Analysis of four achaete-scute homologs in Bombyx mori reveals new viewpoints of the evolution and functions of this gene family
© Zhou et al; licensee BioMed Central Ltd. 2008
Received: 12 October 2007
Accepted: 06 March 2008
Published: 06 March 2008
achaete-scute complexe (AS-C) has been widely studied at genetic, developmental and evolutional levels. Genes of this family encode proteins containing a highly conserved bHLH domain, which take part in the regulation of the development of central nervous system and peripheral nervous system. Many AS-C homologs have been isolated from various vertebrates and invertebrates. Also, AS-C genes are duplicated during the evolution of Diptera. Functions besides neural development controlling have also been found in Drosophila AS-C genes.
We cloned four achaete-scute homologs (ASH) from the lepidopteran model organism Bombyx mori, including three proneural genes and one neural precursor gene. Proteins encoded by them contained the characteristic bHLH domain and the three proneural ones were also found to have the C-terminal conserved motif. These genes regulated promoter activity through the Class A E-boxes in vitro. Though both Bm-ASH and Drosophila AS-C have four members, they are not in one by one corresponding relationships. Results of RT-PCR and real-time PCR showed that Bm-ASH genes were expressed in different larval tissues, and had well-regulated expressional profiles during the development of embryo and wing/wing disc.
There are four achaete-scute homologs in Bombyx mori, the second insect having four AS-C genes so far, and these genes have multiple functions in silkworm life cycle. AS-C gene duplication in insects occurs after or parallel to, but not before the taxonomic order formation during evolution.
Transcriptional factors of the bHLH (basic helix-loop-helix) family play important roles in the development of metazoan, taking part in the regulation of neurogenesis, myogenesis, haemopoiesis and so on [1–5]. The achaete-scute complex (AS-C), a group of four bHLH genes, has been found to be involved in the determination of Drosophila neural precursors [6–9].
AS-C proteins interact with another bHLH protein Daughterless (Da) to form a dimer, and bind Class A E-boxes CAGG/CTG . AS-C/Da heterodimers' binding sites were found in the upstream regions of hunchback (hb)  and phyllopod (phyl) . In Drosophila, they were also found in the upstream region of ac itself, and required for auto- and cross-regulation by ac and sc .
The main functions of AS-C genes are regulating the formation and patterning of precursors of central nervous system (CNS) and peripheral nervous system (PNS). During neural development, AS-C genes are expressed in one or more cells within the proneural clusters, which are committed to the neural precursor fate, and the remaining cells in the cluster differentiate to be epidermal cells or are eliminated by apoptosis [8, 13, 14]. Tribolium castaneum ASH (Tc-ASH) and Hydra vulgaris cnidarian ASH (Cn-ASH) show similar functions during the formation of sensory organs in the transgenic Drosophila [15, 16]. Also, it was found that Cn-ASH protein formed heterodimers with Drosophila Da (Dm-Da) protein in vitro, and these dimers specially bound to the consensus E-boxes . Besides regulation of neurogenesis, AS-C genes have other functions. ac takes part in the development of Malpighian tubule by expressing in the tip cell and regulating the sequential fates of the organ . sc regulates sex determination by its different expression dosage between females and males, but neither ac nor l'sc can substitute for sc in this function [18–20]. l'sc expression is necessary for muscle founder cells segregating from the somatic mesoderm. Loss of expression of l'sc leads to a loss, whereas over expression causes a duplication of muscles and founder cells .
According to the sequence and functional analysis in different species, AS-C genes are conserved during evolution. The four genes of Drosophila AS-C are achaete (ac), scute (sc), lethal of scute (l'sc) and asense (ase). The former three are defined as proneural genes and the last one as neural precursor gene, basing on their expression patterns and functions in proneural clusters. AS- C homologs have been isolated from various invertebrates and vertebrates [15, 16, 22–34]. They all have a conserved bHLH domain and some of them also have a conserved C-terminal motif. Gene duplication events caused a dramatic increase of AS-C gene number during Arthropoda evolution [16, 30]. Within Insecta, AS-C homologs have been studied in Diptera (flies and mosquitoes), Hymenoptera (bees), Coleoptera (the red flour beetle) and Lepidoptera (the butterfly P. coenia). Each of the insects has one ase-like gene. Most of them have only one proneural gene, while the medfly Ceratitis capitata has two, and the fruit fly has three. The four genes in Drosophila were believed to have arisen from a single ancestral gene by three independent duplication events [16, 30]. AS-C homologs in the insects studied are closely linked in the chromosome, for example, the four AS-C genes comprising about 100 Kb in Drosophila, and the two residing about 22 Kb apart in Anopheles, about 55 Kb apart in Tribolium, and about 40 Kb apart in Apis [16, 34].
The silkworm, Bombyx mori, is a model organism of Lepidoptera. With the completion of silkworm genomic sequencing project , Bombyx mori is emerging as an important model lepidopteran. Only one AS-C homolog had been isolated from lepidopteran in previous studies . In the present study, we found there were four AS-C homologs in silkworm, three proneural genes and an ase-like one. We detected the transcriptional activity of the genes by transient expression in Bm-N cells. Expression profiles of the genes in different tissues and expression changes during the development course of silkworm embryo and wing disc/wing were also studied.
Isolation and identification of achaete-scute homologs in Bombyx mori
It is well known that each achaete-scute homolog has a highly conserved bHLH domain which distinguishes them from other bHLH proteins. Using the amino acid sequence of Drosophila Ac (Genbank: AAF45498) to blast the silkworm EST database (see Materials and Methods section), we obtained an EST sequence (GenBank: CK537057) encoding a conserved AS-C bHLH domain. Primers were designed based on the EST sequence, and RACE assay was carried out using the midgut total RNA of 3d 5th instar larva as the template. After sequencing and assembling, we gained a cDNA of 1,332 bp and named it Bm-ASH1 (Genbank: DQ350889). Bm-ASH1 gene contains a 582 bp ORF region (including the stop codon) and encodes a 193 aa protein.
Then we screened the Bombyx mori genome database using the 193 aa Bm-ASH1 protein sequence, and found genes with conserved AS-C bHLH region in four more contigs (Genbank: AADK01030307, AADK01036667, AADK01011379 and BAAB01105243), besides the two (Genbank: AADK01005140 and BAAB01089921) corresponding to Bm-ASH1. BAAB01105243 is part of AADK01011379. Each of them contained a deduced ORF region, and then we cloned the ORF regions by RT-PCR methods and they were sequenced. Primers for 3'-RACE were designed basing on the sequences of the ORF regions, and 3'-RACE assay was processed using the total RNA from 1 d pupal wing as the template. The segments gained by 3'-RACE were sequenced and assembled with each corresponding ORF sequence. The final cDNA sequences were 1,449 bp, 990 bp and 1,695 bp long, and were named Bm-ASH2 (Genbank: EF620927), Bm-ASH3 (Genbank: EF620928) and Bm-ase (Genbank: EF620929) respectively. Bm-ASH2 gene contained a 720 bp OFR region (including the stop codon) and encoded a 239 aa protein, Bm-ASH3 gene contained a 726 bp OFR region (including the stop codon) and encoded a 241 aa protein, and Bm-ase gene contained a 1,215 bp OFR region (including the stop codon) and encoded a 404 aa protein.
Homology comparison of proteins encoded by the Bm-ASH genes with other insect ASH proteins.
Percent Identity (%)
The Bombyx ASH genes could not be assembled into a complex based on current data
Bm-ASH genes regulating promoter activity via E-box in Bm-N cells
Expression distribution of Bombyx ASH genes in larval tissues
Developmental changes of Bombyx ASH genes in the embryo and in the pupal wing
Our results showed that there were four AS-C homologs in Bombyx mori: Bm-ASH1, Bm-ASH2, Bm-ASH3 and Bm-ase. The first three were proneural genes, and the last one was a neural precursor gene. The proneural genes enhanced the activity of the Bm-ASH2 promoter by binding its E-boxes in Bm-N cells, which was one of the AS-C homolog characteristics (Figs. 4 &5). The four genes had various expression profiles in silkworm larval tissues (Fig. 6), and further studies showed that they have important roles during the development of the embryo and the wing (Fig. 7 &8).
AS-C genes duplication in insects occurs after or parallel but not before the taxonomic order formation during evolution
Homologs of AS-C genes exist in various animals from low-grade coelenterate to high-grade mammals, including human being . It has been proposed that AS-C gene has several independent duplication events in Arthropoda, resulting in the plasticity of the gene number. In this model, Cn-ASH, the AS-C homolog in Hydra is supposed as the ancestral one. Parallel but independent duplication events occurred in insects and chelicerates (Fig. 2,a &2b). Within the Diptera two more duplication events happened during evolution. So the present most derived dipteran, Drosophila melanogaster, has the four AS-C genes achaete, scute, lethal of scute and asense [16, 30]; while lower derived dipteran, such mosquito, has only two .
Homology comparison of proteins encoded by the Drosophila AS-C genes with some of the other insect ASH proteins.
Percent Identity (%)
The medfly Ceratitis capitata, a member of family Tephritidae, has three AS-C homolog genes Cc-sc, Cc-l'sc and Cc-ase. The bHLH domains of proteins coded by Ceratitis capitata acheate-scute homologs are highly conserved and display 95%, 91.5% and 90% identity with the Drosophila counterparts, respectively . Only one AS-C homolog, B-ASH1, has been isolated from the Nymphalidae insect P. coenia . B-ASH1 protein has a surprising identity with Bm-ASH2, 100% within the bHLH domains and 90.3% within the whole protein sequences. It is even higher than that between Bm-ASH2 and Bm-ASH3, 91.3% within the bHLH domains and 49.8% within the whole protein sequences (Table 1). Moths first appeared on earth between 100 and 190 million years ago, and butterflies appeared about 40 million years ago, based on fossilized evidence. All above suggest that there are at least three AS-C genes in P. coenia, corresponding with Bm-ASH1, Bm-ASH2 and Bm-ase, respectively, and Bm-ASH3 might be the most recent one among the silkworm proneural genes.
AS-C genes have a broad expression distribution in insect tissues
AS-C genes are important for the development of the nervous system, and have key roles in regulating the formation and patterning of neural precursors. They are specially expressed in most of the proneural clusters during the development of either the central (CNS) or peripheral nervous system (PNS) in arthropod, such as flies, butterflies, beetles, bees, spiders, chilopods, etc. Other functions of these genes have also been found in Drosophila. l'sc participates in the specification of muscle progenitors , sc functions in sex determination  and ac regulates the development of Malphigian tubules .
In the present paper, we studied the expression of Bm-ASH genes in various silkworm larval tissues using RT-PCR methods. These genes are expressed in most organs derived from all three derms, showing they might have multiple functions during silkworm development. They have higher expression levels in the head, wing disc, tracheal cluster, body wall or gonad, and all of them are expressed in the organs though lower at some developmental points. This indicates that the genes might have overlapping functions, just as in Drosophila . Expression profiles during the development of the embryo and the pupal wing also suggest co-operation characters of Bm-ASH genes. During silkworm embryo development, neurogenesis takes place around 3 d-old, and trachea, bristle and appendage occur on 5 d and 6 d. The correspondence between the two stages and those when the two expression peaks of Bm-ASH genes occure in the embryo (Fig. 7) suggests that these genes regulate the development of PNS and CNS. Expressions in wings of all the four genes are significantly higher on 1 d or 2 d after pupation than during other stages. Wing scale precursor cells form around the 2 d-old pupal stage , and our further studies on the scaleless wings mutant silkworm strongly proved the key role of Bm-ASH2 in the formation of wing scales (Zhou et al., unpublished).
Although we have known many important functions of AS-C, the analysis of the structures and evolution of these genes may suggest some of their unknown functions. We compared the protein sequences of 38 AS-C homologs from vertebrate and invertebrate. There are conserved domains besides the bHLH and C-terminal motifs, especially within vertebrates. This conservation even exists between vertebrate and invertebrate organisms. A ~20 amino acid conserved domain (corresponding to PEMRCKRRINFAQLGYNLPQ of Asp-ASH) was found in ASH genes from vertebrates and myriapod animals Lithobius forficatus (Lf-ASH, Genbank: AAT99570) and Archispirostreptus (Asp-ASH, Genbank: AJ536345). Together with the broad expression of Bm-ASH genes in tissues outside of the nervous system, they show that this gene family has other important functions waiting for exploration.
In this work, we isolate and identify four achaete-scute homologs from Bombyx mori. So far, Bombyx mori is the second insect which has been found to have four AS-C genes. During organism evolution, genes are duplicated with conserved domains to gain more special functions. Results of phylogenetic and gene expression analysis show that during evolution, AS-C genes duplication in insects occurs after or parallel to but not before the taxonomic order formation and functions of these genes are broad during insect development.
Insects and cell culture
The silkworm stock Jingsong × Haoyue was maintained in our laboratory. The insects were reared on an artificial diet at 25°C with 70%–80% relative humidity. Bm-N cells derived from silkworm were cultured at 27°C in TC-100 insect medium containing 10% heat-inactivated (56°C, 30 min) fetal bovine serum (Invitrogen). Cell culture details were the same as Summers and Smith .
RNA isolation and RT-PCR
Silkworm tissues were dissected out at different stages and the total RNA was extracted with TRIZOL Reagent (Invitrogen) according to the standard protocol, and whole embryo total RNA was extracted using the acid-guanidine method . One microgramme of total RNA from each sample was used to synthesize first-strand cDNA using M-MLV Reverse Transcriptase (Promega) as the protocol described. PCR with proper program was performed using the reverse transcription product as template. Sequences of all primers used in this paper are available upon request.
Rapid amplification of cDNA ends (RACE)
One microgramme of total RNA was used for RACE cDNA synthesis (BD SMART™ RACE cDNA Amplification Kit, Clontech), according to the user's manual. PCR was performed with primer1 and Universal Primer A Mix (UPM, Clontech), then a nest PCR was processed with primer2 and NUP using the suitable diluted former PCR product as the template. Each PCR reaction was carried out under the following conditions: one cycle of pre-denaturing for 5 min at 95°C; and 30 cycles of 94°C for 40 s, 60°C for 40 s, 72°C for 3 min, then followed by 10 min incubation at 72°C.
Database blast, protein sequences alignment and phylogenetic analysis
We used the amino acid sequence coded by Drosophila achaete (Genbank: AAF45498) to blast the silkworm EST database with the blastx program on the NCBI web site , by limiting the organism with "Bombyx mori". As formerly described , insect ASH protein sequences were aligned with CLUSTALX  and revised manually with Gendoc software. Then a neighbor-joining (NJ) tree based on amino acid sequences was constructed using the PHYLIP software package (100 bootstrap replicates) .
Dual-Luciferase Reporter Assay
Promoter segments were cloned into Luciferase Reporter Vector pGL3-Basic separately, and the ORF region of silkworm ASH and Drosophila da (GenBank: Y00221) genes were cloned into the modified pBacPAK8 vector with an IE1 promoter and a hr3 enhancer . Then the plasmids were transfected into Bm-N cells as described formerly . 0.1 μg of pRL-CMV Vector was co-transfected as an internal control reporter for each transfection. After incubating for 48 hours, the cells were collected by centrifugation at 10,000 rpm for 1 min at 4°C. Then the cell lysates were prepared using the passive lysis buffer, and 10 μg of each lysate was used for the dual-luciferase reporter assay according to the protocol (Dual-Luciferase® Reporter [DLR™] Assay System, Promega). Firefly luciferase activity and Renilla luciferase activity were determined with 20/20n Luminometer (Turner BioSystems, Inc., USA). Each treatment was repeated at least three times.
Introduction of point mutation
Primers P-F and P-R were designed at the terminals of the target segment sequence. Reversed primers m-R and m-F with the mutated bases were designed around the site where the point mutation would be introduced. PCRs were carried out with P-F pairing m-R or m-F paring P-R. After purification, the two PCR products were mixed, and denatured, renatured and extended for three cycles without any primers. Three cycles after running, P-F and P-R were added and the PCR was continued for 30 cycles of amplification. All of the PCRs were processed with pfu DNA polymerase.
Quantitative real-time PCR
Q-PCR was used to determine the changes of silkworm ASH genes expression during embryo development and wing development. Primers were designed based on the cDNA sequence and a segment around 200 bp would be specially amplified for each gene. The housekeeping gene Bm-actin A3 was used as the internal control. A 20 μl volume containing cDNA produced from about 10 ng of total RNA, 5 pmol of each primer, and 10 μl of SYBR Green Realtime PCR Master Mix (Toyobo Co., Ltd., Japan) was used for each PCR reaction. Then the PCR was processed on a Chromo4 Four-Color Real-Time System (Bio-Rad [formerly MJ Research]) under the following program: one cycle of 95°C for 3 min; then 40 cycles of 95°C for 15 sec, 60°C for 15 sec and 72°C for 30 sec. The melting curve was established from 60°C to 95°C. Three independent repeats were carried out for each reaction. Threshold cycle values were used for the further analysis.
Theoretic copy of each sample was calculated with the linear equation for each gene, and trendlines for Bombyx ASH genes during the development course of silkworm embryo and pupal wing were constructed.
The Drosophila daughterless cDNA used in the experiment was presented by Dr. Karen L. Schulze in Prof. Hugo J. Bellen's lab at Baylor College of Medicine. This work was supported by financial grants from the National High Technology Research and Development Program of China ("863" Project No. 2006AA10A119) and the National Natural Sciences Foundation of China (30770279). We thank Ms. Zhenzhen Yang for the language editing of the manuscript.
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