Functional analysis of saxophone, the Drosophila gene encoding the BMP type I receptor ortholog of human ALK1/ACVRL1 and ACVR1/ALK2

Abstract

In metazoans, bone morphogenetic proteins (BMPs) direct a myriad of developmental and adult homeostatic events through their heterotetrameric type I and type II receptor complexes. We examined 3 existing and 12 newly generated mutations in the Drosophila type I receptor gene, saxophone (sax), the ortholog of the human Activin Receptor-Like Kinase1 and -2 (ALK1/ACVRL1 and ALK2/ACVR1) genes. Our genetic analyses identified two distinct classes of sax alleles. The first class consists of homozygous viable gain-of-function (GOF) alleles that exhibit (1) synthetic lethality in combination with mutations in BMP pathway components, and (2) significant maternal effect lethality that can be rescued by an increased dosage of the BMP encoding gene, dpp+. In contrast, the second class consists of alleles that are recessive lethal and do not exhibit lethality in combination with mutations in other BMP pathway components. The alleles in this second class are clearly loss-of-function (LOF) with both complete and partial loss-of-function mutations represented. We find that one allele in the second class of recessive lethals exhibits dominant-negative behavior, albeit distinct from the GOF activity of the first class of viable alleles. On the basis of the fact that the first class of viable alleles can be reverted to lethality and on our ability to independently generate recessive lethal sax mutations, our analysis demonstrates that sax is an essential gene. Consistent with this conclusion, we find that a normal sax transcript is produced by saxP, a viable allele previously reported to be null, and that this allele can be reverted to lethality. Interestingly, we determine that two mutations in the first class of sax alleles show the same amino acid substitutions as mutations in the human receptors ALK1/ACVRl-1 and ACVR1/ALK2, responsible for cases of hereditary hemorrhagic telangiectasia type 2 (HHT2) and fibrodysplasia ossificans progressiva (FOP), respectively. Finally, the data presented here identify different functional requirements for the Sax receptor, support the proposal that Sax participates in a heteromeric receptor complex, and provide a mechanistic framework for future investigations into disease states that arise from defects in BMP/TGF-beta signaling.

Figure 1.—

sax¹ and sax² maternal enhancement of dpp mutant progeny. Females heterozygous for the maternal effect mutations, sax¹ or sax², dominantly interact with dpp^hr alleles. Percentage of survivorship of dpp^hr/+ adults (boxed genotypes) is shown on the y-axis. (A) Females heterozygous for sax¹ or sax², the parental chromosomes (cn bw sp and cn bw) or deficiencies that retain (Df(2R)ST1) or lack (Df(2R)P32, Df(2R)H23) sax function were tested for maternal enhancement of dpp^hr4/+ lethality. The number of progeny examined was ≥577. (B) Survivorship of different dpp^hr alleles when crossed with females heterozygous for a given sax mutation is shown as the percentage of expected dpp^hr heterozygous progeny. The female genotypes are indicated by different bar shading. dpp alleles are listed along the x-axis. The number of progeny examined from each cross was ≥200.

Figure 2.—

Dominant Mad–sax interactions. (A) Mad–sax MEL. The percentage of lethality of progeny from females heterozygous for various Mad and sax alleles crossed to wild-type males is depicted by bars. Transheterozygous (Mad +/+ sax) females show significant maternal effect lethality. Female genotypes are listed on the x-axis. n ≥ 300 embryos scored for control crosses and n ≥ 475 embryos for experimental crosses. (B) Mad–sax zygotic enhancement of the dpp^d12 disk mutation. The number of legs per individual with tarsal claws was quantified in progeny from dpp^d12/+ females crossed to males bearing a Mad⁻ sax⁻ double mutant chromosome (Df(2L)JS17 sax¹/CyO23) (dark bars) or only Mad⁻ (light bars). A lowering of both Mad and sax dosage results in an enhancement of tarsal claw loss associated with a reduction in dpp function.

Figure 3.—

Interallelic complementation between sax alleles. Females (A) and males (B) heterozygous for the listed sax alleles were crossed and the lethality of sax*^A/sax*^B progeny was quantified. Shading of boxes indicates the percentage of progeny exhibiting zygotic viability of each allelic combination (key on right). The percentage of viability was calculated as the number of Cy⁺, divided by the number in the Cy parental class that survived least well (n ≥ 300 adults scored for each cross).

Figure 4.—

Maternal enhancement of dpp mutant progeny by all sax alleles. Test crosses between all sax alleles and dpp^hr4 were performed in both directions, with respect to the genotypes of the females and males. Crosses in which the females were mutant for sax are represented by the solid bars, and the male sax mutant crosses are represented by shaded bars. The genotypes of tested chromosomes are listed below. cn¹ bw¹ sp¹ is the parental chromosome for sax¹, sax^1rv1, and sax^1rv5. nub¹ b¹ pr¹ is the parental chromosome for sax³, sax⁴, sax⁵, and sax⁶. For female sax mutant crosses, the number of progeny scored per cross was ≥550 (except sax^P, n = 417). For male sax mutants crosses, the number of progeny scored per cross was ≥345 (except sax², n = 155; sax^PE4, n = 269).

Figure 5.—

sax⁵ produces more severe phenotypes than sax⁴. (A) Dark field image of a wild-type wing. Longitudinal veins 2 (L2), 4 (L4), and 5(L5) are indicated. (B and C) Wings resulting from sax mutant clones as described in Bangi and Wharton (2006b). Clones marked with shv appear dark in images. (B) A sax⁴ clone encompassing the entire posterior compartment shows no patterning defects. Consistent with previous studies (Singer et al. 1997; Bangi and Wharton 2006b), a small sax⁴ clone in the anterior compartment leads to an ectopic L2 (eL2) vein. (C) A sax⁵ clone in the posterior compartment results in the loss of L4 (arrow) and a narrowing of the L4/L5 intervein, a phenotype never seen in an equivalent sax⁴ clone. The more severe phenotype of sax⁵ suggests that the presence of a defective Sax receptor is more detrimental to BMP signaling during wing patterning than the complete loss of the Sax receptor. (D) A cell-based BMP signaling assay indicates that the sax⁵ mutation is able to negatively affect BMP signaling mediated by Tkv. S2 cells were cotransfected with the Su(H)/brk-lacZ reporter construct, Su(H), and N* constructs to stimulate transcription (sample 1), and tkv, and/or sax and sax⁵ constructs under the control of the actin 5C promoter (samples 2–9). Values depicted are the fold activation of β-galactosidase over the basal activity of the reporter construct alone. All values represent the average of samples measured in triplicate and normalized for transfection efficiency.

Figure 6.—

P-insertion site of sax^P does not disrupt transcription. (A) Genomic structure of sax locus shown with exon (numbered) distribution for the two splice forms of sax mRNAs (RA and RB). The locations of two ATG initiation codons and the TAG termination codon (vertical thick solid lines) indicate the two overlapping open reading frames (speckles) giving rise to the putative protein products PA and PB (shaded speckles). Positions of PCR primers are indicated by arrowheads. The site and junctional sequence of the placW insertion giving rise to sax^P is shown at the top. The endogenous second ATG within the sax transcription unit is bold and underlined. (B) RT–PCR products from different primer pairs generated from RNA isolated from control (yw) (left) and sax^P homozygous mutant flies (right). Lane 1, primers 8103 + 10228; lane 2, primers 9396 + 11119; lane 3, primers placW10648 + 11119; lane 4, primers placW9661 + 10228. Note the insertion of placW disrupts the wild-type transcription unit initiating at RA-1 (lane 1) but allows transcription to initiate within the placW element between primers placW9661 and placW10648 (presence of PCR product in lane 3 of sax^P animals and not in wild type, or in lane 4 of yw or sax^P). Transcription in both genotypes extends through the expected translational termination site (PCR product present in lane 2 of both genotypes). M, marker lane. A, actin control. (C) The predicted amino acid sequence of SaxPB produced by open reading frame initiating at second endogenous ATG (bold and underlined in A) is shown in normal type with potential additional amino acids (italics) if translation initiated at an atg within the 3′ P-element of placW (shown in A).

Figure 7.—

Sequence comparison of Sax (PA and PB isoforms), ACVR1 (ALK2), ALK1 (ACVRL1), Tkv (PA isoform), and ALK3. The extracellular ligand binding domain (yellow), the transmembrane domain (purple), the intracellular GS activation (green), and serine/threonine (blue) kinase domains are shaded. The structural elements of the cytoplasmic GS and kinase domains (based on the TβR1 structure) (Huse et al. 1999) are indicated above the sequence alignment. The positions of mutations associated with the sax alleles discussed are indicated above the sequence alignment. The positions of specific HHT2 and FOP mutations in ALK1 and ACVR1, respectively, are highlighted in red within the sequence. The asterisks mark the invariant Lys and Glu residues critical for stabilization of the catalytic segment with the N and C lobes of the kinase.