Confiabilidad de estudio

In light of these results, new pathways for paired fin and limb developmental evolution can be considered. Hox gene expression dynamics, the independence of Hox gene regulation in paraxial and lateral plate mesoderm, and the fact that morphological transformations can occur in one tissue without altering the other, raise the following possibilities: (a) that paired appendage evolution included a mechanism which staggered Hox gene expression along the lateral plate mesoderm. This is likely to have been achieved by modifying genes which regulate Hox gene expression, rather than the Hox genes themselves; and (b) that paired appendages originated without an obligatory reorganisation of the axial skeleton. Modular control of Hox genes, perhaps by tissue-specific regulatory genes, would enable decoupling of anatomical systems and freedom for variation to occur without inducing wholesale body plan transformations. This model can account for the idea that paired fins appear to have evolved before a regionalized axial skeleton (Carroll, 1988). Independent regulation of Hox gene expression would also allow the position of paired appendages to shift along the body axis without dramatic

reorganization of the axial skeleton (See Agar, 1907, Goodrich, 1930, Thorogood and Ferretti, 1993).

Prior to the evolutionary origin of vertebrates, Hox gene expression was central to regional specialization of the gut (Bienz, 1994, Roberts et al.,

1995). Hox gene expression is staggered in the tetrapod splanchnic mesoderm and is important for regional patterning of the developing gut (Boulet and Capecchi, 1996). Given that somatic and splanchnic mesoderm are both derived from lateral plate, one possibility is that staggered Hox

boundaries in lateral plate mesoderm appeared concomitantly with gut regionalization. Molecular regionalization of the body wall would therefore have provided differential positional values along the body axis before evolutionary emergence of paired fins. Differential Hox expression could have allowed spatially restricted activation of outgrowth signals such as FGF (Care et a/., 1996), and the resultant fins would have inherent differences in their positional identity at different loci along the body axis. The same signaling molecules could therefore operate in different contexts in anterior and posterior appendages, and the resultant differences in signal interpretation during development may explain morphological differences between anterior and posterior appendages. This is consistent with evidence from the fossil record which indicates that evolution of a stomach and pectoral fins was coordinated, and that pelvic and pectoral appendages evolved as morphologically distinct structures (Coates, 1994).

C H A P T E R F IV E : D e v e lo p m e n ta l A n a ly s is o f Limblessness and Axial Patterning in Python Embryos

1. Background

Development of forelimbs and hindlimbs can be induced in chick embryos by FGFs, and the identity of the limb is related to particular combinations of Hox gene expression in the lateral plate mesoderm along the main body axis (Chapters 3 and 4). In snakes, forelimbs and hindlimbs fail to develop and regional specialization of the axial skeleton has been lost, but the developmental basis for these changes is unknown. This chapter aims to investigate the changes that have occurred during evolution of snake development.

Number, pattern and identity of elements within the vertebral column can vary widely across vertebrates. A clear pattern of regionalization is usually evident, in particular among tetrapods (Gadow, 1933, Romer, 1966). In limbed reptiles, the pattern of vertebral identity is generally cervical

vertebrae at the anterior, followed by do rsal (in higher vertebrates subdivided into rib-bearing thoracic and rib-less lumbar), sacral and caudal

(Romer, 1956). Although dorsal vertebrae is the formal name for the vertebrae between the cervical and sacral series, to avoid confusing terminology, I will refer to dorsal vertebrae as thoracic in this thesis. Snakes have between 160 and 400 individual vertebrae, depending on the species, and classification of vertebral type other than "precaudal" versus "caudal" has proved to be a difficult and subjective task because of their apparent uniformity (Bellairs, 1969, Gadow, 1933, Gasc, 1976).

Hox genes are expressed in paraxial mesoderm of vertebrates with specific boundaries corresponding to morphological transitions (e.g., Hoxc6

Oliver et al., 1988a). The pattern of Hox gene expression across vertebrates with different vertebral formulae correlates with morphological identity of the vertebrae rather than with vertebral number (Gaunt, 1994; Burke at a!.,

1995). Gain and loss of function mutations often result in homeotic transformations, in which one type of vertebra is transformed into another.

Hoxc6 and Hoxc8 are expressed in restricted domains corresponding to the thoracic region of the vertebral column in frogs, chicks and mice (Awgulewitsch and Jacobs, 1990, Jegalian and De Robertis, 1992, Kessel, 1992, Oliver at a/., 1988a). Overexpression of Hoxc6 and Hoxc8 each result in anterior transformation of lumbar vertebrae to rib-bearing thoracic vertebrae (Jegalian and De Robertis, 1992, Pollock, Jay, and Bieberich, 1992). Hoxb5 is expressed in paraxial mesoderm with an anterior boundary at the first cervical vertebra (Wall at a/., 1992). Loss of function mutation in the Hoxbô gene causes anterior transformation of thoracic 1 to cervical 6 (Rancourt at a!., 1995).

The signaling molecules that maintain outgrowth and patterning of the limbs after limb initiation in mice and chicks are common to forelimbs and hindlimbs, and the structures that they specify are determined by the context of gene expression in which they operate. The common mechanisms of forelimb and hindlimb development are reflected by several mutations which effect development of both limbs (e.g.. Extra toas, Laglass and Strong’s Luxoid) (Chan at a/., 1995, Masuya at al., 1997, Singh at al., 1991). A number of mutations, however, effect only forelimbs (e.g., Wingless, TBX3

/ulnar-mammary syndrome and TBX5 /Holt-Oram syndrome), or only hindlimbs (e.g., Rim4), which indicates a level of independence in forelimb and hindlimb development (Bamshad at al., 1997, Li at al., 1997, Masuya at al., 1995, Zwilling, 1956b). The ability of forelimbs and hindlimbs to undergo morphological changes without affecting one another has allowed

vertebrates to evolve specializations restricted to one set of limbs.

Modularity during limb development has allowed some vertebrates to lose one set of limbs, as whales have done with hindlimbs and several Australian skinks have done with forelimbs (Greer, 1989). Complete loss of limbs may be caused by failure to initiate limb budding, and this could be related to higher-order changes in the primary body axis, such as loss of regional specialization which may eliminate localized inductive interactions at forelimb and/or hindlimb levels. This hypothesis is supported by the observation that limblessness is often associated with elongation of the body and loss of clear regionalization of the axial skeleton in the region of the lost limb. Most modern snakes have lost both fore and hindlimbs, and determination of regional identity of the vertebrae is difficult (Romer, 1956). Pythons and boas have retained vestiges of hindlimbs, however, suggesting that limb budding is initiated posteriorly but the cellular interactions within the bud have been disrupted. Anteriorly, however, the limb initiation signal appears to have been lost. To understand the developmental basis of the changes that have occurred during evolution of the snake body plan, expression of Hox genes, FGF and several transcription factors involved in chick and mouse limb development was studied in embryonic pythons.

2. Results

Most of the results described in this chapter come from work done on Burmese and Indian pythons. Python molurus molurus and Python molurus bivittatus. Duration of development is fairly uniform within the species; eggs are laid two months after mating, and they hatch after another two months (Ross, 1990). A few specimens of spotted python were used for analysis of skeletal development and scanning electron microscopy. Although the taxonomic classification of the spotted python has not yet been resolved

(either Python antaresia maculosa or Liasis maculosa), the skeletal morphology of the limbs is indistinguishable from the Molurus group. All results in this chapter refer to Python molurus unless otherwise specified.