evolved in the last million years. Using low-coverage genome sequence data, we identified 100 cichlid miRNA genes with mature regions that are highly conserved in other animal species. We computationally predicted target sites on the 3'-untranslated regions (3'-UTRs) of cichlid genes to which miRNAs may bind and found that these sites possessed elevated single nucleotide polymorphism (SNP) densities. Furthermore, polymorphic sites in predicted miRNA targets showed higher minor allele frequencies on average and greater genetic differentiation between Malawi lineages when compared with a neutral expectation and nontarget 3'-UTR SNPs. Our data suggest that divergent selection on miRNA riboregulation may have contributed to the diversification of cichlid species and may similarly play a role in rapid phenotypic evolution of other natural systems.

The odontode explosion: the origin of tooth-like structures in vertebrates.

Essentially we show recent data to shed new light on the thorny controversy of how teeth arose in evolution. Essentially we show (a) how teeth can form equally from any epithelium, be it endoderm, ectoderm or a combination of the two and (b) that the gene expression programs of oral versus pharyngeal teeth are remarkably similar. Classic theories suggest that (i) skin denticles evolved first and odontode-inductive surface ectoderm merged inside the oral cavity to form teeth (the 'outside-in' hypothesis) or that (ii) patterned odontodes evolved first from endoderm deep inside the pharyngeal cavity (the 'inside-out' hypothesis). We propose a new perspective that views odontodes as structures sharing a deep molecular homology, united by sets of co-expressed genes defining a competent thickened epithelium and a collaborative neural crest-derived ectomesenchyme. Simply put, odontodes develop 'inside and out', wherever and whenever these co-expressed gene sets signal to one another. Our perspective complements the classic theories and highlights an agenda for specific experimental manipulations in model and non-model organisms.

Brain diversity evolves via differences in patterning.

Differences in brain region size among species are thought to arise late in development via adaptive control over neurogenesis, as cells of previously patterned compartments proliferate, die, and/or differentiate into neurons. Here we investigate comparative brain development in ecologically distinct cichlid fishes from Lake Malawi and demonstrate that brains vary among recently evolved lineages because of early patterning. Divergence among rock-dwellers and sand-dwellers in the relative size of the telencephalon versus the thalamus is correlated with gene expression variation in a regulatory circuit (composed of six3, fezf2, shh, irx1b, and wnt1) known from model organisms to specify anterior-posterior (AP) brain polarity and position the shh-positive signaling boundary zona limitans intrathalamica (ZLI) in the forebrain. To confirm that changes in this coexpression network are sufficient to produce the differences we observe, we manipulated WNT signaling in vivo by treating rock-dwelling cichlid embryos with temporally precise doses of LiCl. Chemically treated rock-dwellers develop gene expression patterns, ZLIs, and forebrains distinct from controls and untreated conspecifics, but strongly resembling those of sand-dwellers. Notably, endemic Malawi rock- and sand-dwelling lineages are alternately fixed for an SNP in irx1b, a mediator of WNT signaling required for proper thalamus and ZLI. Together, these natural experiments in neuroanatomy, development, and genomics suggest that evolutionary changes in AP patterning establish ecologically relevant differences in the elaboration of cichlid forebrain compartments. In general, variation in developmental patterning might lay the foundations on which neurogenesis erects diverse brain architectures.

Geography disentangles introgression from ancestral polymorphism in Lake Malawi cichlids.

Phenotypically diverse Lake Malawi cichlids exhibit similar genomes. The extensive sharing of genetic polymorphism among forms has both intrigued and frustrated biologists trying to understand the nature of diversity in this and other rapidly evolving systems. Shared polymorphism might result from hybridization and/or the retention of ancestrally polymorphic alleles. To examine these alternatives, we used new genomic tools to characterize genetic differentiation in widespread, geographically structured populations of Labeotropheus fuelleborni and Metriaclima zebra. These phenotypically distinct species share mitochondrial DNA (mtDNA) haplotypes and show greater mtDNA differentiation among localities than between species. However, Bayesian analysis of nuclear single nucleotide polymorphism (SNP) data revealed two distinct genetic clusters corresponding perfectly to morphologically diagnosed L. fuelleborni and M. zebra. This result is a function of the resolving power of the multi-locus dataset, not a conflict between nuclear and mitochondrial partitions. Locus-by-locus analysis showed that mtDNA differentiation between species (F(CT)) was nearly identical to the median single-locus SNP F(CT). Finally, we asked whether there is evidence for gene flow at sites of co-occurrence. We used simulations to generate a null distribution for the level of differentiation between co-occurring populations of L. fuelleborni and M. zebra expected if there was no hybridization. The null hypothesis was rejected for the SNP data; populations that co-occur at rock reef sites were slightly more similar than expected by chance, suggesting recent gene flow. The coupling of numerous independent markers with extensive geographic sampling and simulations utilized here provides a framework for assessing the prevalence of gene flow in recently diverged species.

A role for suppressed incisor cuspal morphogenesis in the evolution of mammalian heterodont dentition.

Changes in tooth shape have played a major role in vertebrate evolution with modification of dentition allowing an organism to adapt to new feeding strategies. The current view is that molar teeth evolved from simple conical teeth, similar to canines, by progressive addition of extra "cones" to form progressively complex multicuspid crowns. Mammalian incisors, however, are neither conical nor multicuspid, and their evolution is unclear. We show that hypomorphic mutation of a cell surface receptor, Lrp4, which modulates multiple signaling pathways, produces incisors with grooved enamel surfaces that exhibit the same molecular characteristics as the tips of molar cusps. Mice with a null mutation of Lrp4 develop extra cusps on molars and have incisors that exhibit clear molar-like cusp and root morphologies. Molecular analysis identifies misregulation of Shh and Bmp signaling in the mutant incisors and suggests an uncoupling of the processes of tooth shape determination and morphogenesis. Incisors thus possess a developmentally suppressed, cuspid crown-like morphogenesis program similar to that in molars that is revealed by loss of Lrp4 activity. Several mammalian species naturally possess multicuspid incisors, suggesting that mammals have the capacity to form multicuspid teeth regardless of location in the oral jaw. Localized loss of enamel may thus have been an intermediary step in the evolution of cusps, both of which use Lrp4-mediated signaling.