Previous work in the laboratory compared the mRNA expression profile of twist loss-of-function embryos to that of wild-type embryos and of embryos that express twist ubiquitously (Furlong et al.,2001). This work led to the identification of hundreds of genes newly linked to mesoderm development. The developmental role of the majority of these genes has not been examined. To evaluate whether these genes are essential for embryonic development, we verified their expression in the mesoderm by in situ hybridization and performed an RNAi-based screen of 26 genes. The 26 genes were selected based on their expression in the embryonic mesoderm, their unknown developmental function, and, in several cases, the presence of protein domains commonly found in signaling proteins. Stage 3 embryos (1 hr after egg lay) were injected with dsRNA probes directed against the coding region of the selected genes and ranging from 500 to 800 bp in length. Embryonic survival was assayed by determining the percentage of embryos that produced motile first-instar larvae able to hatch from the vitelline membrane (Fig. 1). Control-injected embryos show a survival rate of 60–80%. Of the genes tested, only the dsRNA probes targeting CG3983, CG8965, and CG11100 led to significantly lowered survival rates. These lethal effects were verified by injection of a second set of nonoverlapping interfering dsRNA molecules (data not shown). CG3983 is a gene related to mammalian nucleostemin, a gene thought to regulate proliferation of several stem cell populations (Tsai and McKay,2002). Our work on this gene will be reported elsewhere (D. Kaplan, G. Zimmermann, and M.P. Scott, unpublished data). CG8965 has no known function, but is predicted to contain two Ras-association domains. In this study, we focus on CG11100, a gene also identified in a microarray-based screen for genes expressed in discrete dorsal–ventral patterns (Stathopoulos et al.,2002). The gene was named mes2, based on its expression in the early mesoderm, and we shall henceforth refer to it by this name. mes2 has since been identified in several additional microarray-based screens that examined transcript levels in the wing imaginal disc (Butler et al.,2003), glia (Freeman et al.,2003), and the central nervous system (CNS) midline (Kearney et al.,2004).
Mes2 Protein Is a Member of the MADF Family
The mes2 gene is predicted to encode a 437 amino acid protein. Conserved-domain searches of the public database (Marchler-Bauer and Bryant,2004) revealed the presence of a single MADF domain (Myb/SANT-like domain in Adf-1: SMART accession no. 00595) within the N-terminal portion of Mes2 (amino acids 62–149). This domain was originally identified in Adf-1, a transcriptional activator of alcohol dehydrogenase (England et al.,1992). The MADF domain shows weak similarity to Myb domains. MADF domains of two Drosophila proteins, Adf-1 and Dip3, can bind DNA directly (Cutler et al.,1998; Bhaskar and Courey,2002). The Drosophila genome contains 48 predicted proteins that contain at least one MADF domain, including several known or predicted transcriptional regulators such as Adf-1, Dip3, Stonewall, and Regular (England et al.,1992; Cutler et al.,1998; Claridge-Chang et al.,2001; Bhaskar and Courey,2002). In most cases, the domain occurs within the N-terminal portion of the protein.
An alignment of the MADF domains of Mes2, Dip3, and Adf-1 as well as those of two predicted vertebrate proteins reveals the presence of several highly conserved aromatic residues (Fig. 2). An alignment of all Drosophila MADF domains reveals the following consensus motif: [LFI][IVL](2X)[VIY](5X)[LIV][YW][DEN](5X)[YF](9X)[WYF](2)[IVL](14-18X)[WF][KR]X[MLI][R](2X)[YF]. The three aromatic amino acids at positions 12, 29, and 47 (shown in bold) are especially well conserved and form the basis of the similarity to the mammalian Myb domain (England et al.,1992). MADF domains have been identified in diverse organisms ranging from worms to fish, although oddly, no such domain has yet been identified in mammalian genomes. Thus, this is a rare case of a substantial family of genes in multiple branches of the animal kingdom that has never been found in mouse or human. The regions of Mes2 outside of the MADF domain show little or no sequence similarity to other proteins. The closest relative of mes2 is another Drosophila gene (CG12768) that occurs ∼22-kb upstream of the mes2 locus. This is the only protein that shows any similarity to Mes2 outside of the MADF domain. This additional sequence similarity is confined to a 38 amino acid region in the C-terminus of the proteins. In contrast to mes2, we were unable to detect any CG12768 expression in the embryonic mesoderm (data not shown).
Figure 2. Alignment of MADF domains. Sequence alignment of Mes2 (amino acids 62–149) with the MADF domains from Drosophila Adf-1 and Dip3, two proteins known to bind DNA directly. Also shown are the MADF domains from two predicted vertebrate proteins: XP686513 (Danio rerio) and CAG11942 (Tetraodon nigroviridis). The consensus sequence for these domains is indicated. The consensus sequence for all Drosophila MADF domains is as follows: [LFI][IVL](2)[VIY](5)[LIV][YW][DEN](5)[YF](9)[WYF](2)[IVL](14-18)[WF][KR]X[MLI][R](2)[YF].
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To evaluate the expression patterns and the localization of the Mes2 protein during development, we prepared a polyclonal antibody against the full-length protein. Antibody staining of wild-type Drosophila embryos revealed that Mes2 is a nuclear protein first expressed in the trunk and head mesoderm during late gastrulation (Fig. 3A). This antibody specifically recognizes Mes2, as demonstrated by the absence of staining in Drosophila embryos that do not express the gene (Fig. 4E). Mes2 colocalizes with the mesoderm-specific DMef2 protein within cells of the trunk mesoderm (Fig. 3D–F). Expression of mes2 is restricted to the mesoderm until embryonic stage 10. The timing of mes2 expression in the early mesoderm correlates well with that of the twist target genes Dmef2 and tin (data not shown). Our previous microarray analysis (Furlong et al.,2001) revealed that the mes2 transcript is depleted in twist mutant embryos. The ratio of mes2 transcript in these embryos (5–6 hr after egg lay) when compared with stage-matched wild-type embryos was 0.45 (P value of 0.0007). We also see very little Mes2 staining in early (stage 6–10) twist mutant embryos (not shown), which is not surprising given the loss of mesoderm in these mutants. The microarray data also showed enhanced levels of mes2 transcript in early (3–4 hr after egg lay) Toll10B mutant embryos that overexpress twist, with a mutant to wild-type ratio of 3.4 (P value of 0.0003). These data indicate that mes2 is a target gene of twist. We have tested this further by misexpressing twist in the wing disk using the 71B and MS1096 Gal4 drivers. Despite substantial Twist protein production, ectopic production of Mes2 protein is not observed (data not shown). These results suggest that Twist requires additional factors to activate mes2 expression or that repressing influences in the wing disc block Twist from activating mes2.
Figure 3. Mes2 expression in embryonic and larval tissues. A–M: Mes2 protein (shown in red) is detected in wild-type embryos (A–I,M) or larval wing disk (J–L) using a polyclonal anti-Mes2 antibody followed by an Alexa-conjugated secondary antibody. A: Mes2 localizes to nuclei and is first detected in stage 6 embryos during gastrulation. D–F: Double-labeling for Mes2 (red) and the mesoderm marker DMef2 (green) reveals colocalization in the trunk mesoderm of stage 9 embryos. (D, Mes2; E, DMef2; F, merge). B: At embryonic stage 11, Mes2 expression expands to include the neurogenic ectoderm and expression in the trunk mesoderm starts declining. G–I: By stage 16, Mes2 (red) expression in the nervous system is restricted to glial populations and colocalizes with the glial-specific marker Repo (green; G, Mes2; H, Repo; I, merge). C: At these late embryonic stages Mes2 is also expressed in head mesoderm-derived tissues such as the lymph gland, pericardial nephrocytes, and the fat body. M: By embryonic stage 16, Mes2 (red) is no longer expressed in muscle tissues derived from the trunk mesoderm that express DMef2 (green). J–L: During larval stages, Mes2 is expressed in wing-disk myoblasts (J, Mes2; K, DMef2; L, merge) and in central nervous system glia (not shown).
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Figure 4. The KG02901 insertion leads to a complete disruption of mes2 expression. A: Graphic representation of the mes2 locus (green, exons; dark green, translated regions; light green, untranslated regions; black bars, introns). The KG02901 P-element insertion (red) deletes a portion of the mes2 coding region. B,C: In situ hybridization against mes2 demonstrates that the KG02901 insertion results in the complete loss of the mes2 transcript in homozygous mutant stage 8 embryos (C) compared with heterozygous animals (B). D,E: The Mes2 protein cannot be detected in homozygous mutant stage 13 embryos (E). D: Heterozygous embryos are shown for comparison (red, Mes2; blue, DMef2; green, βgalacotsidase carried on balancer chromosome).
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Beginning at stage 11, the expression pattern of mes2 expands to include neurogenic ectoderm (Fig. 3B). As embryogenesis proceeds, the expression of mes2 declines in muscle tissues derived from the trunk mesoderm (Fig. 3C,M), while expression in the neuronal ectoderm becomes restricted to glia (Fig. 3G–I,M). Costaining with the anti-Repo antibody demonstrates that mes2 is expressed in all glial populations of the CNS and peripheral nervous systems (Fig. 3G–I). At these late embryonic stages, Mes2 is also expressed in tissues derived from the head mesoderm, such as the fat body, lymph glands, pericardial nephrocytes, and hemocytes (Fig. 3C). Staining larval tissues with the Mes2 antibody demonstrates expression in the same cell types that produce Mes2 in the embryo, such as myoblasts, which stain for Mes2 in the wing disc (Fig. 3J–L), and CNS glia (not shown). The dynamic expression pattern of Mes2 in two Drosophila germ layers suggests a regulatory function in diverse embryonic and larval tissues.
mes2 Protein Is Essential for Larval Development
To examine the role of mes2 during development, we obtained a fly line carrying a lethal P-element insertion (KG02901) within the mes2 genomic locus. mes2KG02901/mes2KG02901 animals were able to complete embryogenesis and hatch out of the vitelline membrane, but homozygotes die during larval stages. The movement of these first instar (L1) larvae is sluggish, although the animals do respond to touch (data not shown). The growth of the mutant larvae is severely impaired when compared with their heterozygous siblings. The mutant animals arrest during the L1 stage and die during the transition to the second instar (L2) larval stage. A small proportion of mutant larvae survive up to 8 days after egg laying, but even at this late time point, all of the animals remain in the L1 stage. We have never observed a live L2 mutant larva. Many larvae display incomplete ecdysis, a failure to shed the L1 cuticle completely. This developmental arrest was not rescued by feeding ecdysone to the larvae (data not shown), indicating that the inability to complete the transition to L2 is not due to a deficiency in the production of molting hormone. These data show that mes2 is essential for larval development and progression to the L2 stage.
Inverse polymerase chain reaction (PCR) sequencing (see Experimental Procedures section) revealed that the KG02901 insertion is associated with a small deletion that removes a portion of the first exon (including the start codon) and all of the second exon of mes2 (Fig. 4A). The second exon encodes the entire region of the MADF domain. Homozygous mutant embryos have no detectable mes2 transcript (Fig. 4C) or Mes2 protein (Fig. 4E), confirming that mes2KG02901 represents a null allele of mes2. The mutation is not complemented by the Df(3L)ED5017 deficiency, demonstrating that the lethal phenotype maps to the region that contains the P-element insertion.
To conclusively demonstrate that the lethal phenotype observed in these animals is due to the disruption of the mes2 locus, we constructed a genomic DNA rescue plasmid for this gene. Comparing the mes2 locus in Drosophila melanogaster to that in Drosophila pseudoobscura revealed two regions with high conservation in the noncoding portions of the gene (Fig. 5A). One region is located ∼2 kb upstream of the first exon and the other occurs within the large second intron. The conservation of these regions suggests that they may contain regulatory sequences that control the expression of mes2. Based on this information, we generated a rescue construct containing a 12-kb genomic fragment that encompasses all putative regulatory sequences. This fragment includes the entire coding region of the mes2, as well as upstream (3.5-kb) and downstream (1.5-kb) regions (Fig. 5A). Introducing the rescue construct into a mes2 mutant background restores the expression of mes2 in a wild-type pattern and fully rescues the lethal phenotype produced by this mutation, resulting in viable adult flies with no phenotypic defects (Fig. 5B vs. wild-type in 5C). Our data demonstrate that the phenotype associated with the KG02901 insertion is indeed caused by the disruption of the mes2 locus.
Figure 5. The mes2KG02901 allele is completely rescued by a mes2 genomic rescue construct. A: VISTA alignment of the mes2 regions of Drosophila melanogaster and D. pseudoobscura. Regions of high sequence similarity are indicated in pink and include two domains outside of the coding region. Red arrows indicate the limits of the genomic fragment that completely rescues mes2 mutant animals. B,C: Rescued mes2KG02901 flies of the genotype w; rescue; mes2KG02901 (B) are indistinguishable from wild-type CantonS flies (C).
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The survival of zygotic mutant animals up to the L1 larval stage differs from the dramatic embryonic lethality we observed in response to dsRNA injections (Fig. 1). To address the possibility that the maternally deposited mes2 RNA we detect by in situ hybridization (not shown) is able to rescue embryonic lethality, we generated mes2 mutant germline clones (Fig. 6). mes2 is proximal to the available FRT insertion sites that are usually used to generate clones on the left arm of chromosome three, making it necessary to recombine chromosomes another way. We devised a strategy to eliminate the genomic rescue construct from the germline of adult flies that are homozygous for the mes2 mutation (see Experimental Procedures section). To this end, we modified the approach of Luschnig et al. (2004) in which germline clones are detected by the absence of a maternally deposited nuclear green fluorescent protein (GFP; Fig. 6A). The disruption of the mes2 transcript in germline clones was confirmed by carrying out reverse transcription followed by PCR (RT-PCR) with mes2-specific primers with RNA from 1- to 2-hr-old embryos (Fig. 6B). The full maternal-zygotic mutants survive into the L1 larval stage, reproducing the phenotype of the zygotic mutant. These results indicate that maternally deposited mes2 transcript does not mask an embryonic requirement for the gene.
Figure 6. Generating maternal-zygotic mes2KG02901 mutant embryos. Flp-induced recombination produces germline clones that lack mes2 (see Experimental Procedures section for experimental details). A: Mutant clones are detected by loss of maternally deposited green fluorescent protein (GFP). The two embryos on the left are derived from GFP-negative mes2 mutant clones. B: The loss of the mes2 transcript was verified by reverse transcription-polymerase chain reaction using total RNA from 10 pooled embryos (embryonic stage 1–3). GFP-positive embryo pools (+) contain mes2 mRNA, whereas GFP-negative embryo pools (−) do not. To produce full maternal-zygotic mutant embryos, females that produce mutant germline clones were crossed to heterozygous mutant mes2KG02901 males. Loss of mes2 in the female germline does not affect the zygotic mutant phenotype.
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During larval stages, the Mes2 protein is strongly expressed in glia. To examine a requirement for Mes2 in the development of these cells, we adapted the MARCM technique (mosaic analysis with a repressible marker; Lee and Luo,1999) to create mitotic clones that are homozygous for the null mutation. Mitotic recombination on the second chromosome creates YFP-positive mes2-mutant glia by removing the mes2 genomic rescue construct (see Experimental Procedures section). The overall morphology of glia that lack mes2 is normal, and the cells display no obvious developmental defects (Fig. 7B, compared with wild-type in 7A). These results suggest either that Mes2 protein is not essential for glia development or that the requirement is non–cell autonomous. Alternatively, the mes2 mutation may disrupt an essential glia function without affecting the gross morphology of these cells. This possibility could explain the larval death caused by loss of the gene.
Figure 7. Mutant mitotic clones do not disrupt glia morphology in third-instar larvae. Flip-induced recombination produces glial clones that are positively marked by YFP (see Experimental Procedures section for experimental detail). A,B: Control clones (A) and mes2 mutant clones (B) of surface glia in third instar larval brain lobes occur at similar frequencies and display similar overall morphology.
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Mis- and Overexpression of mes2 Is Highly Damaging
To evaluate the effect of mes2 misexpression on embryonic development, we produced transgenic animals that carry an inducible UASt-mes2 construct. We tested many fly strains that produce Gal4 in numerous tissues and patterns (Table 1). We found that increased production of Mes2 in various tissues disrupts normal development and leads to a lethal phenotype (Table 1). We have overexpressed mes2 in cells that express the endogenous protein using glia-specific (repo-gal4 and gcm-gal4) and mesoderm-specific (twist-gal4, Dmef2-gal4, and 24B-gal4) Gal4 drivers. The morphology of the tissues that overexpress the protein was not disrupted, but in all cases, heightened levels of Mes2 resulted in lethality.
Table 1. Misexpression of mes2 Leads to Lethality
|Gal4 Strain||Phenotype (25°C):||Phenotype (18°C)|
|Mesoderm|| || |
| twist2XPE-gal4||Embryonic/Larval (L1) lethal||Viable|
| twist-gal4; 24B-gal4||Embryonic/Larval (L1) lethal||Embryonic/Larval (L1) lethal|
| twist-gal4; Dmef2-gal4||Larval (L1) lethal||Viable|
|Glia|| || |
| gcm-gal4||Larval (L2) lethal||Viable|
| tub-gal4||Embryonic lethal||lethal|
|Ubiquitous|| || |
| NGT4; NGT40||Lethal||N/A|
| da-gal4||Embryonic lethal||N/A|
|Epithelium|| || |
| 69B-gal4||Embryonic lethal||N/A|
| MS1096||Pupal lethal||N/A|
| 71B-gal4||Larval/Pupal lethal||Larval/Pupal lethal|
The ubiquitous production of Mes2 in embryos results in embryonic lethality and causes gross developmental abnormalities. The most striking phenotype was the failure of dorsal closure. Dorsal closure was also disrupted in embryos that specifically misexpress mes2 in the epidermis (Fig. 8C,D). These embryos appear morphologically wild-type up to stage 11. By stage 13, however, the organization of the epidermis is severely disrupted, especially along the leading edge that contacts cells of the amnioserosa. At the time when dorsal closure normally takes place, the cells of the leading edge do not display the normal elongated morphology and the integrity of the epidermis is disrupted, leading to “tears” in the epidermal sheath (Fig. 8C,D). mes2 misexpression also disrupts the development of the wing-disc epidermis (Fig. 8). Most MS1096-gal4; UASt-mes2 animals die during pupal stages, with very few escapers. Those animals that do survive have severely deformed wing and haltere tissues that form a necrotic mass. Even when the MS1096-gal4; UASt-mes2 animals are raised at 18°C to reduce Gal4 function and, therefore, the level of Mes2, the surviving adults display severely disrupted wing tissues (Fig. 8F). In all cases tested, the exogenous Mes2 protein displays a normal subcellular localization within the nucleus (Fig. 8C).
Figure 8. Misexpression of mes2 disrupts epidermal tissues. A–D: A membrane-targeted Src:GFP fusion protein was expressed in the embryonic epithelium either by itself (A,B) or in conjunction with mes2 (C,D) using the 69B-gal4 driver. Embryos were stained with anti-Mes2 antibody (red, Mes2; green, GFP). C,D: Stage 13 embryos that misexpress mes2 display a disruption of the epidermis and amnioserosa. F:mes2 was misexpressed in the larval wing-disk epithelium using the MS1096-gal4 driver. Animals that escape pupal lethality when reared at 18°C display disrupted wings. E: A MS1096-gal4 adult is shown for comparison.
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