Spatiotemporal patterns of cranial feather buds are established by the neural crest
A comparative analysis of integumentary development reveals that the timing and sequence of events during cranial feather morphogenesis are equivalent between stage-matched quail and duck embryos but that species-specific differences in pattern can be observed from early stages onwards. Using epifluorescent illumination of whole-mount embryos stained with ethidium bromide, we find that quail feather placodes can be seen beginning at HH34, in one medial and two lateral rows along the cranial epidermis (). By HH35, additional rows appear over the eyes, and by HH36 they span the entire dorsal surface of the cranial integument (). These quail cranial feather buds are relatively large and widely spaced. Duck feather placodes also first appear at HH34 but they do so in multiple rows over each eye, lateral to the midline (). Duck feather buds are relatively small and positioned close together, and by HH36 they are distributed across the cranial integument ().
Fig. 2 The role of neural crest in the spatiotemporal patterning of cranial feather buds. (A) Starting at HH34, quail feather placodes can be seen in one medial and two lateral rows along cranial epidermis (arrow). (B) By HH35, additional rows appear over the (more ...)
To test the extent to which the dermis regulates feather morphogenesis, we transplanted premigratory cranial neural crest cells destined to form the craniofacial mesenchyme from quail to duck embryos generating chimeric ‘quck’ embryos. We find that quail neural crest cells induce duck host epithelium to form feather buds on a quail-like timetable and spatial pattern. We collected quck chimeras at HH33, which is a stage when cranial feather buds are normally not present in control quail or duck embryos (). Using whole-mount ethidium bromide assays, we observe epidermal placodes that form prematurely across the craniofacial region either individually, in small clusters or as entire rows amid regions of undifferentiated host epithelium (n=13; ). The extent to which these epidermal placodes have developed is equivalent to that observed on control quail embryos at HH36 (). Generally, the placodes on these quck chimeras are widely spaced and large in size like those found on quail. In some locations along the cranial epidermis, rows of placodes are tightly aligned, whereas in other areas they appear asymmetric. Often, the distribution of epidermal placodes is closely correlated with the type of graft (bilateral or unilateral) used to generate the chimeric embryo (compare ). To ascertain if epidermal placodes can be induced at earlier stages, we also collected quck at HH29-HH32. We find that our transplants cause epidermal placodes to form as early as HH31 (n=6; ) and HH32 (n=4; data not shown). In quck chimeras collected prior to HH31, we observe no evidence of epidermal placodes (data not shown).
Fig. 3 Cranial neural crest regulates histogenic and molecular programs of feather morphogenesis. (A) At HH33, there are no cranial feather buds in either duck or quail (not shown) as stained histologically with trichrome (TC). (B,C) However, in chimeric ‘quck’ (more ...)
Thus, our transplants of quail neural crest cells into duck hosts cause the timing of feather morphogenesis to advance by three embryonic stages. This shift reflects the developmental difference separating control quail and duck embryos that are stage-matched for surgery at HH9.5 and incubated for 6 days, which is when feather morphogenesis is initiated in quail (). By HH34 and HH35, quck feather buds are similar to those found on control quail at HH37 (n=10) and HH38 (n=13), respectively (data not shown). By HH36, chimeric quck have patches of long feather buds similar to those found on HH39 quail (n=10; ). Moreover, Japanese quail are pigmented, whereas Pekin duck are white, and some of the HH36 quck chimeras already have pigmented quail-like feathers clustered among unpigmented and less developed feather buds derived from the duck host (n=6). The disparity in developmental stage between donor and host is even more apparent in quck cases collected at HH38, where patches of elongated well-developed brown and black quail-like feathers, which resemble those of control quail at HH41, are arranged among short white duck host feather buds (n=11; ).
Neural crest regulates histogenic programs of cranial feather morphogenesis
To elucidate the cellular nature of these transformations, we compared histological sections of stage-matched control and chimeric quck embryos at key time points during feather morphogenesis. In control quail and duck embryos, dense dermis and epidermal placodes have yet to form in the capital tracts at HH33 but appear from HH34 onwards (; ). Dermal condensations can be detected by HH35, and by HH36 some feather buds have begun to rise above the level of the integument (; ; ). By HH37, the height of these feather buds has become equal to or slightly longer than their width, and by HH39, some of the more mature quail feathers contain brown and black pigment at their distal tips (; data not shown). Quck chimeras collected at HH33, however, have dermal condensations and elevated placodes that resemble those found on control quail at HH36 (n=4; ). Quck at HH32 have dermal condensations and placodes equivalent to those present in HH35 controls (n=4; data not shown), and quck analyzed at HH31 already have dense dermis and placodes like those observed in controls at HH34 (n=3; ). Thus, in quck, the histogenic program of feather morphogenesis is shifted forward by three embryonic stages.
Fig. 4 Donor neural crest can also delay molecular and histogenic programs of cranial feather development. (A) Duck cranial neural crest cells follow their own timetable for differentiation when transplanted into quail hosts. Resultant duail chimeras collected (more ...)
To assess if the premature development and quail-like patterns of quck feathers result from the presence of quail donor neural crest, we processed sections from chimeric embryos for the immunohistological detection of quail cells using the Q¢PN anti-quail antibody (Schneider, 1999
). We find that in all chimeric quck cases with quail-like feather buds collected at HH31, HH32 and HH33, the dermis is derived primarily from quail donor neural crest, while the epidermis originates exclusively from the duck host (n
=11; ; data not shown). We also find that the extent to which quck feather buds are transformed in both size and placement correlates with the amount and distribution of quail donor neural crest cells in duck hosts. The more quail donor cells there are throughout the dermis, the more complete the transformation to a quail-like pattern. In less transformed cases we find fewer and/or more dispersed quail cells (data not shown). Where there is no quail donor mesenchyme (i.e. dermis derived from the duck host), feather buds have yet to form. This also holds true for those quck cases lacking any premature quail-like feather buds; here, the dermis is derived principally from the duck host (n
=6; data not shown). In quck chimeras collected prior to HH31, we find no epidermal placodes or dense dermis, despite abundant quail-derived cells in the mesenchyme of the region of the presumptive capital tracts (n
=6; data not shown). Quail donor neural crest cells also give rise to melanocytes that become secondarily associated with duck host epidermis and ultimately produce feather pigmentation (n
Neural crest regulates expression of the BMP, SHH and Delta/Notch pathways
To test the extent to which the dermis regulates molecular programs for feather development, we performed in situ hybridization to assay for changes in the expression of members and targets of the Bone Morphogenetic Protein, Sonic Hedgehog and Delta/Notch pathways. Our analyses conducted on control embryos collected from HH29 to HH38 demonstrate that the timing of expression for bmp4, bmp2, follistatin, bmpr1a, shh, ptc, delta1 and notch1 in the capital tracts is equivalent between stage-matched quail and duck. In sharp contrast, we find that in chimeric quck embryos the timing of gene expression is accelerated by three stages in both quail donor-derived dermis, and duck host-derived epidermis, which is consistent with our morphological and histological results. Prior to HH34, none of these genes is expressed in either the epidermis or dermis of control embryos (; data not shown), except for bmpr1a and notch1, which are expressed continuously from at least HH29 in most tissues throughout the craniofacial region (data not shown). However, in chimeric quck collected at HH33, all of these feather markers are detected throughout the capital tracts in domains equivalent to those observed in control quail at HH36 (). Specifically, in quck at HH33, we find bmp4 and delta1 expression restricted to the quail donor-derived dermal condensations of short feather buds (), shh in host-derived epidermal placodes (), and bmp2, follistatin, bmpr1a, ptc and notch1 in both tissues (n=4; ). These are the same expression patterns observed in control quail at HH36 (n=3; ). To determine how much earlier these genes could be experimentally induced, we collected chimeric quck at HH32, HH31, HH30 and HH29. We detect transcripts of bmp4, bmp2, follistatin, bmpr1a, shh, ptc, delta1 and notch1 in chimeric quck collected at HH32 (n=4), even though in control embryos ptc, shh, delta1 and follistatin do not appear in developing feather buds prior to HH35 (n=4; data not shown). Similarly, bmp4 and bmp2 are expressed in nascent feather buds of chimeric quck collected at HH31 (n=3), whereas control embryos express these genes in the equivalent region no earlier than HH34 (n=4; ; data not shown). We do not detect any evidence of expression for these genes in the cranial integument prior to HH31 in chimeric quck, despite an abundance of quail donor-derived mesenchyme in the presumptive capital tracts (data not shown).
Donor neural crest can also delay the timing of feather morphogenesis
As another functional test of our hypothesis that the dermis regulates the expression of genes known to play a role during feather morphogenesis, we performed reciprocal transplants of premigratory cranial neural crest cells from duck into quail, generating chimeric ‘duail’ embryos. In general, we find that duck donor neural crest delays the molecular and histogenic programs of feather morphogenesis in quail hosts by three embryonic stages (n=8). Duail chimeras were collected at HH36, which is when stage-matched control quail embryos have consecutive rows of feather buds across the entire dorsal surface of the cranial epithelium (). However, the capital tracts of these duail chimeras contain extensive epidermal regions that lack feather placodes (n=3; ). The absence of epidermal placodes is similar to that observed on duck donor controls prior to HH34 ().
To assess if the delay in duail feather development results from duck donor neural crest-mediated changes in gene expression, we processed sections histologically, with the Q¢PN anti-quail antibody, and for in situ hybridization. We find that in chimeric duail cases collected at HH36, those cranial regions lacking feather buds contain dermis derived from duck donor neural crest, while the epidermis originates from the quail host (n=3). Conversely, where there is no duck donor mesenchyme (i.e. Q¢PN-positive dermis derived from the quail host), feather buds are present (). Molecular analysis of these duail chimeras at HH36 reveals that bmp4, bmp2, follistatin, shh, ptc and delta1 are expressed in feather buds derived from quail host tissues, but not in regions where the dermis is derived from duck donor neural crest (n=3; ; data not shown). After HH37, duck-derived dermis along with quail host-derived epidermis form well developed feather placodes like those found on duck controls subsequent to HH34 (n=4; ). We also find that some pigmented feather buds in duail embryos older than HH38 are derived primarily from duck dermis, which is a source of normally non-pigment-producing melanocytes ().