Canalization by Selection of de Novo Induced Mutations

Abstract

One of the most fascinating scientific problems, and a subject of intense debate, is that of the mechanisms of biological evolution. In this context, Waddington elaborated the concepts of "canalization and assimilation" to explain how an apparently somatic variant induced by stress could become heritable through the germline in Drosophila He resolved this seemingly Lamarckian phenomenon by positing the existence of cryptic mutations that can be expressed and selected under stress. To investigate the relevance of such mechanisms, we performed experiments following the Waddington procedure, then isolated and fixed three phenotypic variants along with another induced mutation that was not preceded by any phenocopy. All the fixed mutations we looked at were actually generated de novo by DNA deletions or transposon insertions, highlighting a novel mechanism for the assimilation process. Our study shows that heat-shock stress produces both phenotypic variants and germline mutations, and suggests an alternative explanation to that of Waddington for the apparent assimilation of an acquired character. The selection of the variants, under stress, for a number of generations allows for the coselection of newly induced corresponding germline mutations, making the phenotypic variants appear heritable.

Keywords: Drosophila; assimilation; canalization; heat shock; transposons.

Figure 1

Schematic representation of experimental design. From each of the Paliano and Carpineto Romano collections, 20 nonvirgin females were allowed to lay eggs to establish F1 stocks. Then, from the F1 offspring, a number of virgin females were crossed to a number of males. The F2 progeny from each population were subjected to 40° for 4 hr during the pupal stage; after eclosion, the treated flies were intercrossed, and their progeny were again treated by heat shock at the pupal stage. The same protocol was repeated for several generations and, in any generation, indicated as F*, where a substantial number of similar phenocopies emerged (F* = fourth for sepia and sixth for forked phenocopies in heat treated Paliano population; seventh for cactus and 12th for singed phenocopies in heat treated Carpineto Romano population, as reported below), these were selected and intercrossed in separate vials, and the progeny were again treated by heat shock at the pupal stage (blue). This procedure was repeated for a number of generations until fixation of the phenotype. Meanwhile, we continued to stress the wild-type lines (green) of both populations looking for phenocopies resembling other classical mutants. We ended the treatments at the generations in which we fixed the last mutant from each population (the ninth generation in Paliano strain with sepia fixation and 12th generation in Carpineto Romano strain with the fixation of cactus and the isolation of singed). Fn represents a number of consecutive treated generations.

Figure 2

Phenotypic variants induced by heat-shock stress. (A) One haltere transformed to a wing. (B) Blistered wings. (C) Abnormal tergites. (D) Abnormal eye morphology and white color. (E) Male with two genital apparatus. (F) Histogram showing the total number of examined flies in both the stressed (HS) and control (CTR) populations, and the number of phenotypic variants (red). In each generation the variants with developmental abnormalities, not resembling heritable phenotypic mutants, were discarded and only wild type flies were allowed to continue to mate.

Figure 3

Fixed phenocopies from Paliano (A and C) and Carpineto Romano (B and D) natural populations. (A) Fly displaying the forked phenotype (right) has shortened, gnarled and bent dorsal bristles compared to the wild-type (left) (arrows). (B) Abnormal shape of dorsal bristles (right) compared to the wild type (left) in the singed mutant (arrows). (C) Eye color phenotypes of wild-type (left) and sepia mutant (right). (D) Phenotype of hypomorphic cactus fixed mutation in third instar larvae (left) and adult flies (right). Melanotic nodules in the hemocoel are seen as black spots, as indicated by arrows.

Figure 4

(A) Frequency of flies showing the sepia phenotype from the fifth heat-shocked generation to se^pal fixation in the ninth generation. (B) Frequency of cactus flies from the eighth heat-shocked generation to cactus fixation in the 12th generation.

Figure 5

Molecular characterization of heat-shock-induced forked mutation. (A) The forked^pal mutation is associated with a 4-nt frameshift deletion in the antepenultimate exon of the gene as determined by Sanger sequencing. Blue bars below the schematic representation the forked gene indicate the overlapped fragments amplified to study the structural organization of forked. (B) The 4-nt deletion was verified by PCR analysis using allele-specific primers for wild-type forked (forked F_forked NO DEL R) that amplify only the wild type DNA but not the forked^pal DNA, and allele-specific primers for the forked^pal mutant allele (forked F_forked DEL R) that amplify only the forked^pal DNA but not the forked wild-type DNA. Rpl32 was used as internal control gene. (C) Partial sequence of the antepenultimate exon of the forked. The primer sequences are highlighted in bold, and the arrows show the primer direction from 5′ to 3′. The 4 bp deletion is underlined. wt^pal = Paliano wild type strain; f^pal= forked Paliano mutant.

Figure 6

Molecular characterization of heat-induced singed mutation. (A) The singed mutation (sn^car) is correlated to a single copy KP-element insertion in the first intron of the sn-RG transcript; the same genomic position corresponds to the 5′-UTR region of another four singed transcripts, sn-RA, sn-RB, sn-RE, and sn-RF. Blue bars below the schematic representation of singed gene indicate the overlapping fragments amplified to study the structural organization of singed gene. The primers indicated by the arrows allowed us to sequence and identify the KP transposon along with its insertion point. (B) PCR analysis of genomic DNA from wt^car, sn^car, and a singed revertant strain (rev^car). The genotyping demonstrates the precise excisions of KP-element. (C) Semiquantitative RT-PCR analysis shows that four sn transcripts are significantly affected by the KP-insertion in singed mutant larvae compared to the control wild-type larvae. cDNA was analyzed using two primer pairs: the first designed to amplify the singed splice variants A, B, E, and F (lanes 1 and 2), and the second specific for sn-RG (lanes 3 and 4). The KP-insertion does not affect the sn-RG transcript in larvae, where it is poorly expressed (C), or in adult heads (D), where it is highly expressed; Rpl32 was used as an internal control. (E) Western blot assay showing an incomplete but significant reduction of SINGED protein levels in sn^car larvae compared to the control strain (HP1 was used as internal control). wt^car = Carpineto Romano wild type strain; sn^car = Carpineto Romano singed mutant.

Figure 7

Molecular characterization of heat-shock-induced sepia mutation. (A) PCR analysis of sepia genomic region, performed using outer primers flanking the deleted area (se2 F_se2 R) and inner primers spanning the deleted region (se1 F_se1 R), reveals that the sepia^pal allele is caused by a large deletion; in the se^pal mutant, PCR with se2 primers amplifies a product of 1388 bp instead of the expected 2989 bp amplicon; PCR with se1 primers instead amplifies a wild-type fragment of only 854 bp in the Paliano wild-type, as expected. Rpl32 was used as an internal control gene. (B) The deletion includes the first and almost the entire second exon of the sepia gene, and the entire second exon of the Gst03 adjacent gene (the 1601 bp deletion mutation is depicted by the shaded area). Blue bars below the schematic representation of Gst03 and sepia genes indicate the amplicons produced by PCR amplification of the Gst03 and sepia genome regions in the wild-type Paliano strain. wt^pal = Paliano wild type strain; se^pal = Paliano sepia mutant.

Figure 8

Molecular characterization of heat-induced cactus mutation. (A) The genetic lesion responsible for the cactus allele is a full-length insertion of a micropia retrotransposon into the 3′-UTR of the gene as demonstrated by PCR analysis (B). Blue bars below the schematic representation of cactus gene indicate the positions of overlapping fragments amplified to study the structural organization of cactus gene. The arrows indicate the oligonucleotides that allowed us to sequence and identify the micropia retrotransposon, along with its insertion point.

Figure 9

PCR analysis of forked gene corresponding to the fixed phenocopies in the wild type Paliano parental flies. (A) Schematic representation of forked gene showing the annealing positions of allele-specific PCR primers (arrows). (B) The forked PCR analysis, genomic DNA purified from each single parental fly was amplified with both allele-specific primers for wild-type forked (forked F_forked NO DEL R) that amplify only the wild type DNA and not the forked^pal DNA, and allele-specific primers for forked^pal mutant allele (forked F_forked DEL R) that amplify only the forked^pal DNA and not the forked wild-type DNA. Rpl32 was used as internal control gene. In all samples from the Paliano parental line, allele-specific PCR amplifies the 400 bp target fragment only with specific primers for the wild-type allele (+), which confirms the absence of the molecular lesion responsible for the stress-induced forked phenotype. Allele specific primers for wild-type and forked^pal are indicated by arrows in (A). The white boxes delimit the part of the gels containing the control PCR reactions performed on genomic DNA purified from wild-type and mutant alleles.

Figure 10

PCR analysis of the genes corresponding to the singed fixed phenocopies in the wild type Carpineto Romano parental flies. (A) Schematic representation of singed^car allele showing the KP-element insertion and the position of PCR primers (arrows). (B) Genomic DNA purified from Carpineto Romano parental flies was amplified using primers flanking (sn2.2 F_sn2.2 R) the KP-element insertion. We obtained a 1.5 kbp amplified product in all parental flies as expected for a wild type genomic sequence. (C) PCR analysis was performed using a forward primer designed on KP-element, and a reverse primer specific for singed 5′ UTR sequence (KP-element F_sn R). These primers amplify only in presence of KP-element insertion (as shown in control PCR on sn^car sample ). Rpl32 was used as internal PCR control. The white boxes delimit the part of the gels containing the control PCR reactions performed on genomic DNA purified from Carpineto Romano wild-type strain and singed^car mutant allele, respectively.

Figure 11

PCR analysis of the sepia gene corresponding to the fixed phenocopies in the wild type Paliano parental flies. (A) Schematic representation of the deletion involving Gst03 and sepia genes. (B) PCR reactions were performed on genomic DNA purified from each single fly of the Paliano parental generation using specific primers flanking the gene regions altered in the sepia stress-induced mutation. se2 outer primers, indicated by arrows in (A), amplify, in se^pal, a product of 1388 bp instead of the expected 2989 bp wild-type amplicon. The white boxes delimit the part of the gels containing the control PCR reactions performed on genomic DNA purified from wild-type and mutant alleles.

Figure 12

PCR analysis of the genes corresponding to the cactus fixed phenocopies in the wild Carpineto Romano parental flies. (A) Schematic diagram showing the cact^car allele and the relative position of primers binding sites (arrows). (B) PCR analysis, using specific primers (cact5.1.3 F_cact5.1.3 R) flanking micropia insertion, shows a 400 bp amplified product in all parental flies as expected for a wild-type genomic sequence; conversely, in cact^car control sample we obtain a 7 kb amplicon. (C) PCR analysis was performed using a forward primer designed on micropia-element, and a reverse specific primer for cactus 3′ UTR sequence (micropia F5_cact5.1.3 R). These primers amplify only in presence of micropia-element insertion (as shown in control PCR on cact^car sample); Rpl32 was used as internal PCR control. White boxes indicate the control PCR reactions performed on genomic DNA purified from wild type and the cactus^car mutant allele.

Figure 13

Transposable element expression profiles from (A) Paliano and (B) Carpineto Romano control and heat-stressed pupae. Fold expression levels of transposon transcripts, determined by quantitative RT-PCR, are shown relative to Rpl32 expression (**P ≤ 0.01).

Figure 14

Models for the assimilation of a stress-induced phenocopy. (A) According to Waddington, the phenocopy induced by stress could be assimilated by the selection of preexisting cryptic mutations at one (monogenic determination) or more (polygenic determination) genes. (B) According to our model, the phenocopy could be fixed by a coselection of a corresponding germline mutation de novo induced by stress. Note that this view is also adaptable to mutant phenotypes with a polygenic determination. As discussed in the text, the assimilation process could take several generations after the mutation induction and before the complete fixation of the mutant phenotype (red). Another important point is that the mutational rate could be higher in stressed phenocopies than in stressed wild-type flies due to increased transposon activity in such variants.