Segmental identity in the python axial skeleton

Pythons have over 300 vertebrae (Fig. 18a), and ribs are present on every segment with the exceptions of the atlas and the caudal vertebrae of the tail (Fig. 18b, d). The morphology of the ribs is approximately uniform between the atlas and the leg/cloacal level, where the ribs abruptly change from long movable structures, to short, forked ribs (lymphapophyses) which are fused to the vertebrae (Fig. 18c). These lymphapophyses contain dorsal and ventral processes which protect the lymph hearts. The transitional vertebra between the free ribs and the lymphapophyses may represent the vestige of a sacral vertebra (Fig. 18d; Gadow, 1933), although a distinct sacral vertebra is not present (Romer, 1956). Posterior to the forked lymphapophyses-bearing series, the vertebrae lack true ribs but have short transverse processes, and are typically referred to as the caudal or tail vertebrae (Fig. 18d). Presence of ribs on all vertebrae anterior to the caudal series suggests that snakes may have undergone anterior and posterior expansion of thoracic identity along the axial skeleton, resulting in what appear to be serial homologues, but are actually secondarily similar structures. The anterior vertebrae do not show any clear transition in regional identity which, in other vertebrates, would indicate forelimb position.

The relationship between Hox gene expression and segmental identity was investigated in the snake axial skeleton in order to determine whether these morphological changes are associated with altered patterns of Hox gene expression. Whole-mount antibody staining of Python molurus

H0XC6, H0XC8 and H0XB5 (Oliver et al., 1988a, Oliver at al., 1988b, Shashikant at al., 1995, Wall at al., 1992). The restricted transverse band of H0XC8 expression seen in the chick thorax (Fig. 19a, b), was not seen in the python, but instead H0XC8 expression was found to extend posteriorly from the anterior limit of the vertebral column (Fig. 19e) throughout the trunk (Fig. 19f). A precise posterior limit of expression can not be determined, but H0XC8 is expressed throughout the lymphapophysis-bearing vertebrae and expression fades posterior to the leg bud in the tail. H0XC6 and H0XB5 expression was examined in the trunks of embryos which had the limbs and head removed for other experiments. H0XC6 expression was detected in a segmental pattern in somites along the entire trunk (Fig. 19g), which contrasts with the restricted thoracic pattern of expression seen in the chick embryo, where the anterior boundary of expression marks the level of the forelimb (Fig. 19c). H0XB5 is expressed in the somites and dorsal root ganglia along the entire trunk (Fig. 19h), which is similar to the expression pattern seen in the chick (Fig. 19d) and mouse (Wall at al., 1992). Thus, both H0XC6 and H0XC8, which are expressed exclusively in the thoracic region of the chick axial skeleton, are expressed throughout the python trunk. H0XB5, which is expressed up to a cervical level in chicks, is also expressed throughout the python axial skeleton. Expansion of rib-bearing vertebrae along the python body axis is tightly correlated with expansion of Hox gene expression domains characteristic of thoracic identity.

Figure 18. Morphological pattern in the python axial skeleton

Alcian blue and Alizarin red stained skeletal preparation of a spotted python embryo at 24 days of incubation. Hindlimb vestiges have been removed to improve visibility of the vertebrae. Anterior is to the left in b-d.

a. Lateral view of complete skeleton, b. High power ventral view of the anterior axial skeleton and base of skull. Note the absence of ribs on the atlas (at). White arrowhead indicates hypapophysis extending ventrally from vertebral body. c. Lateral view of posterior axial skeleton. Arrows indicate lymphapophyses in cloacal region. Note the morphology of the rib on the transitional vertebra (t) that separates the vertebrae with large, movable ribs (left) and the lymphapophyses-bearing vertebrae (right), d. Dorsal view of posterior axial skeleton, showing clear morphological regionalization, d = thoracic/"dorsal" vertebrae, t = transitional vertebra, arrows = lymphapophyses-bearing vertebrae, and c = caudal vertebrae.

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Figure 19. Comparative Hox gene expression in python and chick embryos.

Whole mount antibody staining to show Hox gene expression patterns in stage 25 chick embryos and Python molurus embryos at 1 day of incubation. Anterior is at the top in a-e and g, left in f, and right in h a. Skeletal preparation of a 10 day chick embryo to show the pattern of regionalization of the axial skeleton and position of the limbs, b-d. Expression of H0XC8 (b), H0XC6 (c) and H0XB5 (d) in stage 25 chick embryos. Arrows indicate anterior and posterior expression boundaries, e. Lateral view of anterior trunk and head of python embryo stained with H0XC8 antibody. Note expression extends to the anterior limit of the axial skeleton (arrow), f. Low power view of the embryo shown in panel e. Expression of H0XC8 is detected posterior to the anterior boundary, extending throughout the entire trunk and posterior to the leg bud (arrow with *). Expression fades in the tail,

g. H0XC6 expression in a python embryo. Expression is detected throughout the entire trunk posterior to the head (head removed from this specimen), in a similar pattern to H0XC8. h. H0XB5 expression in a python embryo. H0XB5 expression is detected throughout the python trunk.

H0XC8 H0XB5

HOXC8