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Proximal and distal cell populations that broadly relate to the protopodite and telopodite in the developing limb have the ability to sort themselves from one another based on positional signalling cues. It has been shown that both hth and Dll contribute to this process. Mitotic cell clones that express hth ectopically in the leg can repress Dll and dac. These hth expressing clonal cells migrate towards the protopodite and body wall. In mitotic protopodite cell clones that have lost hth, the proximal leg segments Although the trochanter is considered part of the protopodite by Kukalova-Peck. See Haas, M. S., Brown, S. J. and Beeman, R. W. (2001) 'Homeotic evidence for the appendicular origin of the labrum in Tribolium castaneum', Dev Genes Evol 211(2): 96-102.
fuse. Cell that ectopically express Dll in the proximal part of the limb primordia are excluded from this part of the limb, while cell clones that have lost Dll function cannot remain in the developing leg and migrate out of the leg appendage primordia (Wu and Cohen, 1999; Angelini and Kaufman, 2005; McKay et al., 2009).
Dll is important for both the initial specification of leg primordia (which includes the protopodite) and for differentiating the telopodite from the protopodite in Drosophila. McKay et al. (2009) determined a high resolution cell fate map of the Drosophila leg primordia. The authors showed that the activity of Dll is dependent on several different Dll enhancers at different stages of leg development in the fly embro and is responsible for both telopodite and protopodite progenitors (McKay et al., 2009).
One of these enhancers is Dll304. Cells that have Dll expression activated by the Dll304 enhancer are multipotent and will give rise to both protopodite and telopodite cell populations. Dll304 is activated by wg (providing anterior-posterior positional information) and restricted by Dpp dorsally and by EGFR signalling ventrally. Another enhancer, the LT enhancer (leg trigger enhancer) is activated in cells that give rise to the telopodite, and is activated by both Wg and Dpp (Estella et al., 2008; McKay et al., 2009).
McKay et al. (2009) concluded that Dll304 is responsible for all leg cell populations including the protopodite. Lineage restriction of cell populations between the protopodite and telopodite occurs when the LT enhancer is activated. Cells that express Dll via the LT enhancer become the telopodite. Cells that had activated Dll304 but not the LT enhancer assume protopodite identity.
The trochanter presents an unusual situation. As noted above, the trochanter is marked by both Dll expression and hth-nExd expression late in leg formation. Cells from both the protopodite and the telopodite can contribute to the trochanter.
Therefore the authors note that the protopodite-telopodite division does not constitute a classical definition of a compartment boundary as there is a region, the trochanter, that can incorporate both protopodite and telopodite cell lineages (McKay et al., 2009).
It would be of considerable interest to determine whether a similar division of protopodite and telopodite cell populations occurs in other arthropod taxa. Currently, there are no species that can be practically studied in such detail outside of Drosophila.
But the potential is there to uncover a conserved molecular protopodite-telopodite division mechanism. An understanding of such mechanisms would greatly aid our attempts to homologize limb segments between different limbs and different arthropod taxa, particularly if the protopodite/telopodite division mechanism was studied in a biramous appendage. This would unambiguously relate the protopoditetelopodite cell lineages to the morphological definition of the protopodite and telopodite.
The use of Dll expression as a telopodite marker and the resolution of the gnathobasic or telognathic mandible controversy Studies have used Dll as a marker for the telopodite and have been able to resolve a controversy concerning the gnathobasic or telognathic nature of the mandible in different arthropod groups. The question concerned the mandibular biting edge and whether it had evolved from either the protopodite or the telopodite of the ancestral appendage. Manton hypothesized that the mandible of hexapods and myriapods had evolved from the tip of the ancestral limb, the telopodite, and was therefore telognathic. Whereas the mandible of the crustaceans was less controversially considered gnathobasic, as in numerous representatives of crustaceans the biting edge is present on the base of the mandibular apppendage with the telopodite attached to the protopodite as a palp (Manton, 1964; Manton, 1977).
Manton’s interpretation has been shown to be incorrect through the study of Dll expression which is expressed in the distal tips of developing appendages (Panganiban et al., 1994; Panganiban et al., 1997), but not the developing mandibular appendage (Niwa et al., 1997; Popadic et al., 1998; Scholtz et al., 1998). Dll expression is typically lacking in the mandibular segment of myriapods and hexapods which demonstrates that the functional biting edge of the mandible has a protopodal, and therefore gnathobasic origin, confirming Snodgrass’ gnathobasic interpretation of the insect and myriapod mandible (Snodgrass, 1938; Snodgrass, 1950). The lack of a palp in insects and myriapods is therefore interpreted as a loss of the telopodite, which is still present in numerous crustacean species. In these species, Dll is expressed in the developing palp of mandibles with palps, for example the amphipod Gammarus pulex and the mysid Mysidium columbiae (Browne and Patel, 2000).
1.8 Homology of anterior arthropod segments Hox genes One class of genes that are important for patterning segments in arthropods, and other segmented animals are the Hox genes. Hox genes play a central role in patterning segments and giving them their identity. Hox genes are master regulatory genes that activate downstream targets that pattern segments (Carroll et al., 2004).
Hox genes are segment patterning genes that are expressed in a co-linear manner along the anterior-posterior axis of diverse bilaterians. Hox genes are present in clusters, with varying degrees of organisation from highly compact non-interrupted clusters with genes present in the same orientation as present in vertebrates, less compact but still ordered clusters like in Tribolium (Brown et al., 2002a), to split clusters with genes present in different orientations as in Drosophila (Duboule, 2007).
The gene order (3’ to 5’) of the Hox genes in organised Hox gene clusters mirrors the order of expression from anterior to posterior segments. Posterior Hox genes have dominance over anterior Hox genes when co-expressed in the same segment.
Hox gene null mutants often have one or more segments changing their identity to that of another segment, a transformation known as a homeotic transformation. Homeotic transformations often result in the segment taking the identity of the adjacent anterior segment (Veraksa et al., 2000). In Tribolium, if a segment is lacking Hox gene expression altogether, one result that occurs is transformation of post-antennal appendages into antennal identity (Brown et al., 2002b).
Studies investigating the expression of Hox genes in Arthropods have revealed considerable similarities in anterior expression boundaries. These anterior boundaries have been used to homologize segments between taxa (Hughes and Kaufman, 2002a).
For example, the anterior expression boundary of the hox gene Deformed (Dfd) is expressed in the mandibular segment in all studied Mandibulates. The anterior boundary of Dfd in Chelicerates is expressed in the first leg segment. Considering the correspondence of the anterior boundaries of other Hox genes across different groups (see fig.1.11), the mandibular segment of mandibulates is homologous to the first leg segment of Chelicerates (Damen et al., 1998; Telford and Thomas, 1998a; Hughes and Kaufman, 2002a).
Fig.1.11. Expression of Deformed homologizes the mandibular segment to the first leg segment of Chelicerata. Figure is adapted from Hughes and Kaufman (2002a). The segmental abbreviations are as follows: ocular (Oc), chelicerae (Ch), pedipalps (Ped) and four Leg segments (L1-L4) opisthosomal segments (Op), antennal (ant), intercalary (Int), mandibular (Mn), first maxilla (Mx1), second maxilla (Mx2), maxilliped (Mxpd), labial (Lb), thoracic (T), abdominal (A), Telson (T).
Deformed (Dfd) expression is conserved in the mandibular and maxillary segments across mandibulates. Expression in centipedes also includes the 2 nd maxillary segment. Where Dfd function has been investigated, it is responsible for patterning the mandibular and maxillary segments (Mahaffey et al., 1989; Diederich et al., 1991;
Abzhanov and Kaufman, 1999a; Brown et al., 2000; Hughes and Kaufman, 2002a;
Hughes and Kaufman, 2002b; Rogers et al., 2002; Janssen and Damen, 2006).
There are other Hox genes as well as Dfd that are expressed in the mandibular segment of some mandibulates, such as Hox3 and pb (Shippy et al., 2000b; Hughes and Kaufman, 2002a; Hughes et al., 2004). Although these two genes are typically expressed in the mesoderm of these appendages and therefore are not responsible for patterning the appendage, as appendages are primarily patterned from the ectoderm.
However, to date there has been no discovery of a Hox gene that is responsible for the specific identity of the mandibular segment.
cap’n’collar differentiates the mandibular segment from the maxillary segment One gene that differentiates the mandibular segment from the maxillary segment, at least in Drosophila, is cap’n’collar (cnc). cnc is a basic Leucine zipper family gene (bZIP), members of which are found in all organisms and Orthologues of cnc are found throughout Bilateria.
If the mandible is a synapomorphy of Mandibulata, then genes such as cnc that specify the mandibular segment and differentiate the mandible from other gnathal appendages may be conserved in diverse mandibulate taxa, and not have such a role outside of mandibulates such as chelicerates.
The view from Drosophila genetics
An investigation into the genetics of the fruit fly is a prerequisite in any venture into the genetics of any arthropod. Much work has been done following the work done in Drosophila by comparing classes of developmental genes to other organisms, such as maternal genes, Gap genes, Head gap genes, Pair-rule genes, segment polarity genes, Hox genes and PD domain genes. It was therefore to Drosophila that I turned to in order to chose candidate mandibular patterning genes.
The Drosophila gnathocephalon and pseudocephalon
In a number of morphological respects, Drosophila larvae and adults like other cyclorraphous dipterans are derived (Grimaldi and Engel, 2005). Drosophila larvae are without a head (acephalic) and undergo a derived mode of development known as head involution which involves the anterior ectoderm invaginating and moving to the interior (Jurgens et al., 1986; Finkelstein and Perrimon, 1991; Rogers and Kaufman, 1997; Schinko et al., 2008). Adult Drosophila have lost the gnathal appendages of the mandible, and the maxilla is highly reduced. The feeding appendage is a proboscis derived almost wholly from the labial appendage (Merrill et al., 1987; Chadwick et al., 1990; Abzhanov et al., 2001).
Drosophila larvae do not have gnathal appendages (compare fig.1.12A with fig.1.12B) but do possess gnathal lobes, structures from which appendages are formed in other insects (compare fig.1.12C with fig. 1.12D). The mandibular, maxillary and labial gnathal lobes which form the gnathocephalon, develop into the pseudocephalon at the anterior of the embryo (see fig.1.12A). The pseudocephalon is surrounded by the cuticle from the first thoracic segment (Regulski et al., 1987).
While the gnathocephalon is homologous to the gnathocephalon of other developing mandibulate embryos, the pseudocephalon is highly derived and bears almost no resemblance to other mandibulate larval heads. There are, however, larval structures derived from the mandibular and maxillary segments which have been subject to genetic analysis and laser ablation studies to determine their segmental origin (Jurgens et al., 1986) (see fig.1.12A,C).
The axis of the flattened gnathal lobes has traditionally been described as ventral-lateral to dorsal in Drosophila genetics. The ventral-lateral to dorsal axis is the same as the proximal to distal axis described in other mandibulate arthropods.
Proximal refers to the base and distal refers to the tip of the developing appendage 9.