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The Cellular and Molecular Origins of Beak Morphology

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Science  24 Jan 2003:
Vol. 299, Issue 5606, pp. 565-568
DOI: 10.1126/science.1077827

Abstract

Cellular and molecular mechanisms underlying differences in beak morphology likely involve interactions among multiple embryonic populations. We exchanged neural crest cells destined to participate in beak morphogenesis between two anatomically distinct species. Quail neural crest cells produced quail beaks in duck hosts and duck neural crest produced duck bills in quail hosts. These transformations involved morphological changes to non–neural crest host beak tissues. To achieve these changes, donor neural crest cells executed autonomous molecular programs and regulated gene expression in adjacent host tissues. Thus, neural crest cells are a source of molecular information that generates interspecific variation in beak morphology.

In On the Origin of Species, Darwin skillfully argued that natural selection governs adult beak morphology, but he was hard put to explain how features of different taxa could arise during embryogenesis. After measuring full-grown beaks of pigeon breeds, which varied “extraordinarily in length and form,” and comparing them with those of newly hatched birds, Darwin found that beak proportions of some breeds were already quite distinct in the young. He concluded, “each successive modification, or most of them, may have appeared at an extremely early period … from causes of which we are wholly ignorant” (1). Ever since Darwin's studies, the role of natural selection in beak evolution has been well substantiated, but the developmental basis for interspecific variation has remained elusive.

Here, we investigate the cellular and molecular origins of beak morphology. Beaks among groups of birds are astonishingly variable, and yet at early embryonic stages they all arise from comparable primordia, tissues, and cells. The upper beak is derived from the frontonasal and paired maxillary primordia, whereas the lower aspect forms from paired mandibular primordia. Neural crest cells that emigrate from the rostral neural tube are the principal source of mesenchyme for the facial primordia (2) (Fig. 1, A and B). During beak development, neural crest cells produce skeletal and connective tissues (Fig. 1C), facial ectoderm forms outer cornified layers, nasal placodes give rise to olfactory epithelium, paraxial mesoderm generates blood vessels and voluntary muscles, and pharyngeal endoderm lines part of the oral cavity (3, 4). Beak morphology could originate from any of these components and be conveyed through local molecular signaling centers (5–10). Of these possibilities, we hypothesize that neural crest cells contain patterning information for beak morphology, which they implement by executing autonomous molecular programs and impart by regulating gene expression in adjacent tissues.

Figure 1

Developmental origins of beak morphology. (A) The frontonasal (fn), maxillary (mx), and mandibular (ma) primordia (sagittal view) are surrounded by facial ectoderm (fe) and forebrain neuroepithelium (fb) and contain contributions from the neural crest, nasal placode (np), and cranial nerves (V, VII, IX). (B) Neural crest cells (dorsal view) migrate from the forebrain (fb) and midbrain (mb) and produce frontonasal structures (pink). Cells from the midbrain and first two rhombomeres (r) of the hindbrain (hb) give rise to maxillary and mandibular derivatives (orange). The hyoid (hy) arises from r4 (purple). (C) Neural crest cells produce skeletal elements such as the premaxilla (pm), nasal capsule (nc), nasal bone (na), frontal (f), palatine (p), dentary (d), and Meckel's cartilage (Mk) [based on (3, 4,23)]. (D and E) Beaks of ducks and quails are morphologically distinct. Scale bars, 2 mm. (F) Quail and duck embryos stage-matched for surgery at HH9.5 subsequently diverge in stage because of their different rates of maturation.

To test this hypothesis, we exchanged neural crest cells of the presumptive beak region between quail and duck (Fig. 1, D and E). Our approach exploits three properties that separate these species. First, quails have beaks that are short, narrow, and convex in comparison to the long, broad, and flat bills of ducks. Such traits offer a direct way to establish whether facial structures that result from our transplants more closely resemble the donor or host. Second, quail and duck embryos have considerably different rates of maturation (Fig. 1F). If donor cells maintain their timetable for development in the host environment, then we can determine the extent to which neural crest regulates gene expression in other tissues involved in facial patterning. Third, quail cells can be detected on the basis of a ubiquitous nuclear marker not present in ducks. This allows donor and host-derived structures to be distinguished.

Neural crest cells fated to give rise to the beak (3) were grafted from either quail to duck (“quck”) or duck to quail (“duail”) (Fig. 1, A to C) (11). Donor and host embryos were incubated until each reached Hamburger and Hamilton (HH) stage 9.5 (11, 12). Precise boundaries of excised tissue were based on morphological landmarks (13) as well as molecular markers. Donor graft tissue was inserted into a host with the corresponding region of tissue removed. For controls, we performed grafts within each species as well as sham operations.

Our results demonstrate that neural crest cells provide patterning information for beak morphology. Not only do neural crest cells direct their own morphogenesis, they also pattern non–neural crest beak tissues in a manner characteristic of the donor species (Fig. 2). When transplanted into duck embryos, quail neural crest cells give rise to beaks like those found in quails (n= 6). The use of an anti-quail antibody (11) reveals that the extent to which beaks are transformed in both size and shape depends on the location and distribution of quail donor neural crest cells in duck hosts. In complete transformations, quail donor cells are found throughout the jaw, nasal capsule, and orbital region (fig. S1, A and B). In less transformed cases, quail cells are localized to distal portions of the beak. Reciprocal transplantations of duck presumptive upper beak neural crest into quail hosts lead to analogous transformations (n = 3). The bills of chimeric duails resemble those of ducks in shape and relative length and are composed of elements derived primarily from duck donor neural crest (fig. S1, C and D).

Figure 2

Transforming beak morphology by swapping neural crest cells. (A) Quail neural crest cells generate quail-like beaks in duck hosts. Note the webbed foot of the quck. (B) Reciprocal transplantations of duck presumptive upper beak neural crest into quail hosts lead to analogous transformations. (C to F) These transformations are seen in sagittal sections (see also fig. S1) and affect elements throughout the jaw, nasal capsule (nc), and orbital region (ey) such as the premaxilla (pm), prenasal process (pp), dentary (d), and Meckel's cartilage (Mk). Trigeminal (V) sensory neurons, which are normally widespread along the surface of the duck bill, are reduced along the dorsal margins of the quck beak. In the duail, they are distributed like that normally observed in ducks. Non-neural crest tissues such as the egg tooth (et) and nasal passage (np) are also modified. Scale bars: 2 mm in (A) and (B); 1 mm in (C to F).

The central regulatory role that neural crest cells play in determining beak morphology was further demonstrated by our finding that donor neural crest cells also transform host structures. Normally, the duck egg tooth is a flat epidermal nail at the tip of the bill, whereas the quail egg tooth is a conical body of hard keratin (14). The quck egg tooth, despite being derived from nontransplanted host tissue, resembles that found in quails. The duail egg tooth has acquired a phenotype reminiscent of the duck. Instead of being thin and leathery like that in ducks, quck beak epidermis is thick and rigid like that of quails. Other non–neural crest derivatives appear quail-like in qucks. The nasal epithelium closely contours the neural crest–derived nasal capsule cartilage, which is dome-shaped in contrast to being flattened as in ducks. Additionally, the external nasal openings, which in ducks are within the keratinized portion of the bill, are relocated near the distal tip of the beak, which is characteristic of quails. The nasal passages, however, maintain a slightly duck-like sigmoidal shape.

Morphometric analyses confirm our qualitative observations that quck beaks are similar to those of quails while duail bills resemble those of ducks. We quantified changes in beak morphology by employing a landmark-based approach used previously to compare breeds of dogs and wild canids (11, 15). Landmark points in the upper beak were evaluated with resistant-fit theta-rho analysis (RFTRA), which provides a distance coefficient summarizing vector differences from pairwise comparisons (fig. S2). We used these distance coefficients in a cluster analysis to reveal trends in the morphometric data. On the basis of overall similarity in beak morphology, quail and quck embryos cluster together and are distinct from the group that includes duck and duail embryos (Fig. 3).

Figure 3

Morphometric analysis of beak shape. UPGMA (unweighted pair group method with arithmetic mean) cluster analysis summarizes quantification of beak shape in quails, in ducks, and in chimeras resulting from neural crest transplants. This tree was generated from a matrix of RFTRA distance coefficients representing vector changes in shape (fig. S2). Specimens are grouped on the basis of overall similarity, and branch lengths represent amounts of shape change among specimens.

The molecular basis for these beak transformations is revealed by our discovery that donor neural crest cells execute autonomous molecular programs and regulate gene expression in adjacent host tissues such as facial ectoderm (11). We focused on genes that are involved in facial patterning and also exhibit well-defined periods of expression (16–18). Although control and chimeric embryos were incubated for equal amounts of time following surgery, they exhibit distinct stage-dependent patterns of gene expression due to their different maturation rates (Fig. 1F).Barx1 and msx1 are detected in neural crest mesenchyme of control quails but not control ducks (Fig. 4, A to F). These genes are expressed in quck mesenchyme derived from quail but not in mesenchyme derived from the duck host (fig. S1E). Control quails express shh but notpax6 in facial ectoderm (Fig. 4, G to L). Control ducks express pax6 but not shh. In quck chimeras, we detect shh in duck host facial ectoderm but notpax6, which is the pattern observed in quails. These temporal shifts in the initiation and cessation of gene expression provide evidence that quail neural crest cells create quail beaks on ducks by maintaining their own molecular programs and by altering patterns of gene expression in non–neural crest host tissues.

Figure 4

Neural crest cell–autonomous molecular programs and regulation of gene expression in host facial ectoderm. (A to F) Twenty-four hours after surgery, control quails express barx1 (green) and msx1 (orange) in neural crest (arrows) of the maxillary (mx), mandibular (ma), and hyoid (hy) primordia, but control ducks do not yet express these genes. Quck chimeras at a stage equivalent to control ducks expressbarx1 and msx1. Expression of these genes was not detected in quck hyoid cells (asterisks). (G toL) Forty-eight hours after surgery, control quails expressshh (yellow) but not pax6 (pink; arrows) in facial ectoderm (fe). Control ducks express pax6 (arrows) but not shh. In quck chimeras stage-matched to control ducks, shh is detected (arrows) in duck host facial ectoderm but pax6 (asterisk) is not. fb, forebrain neuroepithelium. Scale bar, 200 μm.

This study demonstrates that patterning information for beak morphology comes from the neural crest. Our results are consistent with previous grafting experiments, but they add mechanistic insights by incorporating cellular and molecular analyses. Classic transplants between salamanders and frogs, which suggested that neural crest influences jaw morphology (19, 20), were limited by the lack of a technique to identify all derivatives of donor neural crest. Transplants between quails and chickens, which have been invaluable for studying embryogenesis, have revealed little in the way of interspecific patterning because of the morphological similarities and comparable rates of growth between these birds. Grafting experiments in chickens and ducks have shown that chimeras can be produced with the eyes, forebrain, and face of the donor species (21). In these surgeries, however, the entire rostral head, including the neuroepithelium, paraxial mesoderm, neural crest, and facial ectoderm, was grafted between chicken and duck embryos, which precluded the possibility of discerning the specific effects of any one tissue on the establishment of beak morphology. Moreover, these experiments examined motility and hatching behavior, so there were minimal evaluations of resulting facial transformations. By design, our experiments have allowed us to understand the role of neural crest cells in generating interspecific beak morphology and to determine the extent to which they influence nontransplanted host tissues.

Beak evolution likely involves changes to the neural crest, changes in the regulation of surrounding tissues by the neural crest, and changes in the regulation of neural crest cells by surrounding tissues. In our experiments, the host environment may facilitate beak transformations by providing inductive signals that activate certain species-specific programs intrinsic to donor neural crest. Such a mechanism was anticipated by Hans Spemann's 19th-century work on inductive interactions of tissues. Spemann explains that after an interspecific transplant, the responding grafted tissue says to its inducer, “you tell me to make a mouth; all right, I'll do so, but I can't make your kind of mouth; I can make my own and I'll do that” (22). Thus, in terms of beak evolution, the neural crest appears to act as a conduit through which species-specific adaptations are implemented, and in this capacity neural crest cells may play an essential role by serving as responsive targets of natural selection.

Supporting Online Material

www.sciencemag.org/cgi/content/full/299/5606/565/DC1

Materials and Methods

Figs. S1 and S2

References and Notes

  • * To whom correspondence should be addressed. E-mail: helms{at}itsa.ucsf.edu

REFERENCES AND NOTES

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