Feather morphogenesis and feather pattern in normal and talpid3 mutant chick embryos

0 Author's address: Department of Zoology, University of Edinburgh , King's Buildings, West Mains Road, Edinburgh, 9, U.K. 5 EMB 25 1 Author's address: Department of Zoology, University College of Wales , Penglais, Aberystwyth, U.K 2 Author's address: Poultry Research Centre , King's Buildings, West Mains Road, Edinburgh, 9, U.K 3 By D. A. E D E \ J. R. HINCHLIFFE SUMMARY A comparative study of feather morphogenesis and the development of feather pattern in normal and talpid3 embryos has been carried out. The development of talpid* CAM grafts shows that the effect of the gene is autonomous in the skin. The most striking effect of the gene upon feather morphogenesis is the failure of normal feather germ condensations to appear within the dermis. This is reflected in the abnormal distribution of alkaline phosphatase through the dermis. Dermal cells within and between condensations are not orientated in the mutant as they are in normal embryos, probably owing to the same defect in cell behaviour which causes condensation failure in talpid3 precartilage mesenchyme. The role of dermal cell orientation and movement in generating the overall feather pattern is examined in both normal and talpid3 embryos. - The three talpid mutants of the fowl are characterized by widespread pleiotropic abnormalities of which the most remarkable is extreme polydactylism. All three talpid genes are autosomal recessive lethals and death of homozygotes usually occurs at 8-10 days (d) in talpid1, 10-14 d in talpid2 and at about 6 d in talpid3. The complex pattern of developmental abnormalities has been described in talpid1 by Cole (1942) and Inman (1946) and in talpid2 by Abbott, Taylor & Abplanalp (1960) and Goetinck & Abbott (1964). The development of talpid3 embryos has been described in detail by Ede & Kelly (1964a, b) and by Hinchliffe & Ede (1967, 1968), who found that the major abnormalities originated in the failure of precartilaginous mesenchyme to form distinct condensations on the normal pattern. Abnormalities in cell behaviour which probably account for this failure were found by Ede & Agerbak (1968) in studies on the reaggregation of normal and mutant cells in culture. Feather morphogenesis is arrested in all the talpid mutants. In talpid1 there is no sign of elongation of the feather papillae even at 17 d, and in talpid2 Abbott et al. (1960) report that 'feathering is retarded' at 10 d. In talpid* the appearance of papillae is retarded, they remain flattened and unelongated and their overall pattern is disrupted (Ede & Kelly, 19646). Since mesenchymal cell condensation plays a prominent part in the initial stages of feather formation the present investigation was undertaken in order to see whether the cellular abnormality that causes defects of condensation in precartilage mesenchyme might also account for the defective feather formation. Comparison of the mutant with normal development also throws light on problems of feather morphogenesis and feather pattern formation raised by Stuart & Moscona (1967), Wessells & Evans (1968) and Sengel & Rusaouen (1968). Living normal and talpid* embryos were fixed at 6-11 d in Bouin's or Carnoy's fluid, photographed and then used for whole-mount preparations of skin or serially sectioned, staining routinely with iron haematoxylin, haematoxylin and eosin and alcian blue/chlorantine fast red, and in some cases Masson's Ponceauacid fuchsin-light green or iron haematoxylin with van Gieson to show collagen more clearly. Since the majority of talpid3 embryos die at 6 d, before feather development has begun, material obtained from whole embryos was supplemented by material maintained to the required age as grafts on the chorioallantoic membrane (CAM) by the method of Hamburger (1960). Skin was removed from embryos at talpid3 1 7 36 21 4-}-5 d (1) from the back region, with underlying muscle and vertebral column still attached, and (2) from the flank area between the anterior and posterior limb, then grafted on to the chorioallantoic membrane of 8-10 d embryos. Graft material was sectioned and also used for the preparation of dermis whole mounts. The latter were made by placing the skin in Ca-Mg-free Tyrode External appearance of feather papillae Normal. On the back the first feather papillae appear at 7 d, in a single file directly over the line of the vertebrae in the posterior (saddle) region and in two files over the myotome muscles, one on each side of this line, more anteriorly, producing a tuning-fork pattern. Additional files of papillae are formed in sequence laterally, each new papilla arising equidistant from its nearest neighbours so that a series of equilateral triangles is marked out, forming eventually an arrangement of points on a rhomboidal lattice (Fig. 1 A). From 9 to l i d the papillae, starting with those nearest the mid-line and in sequence laterally, elongate in a caudal direction (Fig. IB) and barb-ridges are formed within them. On the flank the development of the papillae is similar but the first files, which appear ventrally, do not appear until 8 d. Feather papillae are formed in the same way in grafts but development is retarded by up to half a day and they do not elongate well. talpid2. The appearance of feather papillae is delayed and their progressive production in files from the mid-line is much less marked. There are none visible in embryos at 7 d or in most grafts at 8 d; in 8 d embryos and 9 d grafts there are broad flattened papillae over the whole area, arranged much less regularly than normal, and rather close together, at the points of a very roughly rhomboidal lattice (Fig. 1C). The boundaries of the papillae are less well defined than in normal embryos. At 10 d the appearance of the skin is not much altered since there is little elongation and no caudal orientation of the papillae (Fig. 1D). Development of talpid3 papillae on the flank is similar except that in this region they are more widely separated than in normal embryos and grafts. Fig. 1. Normal (A, B) and talpid* (C, D) embryos at 8 d and 10 d. Fig. 2. Transverse sections of embryos in mid-back region. (A) Normal 6 d, (B) talpid3 6 d (stained haematoxylin and eosin). (C-E) Normal 7 d, with increasing magnification of right-hand condensation region. (F-H) talpid31 d, with increasing magnification of right-hand condensation region. (Stained iron haematoxylin.) Histology of early feather development A. 6-11 days in back skin in embryos and grafts 6 days Normal (Fig. 2 A) and talpids (Fig. 2B) skin are almost indistinguishable at this stage. The epidermis consists of a single layer of cells which are not orientated in any particular way. Beneath it the dermal mesenchyme cells are loosely packed, not orientated, and there is no collagen present. The neural tube is sunk more deeply in the mesoderm in talpid3 than in normal embryos. 7-days Normal (Fig. 2C, D, E). Mesenchyme cells immediately beneath the epidermis show some orientation transvers (...truncated)