Mutational Analysis of the Drosophila homothorax Gene

Corresponding author: Adi Salzberg, Unit of Genetics, Rappaport Faculty of Medicine, Technion, P.O. Box 9649, Haifa 31096, Israel., adis{at}tx.technion.ac.il (E-mail)

Communicating editor: T. C. KAUFMAN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The homothorax (hth) gene is involved in multiple aspects of embryonic and adult fly development. It encodes a homeodomain-containing protein of the MEIS family and was shown to regulate the subcellular localization of the homeotic protein cofactor Extradenticle (EXD). The HTH protein contains a TALE class homeodomain and a conserved MH domain, which is required for its interaction with EXD. In this work, we describe the structure of the hth locus, characterize at the molecular level a collection of mutant alleles of hth, and discuss the correlation between the identified structural defects and their consequent phenotypes. The hth locus spans more than 100 kb and contains 14 exons. Several of the exon-intron boundaries within the homeodomain and the MH domain-coding regions are conserved between Drosophila and Caenorhabditis elegans. The analysis of hth mutations demonstrates that the homeodomain of HTH is not required for nuclear localization of EXD and that the MH domain-containing first 240 residues are sufficient for nuclear localization of both EXD and HTH. Mutations that alter or delete the homeodomain cause only partial homeotic transformations in the PNS, whereas mutations affecting the MH domain cause distinct and more severe PNS phenotypes. These observations may suggest that driving nuclear localization of EXD is the main role of HTH in patterning the embryonic PNS. They may also suggest that homeodomain-defective HTH protein retains some of its transcription-regulating functions by binding DNA via its interaction with EXD.

THE hth gene encodes for a homeodomain-containing protein of the MEIS family (PREP and MEIS in mammals, HTH in Drosophila, Ceh 25 in Caenorhabditis elegans; BURGLIN 1997 ). The MEIS proteins form stable DNA-independent heterodimers with other TALE class homeodomain proteins of the PBC family (PBX in mammals, EXD in Drosophila, and Ceh-20 in C. elegans; CHANG et al. 1997 ). Heterodimerization results in nuclear localization of the PBC proteins (RIECKHOF et al. 1997 ; KURANT et al. 1998 ; PAI et al. 1998 ), which is thought to be controlled by antagonizing active nuclear export machinery (ABU-SHAAR et al. 1999 ; BERTHELSEN et al. 1999 ). When nuclear, the PBC and MEIS proteins are able to form stable DNA-dependent ternary complexes with HOX proteins altering their in vivo promoter selectivity and functional specificity (e.g., KNOEPFLER et al. 1997 ; BERTHELSEN et al. 1998 ; FERRETTI et al. 1999 ; GOUDET et al. 1999 ; JACOBS et al. 1999 ; PASSNER et al. 1999 ; RYOO et al. 1999 ; PENKOV et al. 2000 ).

Proteins of the MEIS family share several well-conserved structural motifs implicated in their various functions. All MEIS proteins contain an atypical homeodomain of the TALE superclass, which has three extra residues in the loop between helix 1 and 2 (BURGLIN 1997 ). This loop is highly conserved among members of the MEIS family and it also contains a proline-tyrosine-proline sequence that is common to all members of the TALE superfamily (BURGLIN 1997 ). In addition to the highly conserved homeodomain, MEIS proteins share structural similarity in a 120- to 130-amino-acid bipartite domain (Meis-homology domains HR1 and HR2), which is located within their amino terminus (BURGLIN 1997 ). This domain was shown to be required for the interaction of MEIS proteins with PBC proteins and for nuclear translocation of the latter (KNOEPFLER et al. 1997 ; BERTHELSEN et al. 1998 ; RYOO et al. 1999 ).

Most of our knowledge about the molecular function of various structural domains in MEIS proteins comes from in vitro assays and from work done in tissue culture (ABU-SHAAR et al. 1999 ; BERTHELSEN et al. 1999 ). Little is known about these domains from in vivo studies. A first attempt to address these structure-function relations in vivo was described recently by RYOO et al. 1999 and JAW et al. 2000 , who established which portions of the HTH protein are required for the generation of various gain-of-function phenotypes by ectopically expressing truncated variants of HTH in transgenic flies.

Some of the structural motifs, mainly those involved in nuclear import and export, seem to work in a context-dependent fashion (BERTHELSEN et al. 1999 ) and some discrepancy still exists among results of different experiments (reviewed by AFFOLTER et al. 1999 ). The available collection of hth mutant alleles provided an opportunity to test the current hypotheses in vivo in the context of the developing Drosophila embryo.

We have generated and collected from other laboratories numerous mutations in the hth locus. These mutations were induced by ethyl methanesulfonate (EMS), ethyl nitrosourea (ENU), P-element insertions, and excision mutagenesis. Here we describe the molecular analysis of nine chemically induced hth alleles. In addition we describe some of the phenotypes caused by each of these mutations and discuss the correlation between the observed structural and functional defects.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Fly strains:
Five EMS-induced hth alleles were sequenced: hth^H321 and hth^J186 are described in SALZBERG et al. 1994 , the hth^B2 and hth^C1 alleles are described by RIECKHOF et al. 1997 , and the hth^5E allele is described by JURGENS et al. 1984 . The ENU-induced alleles hth^100-1, hth^100-2, hth^100-4, and hth^100-69 were generated by R. EMMONS, P. KIEFEL and I. DUNCAN (unpublished results).

Molecular techniques:
Exon/intron boundaries of exons 1, 6, 11, and 12 were mapped by sequencing genomic clones that hybridized to hth cDNA probes. The genomic sequence published recently confirmed our results (ADAMS et al. 2000 ). The borders of exons 2, 3, 4, 5, 7, 8, 9, 10, 12, 13, and 14 were deduced only from sequences published by the Berkeley Drosophila Genome Project (FLYBASE 1999 ; ADAMS et al. 2000 ).

To sequence the chemically induced alleles of hth, each exon was amplified by PCR from each mutant (and control) strain. PCR reactions were performed on extracts of single heterozygous flies (GLOOR et al. 1993 ). The primers used for amplifying each exon are listed in Table 1. For most exons, the amplification product included the entire exon plus short intronic sequences on both sides (the exceptions are indicated in Table 1). The same primers were used for sequencing. Amplified exons were cleaned using the Qiaquick gel extraction kit (QIAGEN, Valencia, CA) and sequenced using the Big Dye terminator cycle sequencing kit (PE Applied Biosystems, Foster City, CA) and capillary electrophoresis on an ABI PRISM 310 automated sequencer.

Immunohistochemical staining:
For the analysis of EXD subcellular localization and the evaluation of peripheral nervous system (PNS) phenotypes, males from each hth mutant strain were crossed to Canton-S virgin females. Heterozygous hth/+ progeny were crossed and embryos were collected overnight. Staining of whole-mount embryos with monoclonal antibody (mAb) 22C10 (GOODMAN et al. 1984 ) and anti-EXD mAb B11M (ASPLAND and WHITE 1997 ) was performed using standard techniques (PATEL 1994 ). For the analysis of HTH distribution each mutant allele was balanced over a TM6B, P[w⁺, abdA-lacZ] balancer. Embryos were collected and stained with anti-HTH (AS1924; KURANT et al. 1998 ) and anti-ß-galactosidase (Promega, Madison, WI). Embryos were viewed using bright field and confocal microscopy (Radiance2000 confocal system).

Western analysis:
Protein samples were prepared from third instar larval brains, five brains for each sample, by boiling the dissected brains for 4 min in Laemmli buffer without ß-mercapto-ethanol (LAEMMLI 1970 ). Protein samples were separated on a 10% SDS PAGE and transferred to a Hybond enhanced chemiluminescence nitrocellulose membrane (Amersham Pharmacia Biotech). Membranes were blocked with phosphate buffer saline + 2% BSA + 2% normal goat serum. Signal detection was performed with horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma, St. Louis) using SuperSignal chemiluminescence substrate (Pierce, Rockford, IL). The AS1924 anti-HTH serum was preabsorbed to embryos overnight at 4° and was used in a final concentration of 1:5000.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Structure of the hth locus:
The hth locus is quite large and complex. It spans more than 103 kb and contains 14 exons, 24–1266 bp long, which are separated by 208- to 23,700-bp-long introns (Table 1 and Fig 1A). The homeodomain and MH box are not encoded by single exons. The homeodomain is encoded by 3 different exons (11, 12, and 13) and the MH domain is encoded by 5 exons (2, 3, 4, 5, and 6). The splicing sites within the exons encoding the homeodomain are fully conserved between hth and its C. elegans homologue ceh25 (BURGLIN 1997 ; Fig 1B). In addition, the splicing sites between exons 2 and 3 and exons 5 and 6, which encode the MH domain, as well as exons 1 and 2, are conserved between hth and ceh25 (Fig 1B).

View larger version (77K): In this window In a new window Download PPT slide   Figure 1. Structure of the hth locus. (A) The hth gene contains 14 exons represented by black boxes. The insertion sites of three P elements within the hth locus are also indicated [P1422-4 (DEAK et al. 1997 ; SALZBERG et al. 1997 ); l(3)05745 (FLYBASE 1999 ); P1-K1-8 (PAI et al. 1998 )]. (B) Alignment of the HTH and CEH-25 protein sequences reveals the conservation of certain splicing sites. Splicing sites are indicated by arrows (pointing down for HTH and pointing up for CEH-25) and the exons are numbered E1–E14. The MH and homeodomains are underlined.

The molecular nature of hth mutations:
Nine chemically induced alleles of hth were sequenced. In seven of these alleles we identified a point mutation that can lead to the production of an aberrant protein (Table 2).

hth^H321 and hth^J186 are EMS-induced alleles generated by SALZBERG et al. 1994 in a screen for mutations that affect pattern formation in the embryonic PNS. The hth^H321 allele carries an A to T tranversion in nucleotide number 2108, changing Lys²⁸⁶ into a stop codon (Fig 2A). We were unable to identify any point mutation in the coding sequence of the hth^J186 allele using the described method. hth^B2 and hth^C1 are EMS-induced alleles generated by RIECKHOF et al. 1997 . The hth^C1 allele bears a G to A transition in nucleotide 1793, which transforms the conserved Val¹⁸¹ within the MH domain into an Ile. The only mutation identified in the hth^B2 allele is a G to A transition at the 3' splice site of intron 6, which alters the consensus AG dinucleotide into an AA sequence (Fig 2A and Fig B). A failure of proper splicing in this position is expected to alter the translated protein after the Val²⁴² residue. hth^5E is an EMS-induced allele isolated by JURGENS et al. 1984 in their screen for mutations affecting embryonic patterning. We did not identify any point mutation in the coding sequence of the hth^5E allele.

View larger version (40K): In this window In a new window Download PPT slide   Figure 2. Mutations identified in chemically induced hth alleles. (A) Amino acid sequence of the HTH protein. The MH domain and homeodomain are framed. Exon boundaries are indicated by thin arrows above the appropriate residues and the exons are numbered E1–E14. Sites of predicted alterations caused by each of the alleles are doubly underlined. The name of the relevant allele is indicated above it. (B) The hth100-4 and the hthB2 alleles are predicted to affect the 3' splicing site of the introns between exons 10 and 11 and exons 6 and 7, respectively (the affected nucleotides marked with arrows). Exons are boxed and the amino acid sequence is written below the coding triplets.

Four ENU-induced alleles, hth^100-1, hth^100-2, hth^100-4, and hth^100-69, were isolated in a screen for enhancers of spineless (R. EMMONS, P. KIEFEL and I. DUNCAN, unpublished results). All of these alleles contain a C to T or an A to G transition. Two of the alleles, hth^100-1 and hth^100-2, were found to harbor an identical mutation and are likely to represent a clonal event. The hth^100-1/hth^100-2 mutation is a C to T transition in position 2213 that changes Arg³²¹ to a stop codon (Fig 2A). The only mutation identified in the hth^100-4 allele is an A to G transition in the 3' splice site of intron 10, which alters the consensus AG dinucleotide into a GG sequence (Fig 2A and Fig B). A failure of proper splicing in this position is expected to alter the translated protein after the Gly³⁴⁰ residue. The hth^100-69 mutation is a C to T transition in position 2424 that changes the conserved Pro³⁹², which is located between the first and second helix of the homeodomain, into a Leu.

In addition to the chemically induced mutations, we have mapped the P[lacW] insertion in the hth^K1-8 allele. hth^K1-8 is derived from the hth^P1 allele by imprecise excision and local hop of the P[lacW] (PAI et al. 1998 ). The insertion was mapped by amplifying a 190-bp genomic fragment by inverse PCR (GLOOR et al. 1993 ) and sequencing the PCR product. The insertion maps to intron 11, 1900 bp downstream of exon 11 and 4700 bp upstream of exon 12. Since hth^K1-8 homozygous embryos exhibit very low levels of hth RNA and protein expression, it is possible that this insertion affects a regulatory element in the hth locus.

To further characterize the identified mutations we tested whether they lead to the production of aberrant HTH polypeptides that can be detected on Western blots. We performed the analysis on protein samples prepared from third instar larval brains, which contain high levels of the HTH protein (A. SALZBERG, unpublished results). The AS1924 anti-HTH serum identifies a 52-kD band in protein extracts made from embryos (KURANT et al. 1998 and Fig 3). However, in protein extracts made from larvae or adults this band was not evident and the anti-HTH serum identified a 65- to 68-kD polypeptide (Fig 3). To determine which band corresponds to the HTH protein we performed Western analysis on protein extracts made from hs-hth transgenic flies, with or without heat shock. This experiment demonstrated that the HTH protein, extracted from larvae or adults, runs on SDS PAGE as a 65- to 68-kD polypeptide, higher than its expected molecular weight (Fig 3). Similar results were obtained from da-GAL4/UAS-hth embryos overexpressing HTH (data not shown). The nature of the smaller band, which is unique to embryonic extracts, is not known.

View larger version (23K): In this window In a new window Download PPT slide   Figure 3. Western analysis of HTH: The blot on the left side (lanes 1–6) demonstrates that HTH appears on Western blots as an 65-kD protein (arrow). (Lanes 1–4) Protein extracts were made from hs-hth transgenic flies (PAI et al. 1998 ) without heat shock (lanes 1 and 3) or following a 1-hr heat shock at 37° and 90 min recovery at 24° (lanes 2 and 4). Lanes 1–2 were reacted with preimmune serum (1:1000), and lanes 3–4 were reacted with AS1924 anti-HTH serum produced from the same rabbit (1:5000). The 65-kD band is also present in wild-type larval (lane 5) and embryonic (lane 6) extracts. An 52-kD band of unknown nature is evident in embryonic extracts only (arrowhead). The blot on the right side demonstrates the presence of truncated forms of HTH in heterozygous hth100-1 and hth100-4 larvae (marked with asterisks).

Western blot analysis performed on heterozygous hth^100-1 larvae and embryos demonstrated the presence of an 45-kD truncated form of HTH (Fig 3 and data not shown). Western blots performed on heterozygous hth^100-4 larvae revealed a 43- to 48-kD polypeptide not seen in wild-type larvae or in other mutants. We did not detect abnormal protein products in the other mutant strains; however, in hth^B2 and hth^H321 the intensity of the normal HTH band was reduced as compared to wild type (not shown).

Phenotypic analysis of hth mutations:
Embryos from each mutant strain were examined for two phenotypes: abnormal localization of the EXD and/or HTH proteins and abnormal patterning of the peripheral nervous system. In general, most of the described chemically induced alleles (hth^H321, hth^J186, hth^100-1, hth^100-2, hth^100-4, hth^100-69, and hth^B2) caused a relatively mild phenotype in the PNS, which consisted mainly of dorsal localization of the LCh5 neurons in 20–58% of the abdominal segments (Fig 4). In addition, embryos homozygous for the hth^5E allele or the hth^C1 allele exhibited a reduction in the number of LCh5 neurons, loss of thoracic DCh3 neurons, and an abnormal pattern of the axonal trajectories in the PNS (Fig 4). The insertion/excision allele hth^K1-8 is characterized by a more severe loss of neurons, dorsal localization of most Lch5 neurons, and general disorganization of the PNS (KURANT et al. 1998 ).

View larger version (130K): In this window In a new window Download PPT slide   Figure 4. PNS phenotype of hth mutant embryos. Lateral view of stage 15–16 embryos homozygous for various alleles of hth. Embryos were immunohistochemically stained with Mab 22C10. (A) A wild-type embryo. The lateral chordotonals of segments A1–A3 are indicated by small arrows. (B) hth100-2. (C) hthH321.(D) hthB2.(E) hth100-69.(F) hthC1.(G–H) High and low magnification of a hth5E homozygous embryo. Note the reduced number of lateral neurons (asterisks) and pathway defects (arrows). In B–H dorsal chordotonal neurons are indicated by arrowheads and pathway defects are indicated by arrows.

To examine the subcellular localization of EXD in the various hth mutants we doubly stained embryos from each strain with Mab 22C10 and anti-EXD antibody. Homozygous mutant embryos were identified unambiguously on the basis of their PNS phenotype. Most of the mutations, including hth^H321, hth^J186, hth^B2, hth^100-1/100-2, hth^100-4, and hth^100-69, did not affect the subcellular localization of EXD, which exhibited the normal pattern of nuclear localization (Fig 5 and data not shown). The HTH protein itself was also nuclear in mutant embryos homozygous for these alleles (data not shown).

View larger version (116K): In this window In a new window Download PPT slide   Figure 5. EXD remains nuclear in hth mutants lacking the homeodomain. Confocal images showing the distribution of EXD in hth mutant embryos. Embryos were doubly labeled with Mab 22C10 and anti-EXD, which are shown here in a single channel. Mutant embryos were identified on the basis of their PNS phenotype. (A) A wild-type embryo exhibiting nuclear localization of EXD. (B) An hthK1-8 homozygous embryo exhibiting strictly cytoplasmic localization of EXD. (C–D) An hthH321 homozygous embryo shown in two focal planes, one that focuses on the PNS (D) and one that focuses on the epidermis (C). (E–F) hthB2 homozygous embryo shown in two focal planes, one that focuses on the PNS (F) and one that focuses on the epidermis (E). Abnormally positioned chordotonal neurons are indicated by asterisks; the arrows point to regions where the subcellular localization of EXD can be visualized clearly.

Embryos homozygous for the hth^C1 allele exhibited cytoplasmic localization of EXD. However, unlike embryos carrying the severe hypomorphic hth^K1-8 allele, EXD was not completely excluded from the nucleus (Fig 6). The HTH protein was not confined to the nucleus in hth^C1 embryos, but appeared uniformly distributed within most of the cells (Fig 6). Embryos homozygous for the hth^5E allele exhibited a more uniform distribution of EXD and significantly reduced levels of the HTH protein. Although the level of HTH was low, it clearly exhibited a nuclear localization (data not shown).

View larger version (143K): In this window In a new window Download PPT slide   Figure 6. hthC1 mutant embryos exhibit abnormal subcellular localization of HTH and EXD. (A–B) Thoracic segments of stage 11 embryos immunohistochemically stained with anti-HTH. HTH accumulates in nuclei of heterozygous hthC1 embryos (A) but remains largely cytoplasmic in homozygous hthC1 embryos (B). Arrows point to the tracheal pits. (C–F) Dissected stage 15 embryos immunohistochemically stained with anti-EXD shown in two focal planes: central nervous system (CNS; C and D) and ectoderm (E and F). EXD is nuclear in the CNS and ectoderm of heterozygous hthC1 embryos (C and E) but remains largely cytoplasmic in homozygous hthC1 embryos (D and F).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Correlation between structural elements and functional elements in HTH:
It was previously demonstrated that HTH contains two separable functional domains: the N-terminal MH domain, which interacts with EXD and is required for EXD nuclear localization (RYOO et al. 1999 ; JAW et al. 2000 ), and the homeodomain, which is thought to bind DNA when HTH participates in trimeric HTH/EXD/HOX transcriptional regulating complexes.

Most of the mutations identified in this work alter the homeodomain itself (hth^100-69) or cause a truncation of the HTH protein upstream to the homeodomain (hth^100-1/2, hth^100-4, hth^B2, and hth^H321). The subcellular localization of the EXD and HTH proteins was not affected in embryos homozygous for any of these mutations. This observation indicates that the HTH homeodomain is not required for nuclear localization of EXD, supporting previous observations made in transgenic flies expressing mutated or truncated forms of the HTH protein (RYOO et al. 1999 ; JAW et al. 2000 ).

hth^100-69 is the only allele containing a missense mutation within the homeodomain. Interestingly, it substitutes the first proline in the conserved proline-tyrosine-proline sequence, in the loop between helixes 1 and 2, into leucine. The conserved proline-tyrosine-proline motif is common to all members of the TALE homeodomain superfamily (BURGLIN 1997 ). The hth^100-69 mutation demonstrates the importance of this motif in vivo, because it causes a phenotype that is similar to the phenotype caused by a truncation of the entire homeodomain.

Only one mutation that affects the MH domain was identified (hth^C1). This mutation substitutes a valine residue into isoleucine, a highly conservative substitution. However, the affected valine residue is located within the highly conserved HR2 / HM^B domain (BERTHELSEN et al. 1998 ; RYOO et al. 1999 ) and it is present in all proteins of the MEIS family. The phenotype of hth^C1 homozygous embryos is somewhat more severe and is clearly distinct from the phenotypes caused by mutations in the homeodomain. Although we cannot exclude the possibility that the hth^C1 allele carries another unidentified mutation in intronic or regulatory sequences, the observation that EXD remains largely cytoplasmic in this mutant suggests that the MH domain is not functioning. It was previously shown that HTH is not stable in the absence of EXD (ABU-SHAAR and MANN 1998 ; KURANT et al. 1998 ). Therefore, further experiments are required to establish whether the partly cytoplasmic localization of HTH in hth^C1 embryos reflects a genuine defect in nuclear import of HTH or whether it is secondary to the cytoplasmic localization of EXD.

By amplifying and sequencing single exons we were unable to reveal the molecular nature of two hth alleles: hth^5E and hth^J186. These alleles may harbor an intronic mutation that affects splicing, mutations in the 5' untranslated region, mutations in regulatory sequences that affect the expression of hth, or mutations that prevent amplification of the affected exons due to a deletion or an alteration of the sequence that anneals with the PCR primers.

The hth^100-1/2, hth^100-4, hth^B2, and hth^H321 mutations cause indistinguishable and relatively mild PNS phenotypes. Since all these mutations affect the homeodomain of HTH this may suggest that driving nuclear localization of EXD is the main role of HTH in patterning the embryonic PNS. However, it may also suggest that homeodomain-defective HTH protein can retain some of its transcription-regulating functions by being tethered to DNA-binding complexes via its interaction with EXD. The observation that the nuclear form of EXD could not rescue the PNS phenotype of hth mutants (E. KURANT and A. SALZBERG, unpublished results) supports the latter assumption. Similarly, RYOO et al. 1999 have suggested that, in the context of the lab550 enhancer element, the interaction between HTH and EXD is more significant for the HOX protein complex stability than the interaction of HTH with the DNAs.

	ACKNOWLEDGMENTS

We thank Richard Mann, Henry Sun, and Ian Duncan for sending us fly strains and Richard Emmons and Ian Duncan for sharing unpublished data with us. We also thank Cai Ayjun and Naomi Halachmi for technical assistance, Reymonde Szargel for helping us with the sequencing, and Adi Inbal for critical reading of the manuscript. This work was supported by a Research Career Development Award to A.S. from the Israel Cancer Research Fund and a grant from the Israel Science Foundation founded by the Israel Academy of Sciences and Humanities-Charles H. Revson Foundation.

Manuscript received July 24, 2000; Accepted for publication October 31, 2000.

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