Analysis of the Tribolium homeotic complex: insights into mechanisms constraining insect Hox clusters

Abstract

The remarkable conservation of Hox clusters is an accepted but little understood principle of biology. Some organizational constraints have been identified for vertebrate Hox clusters, but most of these are thought to be recent innovations that may not apply to other organisms. Ironically, many model organisms have disrupted Hox clusters and may not be well-suited for studies of structural constraints. In contrast, the red flour beetle, Tribolium castaneum, which has a long history in Hox gene research, is thought to have a more ancestral-type Hox cluster organization. Here, we demonstrate that the Tribolium homeotic complex (HOMC) is indeed intact, with the individual Hox genes in the expected colinear arrangement and transcribed from the same strand. There is no evidence that the cluster has been invaded by non-Hox protein-coding genes, although expressed sequence tag and genome tiling data suggest that noncoding transcripts are prevalent. Finally, our analysis of several mutations affecting the Tribolium HOMC suggests that intermingling of enhancer elements with neighboring transcription units may constrain the structure of at least one region of the Tribolium cluster. This work lays a foundation for future studies of the Tribolium HOMC that may provide insights into the reasons for Hox cluster conservation.

Fig. 1

Embryonic transcription across the complete Tribolium Hox complex. The tiling array consists of ∼50,000 50 bp probes that estimate degree of transcription. Relative intensities for each probe are represented as peaks correlated with a consensus annotation of the Tribolium Hox complex (below). Peak height, shown as Percentile Probe Intensity (PPI), corresponds to the level of transcription for a particular probe. The nucleotide position for each segment is displayed in the upper left and right corners of the panel (numbers correspond with linkage group 2, release Tcas_2.0). New ESTs (cyan) are displayed in the annotation track along with transposable elements (gray). For annotated genes and ESTs, the arrow indicates the direction of transcription. Red arrows indicate the location of two RNA-FISH probes

Fig. 2

Expression pattern of two HOMC noncoding transcripts in Tribolium embryos. Probe positions are shown in Fig. 1. a Expression pattern from a 1-kb probe located ∼86 kb 5′ of the start of ptl/Tc-Antp. b The expression pattern of the Tribolium homolog of the iab-4 miRNA (tca-miR-iab-4)

Fig. 3

Rearrangements of the HOMC. In these schematic diagrams, the positions of cloned breakpoint fragments are underlined. Wild-type chromosomal position (not to scale) on LG2 (purple) is indicated by a gradient of color to illustrate the effects of inversions. a In the mxp^Dch-3 rearrangement, an approximately 150-kb fragment of the HOMC has been transposed between fragments of LG9 and LG2. b The ptl^D60 mutation is a large inversion that splits the Hox cluster into two parts. Small fragments at each end of the inversion appear to have been deleted, including part of the ptl/Tc-Antp locus

Fig. 4

Cuticle and enhancer trap phenotypes of ptl/Tc-Antp mutations. The antennae (ant) and the labial (lab) and thoracic (T1–T3) segments are denoted where relevant. a–e Cuticle preps displaying the phenotypes of wild-type (Ga-1; a), ptl/Tc-Antp RNAi (b), ptl^D60/ptl^D60 (c), ptl^KT76/ptl^KT76 (d), and ptl^KT76/ptl^D60 (e) first instar larvae. Enhancer trap-driven EGFP expression in a ptl^KT76 embryo (f) appears in a very similar pattern to Cx/Tc-Scr expression (purple) in a wild-type embryo (g). A ptl^KT76 larva (h) and pupa (i) also display EGFP enhancer trap expression in parts of the labial and first thoracic segments

Fig. 5

Overlap of Cx/Tc-Scr regulatory elements with the ptl/Tc-Antp locus. The gene structure of ptl/Tc-Antp (coding sequence is shaded gray) and the positions of mutant lesions are shown in the diagram. The inferred positions of Cx/Tc-Scr regulatory elements in the ptl/Tc-Antp region are indicated below the diagram