Abstract
In insects, there is a considerable diversity in leg distribution on the body, including number, segmental arrangement, morphological identity and consequent function, but the genetic basis for these differences is not well understood. Here by positional cloning, we showed that a ~355 kb region, including Bombyx mori Ultrabithorax (BmUbx) and abdominal-A (Bmabd-A), was responsible for the silkworm mutant Kh-extra-crescents-like (EKh-l) that displayed additional thoracic limb-like legs on the first abdominal segment (A1) and occasionally on the second abdominal segment (A2). We found that BmUbx gene was downregulated at both messenger RNA level and protein level in EKh-l embryo, while its expression domain in the EKh-l embryo was almost the same as that in the wild type. Whereas Bmabd-A was upregulated at both levels and was ectopically overexpressed on the supernumerary leg-bearing segments in EKh-l. Compared with the previously reported Ecs-l mutant in which increased expression of both BmUbx and Bmabd-A gave rise to ectopic proleg-like appendages on the same segments, we propose that overexpressed Bmabd-A gene is capable to promote the outgrowth of extra leg appendages on A1 and A2 segments, whereas BmUbx gene is required to specify accurate morphologies of the ectopic legs in a dosage-dependent manner in silkworm. These results provide insights into how these hox genes regulate the leg morphologic diversity on the same segments.
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Introduction
In insects, there are several sets of serially homologous appendages distributed on the repeated segments, showing a considerable diversity in number, position and morphology. For example, each thoracic segment carries a pair of appendages called thoracic limbs (Jockusch et al., 2004), whereas legs have been suppressed in the abdomen of most insects. However, still some insects possess abdominal appendages called prolegs in several groups, such as the basal hexapod Collembola and larval Lepidoptera (Angelini and Kaufman, 2005). The type of appendage depends on the identity of the segment where they appear, indicating that the Hox genes contribute to appendage specification (Lawrence and Morata, 1994; Morata and Sanchez-Herrero, 1999), through acting on targets downstream of the original proximodistal axis genes (Jockusch et al., 2004).
Hox genes Antennapedia (Antp), Ultrabithorax (Ubx), abdominal-A (abd-A) and Abdominal-B (Abd-B) are expressed along the anterior–posterior body axis to determine whether the leg develop on these segments or not and to specify the leg identity in insects. The Antp is expressed in the first three thoracic segments and regulates limbs developing on the thorax (Abbott and Kaufman, 1986). A particularly marked example is provided by the Antp gain-of-function mutations in Drosophila melanogaster, which transform the antennae into second thoracic legs (Kaufman et al., 1990). Antp loss-of-function mutation in silkworm produced the fused thorax and defects in limb development (Chen et al., 2013a). The Hox gene Ubx is normally expressed in a complex pattern in the posterior thoracic and anterior abdominal segments of insects, where it has multiple roles in modifying specific morphological differences between the mid-legs (T2) and meta-legs (T3) on the thorax, including appendage size, shape and function (Averof and Patel, 1997; Akam, 1998; Roch and Akam, 2000; Tomoyasu et al., 2005), and inhibit the development of abdominal prolegs via repressing the Distal-less (Dll) expression (Gebelein et al., 2002; Ronshaugen et al., 2002). Although insects expressed abd-A in a similar pattern from the posterior compartment of the first abdominal segment to the remaining abdominal segments (Kelsh et al., 1994; Shippy et al., 1998), there is considerable diversity in the prolegs distribution on the abd-A expressed segments among various insects, with a wide range of variation in both segmental arrangement and number. In Diptera Drosophila (Vachon et al., 1992) and Coleoptera Tribolium (Lewis et al., 2000), abd-A suppressed leg development by inhibiting expression of the gene Dll leading to the deficiency of abdominal limbs in larvae, however, abd-A promotes the development of prolegs in silkworm (Chen et al., 2013a). The Abd-B gene is expressed in the last few segments of the abdomen and is required for proleg suppression in these segments (Tomita and Kikuchi, 2009; Xiang et al., 2011). It is therefore of great interest from an evolutionary point view to understand how the Hox genes affect the leg morphologic variation, including the identity, number and segmental arrangement, among variety insects, or even between the limbs and proleg within the same species.
In silkworm, Bombyx mori, >30 homeotic mutants belong to pseudoallele group E exhibiting various anomalies in legs development, including number, position and identity (Ueno et al., 1992; Banno et al., 2005). The silkworm E loci were located on the chromosome 6 and were presumed to be analogous to the D. melanogaster bithorax complex (BX-C; Ueno et al., 1992; Yasukochi et al., 2004). Over 20 mutants of them present extra legs on A1 or A2 or on both of the two abdominal segments in the larval stage. Furthermore, the identity of these supernumerary legs were thoracic limb-like in some mutants but were abdominal proleg-like in others. Thus, it raises an intriguing question of how these Hox genes regulate the leg morphologic variation on the same segments. To address these questions, here we reported the molecular identification and characterization of the gene responsible for the spontaneous Kh-extra-crescents-like (EKh-l) mutant that presents extra thoracic limb-like legs on the first abdominal (A1) segment. Our results indicated that EKh-l locus was restricted within a ~355 kb region including the BmUbx and Bmabd-A gene. Furthermore, we analyzed the expression pattern of BmUbx and Bmabd-A in the wild type (WT; Dazao) and the EKh-l mutant to determine their role in generating the extra limb-like legs on abdomen.
Materials and methods
Silkworm strains
The EKh-l and Dazao (WT) stains were obtained from the Silkworm Gene Bank at Southwest University (Chongqing, China). EKh-l and Dazao were used as parent strains to produce F1 offspring. For no recombination in female silkworms, 22 progeny from a single-pair backcross between an F1 female and a Dazao male (BC1F) were used for the linkage analysis, whereas 1724 progeny from the cross between Dazao female and F1 male (BC1M) were used for the recombination analysis. The developing embryos were incubated at 25 °C with adequate humidity and larvae were reared with fresh mulberry leaves under a photoperiod of 12 h light and 12 h dark at 25 °C.
DNA extraction
Total genomic DNA of parental strains and BC1 individuals described above were extracted from adults and larvae, respectively. All the samples for DNA extraction were the whole body of the individuals. The samples were disrupted in liquid nitrogen and suspended in DNA extraction buffer (pH 8.0, 10 mm Tris-HCl, 0.1 m EDTA, 0.5% SDS) with 100 μg ml−1 proteinase K. Phenol–chloroform extraction was performed after digestion at 55 °C for 5 h. The DNA was precipitated with freezing dehydrated alcohol and dissolved in TE buffer (pH 8.0, 10 mm Tris-HCl, 1 mm EDTA).
Mapping and linkage map construction
Based on the previous studies, the E loci are located at the 21.1 cM on the sixth linkage group (Banno et al., 2005), including the homeotic genes BmUbx, Bmabd-A and BmAbd-B (Ueno et al., 1992), which were located scaffold nscaf2853. We searched the PCR-based amplicon length polymorphisms on nscaf2853 between the Dazao strain and the EKh mutant as markers for recombination analysis. The primer sequences and position information of the DNA markers for genetic analysis in this study were listed in Table 1.
The markers were first confirmed to be linkage with the EKh locus by using 20 BC1F progeny (10 WT and 10 mutant), and subsequent were used for recombination analysis with a large BC1M mapping populations (1724 individuals). The segregation patterns were analyzed using JoinMap 4.0 software (Van Ooijen, 2006), with a logarithm of the odds threshold of 4.0 and the Kosambi mapping function (Kosambi, 1944).
Quantitative real-time PCR
The relative expression levels of genes, BmUbx and Bmabd-A, were examined using Quantitative real-time (qRT-PCR). The embryos of EKh-l, Ecs-l and Dazao were dissected at the 7th day of embryonic developmental stage when the phenotype could be easily distinguished. Each sample contained eight individual embryos and was disrupted with a mini homogenizer (Polytron PT1600E, Kinematica, Lucerne, Switzerland), for RNA extraction using MicroElute Total RNA kit (mega Bio-Tek, Norcross, GA, USA) according to the manufacturer’s instructions. The complementary DNA was synthesized from total RNA sample using PrimeScript RT reagent Kit with gDNA Eraser (Takara, Dalian, China) according to the manufacturer’s instructions. qRT-PCR experiments were performed using the StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA, USA) with a SYBR Premix EX Taq_kit (Takara), according to the manufacturer’s recommended procedure. Referring to the full-length complementary DNA sequence of Bmabd-A and BmUbx (GenBank Accession number: AB461860 and AB505052), we designed gene-specific primers for qRT-PCR. The eukaryotic translation initiation factor 4A (silkworm microarray probe ID: sw22934) was used as an internal control. The primers used for qRT-PCR were listed in Table 1. The qRT-PCR condition was denaturation at 95 °C for 4 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 31 s, 95 °C for 15 s, 60 °C for 20 s and 95 °C for 15 s. All experiments were performed in three independent biological sets and results were presented as mean±s.d. Statistical analyses were performed using Student’s t-test (n=3), and P-values <0.05 were considered statistically significant.
Protein isolation and western blotting
Proteins were isolated from embryos at the 7th day of embryonic developmental stage. Embryos were grinded in liquid nitrogen and boiled in 0.02 m phosphate-buffered saline, pH7.4, containing 2% SDS for 30 min. After centrifuging twice at 12 000 g for 15 min at 4 °C, we collected the supernatants. The concentration of proteins was determined using a BCA Protein Assay kit (Beyotime, Jiangsu, China) followed the manufacturer’s instructions.
For western blotting, proteins were separated by electrophoresis on SDS-polyacrylamide gel electrophoresis. The proteins were then transferred onto polyvinylidene fluoride membranes and probed with rabbit anti-Bmabd-A antibody (Chen et al., 2013b) or rabbit anti-Ubx antibody (Tong et al., 2014). After incubation with horseradish peroxidase-conjugated secondary antibodies (Beyotime) the signals were visualized with Lumi-Light PLUS western blotting substrate (Roche, Mannheim, Germany). Specific protein expression levels were normalized to tubulin for total protein measurements.
Immunofluorescence
Expression pattern of BmUbx and Bmabd-A was assessed using immunostainings in embryos of embryos of EKh-l and Dazao following the protocol as previously described (Chen et al., 2013b; Tong et al., 2014). The embryos were dissected at 2 days old. We used a rabbit anti-Ubx antibody (at 1:500; a gift from L Shashidhara), and rabbit polyclonal anti-Bmabd-A (at 1:200) antibody. The images were captured on a fluorescence microscope BX51TRF (Olympus, Tokyo, Japan).
Results
Phenotypic characteristics of the EKh-l mutant
Compared with the WT strain (Figure 1a), the larva of EKh-l mutant exhibited a pair of extra rudimentary legs on the first abdominal (A1) segment and ~12% (13 out of 106) presented a pair of smaller ones on the second abdominal (A2) segment (Figure 1b). Advanced magnified observation of the supernumerary legs indicated that they were varied in morphology and length, and showed some bristles and dark pigmentation but typed to neither limb nor proleg based on their morphology at this stage (Figures 1c–e). To further explore the identity of the extra legs, we dissected the embryos in the late developmental stage (7–8 days after oviposition) and found that the EKh-l individuals presented additional legs similar to thoracic limb on the A1 segment according to the similar proximal/distal patterning module (Figures 1f–g). Furthermore, ~5% (5 out of 106) of the EKh-l moths had four pairs of legs compared with three pairs in the WT. The extra legs on the first abdominal segment were much smaller than the normal legs and were almost completely immovable (Figures 1h–i). Considering the adult legs of B. mori develops from the larval thoracic legs (Singh et al., 2007), therefore, the supernumerary legs of EKh-l homozygotes were completely thoracic typed legs on the first abdominal (A1) segment.
Positional cloning of EKh-l locus in Bombyx mori
Previous linkage analysis efforts mapped the E complex in B. mori to BX-C complex on the sixth linkage group (LG6, nascaf2853; Ueno et al., 1992; Xiang et al., 2008; Chen et al., 2013b). To identify the candidate region responsible for the EKh-l locus, we searched for the genomic polymorphism by PCR between the recurrent parents, Dazao strain and the EKh-l mutant in the scaffold nscaf2853 on chromosome 6. We found that eight pairs of newly designed markers exhibited polymorphism between them. Then, we performed genetic analysis using 20 BC1F progeny (10 WT and 10 mutant) that were generated from cross (BC1F) between the F1 females and the males of WT strain Dazao, and subsequent fine mapping with a large BC1M mapping populations (1724 individuals) from cross between F1 heterozygote males and the females of Dazao strain. The genotyping showed that the EKh-l locus was delimited between markers L1048 and L1391, and three markers, L1779, L1011 and L1153 were completely linked to the EKh-l locus (Figure 2a); therefore, the EKh-l locus was localized within a 355 kb on the nscaf2853 scaffold according to the SilkDB database (Duan et al., 2010). Five predicted genes (BGIBMGA006389, BGIBMGA006487, BGIBMGA006388, BGIBMGA006387 and BGIBMGA006386) were located within this region (Figure 2b). Further bioinformatics analysis revealed that BGIBMGA006386, BGIBMGA006387 and BGIBMGA006388 were assembled to be Bmabd-A gene, and BGIBMGA006389 was the first exon of BmUbx, whereas BGIBMGA006487 was a wrongly predicted gene in SilkDB. Thus, we concluded that the interval of 355 kb containing the first exon of BmUbx and Bmabd-A was responsible for the EKh-l phenotype (Figure 2c).
To determine whether sequence variation exists in the transcripts of BmUbx and Bmabd-A genes in EKh-l mutant, we cloned the two genes in both EKh-l mutant and Dazao strains. The result showed that no sequence difference was observed between EKh-l and Dazao in the full-length complementary DNA of both genes. Considering the location of the EKh-l candidate region and the essential function of BmUbx and Bmabd-A genes in body planning, we next investigated the expression profiles of the two candidate genes in EKh-l mutant and Dazao strains.
Expression patterns of BmUbx and Bmabd-A gene
To determine the expression profiles of the BmUbx and Bmabd-A genes in the EKh-l mutant, we detected the expression levels of Bmabd-A and BmUbx at both messenger RNA and protein levels in the embryos (7 days old). The qRT-PCR analysis data showed that the EKh-l mutant embryos exhibited a decreased level of BmUbx expression (Figure 3a), but a significant increased level of Bmabd-A expression compared with the WT (Figure 3b). Then, we examined the protein level of BmUbx and Bmabd-A. The western blotting results revealed that BmUbx protein was markedly decreased (Figure 3c), whereas Bmabd-A protein was obviously augmented (Figure 3d) in the EKh-l mutant, which was consistent with their transcriptional level. These results strongly indicated that the BmUbx was downregulated and Bmabd-A gene was upregulated at both transcriptional and translational levels in the EKh-l embryos.
Previous reports have showed that the specific regional functions of individual Hox genes were largely restricted to their expression patterns, the disruption of which can lead to developmental defects (Masumoto et al., 2009; Xiang et al., 2011). To assess whether the expression domains of BmUbx and Bmabd-A were diverged between the EKh-l mutant and Dazao, we performed the antibody staining in those embryos (2 days old). In the control strain Dazao, the main expression domain of the BmUbx was in the A1 and A2 segments where no leg appendage appeared (N=7; Figure 4a; Supplementary Figure 2A), whereas it was expressed in the same region but in a seemingly reduced level in the EKh-l mutant (N=12; Figure 4b; Supplementary Figure 2B). Bmabd-A was detected strongly in the leg primordias of the A3–A6 and A10 segments in WT (N=9; Figure 4c; Supplementary Figure 2C). However, the EKh-l mutant ectopically overexpressed Bmabd-A in the leg primordias of A1 and A2 segments where extra legs growth out besides the normal expression domain (N=6; Figure 4d; Supplementary Figure 2D). These results suggested that the reduction of BmUbx and the ectopic expression of Bmabd-A on the A1 and A2 segments were responsible for the appearance of the supernumerary limb-like legs in the EKh-l mutant.
Discussion
EKh-l, one of the silkworm E loci mutants, exhibits additional thoracic limb-like legs on the A1 segment and occasionally on the A2. In this study, we performed positional cloning and narrowed the EKh-l candidate region to a ~355 kb including the first exon of BmUbx and Bmabd-A gene using a large mapping population (1724 individuals) in the B. mori. By cloning the BmUbx and Bmabd-A in EKh-l, we did not find any mutation in their transcriptional sequences. The EKh-l mutant embryos expressed lower level of BmUbx but higher level of Bmabd-A compared with the WT. The previous evidences have reported that many noncoding RNAs, such as intergenic regulatory transcripts and evolutionarily conserved microRNAs, were mapped to the intergenic regions of Hox gene clusters to coordinate their expression through a variety of cis- and trans-regulatory mechanisms in various organisms (Lemons and McGinnis, 2006; Gummalla et al., 2012). However, we did not found any other potential functional transcript within the mapped region by search against the SilkTransDB (http://124.17.27.136/gbrowse2/; Li et al., 2012). The misexpression of BmUbx and Bmabd-A in EKh-l is probably regulated by some unidentified cis-regulatory elements or transcripts in the mapped region, respectively. Another possibility is that the overexpressed Bmabd-A induced the downregulated BmUbx by the posterior dominance rule in which a more posterior homeotic gene suppresses the expression of the more anterior Hox gene in general. And a third possibility is that the downregulation of BmUbx is causal and leads to the expansion of Bmabd-A, as it has been shown that downregulation of BmUbx through RNAi leads to the development of thoracic-like appendages on A1 (Masumoto et al., 2009). As only two genes were involved in the mapped region, we proposed that the abnormal expressed BmUbx and Bmabd-A were associated with the EKh-l mutant, but we could not exclude the possibility that other Hox genes, particularly BmAntp, which influences leg development in the thorax, might also be altered in expression level or pattern by indirect regulation of BmUbx misexpression or by regulatory changes in the mapped region.
Previous studies have reported that above 30 homeotic mutants located on chromosome 6 are related to B. mori Hox genes, dozens of them were displayed diversity of supernumerary legs on the first two abdominal segments, including number and morphologic identity (Ueno et al., 1992; Xiang et al., 2008; Chen et al., 2013b). Among them, only two mutants Ekp-1 and ECs-l that present supernumerary abdominal prolegs in the A2 segment have been identified to date (Xiang et al., 2008; Chen et al., 2013b). Ekp-1 locus was delimited to a region that included the Bmabd-A and a noncoding RNA, miR-iab-4. Whereas ECs-l was restricted to a ∼68 kb sequence in the upstream intergenic region of Bmabd-A containing the miR-iab-4 as well and displayed remarkable ectopically expressed Bmabd-A in the supernumerary prolegs in the A2. In the present study, the Bmabd-A expression was also prominently increased in the EKh-l mutant, not only the messenger RNA but also the protein. In particular, the immunofluorescence experiment revealed that the Bmabd-A protein was also ectopically overexpressed in the supernumerary legs on A1 and A2 in the EKh-l mutant, indicating that the Bmabd-A promote the development of extra legs on these two segments. However, the extra legs were limb-like in the EKh-l instead of proleg-like in Ekp-1 and ECs-l. These results suggested that the overexpressed Bmabd-A was sufficient to promote the outgrowth of additional appendages on A1 and A2, but insufficient to determine their morphological identity. In addition, it is noteworthy that the BmUbx expression level was changed in opposite directions in EKh-l and ECs-l even they both overexpressed Bmabd-A on the same segments. The BmUbx expression was significantly increased in the ECs-l but was decreased in the EKh-l mutant (Supplementary Figure 1). We speculate that the reduced BmUbx level specified the additional legs to limb-like in the EKh-l, whereas the improved BmUbx level specified them to proleg-like identity in ECs-l. Thus, we proposed that the BmUbx was required to specify accurate morphologies of the extra legs in A1 and A2 in a level-dependent manner (Figure 5). In contrast to the case in D. melanogaster, Ubx and abd-A inhibit proleg development in the abdomen by suppressing Dll expression (Vachon et al., 1992), in silkworm, the two genes determine the number, position and identity of legs in a dosage-sensitive manner.
In addition, even the additional legs displayed morphological similarities with the normal thoracic limb in EKh-l, they were expressed different Hox genes combinations. The promotion of normal thoracic limbs was carried out primarily by Antp, and in T3 either BmAntp or BmUbx can apparently execute this function, whereas the additional legs in EKh-l mutant were due to the increased Bmabd-A and decreased BmUbx, suggesting that morphological similarities was not required similar Hox gene expression.
Many insects possess both thoracic limbs and abdominal prolegs, but the molecular origins of the morphological diversity are not well understood. In Panorpidae (Insecta: Mecoptera), the histological and morphological evidence of the embryonic development suggested that the abdominal prolegs of Panorpidae were not serially homologous with the thoracic limbs (Yue and Hua, 2010). The morphological and gene expression analyses of decapentaplegic and Distal-less genes, two genes required for the limb development, indicated that the abdominal prolegs were evolved independently with the limb in the sawfly as well (Oka et al., 2010). So far, it is still unclear the relationship between the two types of legs. In the study, the same segments of silkworm have potential to develop either limbs or prolegs, which was determined by the different combination of Hox genes expression patterns, including expression level and domain. Thus, our findings offer a probable approach to answer the above question by using genetic techniques to modify the expression patterns of Hox genes. Further comparative analysis of the morphological changes in appendages and different expressed genes will contribute a lot to reveal the molecular origins of limbs and prolegs. In conclusion, our results show that BmUbx and Bmabd-A were the candidate genes of the EKh-l mutant. The outgrowth of extra limb-like legs in the A1 and A2 was attributed to the altered expression patterns of the two Hox genes. To be specific, the overexpression of Bmabd-A on the A1 and A2 segment caused the outgrowth of the extra legs, and the decreased expression of the BmUbx on the A1 segment gave rise to the thoracic limb-like leg phenotype. These results provide insights into how these Hox genes regulate the leg morphologic variation on the same segment.
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Acknowledgements
We thank Dr Tingting Gai and Dr Chunlin Li for their assistant of the genetic mapping. This work was funded by Hi-Tech Research and Development 863 Program of China Grant (no. 2013AA102507), National Natural Science Foundation of China (nos 31472153 and 31372379), Chongqing Youth Science and Technology Talent Training Project (cstc2014kjrc-qnrc80001) and Fundamental Research Funds for the Central Universities in China (no. XDJK2013A001).
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Tong, X., Fu, M., Chen, P. et al. Ultrabithorax and abdominal-A specify the abdominal appendage in a dosage-dependent manner in silkworm, Bombyx mori. Heredity 118, 578–584 (2017). https://doi.org/10.1038/hdy.2016.131
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DOI: https://doi.org/10.1038/hdy.2016.131
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