A genomewide survey of basic helix-loop-helix factors in Drosophila

Abstract

The basic helix-loop-helix (bHLH) transcription factors play important roles in the specification of tissue type during the development of animals. We have used the information contained in the recently published genomic sequence of Drosophila melanogaster to identify 12 additional bHLH proteins. By sequence analysis we have assigned these proteins to families defined by Atonal, Hairy-Enhancer of Split, Hand, p48, Mesp, MYC/USF, and the bHLH-Per, Arnt, Sim (PAS) domain. In addition, one single protein represents a unique family of bHLH proteins. mRNA in situ analysis demonstrates that the genes encoding these proteins are expressed in several tissue types but are particularly concentrated in the developing nervous system and mesoderm.

Figure 1

Neighbor-joining plot of bHLH domains. A representative neighbor-joining plot was constructed by using the bHLH domain. To simplify the tree, nodes with a bootstrap value of less than 5% have been removed and the lower branch has been increased in length accordingly. Nodes with a bootstrap value of less than 50% are unmarked, those at 50–75% are marked +, 75–95% are marked ++, and 95–100% are marked +++. Note that the majority of lower (right-hand side) nodes in the tree are of very low predictive value and, hence, are unlikely to give a true indication of the phylogenetic relationship between sequences. On the other hand, upper nodes in the tree (left-hand side) are of a high predictive value. The approximate position of the genes of the AS-C has been indicted on the plot by comparison with trees drawn with the same data set, with the genes of the AS-C included (dashed line). A colored background marks subfamilies of bHLH proteins: bHLH-PAS (red), HES (gray), bHLH-zip (yellow), Atonal-related (dark blue), Mesp-related (orange), Hand family (light blue), and p48-related (green).

Figure 2

Amino acid sequence lineups of bHLH families. Residues with predicted structural function are in bold (5); 1, DNA binding; 2, helix formation. Residues with amino acid identity between the family members illustrated are shaded. (a) Atonal-related. (b) p48-related. (c) Hand-related. (d) HES family; omission of residues in the loop region of this family is marked with two slashes (//). (e) Mesp-related. (f) bHLH-PAS. (g) CG11450/Shout. (f) USF family (note: the loop and the second helix domains of CG17592 are extended with respect to USF2 and contain multiple serine residues).

Figure 3

mRNA expression in Drosophila embryos. Except where indicated, ventral is down and anterior is to the left. (a) CG5545/doli widespread in the CNS at stage 12. (b) CG8667/mistr in the VNC and parts of the ring gland (arrow) at stage 16. (c) CG10066/fer1, over the whole surface of the embryo but concentrated in two posterior dorsal pads (arrowhead) and around the forming denticle belts (arrows) at stage 16. (d) CG5952/fer2 stg12 expression in the VNC and brain at stage 12. (e) CG10046/side in the CNS at stage 12. (f) CG12952/sage dorsal view in the salivary glands at stage 15. (g) CG17592/Dm usf in the VNC and proventriculus (*) at stage 15. (h) CG6211/gce in a subset of germ cells (arrow) stage 9. (i–k) CG11450/shout at stage 5 (i), stage 8 (j), and stage 11 (k). At the cellular blastoderm stage, CG11450/shout transcript is expressed first in the dorsal and then in the ventral blastoderm (i, arrow). This expression becomes hemisegmentally repeated in the dorsal and ventral blastoderm but is not present in the procephalic region or telson and is not present in the lateral regions of the embryo. During gastrulation, all of the CG11450/shout-expressing cells on the ventral surface of the embryo move through the ventral furrow (j, arrows). It then is expressed in myoblast cells as they migrate underneath the body wall of the embryo (k, arrows). CG18144/Dm hand expression at stage 9 (l), stage 13 (m), stage 14 (n), and stage 16 (o); Dm hand expression is seen first at stage 10 of embryo development. It occurs in bilateral stripes in the ventral mesoderm (l, arrow). These stripes represent the precursors of the forming dorsal vessel. Dm hand continues to be expressed in this structure, including the latest stage we have investigated. As germ-band retraction proceeds, a second bilateral strip of Dm hand expression becomes apparent in the ventral mesoderm (m, arrowhead). These cells are the precursors of the circular visceral musculature. Dm hand continues to be expressed in the cells of the circular visceral musculature of the gut as they migrate over the gut surface (m–o). Additionally, there is a high level of Dm hand expression in the external portion of the proventriculus (n–o, *). At late stage 13, Dm hand expression comes on in the VNC and brain. In the VNC, Dm hand is expressed in every hemisegment in a small group of cells at the lateral–dorsal part of the hemisegment adjacent to the perineurium (n).