Species-specific developmental timing dictates expansion of the avian wing skeletal pattern

Holly Stainton, Matthew Towers

Preprint posted on 3 May 2021

Article now published in Cell Reports at

It’s about birds time! Temporal scaling of proximo-distal wing patterning can be set by retinoic acid in quail and chick wing buds.

Selected by Teresa Rayon

Categories: developmental biology

In this preprint, Holly Stainton and Matthew Towers investigate the differences in developmental pace in proximo-distal wing patterning across birds. In the developing wing of quail and chick, the downregulation of Fgf8 expression, Sox9 onset of expression, and necrotic changes occur at equivalent Hamburger-Hamilton (HH) stages, which correspond to 46 chronological stages in bird development. Similarly, Hoxa11 and Hoxa13 follow equivalent dynamics in both species. However, there is a sustained 12-hour difference in the dynamics of expression of these genes between quail and chick that indicates that quail proximo-distal wing patterning progresses faster in comparison to chick. Strikingly, the growth rates are comparable between the species during the patterning phase.

Figure 1.Schematics depicting the timing of gene expression during the proximo-distal specification and differentiation of the wing in quail and chick


In this preprint, the authors perform a series of homochronic transplants to investigate what sets the tempo. As the offset in timing is initiated 12 hours after the HH18/19 stage, the authors perform interspecies polarising region grafts prior to any offset in developmental timing. Whereas Shh expression is downregulated after 48 hours in normal quail wing development, Shh expression in quail grafts placed on chick hosts is extended for 48 hours, matching Shh chick expression at HH26. Likewise, when they perform the reverse transplants grafting chick cells on quail, they are able to show an accelerated decrease in SHH expression of the chick graft transplanted in the quail host at 48 hours later. These experiments indicate that the pace of expression is set at the onset of proximo-distal patterning in wing buds, at the time that the chick and quail HH stage is equivalent.

The authors then looked into retinoic acid (RA) signaling, as it is cleared in the distal region of the wing bud by HH21, and could cause the different behaviour between the HH18/19 stage and the subsequent time points. They found that Cyp26b1 levels rise significantly faster in the quail compared to the chick. As Cyb26b1 is the major retinoic acid degrading enzyme, these findings indicate a decreased degradation rate of retinoic acid in the wing bud of larger species. Moreover, by transiently prolonging retinoic acid signalling, the authors elegantly demonstrate that Shh expression timing is delayed in the quail polarising region graft, as the expression is maintained for approximately the same duration as in the host chick polarising region.

Figure 2. Resetting of developmental timing in polarising region xenografts. In interspecies polarising region grafts between 0-hour quail and chick wings (HH18/19 in both species) (f, h), Shh expression is reset according to host timing (g, i). The image is taken from Figure 3.


To conclude the paper, the authors characterize the dynamics of wing development in turkey. Turkey wings are larger in size than chick, and the preprint shows that they progress with a 12-hour delay in the expression of Shh, Hoxa11 and Hoxa13 compared to chick. As predicted from their findings of the involvement of RA in setting the pace, culturing chick wings with RA from HH18/19 delayed the pace of chick development and matched the length of the chicken wing to that of the turkey at HH21. Altogether, the preprint shows that there is a proportional patterning and temporal scaling in wing bud development between quail, chick, and turkey that can be reset by addition of retinoic acid within the first 12 hours of wing development.

Why I chose the paper and how it moves the field forward:

I’m interested in understanding how developmental timing operates at the cellular level, and this work studies developmental timing in vivo during proximo-distal limb development in birds. Limbs are one of the best characterized developmental systems, and they show a well-defined sequence of temporal events that can be used to investigate developmental tempo. In addition, limbs have undergone extensive evolutionary diversification across species. Moreover, birds are a great model in developmental biology as eggs can easily be collected and cultured, and are amenable for numerous embryology perturbations. Even though chick is the dominant model, reports in quail, zebra finch and emus have provided some interesting developmental evolutionary perspectives (Uygur et al., 2016; Young et al., 2019). I find particularly useful for the of study developmental pace the fact that HH staging is based on morphological features that correspond quite neatly with changes in gene expression programs.

Notably, Stainton and Towers show that developmental timing is proportionately scaled across turkey, chick and quail and demonstrate that RA can delay the timing of wing bud development in quail and chick, suggesting that the degradation rate of RA sets the tempo in the system. This finding resembles the differences in protein degradation observed in mouse and human cells that associate with differences in developmental tempo (Matsuda et al., 2020; Rayon et al., 2020). It would be really exciting to know how Cyp26b1 levels are regulated to modify the degradation rate of RA in their system. Looking forward to their future work!



Posted on: 4 May 2021


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Questions to the authors

Holly Stainton shared


  1. RA addition induces a delay in developmental timing, but it seems that quail wings are still able to develop slightly faster compared to chick, as do chick wings treated with RA in comparison to turkey development. How much can RA delay the timescales of the process, where is the limit in the process, and does it depend on the concentration and/or duration of signaling?

We have been limited in how far we can test this in vivo because increasing the concentration/duration of retinoic acid signaling causes the apical ectodermal ridge to flatten and results in defective outgrowth. To overcome this, and to see how far we can push the delay in developmental timing, we are currently working on developing an in vitro explant system that will allow us to have better control over manipulating the concentration and duration of retinoic acid signalling.

  1. In motor neuron differentiations from embryonic stem cells, we add signaling molecules at high concentrations and for long durations directly into the medium. Would the authors expect to detect a similar protracted expression of Shh in response to RA in chick explants incubated with RA?

 Good point! As mentioned in response to question 1, this is something that we’re currently working on in the lab for a separate publication, so hopefully we’ll be able to provide an answer to this question in the near future.

  1. Does increasing RA degradation by overexpressing Cyp26b1 in quail, chick or turkey accelerate developmental progression? Did you check if an RA antagonist accelerated the process?

Unfortunately, inhibiting retinoic signaling alone doesn’t accelerate developmental timing because the activation of Hoxa13 expression also requires a permissive epigenetic environment as shown by Rosello-Diez, Development 2014. In this paper they show that combining retinoic acid signaling inhibitors and histone deacetylase inhibitors is capable of precociously activating the expression of Hoxa13 in the chick limb. However, histone deacetylase inhibitors (eg. Trichostatin A) also affect proliferation and limb growth (shown in Towers et al Nature 2008), and so this would have an effect on how we assess other aspects of developmental timing, such as the accurate staging of wings, and growth rates.

  1. Do the authors think that differences in cell cycle length may play a role at the onset of the differences in developmental timing between quail and chick, prior to HH21?

This is a possibility since faster cell cycle rates could account for the faster outgrowth of the quail wing bud. Our results using flow cytometry are consistent with this idea, but differences in the cell cycle are likely to be very small and this will need further investigation.

However, this accounts for only a small difference in cell cycle rates and so at the moment it seems unlikely, but we’d like to explore this possibility further.

  1. Emus are big birds with small wings, and it has been shown that the heterochrony in wing bud outgrowth is due to an attenuation on FGF signalling that pauses proliferation of the limb mesenchyme at HH18 (Young et al., 2019). Do the authors think that RA may have a role in setting the pace of wing patterning in emus?

This is a really interesting question. I think it’s quite possible that retinoic acid signaling can set the pace of development in emu wings, since it is likely to have a general role in controlling the tempo of 5’Hox gene activation. However, this probably occurs alongside the mechanism described in Young et al, 2019, where the emu wing employs an additional process to further reduce wing size through reduction of Fgf8 signalling (and therefore decreasing proliferation and the progenitor population). I’d be really interested in analysing if reduced Fgf signalling affects retinoic acid clearance/cyp26b1 expression, and the tempo of 5”Hox gene expression in the vestigial emu wing.

  1. Given the high degree of conservation in limb development between tetrapods, do the authors think that the same mechanism operates in the scaling of forearms in mammals? 

This is definitely an avenue of research which we would like to explore. We’re currently working with the HDBR (Human Developmental Biology Resource) to look at the timing of patterning in human limbs and have recently got some interesting preliminary data. At the moment we’re waiting on some cell cycle analysis for the human limb which will be really exciting to see, and is something which we hope to publish in the near future. It would also be great to compare this data to timing in other mammals, including the mouse.


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