A positional information gradient of sonic hedgehog is required for flight feather formation in avian wings

Flight is a triumph of evolution that enabled the radiation and success of birds. A crucial step was the development of forelimb flight feathers that may have evolved for courtship or territorial displays in ancestral theropod dinosaurs. Classical tissue recombination experiments performed in the chick embryo provide evidence that signals operating during early limb development specify the position and identity of feathers. Here we show that a positional information gradient of Sonic hedgehog (Shh) signalling in the embryonic chick wing bud specifies the pattern of adult flight feathers in a defined spatial and temporal sequence that reflects their different identities. We reveal that the Shh signalling gradient is interpreted into specific patterns of flight feather-associated gene expression. Our data suggests that flight feather evolution involved the co-option of the pre-existing digit patterning mechanism and therefore uncovers an embryonic process that played a fundamental step in the evolution of avian flight.


Introduction
Flight feathers provide most of the flapping, gliding and soaring ability required for airborne locomotion in birds. Three identities of flight feather-based on differences in size, shape and location-are present in bird wings: primaries along the posterior margin of digits 2 and 3, secondaries along the posterior margin of the ulna, and alulars along the posterior margin of digit 1 1 (Figs. 1a and b). Flight feathers are much longer and more rigid than other feathers, including covert feathers that adorn most of the surface of a bird, and down feathers that lie close to the body to provide insulation 1 . They are also unique, both in being bilaterally asymmetric across the central midvein, and in forming strong ligamentous connections with the skeleton, which aids their independent movement during flight 1,2 ( Fig. 1b-primaries to digits 2 and 3; secondaries to the ulna; alulars to digit 1). The presence of 'flight feathers' on the posterior margins of forearms of flightless bipedal theropod dinosaurs provides strong evidence that they evolved to fulfil another function such as defence or courtship, thus making it likely that they played an early and important step in the evolution of flight in later birds [3][4][5] .
Although much is known about the molecular pathways involved in the induction, positioning and morphogenesis of feathers 6 , little is known about how different types of feathers are specified. The analyses of naturally occurring mutants provides evidence of a genetic pathway for flight feather development 7,8 . In support of this proposal, it was recently revealed that the gene encoding the Sim1 transcription factor is a specific marker of the flight feather-forming regions of the bird wing 9 . In addition, classical tissue recombination experiments in chickens provide evidence that signals acting at the earliest stages of wing bud development at around day 3 of incubation (HH20-see methods for staging) specify feather position and identity 10,11 . An important signal known to function at this stage is Sonic hedgehog (Shh)-a protein that emanates from a transient signalling centre called the polarising region (also known as the zone of polarising region activity), which is located at the posterior margin of the limb bud 12 . Shh is involved in the specification of antero-posterior positional values in the chick limb in a concentration-dependent manner between HH19 and HH22 13 14 (thumb to little finger) and in stimulating proliferative growth along this axis 15 (reviewed in 16 ). However, it is unclear if Shh has a direct role in the specification of feather pattern, because although grafts of polarising region cells made to the anterior margin of host HH20 wing buds at incubation day 3.5 can duplicate all feather buds as shown at day 13 ( Fig. 1c-primaries flight feathers in Roman numerals, secondaries in Arabic numerals), this could be an indirect consequence of all tissues being duplicated across the anteroposterior axis ( Fig. 1c-ulna, digits 1, 2 and 3) 17 .
In this study, we show that Shh signalling by the embryonic chick wing polarising region is required for the specification and formation of adult flight feathers. Our data provide evidence that a positional information gradient of Shh signalling integrates digit and flight feather patterning, and thus provides insights into the co-evolution of these important structures.

Shh signalling is required for flight feather bud formation
During our extensive analyses of chick embryos in which the Shh signalling pathway was transiently inhibited with cyclopamine at day 3 of incubation (HH19/20) for approximately 72 h 18 , we often noticed abnormal flight feather bud development.
Thus, raised flight feather buds expressing Ptch1-a direct target of Shh signalling that is involved in feather morphogenesis [19][20][21] -are found along the posterior margin of untreated day 13 wings (arrow in Fig. 1d), but not in the wings of embryos which were systemically treated with cyclopamine at HH19 (arrow in Fig. 1e), in which all of the feather buds have a similar morphology. Therefore, this observation demonstrates that it is the earlier loss of Shh signalling by the polarising region that perturbs flight feather bud formation, rather than the loss of Shh signalling within the buds themselves.
Flight feather buds form along the dorsal-ventral boundary of the wing, which if disrupted can result in abnormal flight feather bud development 22 . Therefore, to examine if the loss of Shh signalling affects dorso-ventral patterning of the wing bud, we examined the expression of Wnt7a, which is expressed in the dorsal epithelium. In both the wing buds of untreated (arrows in Fig. 1f) and HH19 cyclopamine-treated embryos, the expression of Wnt7a reveals that the dorsal-ventral boundary remains intact after 16 h (arrows in Fig. 1g). Therefore, abnormal flight feather bud formation following the earlier transient loss of Shh signalling is not a consequence of defective dorso-ventral patterning.
Developing flight feather buds become morphologically distinct during late incubation stages by growing inwards to make ligamentous connections with the skeleton, and by displaying bilateral asymmetry 1 2 . Therefore, we used these morphological characteristics to explore if Shh signalling is required for advanced stages of flight feather bud development. H&E staining on transverse sections of untreated forewings reveal that flight feather buds grow away from the posterior margin of the wing, and also invaginate into deeper tissues until they reach the ulna by day 13 2 (Figs. 1h-j). In addition, developing flight feather buds can also be identified in transverse section at day 15 by their asymmetric pattern of Shh expression 2 (Fig. 1k). However, in embryos treated with cyclopamine at HH19, flight feather buds frequently fail to form along the posterior border of the wing, although it remains covered with natal down buds, none of which invaginate deeply towards the skeleton (Figs. 1l-n- Supplementary Table 1). Furthermore, only developing feather buds with symmetric expression of Shh are observed in forewing regions of wings at day 15, thus again demonstrating that flight feather buds are selectively missing (Fig.   1o).
Therefore, these observations demonstrate a transient and specific requirement for Shh signalling by the polarising region for later flight feather bud development.

Shh signalling is required for flight feather-associated gene expression
Recently, molecular markers of the flight-feather forming regions of the chick wing have been identified which include Sim1 9 and Zic1 23 . Notably, Sim1 has an avian-specific forelimb expression pattern in the dermis 24 9 . Therefore, to examine if the inhibition of Shh signalling affects flight feather bud-associated gene expression, we performed a series of RNA sequencing experiments on tissue dissected from day 10 wings. This stage was selected because it is when flight feather buds become morphologically distinct from other feather buds (see expression of a general marker, We performed a hierarchical clustering analysis to identify genes that behave similarly (genes included are expressed at a >2-fold difference between at least one contrast-p-value 0.005). This produced four clusters (Supplementary Information), and we focussed on cluster four that comprises twenty-six known genes including Sim1 and Zic1 (Figs. 2d, e and j-note, blue is down-regulated; red is up-regulated).
As reported previously, Sim1 (Fig. 2f) and Zic1 Fig. 2k) are expressed in the flight feather-forming regions of the chick wing 9 23 , although Zic1 is only weakly expressed along the posterior margin of digit 1 (Fig. 2k). However, both Sim1 (Fig. 2g) and Zic1

Shh signalling is required for flight feather formation in mature birds
Chicks that were treated with cyclopamine at HH19 did not survive beyond hatching, which prevented the study of their mature wing plumage. Therefore, since our analyses of hatched chickens shows that dorsal major coverts-which are closely associated with developing flight feathers-are also absent, this raised the possibility that Shh inhibition could affect the later development of other feathers that had not yet replaced the natal down.
To analyse feather development in mature bird wings, we treated embryos at HH19/20 with cyclopamine and then grafted their right-hand wing buds in place of the right-hand wing buds of untreated embryos (Fig. 5a -see control experiment showing that the grafting procedure does not affect feather development -Supplementary Figure 4). This procedure enabled chicks to survive beyond hatching ( Fig. 5b), and they displayed the same patterns of flight feather loss as hatched chicks that were systemically treated with cyclopamine as embryos (Supplementary Tables 1   and 2). Several birds were allowed to progress to later stages of development and their patterns of flight feather loss remained the same as at hatching (Supplementary Table   2). Thus, one such example of a postnatal day 22 bird shows that the flight feather pattern is normal in its untreated left-hand wing, but that there is a loss of distal primary flight feathers in its cyclopamine-treated right-hand wing (arrow, Fig. 5c).
This bird was allowed to survive until postnatal day 66, so that its adult feather pattern could be studied in more detail (Figs. 5d-g). Manual examination of its untreated left-hand wing reveals that the natal down has been replaced by defined rows of mature feathers (dorsal view Fig. 5d, ventral view Fig. 5f). Thus, eighteen secondary flight feathers develop from its ulnar region (green asterisks Figs. 5d and f, note -not all feathers can be seen because they overlap) and ten primary feathers develop from its digital region (eight primaries from digit 3-orange asterisks, and two from digit 2-blue asterisks, Figs. 5d and f, note, one feather was broken). Three  -Fig. 5e). The remaining feathers are of the same identities as those present in its untreated wing, although some of the secondary flight feathers are shorter (green asterisks - Figs. 5e and g). In addition, the development of ventral major covert feathers is unaffected by cyclopamine treatment (Fig. 5g). Interestingly, the pattern of feather loss in the wing of this bird is consistent with the pattern of Sim1 expression in the wings of embryos that were treated with cyclopamine at HH19 (Fig.   3b). These results demonstrate that the inhibition of Shh signalling in the embryo causes the selective loss of mature flight feathers and their overlying dorsal major coverts in a defined spatial and temporal sequence.

Embryonic Shh signalling is required for flight feather formation
We have revealed that Shh signalling by the embryonic chick wing polarising region is required for specifying the adult pattern of flight feathers and their associated dorsal major covert feathers. This process is independent of the later role that Shh signalling fulfils in feather morphogenesis [19][20][21] . Thus, the transient ablation of Shh signalling between HH18 and HH22, but not later, causes the loss of bilaterally asymmetric flight feathers that make ligamentous connections to the skeleton, and also the loss of molecular markers associated with the flight feather forming regions of wing. However, the Shh signalling pathway (Ptch1) is still active during later feather bud morphogenesis, thereby showing that this general process is unaffected.

Detailed fate mapping experiments have shown that chick wing bud cells
contribute to the development of distal structures when Shh signalling by the polarising region is transiently blocked 15 14 . Taken together with the finding that apoptosis is also suppressed in the posterior part of the wing bud 18 , this provides evidence that the loss of both dorsal major covert and flight feathers is not caused by the selective loss of cells. We also revealed that the dorso-ventral boundary, which is important for flight feather development 22 , remains intact following the inhibition of Shh signalling. In addition, we demonstrated that feather buds, which produce other feather types, still form along the posterior border of the ulna and digit skeleton. Therefore, our data provides molecular insights into classical tissue recombination experiments, which showed that feather position and identity are determined by signals acting at around HH20 10,11 .

A positional information gradient of Shh specifies flight feather identity
Our findings can be explained by the classical positional information model of antero-posterior patterning, in which Shh signalling specifies limb bud cells with a positional value, which when interpreted at a later stage of development, allows them to differentiate into the appropriate structure 16,17 . Thus, the temporal requirement for Shh signalling in specifying the anterior to posterior pattern of Sim1 expression and flight feathers closely follows that for specifying the anterior to posterior pattern of digits 13 14 16 (Fig. 5h): digit 1 and alular flight feathers are specified first by a low concentration/short duration of Shh at HH18, and then increasing concentrations of Shh over time specify the other skeletal elements and flight feathers in the order; digit 2 and distal primaries at HH19; the ulna and secondaries at HH21, and digit 3 and proximal primaries at HH22 (Fig. 5h). It is of note that the pattern of Sim1 expression in the wings of embryos treated at HH19, precisely matches the pattern of flight feathers present both at hatching and in mature bird wings (Fig. 5h). Therefore, eight proximal primaries-which normally form along the border of digit 3-are absent; yet two distal primaries are present along the border of digit 2 (Fig. 5h). This pattern of flight feather loss is also accompanied by the loss of the associated overlying row of dorsal major covert feathers. However, the inhibition of Shh signalling does not affect the development of the remaining feathers in the wing, thereby implying that their identities are specified by other signals. Our findings therefore indicate that flight feathers and dorsal major covert feathers have similar developmental programmes, the study of which could warrant further investigation. Taken together, these observations reveal that the evolution of the flight feather programme involved the co-option of the pre-existing positional information gradient of Shh signalling used in forewing/digit patterning (Fig. 5h).

Interpretation of the Shh gradient into flight feather identity
The interpretation of positional information, in which cells memorise their positional value to give rise to appropriately patterned and positioned structures at a later stage of development, is generally an unknown process in developmental biology 28

RNA sequencing analyses and clustering
Tissue used for making RNA was manually dissected using fine forceps. Three replicate experiments were performed from each condition and the tissue was pooled before the RNA was extracted using Trizol reagent (Gibco). Sequencing libraries were prepared using Illumina TruSeq library preparation kit. Samples were sequenced on a HiSeq 2000 (Paired end readings of 50 bp -Instrument: ST300). Reads were aligned to the chicken genome, assembly Gallus_gallus-5.0, using STAR aligner.