Sequence of ossification in the skeleton of growing and metamorphosing tadpoles of Rana pipiens.

The order o f ossification of bones in the skeleton of Rana pipiens during larval growth and metamorphosis has been determined from observations on specimens fixed in 70% alcohol and stained with alizarin red S . The axial skeleton ossifies i n a generally cephalo-caudal sequence, beginning with the parasphenoid bone at Taylor-Kollros stages IV-IX, followed by vertebrae (V-IX) and then the urostyle (IX-XIV). Exoccipitals (VII-IX), frontoparietals (XI-XII) and prootics (XIII-XVII) are additional cranial bones which successively ossify before metamorphosis. With the onset of metamorphosis a t stage XVIII jawbones and rostra1 bones of the skull ossify in the following succession: premaxilla, maxilla, septomaxilla, nasal, dentary, angular, squamosal, pterygoid, prevomer, mentomeckelian, quadratojugal, palatine, columella, posteromedial process of "hyoid." The sphenethmoid does not ossify until after metamorphosis. Ossification of limbbones begins with the femur or humerus at stages X-XI1 and progresses proximo-distally to the phalanges by stages XIII-XV. Carpals, however, do not ossify until stage XXV or after metamorphosis. The ilium of the pelvic girdle begins to ossify at stages X-XII, but the ischium is delayed until stages XX-XXIII. Scapula and coracoid of the pectoral girdle undergo initial ossification at stages XIIXIV, suprascapula and clavicle at stages XIII-XV. The sternum does not begin to ossify until stage XXIV. The possible role of thyroid hormones in stimulating osteogenesis is dicussed. Bone is a vertebrate tissue which develops after larval digerentiation of connective tissue and cartilage. Once osteogenesis commences, bones ossify in a sequence which is characteristic for the species. While studying the influence of thyroxine on development of limb and skull bones in Rana pipiens (Kemp and Hoyt, '65a,b,c), we became aware of the need for a reliable reference on the chronology of ossification in this species. This paper is designed to provide such a reference by correlating skeletal development with the developmental stages of Taylor and Kollros ('46 j . We also wish to discuss the general problem of sequential morphogenesis of bones. Skeletal development in anuran amphibians has been treated extensively in publications by Parker (1871, 1876, 1881), Gaupp (1896, 'OS), Kingsley ('25), Erdmann ('33), DeBeer ('37) and Goodrich ('58). Most of the published work on development of the frog skeleton has dealt with the European species Rana fusca (synonym J. MORPW., 129: 415-444. temporaria) or Rana esculenta, although Parker ( 1881 has compared developing skulls of several anurans, including Rana pipiens. Erdrnann's ( '33) careful study of the order of ossification of skeletal elements both in Triton vulguris and in Rana temporaria provided the basis for DeBeer's ('37) account of the sequence of osteocranial differentiation in Rana. Sequential ossification of skull bones has also been reported for two hylid species, Pseudacris nigrita triseriuta (Stokely and List, '54) and Hyla septcntrionalis (Trueb, '66). Ossification of the skeleton of Rana pipiens begins in the parasphenoid bone at stages IV-IX (Kemp and Hoyt, '65a,b) and progresses cephalo-caudally along the vertebral column. Limb bones ossify in proximo-distal sequence, starting with the femur (Kemp and Hoyt, '65c) or the humerus at stages X-XII. Jawbones develop in medio-lateral sequence during 1 Supported by research grants from the National Science Foundation (GB 4317) and the U. S. Public Health Service (GM 05867-10).