Development of the Tibial1 Ti1 Pathway

A. Grasshopper Limb Development

Grasshopper embryogenesis is divided into stages based on the percentage of development. Embryos develop roughly 5% per day with the earliest born neurons in the peripheral nervous system (PNS) and central nervous system (CNS) appearing at approximately 30% of development. The Ti1 pathway is established from 30 to 35% of embryonic development. During this time, the developing limb bud also undergoes molecular and morphological changes. At 30% of development, during the time when the Ti1 cell bodies are differentiating and delaminating from the distal tip of the limb bud, the limb bud consists of an epithelial tube that is loosely filled with mesoderm. A basal lamina separates the epithelium from the mesoderm. At this stage, the mesoderm is relatively undif-ferentiated, consisting largely of various cell specific precursors (e.g., muscle precursors) and hemocytes. The epithelium is rapidly dividing at this stage and is unsegmented. Over the next 5% (24 h) of embryonic development, as the Ti1 pathway is established, muscle cells begin to differentiate (Ho et al., 1983), limb segmentation proceeds, and an abundant heterogeneous basal lamina is produced (Anderson and Tucker, 1988, 1989).

B. Anatomy of Ti1 Neurons

Neurons are highly polarized cells whose cell bodies may lie great distances from their terminal processes. As a result, neurons have specialized compartments. When a neuron differentiates from the surrounding epithelium, it consists solely of a cell body. During axonogenesis, the plasma membrane extends asymmetrically to generate a growth cone and axon. The growth cone is the most distal structure of the neuron; it is highly motile and "reads" the surrounding environment for directional cues. Emanating from the growth cone are long finger-like processes called filopodia (Fig. 1A) and dynamic veil-like membranous protrusions called lamellipodia, which lie between individual filopodia. Elongation of the axon occurs as the growth cone extends and the most proximal region of the growth cone is converted to an axon. In general, the growth cone exhibits a consistent size, although predictable changes occur in growth cone shape and size depending on the substrate encountered (O'Connor et al., 1990). Not surprisingly, the growth cone and the axon contain distinct cytoskeletal elements. The relatively immobile axon consists mainly of highly packed microtubules (Fig. 1A; Sabry et al., 1991), whereas the highly motile growth cone is composed of both actin filaments and microtubules (Fig. 1A; Sabry et al., 1991; O'Connor and Bentley, 1993). Filopodia and lamellipodia are composed of F-actin bundles (O'Connor and Bentley, 1993). In the central core of the growth cone, microtubule bundles extend from the axon shaft (Fig. 1A). Unlike the tightly packed array of microtubules in the axon, microtubules are splayed out in the growth cone and often form transient microtubule loops that may extend into lamellipodia (Sabry et al., 1991).

C. The Path of Til Pioneer Neurons: From the Periphery to the Central Nervous System

Consisting of two neurons, the Ti1 pathway is established early in development when the limb bud is relatively undifferentiated. Ti1 neurons are considered to be pioneer neurons because they are the first peripheral neurons to establish a projection into the CNS. The growth cones of migrating Ti1 axons make contact with several substrates, including the basal lamina (Anderson and Tucker, 1988; Bonner and O'Connor, 2001), intrasegmental epithelium, segment boundary epithelium, and several intermediate neuronal targets (Keshishian and Bentley, 1983; O'Connor, 1999). At 30% of development, Ti1 cell bodies delaminate from the epithelium and initiate axon extension toward the CNS. Ti1 cell bodies are located at the distal tip of limb epithelium when they first initiate axons. As the morphology of the limb changes and the segmental boundaries are generated, the cell bodies typically end up in the tibia, just distal to the femur-tibial segment boundary (hence the Ti1 designation; Fig. 1B). Much of the pathway is pioneered along epithelium that forms the femur limb segment. As the Ti1 growth cones extend along the midfemur, they may contact a neuronal guidepost cell, the Fe1 cell, although often this cell has not differentiated at this stage. After proximal extension across the femur, Ti1 growth cones contact another preaxonogenesis neuron, the Tr1 cell, which is located in the trochanter epithelium. After contact with the Tr1 cell, the Ti1 axons make an abrupt ventral turn (Fig. 1B). Near the ventral midline of the limb bud, the growth cones contact another intermediate target, the Cx1 preaxonogenesis neurons, which lie proximal to the trochanter limb segment in the coxa limb segment. Upon contact with the Cx1 neurons, the Ti1 axons cease

Grasshopper Limb Bud Axon

Fig. 1 Schematic representation of a growth cone and the Ti1 pioneer neuron pathway. (A) The growth cone extends from the distal axon shaft and consists of lamellipodia and filopodia, which are motile. The axon shaft, as well as the proximal growth cone, is filled predominantly with microtubules (white), whereas the filopodia and lamellipodia consist of actin filaments (gray). (B) The Ti1 pioneer pathway in the developing grasshopper limb bud is depicted at approximately 34% of development. Limb segments and the axis are given; details on the pathway are found in the text. Do, dorsal; Di, distal; P, proximal; V, ventral.

Fig. 1 Schematic representation of a growth cone and the Ti1 pioneer neuron pathway. (A) The growth cone extends from the distal axon shaft and consists of lamellipodia and filopodia, which are motile. The axon shaft, as well as the proximal growth cone, is filled predominantly with microtubules (white), whereas the filopodia and lamellipodia consist of actin filaments (gray). (B) The Ti1 pioneer pathway in the developing grasshopper limb bud is depicted at approximately 34% of development. Limb segments and the axis are given; details on the pathway are found in the text. Do, dorsal; Di, distal; P, proximal; V, ventral.

ventral extension along the trochanter and turn proximally to extend into the CNS (Fig. 1B; Keshishian and Bentley, 1983; Bentley and O'Connor, 1992.) Later in development the Ti1 pioneer pathway serves as a scaffold for the axons of sensory neurons that are born in the periphery. Distinct from pioneer neurons, these later arising neurons fasciculate with the established Ti1 pathway to gain entry into the CNS (Klose and Bentley, 1989; Wong et al., 1997; O'Connor, 1999).

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