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Fig.3.5. Expression of Tc ser and the PD domain genes in the developing mandible, maxilla and leg appendages. Gene expression was detected by in situ hybridisation. All views are distal to the top and lateral to the right unless otherwise indicated. Expression of Tc ser and the genes Tc Dll, Tc dac, and Tc hth in dissected mandibles (A-C), dissected maxillae (D-H), Dissected legs (I-K). Domains of serrate are numbered 1 to 5 from proximal to distal. (A) lateral view of Tc ser expression in the mandible. There is a spot domain in the outer lobe (white arrowhead) and a subcoxa domain that is expressed proximal to the subcoxal groove visible in SEMs. (B) Expression of Tc ser (blue) and Tc dac (red) in a dissected mandible. Tc dac expression is strongest in the outer lobe and coxa of the mandible. (C) Expression ot Tc hth (blue) and Tc dac (red) in a dissected mandible. Tc hth is expression is faint in the inner lobe and stronger at the distal part of the inner lobe. Tc dac is expressed strongly in the outer lobe. There is faint expression in the distal part of the outer lobe in to which it appears the spot domain of Tc ser fits neatly (Arrowhead). (D) Expression of Tc ser in a dissected maxilla. (E) Expression of Tc ser (blue) and Tc dac (red) in a dissected maxilla. There are two domains of Tc dac: a proximal domain (star) and distal (arrow) domain. (F) Expression of Tc hth (blue) and Tc dac (red) in a dissected maxilla.
The cardo/stipes boundary in the larva is shown in fig.3.7G,H and also in a very late embryo shown in fig.2.4D from the previous chapter.
The expression of Tc ser is particularly faint and hard to interpret in the labial appendages. Tc ser appears to be expressed in the labial appendages in a manner similar to that in the maxilla, with a subcoxal-1 and coxal -2 domain (fig.3.4B,D and fig.3.3A). Labial appendages fuse during late embryogenesis.
There are two rings of Tc ser expression in the antenna (see fig.3.3A and fig.3.6). Tribolium first instar larvae have an antenna that is made of four segments in proximal to distal order, the antennifer, scapus, pedicellus and flagellum (Toegel et al., 2009). The two rings of Tc ser expression to appear first demarcate three segments.
The proximal ring is expressed in the distal part of the developing antennifer. The distal ring is expressed in the distal part of the developing scapus. It appears based on the morphology of the appendage that the flagellum develops after the other segments have formed. Expression of Tc ser has more complex expression in the antenna distal of the second Tc ser domain (fig.3.6B,C), the significance of which is not understood.
Fig.3.6. Expression of Tc ser and the PD domain genes in developing antennae. Gene expression was detected by in situ hybridisation. (A) Expression of Tc ser in a dissected antenna. (B) Expression of Tc ser (blue) and Tc Dll (red) in a dissected antenna. (C) Expression of Tc ser (blue) and Tc dac (red) in a dissected antenna. (D) Expression ot Tc hth (blue) and Tc dac (red) in a dissected antenna. These expression domains are illustrated E,F (E) Schematic of Tc dac and Tc Dll expression (F) Schematic of Tc dac and Tc hth expression in the antenna. Tc ser domains are numbered 1 and 3. There are addition Tc ser expression patterns distal to the second ring domain that appear after the second ring domain has formed. The significance of this expression is unknown.
Fig.3.7. Two proximal domains of Tc ser expression in the developing maxilla could relate to the future segment boundary between the cardo and stipes. It is not obvious where the cardo/stipes segment boundary develops in the embryonic maxillary appendage. The subcoxal-1 domain of Tc ser is clearly associated with a segment boundary. The cardo of the first instar larva is small and the segment boundary with the stipes is close to the base of the maxilla appendage. The appearance of a proximal-0 stripe suggests that this may form the boundary between the cardo and stipes. If this is the case, then the significance of the subcoxal-1 domain of Tc ser is unknown. All views are proximal to the top and lateral to the right. Gene expression was detected by in situ hybridisation. (A) Tc ser expression in a germ band retracted stage embryo. Tc ser is strongly expressed in the subcoxal-1 domain (arrow) and very faintly expressed proximal-0 stripe domain (star) more proximal to the subcoxal-1 domain. (B) SEM of a maxilla at a similar stage to that of A. The subcoxal/coxal boundary is indicated with an arrow. (C) Later stage maxilla from an embryo undergoing dorsal closure. The subcoxal-1 domain of Tc ser is strongly expressed (arrow). The proximal-0 stipe domain (star) is more strongly expressed than earlier stages. (D) SEM of a maxilla of a similar stage to C. The subcoxal/coxal segment boundary is visible and marked by an arrow. A smaller groove (star) is also visible and is in a corresponding position to the proximal stipe visible in the maxilla in C. (E,F) The subcoxal-1 domain of Tc ser is clearly associated with a cell boundary marked by an arrow. (G) Close up of a cuticle preparation from a first instar larval maxilla.
The segment boundary between the cardo and stipes is indicated with an arrow. A diagram of the outline of the maxilla is shown in (H).
Tc ser expression in the mandibular limb bud
There are two domains of Tc ser expression in the mandibular limb bud, a ring and spot domain (see fig.3.3C and fig.3.5A-C). The spot domain is present on the distal tip of the mandible limb bud, more specifically on the outer lobe of the developing endite that relates to the developing incisor process. Tc dac expression appears to be fainter in a similar region to the location of the spot domain (see arrowhead in fig.3.5C). The ring domain of Tc ser is situated proximal to the endite (see fig.3.4B-D), and lies proximal to the lateral furrow visible on the scanning electron micrographs of Tribolium embryos (compare fig.3.3C with fig.3.3D).
On the lateral part of the mandible, a groove can be seen below the incisor lobe (fig.3.2). The significance of this groove is not easy to determine as in the first instar larva there is no evidence of segmentation or of any groove present on the lateral sheath of the mandible (cuticle on the lateral side of the mandible) that corresponds to that position (see fig.2.4H,I from chapter two). In consideration of the expression of Tc ser expression in the developing mandibular limb bud, this structure is interpreted to mark the division of the mandible into subcoxa and coxa. The evidence in support of this interpretation is detailed below in later sections of this chapter.
Tc prd expression relative to Tc ser expression
Tc prd is expressed in the developing endites in the mandibular, maxillary and labial segments (see fig.3.3A). There is also expression of Tc prd in the developing mesoderm of the mandible, maxillary, labial and leg appendages (fig.3.3A). This expression is probably located to the developing muscles. Tc prd is expressed in the coxa segment, the distal segment of the protopodite. Expression of Tc prd is more distal to the subcoxal-1 Tc ser domain of the mandible, maxilla and labial protopodite (see fig.3.4B-D).
PD domain gene expression relative to Tc ser expression
In order to determine the identity of the ring domains of Tc ser expression in all post-antennal appendages, double in situ hybridizations of Tc ser with Tc dac and Tc Dll were performed (see fig.3.5, fig.3.8, fig.3.9 and fig.3.10).
In the leg, Dll expression clearly overlaps the third, fourth and fifth domains of Tc ser (fig.3.5I, fig.3.10A,E,F). These relate specifically to the trochanteral-3, femoral-4 and tibial-5 domains of Tc ser. Tc Dll is expressed in two domains, a ring and sock domain (seen clearly in fig.2.3D in chapter two). The ring domain overlaps with the trochanteral-3 Tc ser domain, the sock domain is co-expressed with the tibial-5 domain (see fig. 35I and fig.3.10A,E,F).
Tc dac has two domains of expression, a proximal domain in the protopodite and a distal domain in the medial region of the telopodite (see fig.3.5J and fig.3.9J).
The distal domain of Tc dac is coexpressed with the femoral-4 Tc ser domain. The proximal domain of Tc dac is co-expressed with the coxal-2 Tc ser domain. There is a spot of Tc dac expression that is present with the subcoxal-1 Tc ser domain.
Comparison of Tc hth to Tc dac expression shows that Tc hth is expressed in the subcoxal-1, coxal-2 and trochanteral-3 Tc ser domains of the developing leg (fig.3.5K).
Tc hth expression abuts Tc dac expression in the leg. There is no apparent overlap of expression of Tc hth and Tc dac. The distal domain of Tc dac expression relates to the femoral-4 ring of Tc ser expression in the leg and is expressed in the developing femur and tibia segments (fig.3.5J). The proximal domain of Tc dac expression, that relates to the coxal-2 ring of Tc ser expression, is visible well within the domain of Tc hth expression (which encompasses Tc ser ring domains 1-3). This is consistent with Tc hth being co-expressed with the trochanteral-3 domain of Tc ser expression.
In the maxilla of Tribolium, the proximal domain of Tc dac is expressed before the distal domain in all post-antennal appendages (see fig.3.8 and fig.3.9). Initially, Tc dac is co-expressed with the coxal-2 Tc ser domain (fig.3.9A). Later in development, the expression of Tc dac shifts proximally towards the subcoxal-1 Tc ser domain (see fig.3.9D,F,I). Tc dac is expressed in both endites, though significantly more strongly in the proximal endite, the lacinia, and expression forms a ring around the coxal-2 segment of the maxilla. There appears to be overlap of the proximal domain of Tc dac expression with the first subcoxa domain of Tc ser on the lateral side of the maxilla (see fig.3.9I and fig.3.5E). However, this could be an artefact of the manner in which the dissected maxilla was mounted, and reveals the limitations of bright field microscopy. If for example different regions of the maxilla, the subcoxa and coxa, overlap through the line of sight through the microscope, then mutually exclusive expression domains will appear to be co-expressed.
Tc hth can be related to Tc ser expression in the maxilla by comparison with Tc dac expression. Tc hth is expressed throughout the developing protopodite of the maxilla and extends into the proximal region of the palp abutting Tc dac expression (see fig.3.5F). Relating Tc hth expression to the Tc ser ring domains by consideration of the Tc dac distal domain in the maxillary palp (fig.3.5E) shows that Tc hth is expressed up to the trochanteral-3 domain of Tc ser expression. This is reminiscent of the expression of Tc hth and Tc dac in the leg appendage. Tc hth is therefore co-expressed with the third Tc ser domain in the maxilla, similar to the leg (compare fig.3.5F with fig.3.5K).
Tc hth is expressed throughout the mandible lobe but is weaker in the inner lobe and the proximal ventral medial region to the molar lobe. Tc hth is more strongly expressed in the distal part of the inner lobe (fig.3.5C).
Early expression of Tc ser and Tc dac In Drosophila, it has been shown that the first ser ring to appear is the coxa ser domain (Rauskolb, 2001). In order to compare the proximal rings of Tc ser in Tribolium, the onset of Tc ser expression was compared in different appendage types to the expression to the PD domain gene Tc dac (shown in fig.3.8, fig.3.9). The proximal domain of Tc dac is upregulated earlier than the distal domain of Tc dac (see fig.3.8B) and occurs in the gnathal appendages as soon as the limb buds form (see fig.3.8A).