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Scanning electron micrographs of Tribolium embryos Scanning electron microscopy (SEM) were taken of Tribolium to have higher resolution of mandibular morphology, to relate the inner and outer lobe found in other embryonic taxa to expression of the PD domain genes, endite. The developing mandible has two lobes perpendicular to the anterior posterior axis. The proximal lobe, the inner lobe, relates to the molar process and the distal lobe, the outer lobe, relates to the incisor process are distinguishable in the developing mandible (see fig.2.2).
Development of the mandibular endite as revealed by expression of Tc prd Tc prd, in addition to its role as a pair rule gene, is expressed in the endites during embryogenesis (Aranda et al., 2008). There are two domains of expression in the maxilla that relate to the two endites, the presumptive lacinia and galea (fig.
2.3C,D). These two endites fuse to form the ventral branch (fig.2.3.D,E). There is one domain of expression in the mandible which is significantly larger than the domains found in either the maxillary or labial appendages. There is one domain of Tc prd expression in the developing mandible, at all stages of embryogenesis (fig.2.3B-E).
Tc prd expression is present in the developing endites soon after formation of the limb buds (fig2.3B). Tc Dll is expressed in all limb bud primordia except the mandible (fig.2.3A). Tc Dll is also expressed in the developing proximal maxillary endite, the lacinia endite (fig.2.3C,D).
In very late stages, immediately prior to the formation of the cuticle, the single domain of Tc prd expression can be seen to encompass the entire gnathal edge including the developing incisor and molar processes (see fig.2.4A-E). The morphology of these structures clearly resembles the structure of the mandible present in the first instar larva (compare fig.2.4A-E with fig.2.4H-J). This result suggests that the single domain of paired expression is a valid marker for the entire mandible endite that includes the incisor process and the molar process. Tc prd expression includes the developing incisor (see arrow in fig.2.4E). The Tc prd expression domain does not appear to extend to the proximal boundary of the inner lobe (the developing molar).
Fig.2.2. Scanning electron micrographs (SEMs) of developing gnathal appendages of Tribolium embryos showing the the inner and outer lobes. All views are ventral with anterior to the left unless otherwise indicated. Inner lobe is indicated with a star. The outer lobe is indicated with an arrowhead.
The labial endite is indicated with an arrow in C. (A) Embryo at germ band retracting stage. Endites are visible on the maxillary appendage (La and Ga) and labial appendage (arrow). The labial appendages have not yet fused at the ventral midline. The inner and outer lobes of the mandible are faintly distinguishable. (B) (C) Lateral view of mandible, maxilla and labial appendages of an embryo at a similar stage to A. (D) Close up of mandibular limb bud with clearly distinguishable inner and outer lobes present. Anterior is bottom, lateral is to the right. Mandibular (Mn), maxillary (Mx) and labial (La) segments are indicated, as are the lacinia (Lc), galea (Ga), and maxillary palp (P).
Fig.2.3. Development of the mandible and maxillary endites in the gnathal appendages of Tribolium embryos as revealed by expression of Tc Dll and Tc prd. All views are ventral with anterior to the left unless otherwise indicated. The mandible is indicated with an arrowhead. (A) Germ band extending stage embryo after formation of the limb buds, which are marked by Tc Dll expression. (B) Later germ band extending stage embryo. Tc prd expression is visible in the mandible. Expression of Tc Dll and Tc prd is present the maxillary lacinia endite (asterisk). Tc Dll is expressed in the maxillary palp (arrow). (C) Fully germ band extended stage embryo. Both maxillary endite lobes, the mandibular endites and the labial are marked with Tc prd expression. Tc Dll expression is present in the lacinia endite lobe (asterisk) but absent from the galea (star). The mandible has one domain of Tc prd expression which is significantly larger than either of the maxilla endite expression domains. (D) Germ band retracting stage embryo, Expression of Tc prd and Tc Dll is maintained in the same domains as in C. (E) Very late stage embryo prior to hatching as a larva with Tc prd detected by in situ hybridisation. Tc prd expression is present in the mandibular endites (arrowhead) and the maxillary endites (arrow). The lacinia and galea have fused to form the ventral branch (arrow). (F) Cuticle preparation of a first instar larva. The four segmented maxilla palp (P) is attached to the protopodite which consists of two segments, the cardo (ca) and stipes (st). The labial appendage consists of two appendages that have fused protopodites. (G) Mandibles of a first instar larva visualized by fluorescent microscopy. The mandible is unsegmented, with the gnathal edge consisting of an incisor process and molar process. One condyle of the mandible is visible (asterisk).
Fig.2.4. Expression of Tc prd in the mandibular endite relates the inner and outer lobe to the incisor and molar processes. The shape of the mandibular limb bud at this stage resembles the shape of the mandible in the larva. The distal tip of the mandible forming the incisor process and the area at the proximal limit of Tc prd expression forming the molar process. Labrum (arrow), maxillae (arrowhead) and antennae (star) cover the mandibles. Gene expression was detected by in situ hybridisation. (A-E) Expression of Tc prd in the mandible endite of a very late stage embryo prior to hatching. Anterior is left, dorsal is up. (A) View of entire larvae. (B-E) Different focal planes of A at higher magnification. (B) Gnathal appendages of A. Mandibular endite (arrow) and maxillary ventral branch consisting of fused lacinia and galea (arrowhead) are marked by Tc prd expression. (C) Antennae, labrum and maxilla are visible, mandible is visible as a purple blur (out of focus). (D) Possible cardo/stipes boundary (arrow), cardo is marked as an arrowhead. (E) Expression is visible in the pointed distal tip, the presumptive incisor process, of the mandibular endite. (F,G) SEM of late stage embryos. (H) Cuticle preparation of a fourth or later stage instar larval head. The mandible gnathal edge is more developed than the first instar larva shown in I. (I) mandibles of first instar larva with the expression domain of Tc prd highlighted on the gnathal edge based upon the position of the expression domain from A-E (J) Diagram outlining the shape of both the mandible and maxilla from A with Tc prd expression marked in purple. The expression of Tc prd is in one domain which encompasses the majority of the gnathal edge.
Comparison of the expression patterns of Tc prd, the PD domain genes, and Tc wg in the endites of the mandible and maxilla suggest the mandible is composed of one endite.
The expression of Tc dac in the mandible relative to Tc prd is similar to that seen in the maxilla endite lobes (fig.2.5). The proximal domain of Tc dac is expressed in the distal half of each maxillary endites whilst Tc prd is expressed throughout each endite lobe (fig.2.5C,F,G). In the mandible limb bud, Tc dac is expressed in the outer lobe with Tc prd expressed more or less continuously through both inner and outer lobes (fig.2.5A-C,E). The similarity of the relationship of Tc dac expression to Tc prd expression in the maxilla endites suggests that the mandible has only one endite (fig
Tc wg is expressed in a stripe that runs through the middle of the ectoderm of all appendages (fig 2.6A,C). Tc wg expression retracts from the endites of the maxilla in developing embryos to form two gaps in Tc wg expression. In the mandible, only one gap in Tc wg expression develops which suggests that there is only one endite present (fig 2.6B,D).
Fig 2.5. Expression of Tc dac and Tc prd in the mandible and maxillary endites. All views are ventral with anterior to the left unless otherwise indicated. Gene expression was detected by in situ hybridisation. (A) Expression of Tc Dll and Tc prd in a germ band retracting stage embryo. (B) Expression of Tc dac and Tc prd in a germ band retracting stage embryo. There are two domains of Tc dac expression, a proximal domain (arrowhead) and distal domain (asterisk). In the legs, the distal domain on Tc dac is more strongly expressed than the proximal domain. In the gnathal appendages, the proximal domain is more strongly expressed than the distal domain. The mandible domain of Tc dac is likely to represent the proximal domain (Prpic et al., 2001). (C) Gnathal appendages of B (D) SEM of mandible limb bud with inner lobe (star) and outer lobe (arrowhead) and subcoxa/coxa boundary (arrow) visible. Anterior is to the bottom, lateral is to the right. (E-G) dissected mandibles and maxillae from germ band retracting embryos. (E) Expression of Tc prd and Tc dac in a dissected mandible. Lateral is to the right, distal is to the top. (F) Expression of Tc dac and Tc prd in the mandible. (G) Expression of Tc dac and Tc prd in a dissected maxilla. Ventral view with anterior to the top. (H) Diagram illustrating the expression domains of Tc dac and Tc prd in the mandible and maxilla appendages. Tc dac is expressed in the distal half of the maxilla endites and the outer lobe of the mandible. Tc dac expression overlaps with Tc prd in the distal half of the endites (arrowheads). Tc dac expression is lacking from the proximal half of the endites and the inner lobe of the mandible. These expression patterns suggest that the mandible consists of one endite, the outer lobe of which is homologous to the distal half of the endites marked by Tc dac expression. The inner lobe is homologous to the proximal half which is marked by Tc prd but not Tc dac expression.
Fig.2.6. Expression of Tc wg in the mandible and maxilla suggest the the mandible has one endite. All views are ventral with anterior to the left. Gene expression was detected by in situ hybridisation. (A) Fully extended germ band stage embryo. Expression of Tc wg is in the posterior of each segment and in the medial region of each developing appendage. (B) Germ band retracting stage embryo. Tc wg expression is continuous in the medial region of the legs. In the gnathal appendages, there are spots of Tc wg expression in the ectoderm of the protopodite of the gnathal appendages with gaps of expression in the endites. (C) Close up of the gnathal appendages of the embryo shown in A. Expression is continuous throughout the medial part of each appendage including the developing endites. (D) Close up of the mandible and maxilla of the embryo. There is Tc wg expression in the ectoderm adjacent to the endites in the mandible and maxilla. Tc wg expression is lacking in the mandibular endite (white arrowhead) and the maxillary endites (arrows).
Investigation of the expression of Tc prd in late stage Tribolium embryos shows that the inner and outer lobes that make up the mandibular endite develop into to the future incisor and molar processes. Study of the expression of the PD domain gene Tc dac, the endite marker Tc prd and Tc wg shows that the inner and outer lobes are derived from a single endite. The gnathal edge of the Tribolium mandible is therefore derived from one endite which disproves Machida’s hypothesis that the mandible incisor and molar processes are derived from two separate endites which are homologous to the maxillary galea and lacinia.
The mandibular inner and outer lobe are the developing molar process and incisor process respectively The developing mandibular appendage has two lobes which are visible in both SEMs (fig.2.2 and fig 2.5D) and in situ hybridizations of fixed Tribolium embryos (fig
2.5E). These two lobes are orientated perpendicularly to the anterior/posterior axis of the Tribolium embryo. The proximal or inner lobe relates to the molar process and the distal or outer lobe relates to the incisor process. The identity of these lobes has been suggested in other analyses.
This conclusion is confirmed in this study by the position of the expression domain of Tc prd in a late stage Tribolium embryo. Late Tc prd expression shows that the mandibular expression domain is in the developing incisor and molar processes (fig.2.4). The outer lobe is developing into the characteristic tooth morphology of the incisor process. Expression of Tc prd is present in the developing incisor process (fig.