The Tribolium castaneum ortholog of Sex combs reduced controls dorsal ridge development

Abstract

In insects, the boundary between the embryonic head and thorax is formed by the dorsal ridge, a fused structure composed of portions of the maxillary and labial segments. However, the mechanisms that promote development of this unusual structure remain a mystery. In Drosophila, mutations in the Hox genes Sex combs reduced and Deformed have been reported to cause abnormal dorsal ridge formation, but the significance of these abnormalities is not clear. We have identified three mutant allele classes of Cephalothorax, the Tribolium castaneum (red flour beetle) ortholog of Sex combs reduced, each of which has a different effect on dorsal ridge development. By using Engrailed expression to monitor dorsal ridge development in these mutants, we demonstrate that Cephalothorax promotes the fusion and subsequent dorsolateral extension of the maxillary and labial Engrailed stripes (posterior compartments) during dorsal ridge formation. Molecular and genetic analysis of these alleles indicates that the N terminus of Cephalothorax is important for the fusion step, but is dispensable for Engrailed stripe extension. Thus, we find that specific regions of Cephalothorax are required for discrete steps in dorsal ridge formation.

Figure 1.—

Dorsal ridge development in wild-type embryos. En expression in the dorsal ridge is indicated by an arrow in A–I. Relevant segments are labeled as follows: maxillary (Mx), labial (Lb), prothoracic (T1). (A–F) En expression (purple) in the wild-type dorsal ridge. (A) An extended germband-stage embryo viewed from the yolk side to provide a clearer view of Engrailed expression along the lateral edges of the labial segment. (B) As dorsal closure begins, a patch of En expression (apparently representing a fusion of the maxillary and labial En stripes) extends dorsally. (C) As dorsal closure continues, the dorsal ridge En stripe becomes significantly narrower than the other extending En stripes. (D) An enlarged view of the region boxed in A. (E) An enlarged view of the region boxed in B. (F) An enlarged view of the region boxed in C. (G–I) Cx expression (purple) and En expression (brown) in the wild-type dorsal ridge. (G) Early in the dorsal closure process, Cx is expressed in a subset of the En-expressing dorsal ridge cells. (H) An enlarged view of G showing the dorsal ridge cells that coexpress Cx and En (arrowhead). (I) As the dorsal ridge En stripe extends and narrows, Cx expression is confined to the posterior half of the En stripe.

Figure 2.—

Scanning electron micrographs of wild-type and Cx mutant Tribolium larvae. The boundary between head and thorax (approximated in cephalized mutants) is denoted by an arrowhead in A–L, and the labial appendages are indicated by an arrow in all ventral views. (A) Wild type, ventral view. (B) Wild type, dorsal view. (C) Cx⁶ homozygote, ventral view. Note the transformation of labial appendages to antennae (arrow) and the fusion of T1 with the head. (D) Cx⁶ homozygote, dorsal view. T1 is fused with the head dorsally as well as ventrally. (E) Cx²⁰ homozygote, ventral view. The phenotype of Cx²⁰ homozygotes is variable. In this mildly affected individual, the labial appendages are more posteriorly located than in wild type and only the most proximal segment of each appendage is fused. The extra dorsal segment between the head and T1 is not visible from the ventral side. (F) Cx²⁰ homozygote, ventral view. In a more severely affected individual, the labial appendages completely fail to fuse. The extra dorsal segment between the head and T1 (bracket) can be seen from the ventral side. (G) Cx²⁰ homozygote, dorsal view. Note the extra segment (bracket) between the head and T1. (H) Cx²⁰ hemizygote [Cx²⁰/Df(HOMC)], dorsal view. An extra segment (bracket) is present, although partly obscured by dorsal closure defects. (I) Cx^E homozygote, ventral view. The labial appendages are positioned laterally, in a similar orientation to the maxillary appendages. Note the triangular head shape. (J) Cx^E homozygote, dorsal view. A segmental groove is present between the head and thorax, but does not appear as deep as in wild type. (K) Cx^E hemizygote [Cx^E/Df(HOMC)], dorsal view. T1 is fused with the head. The head has the abnormal triangular shape seen in Cx^E homozygotes. (L) Cx^E/Cx²⁰, dorsal view. An extra segment is present (bracket) although not as distinct as in Cx²⁰ homozygotes. Head shape is apparently normal.

Figure 3.—

Molecular lesions associated with Cx mutant alleles. The sequence of the Cx 5′-UTR and coding region is annotated with locations of Cx mutant lesions. The encoded amino acids are written above the nucleotide sequence. Significant motifs are color coded: octapeptide (purple) and homeodomain (blue). The nucleotides deleted in various Cx mutant alleles are underlined in red. Two methionine-encoding codons (ATG) that are in frame in the Cx²⁰ allele and could serve as alternative translation initiation sites are boxed in black.

Figure 4.—

Expression patterns of En and Cx in wild-type and Cx mutant embryos. All embryos are shown in ventral view and have begun the process of germband retraction. In schematic views, brown represents En expression and blue represents Cx. Segments are labeled as follows: maxillary (Mx), labial (Lb), and prothoracic (T1). (A) Wild type, En (brown), and Cx (purple) expression. The maxillary and labial En stripes have fused and the dorsal ridge En stripe (black arrow) extends dorsally. Cx is expressed ventrally in the labial appendages, the anterior compartment of the labial segment, and the posterior compartment of the maxillary segment, as well as dorsolaterally in the anterior compartment of T1. Cx is also expressed in some cells of the developing dorsal ridge. (B) Enlarged view of A (top) and schematic representation of En and Cx expression (bottom). (C) Cx⁶ homozygote, En expression (purple). The maxillary (purple arrowhead) and labial (black arrowhead) En stripes have not fused and neither extends dorsally. The apparent contact between stripes is likely due to continued proliferation of En-positive cells, resulting in thickening of the ends of the En stripes. (D) Enlarged view of C (top) and schematic representation of En and Cx expression (bottom). (E) Cx²⁰ homozygote, En (brown), and Cx (purple) expression. The maxillary (purple arrowhead) and labial (black arrowhead) En stripes have not fused. Instead, two stripes extend to the dorsal edge of the embryo. Cx is expressed within its normal domain, and additionally in the ectopic dorsolateral material between the two En stripes. (F) Enlarged view of E (top) and schematic representation of En and Cx expression (bottom). (G) Cx^E homozygote, En (brown), and Cx (purple) expression. The maxillary (purple arrowhead) and labial (black arrowhead) En stripes have not fused. Only the labial En stripe extends dorsally. Cx is expressed in the CNS of the maxillary and labial segments, but is absent or very faint in the ectoderm. (H) Enlarged view of G (top) and schematic representation of En and Cx expression (bottom).

Figure 5.—

Expression of En and Tc hh in Cx²⁰ embryos. (A) En expression in a Cx²⁰ homozygote. The labial En stripe extends to the dorsolateral edge of the embryo (arrow). A second stripe of En expression (arrowhead) appears anterior to the labial En stripe. This new expression domain is discontinuous with but roughly parallel to the maxillary En stripe. (B) Tc hh and En expression in a Cx²⁰ homozygote. Tc hh (purple) and En (brown) are coexpressed in the labial segment (arrow) and in the more anterior expression domain (arrowhead).