In 1916, E.S. Russell posed the question in his now classic book, Form and Function[18], “Is function the mechanical result of form, or is form merely the manifestation of function or activity? What is the essence of life, organisation or activity? (p.v).” A broad range of experimental strategies across different model systems have revealed that NCM is an essential player in most, if not all, of the decisive events that generate the primary organization of the amniote jaw complex. NCM not only provides the raw materials for the cartilages, bones, and other essential components that comprise the jaws, but NCM is also required for the critical signaling interactions that imbue these tissues with the multidimensional aspects of patterning from which their form is derived. Deficiencies in NCM or perturbing these interactions on the molecular or cellular level alters the form of the jaw complex in profound ways, which illuminates why the jaw complex is both highly evolvable and extremely susceptible to developmental defects [164]. Moreover, while NCM and neighboring epithelia typically collaborate to pattern the cartilages and bones of the jaws, and while NCM and mesodermal mesenchyme work together to pattern the jaw muscles, NCM seems to act as the dominant source of information that gives all of these jaw structures their species-specific size and shape. In this role, NCM is the common denominator that underlies the structural integration of the jaw apparatus, generates species-specific variation, and likely serves as a responsive target of natural selection during evolution [7, 37, 138, 140, 191]. Moreover, NCM has augmented the evolutionary potential (i.e., adaptability) of the pharyngeal and rostral portions of the head and imparts the jaw skeleton with developmental plasticity, as evidenced by the ability of the NCM-derived skeleton to respond to mechanical forces like in the case of secondary cartilage. Initially, the form of the jaw appears to dictate function, but then through embryonic motility, function modulates form. In other words, NCM sets up the species-specific “organisation” of the jaw apparatus prior to the onset of muscle “activity.” But once jaw activity starts, the form of the skeleton adapts to support its functional needs. The species-specific form of the duck jaw apparatus, especially the geometry of the NCM-mediated muscle attachments, produces mechanical forces that differentially regulate FGF and TGFβ signaling and cause secondary cartilage to form on the coronoid process. In this regard, NCM not only mediates form but also helps shape the biomechanical environment. Additionally, the patterning abilities and plasticity found in NCM-derived jaw progenitors facilitate seamless integration of form and function during embryonic development and evolution. These same processes are likely perturbed in cases of injury or disease. Overall, elucidating the molecular and cellular mechanisms through which NCM governs the species-specific patterning of cartilage, bone, tendon, and muscle has shed light on the evolutionary integration of form and function in the amniote jaw complex, and in the near future could help remedy an unmet clinical need to repair and regenerate jaw tissues affected by birth defects, disease, or injury.

Background

Vertebrates possess a diverse range of integumentary epithelial appendages, including scales, feathers and hair. These structures share extensive early developmental homology, as they mostly originate from a conserved anatomical placode. In the context of avian epithelial appendages, feathers and scutate scales are known to develop from an anatomical placode. However, our understanding of avian reticulate (footpad) scale development remains unclear.

Results

Here, we demonstrate that reticulate scales develop from restricted circular domains of thickened epithelium, with localised conserved gene expression in both the epithelium and underlying mesenchyme. These domains constitute either anatomical placodes, or circular initiatory fields (comparable to the avian feather tract). Subsequent patterning of reticulate scales is consistent with reaction–diffusion (RD) simulation, whereby this primary domain subdivides into smaller secondary units, which produce individual scales. In contrast, the footpad scales of a squamate model (the bearded dragon, Pogona vitticeps) develop synchronously across the ventral footpad surface.

Conclusions

Widely conserved gene signalling underlies the initial development of avian reticulate scales. However, their subsequent patterning is distinct from the footpad scale patterning of a squamate model, and the feather and scutate scale patterning of birds. Therefore, we suggest reticulate scales are a comparatively derived epithelial appendage, patterned through a modified RD system.

Overall, we demonstrate that the development of avian epithelial appendages, including feathers, scutate and reticulate scales, is regulated by the signalling of conserved developmental genes. During reticulate scale development, circular domains of localised gene expression are observed along the ventral footpad at E10.5, constituting either anatomical placodes or circular initiatory fields. These domains subsequently subdivide into individual reticulate scales, following a patterning mechanism consistent with RD simulation. This is distinct from the patterning of squamate (P. vitticeps) ventral footpad scales. Therefore, we suggest that reticulate scales are derived epithelial appendages patterned through a modified RD system.

Background

Unravelling the genetic basis of polymorphic characters is central to our understanding of the origins and diversification of living organisms. Recently, supergenes have been implicated in a wide range of complex polymorphisms, from adaptive colouration in butterflies and fish to reproductive strategies in birds and plants. The concept of a supergene is now a hot topic in biology, and identification of its functional elements is needed to shed light on the evolution of highly divergent adaptive traits. Here, we apply different gene expression analyses to study the supergene P that controls polymorphism of mimetic wing colour patterns in the neotropical butterfly Heliconius numata.

Results

We performed de novo transcriptome assembly and differential expression analyses using high-throughput Illumina RNA sequencing on developing wing discs of different H. numata morphs. Within the P interval, 30 and 17 of the 191 transcripts were expressed differentially in prepupae and day-1 pupae, respectively. Among these is the gene cortex, known to play a role in wing pattern formation in Heliconius and other Lepidoptera. Our in situ hybridization experiments confirmed the relationship between cortex expression and adult wing patterns.

Conclusion

This study found the majority of genes in the P interval to be expressed in the developing wing discs during the critical stages of colour pattern formation, and detect drastic changes in expression patterns in multiple genes associated with structural variants. The patterns of expression of cortex only partially recapitulate the variation in adult phenotype, suggesting that the remaining phenotypic variation could be controlled by other genes within the P interval. Although functional studies on cortex are now needed to determine its exact developmental role, our results are in accordance with the classical supergene hypothesis, whereby several genes inherited together due to tight linkage control a major developmental switch.

Here, we developed the first transcriptome resource for H. numata and performed differential expression analyses during wing development to identify genes involved in forming the supergene and contributing to the expression of differentiated phenotypes. Our analyses confirm the role of cortex in the formation of black wing pattern elements. Our results are consistent with the hypothesis that other genes in the P region may play a role in colour variation in H. numata. Functional studies on the gene cortex and exploration of longer developmental time series will now be required to conclude whether the supergene P is a classical supergene, or whether cortex alone fully controls the developmental switches involved in colour pattern polymorphism in H. numata.

Background

Many traits evolve by cis-regulatory modification, by which changes to noncoding sequences affect the binding affinity for available transcription factors and thus modify the expression profile of genes. Multiple examples of cis-regulatory evolution have been described at pattern switch genes responsible for butterfly wing pattern polymorphism, including in the diverse neotropical genus Heliconius,but the identities of the factors that can regulate these switch genes have not been identified.

Results

We investigated the spatial transcriptomic landscape across the wings of three closely related butterfly species, two of which have a convergently evolved co-mimetic pattern and the other having a divergent pattern. We identified candidate factors for regulating the expression of wing patterning genes, including transcription factors with a conserved expression profile in all three species, and others, including both transcription factors and Wnt pathway genes, with markedly different profiles in each of the three species. We verified the conserved expression profile of the transcription factor homothorax by immunofluorescence and showed that its expression profile strongly correlates with that of the selector gene optix in butterflies with the Amazonian forewing pattern element ‘dennis.’

Conclusion

Here we show that, in addition to factors with conserved expression profiles like homothorax, there are also a variety of transcription factors and signaling pathway components that appear to vary in their expression profiles between closely related butterfly species, highlighting the importance of genome-wide regulatory evolution between species.

Our understanding of the regulatory evolution of wing pattern in butterflies is dependent on a clear picture of the expression of developmental factors around the time of wing pattern specification. This study has provided a picture of gene expression along one axis of developing wings in a manner unbiased by our understanding of wing development in non-lepidopteran systems. At the within-species level, we can broadly rule out the hypothesis that trans-regulatory factors change their expression profiles in different pattern forms (Fig. 1d) based on genetic mapping, but we are not able to rule out this phenomenon at the between-species level—it is likely that both processes play a role, either through selection or drift. Our deeper understanding of factors that are expressed in the wing in correlation with pattern elements will permit us to decode the regulatory linkages that lead to the differential expression of pattern switch genes like optix, WntA and cortex, and it is clear that we should look to both conserved and diverging regulatory factors as the causative agents of cis-regulatory evolution.

Background

Melanic patterns such as horizontal stripes, vertical bars and spots are common among teleost fishes and often serve roles in camouflage or mimicry. Extensive research in the zebrafish model has shown that the development of horizontal stripes depends on complex cellular interactions between melanophores, xanthophores and iridophores. Little is known about the development of horizontal stripes in other teleosts, and even less is known about bar or spot development. Here, we compare chromatophore composition and development of stripes, bars and spots in two cichlid species of sand-dwellers from Lake Malawi—Copadichromis azureus and Dimidiochromis compressiceps.

Results

(1) In D. compressiceps, stripes are made of dense melanophores underlaid by xanthophores and overlaid by iridophores. Melanophores and xanthophores are either loose or absent in interstripes, and iridophores are dense. In C. azureus, spots and bars are composed of a chromatophore arrangement similar to that of stripes but are separated by interbars where density of melanophores and xanthophores is only slightly lower than in stripes and iridophore density appears slightly greater. (2) Stripe, bar and spot chromatophores appear in the skin at metamorphosis. Stripe melanophores directly differentiate along horizontal myosepta into the adult pattern. In contrast, bar number and position are dynamic throughout development. As body length increases, new bars appear between old ones or by splitting of old ones through new melanophore appearance, not migration. Xanthophore and iridophore distributions follow melanophore patterns. (3) Metamorphic pigmentation arises in cichlids in a fashion similar to that described in zebrafish: melanophore progenitors derived from the medial route of neural crest migration migrate from the vicinity of the neural tube to the skin during metamorphosis.

Conclusion

The three pigment cell types forming stripes, bars and spots arise in the skin at metamorphosis. Stripes develop by differentiation of melanophores along horizontal myosepta, while bars do not develop along patent anatomical boundaries and increase in number in relation with body size. We propose that metamorphic melanophore differentiation and migratory arrest upon arrival to the skin lead to stripe formation, while bar formation must be supported by extensive migration of undifferentiated melanophores in the skin.

The three same chromatophore types make up D. compressiceps and C. azureusbody coloration: melanophores, iridophores and xanthophores. In D. compressiceps,stripes are made of dense melanophores underlaid by xanthophores and overlaid by iridophores. Melanophores and xanthophores are either loose or absent in interstripes, and iridophores are dense. In C. azureus, spots and bars are composed of a chromatophore arrangement similar to that of stripes but are separated by interbars where density of melanophores and xanthophores is only slightly lower than in stripes and iridophore density appears slightly greater.

Differences in pigmentation between C. azureus and D. compressiceps arise at metamorphosis when melanophores appear in the skin as arrangements prefiguring stripes, bars or spots. In D. compressiceps, stripe melanophores differentiate along horizontal myosepta and the adult pattern is essentially in place by the end of the larval period. In contrast, the number of bars developing in C. azureus larvae is smaller than in adults. Bars appear as a series of lateral melanophore patches that progressively elongate dorso-ventrally by new melanophore additions. New bars appear either between old ones, or through splitting of old ones. The place of new melanophore appearance, not migration, determines new bar formation. Increase in xanthophore density in developing bars and stripes follows that of melanophores. Iridophores appear in interstripes after stripe formation, while their appearance in interbars is concomitant with bar formation.

Analysis of neural crest migration by ISH indicates that melanophore progenitors migrate from the vicinity of the neural tube to the skin along myosepta at metamorphosis, as previously described in zebrafish [13]. Time-lapse analysis of bar development during metamorphosis shows that melanophores do not migrate after differentiation. We thus propose that metamorphic melanophore differentiation and migratory arrest upon arrival to the skin lead to stripe formation, while bar formation must be supported by extensive dorsal and/or ventral migration of undifferentiated melanophores in the skin.