Method of detecting expression of MAB-21

This invention provides vectors comprising isolated polynucleotides comprising a member selected from the group consisting of: (a) a polynucleotide encoding MAB-21 protein; (b) a polynucleotide capable of hybridizing to and which is at least 60% identical to the polynucleotide of (a); and (c) a polynucleotide fragment of the polynucleotide of (a) or (b). This invention further provides host-vectors system for the expression of MAB-21 protein and protein products of mab-21 homologs. This invention further provides MAB-21 protein and protein products of mab-21 homologs. This invention also provides antibodies capable of specifically binding to the MAB-21 protein or protein products of mab-21 homologs. Finally, this invention also provides methods for identifying various suppressor, enhancer and modifier genes of the mab-21 and its homologs.

Within this application, publications are referenced within parentheses.
 Full citations for these references may be found at the end of each series
 of experiments. The disclosures of these publications in their entireties
 are hereby incorporated by reference into this application in order to
 more fully describe the state of the art to which this invention pertains.
 BACKGROUND OF THE INVENTION
 The C. elegans gene mab-21 is required for the choice of alternate cell
 fates by four epidermal and neuronal cells present in the lateral
 epidermis on each side of the male tail. Three of the four cells are
 products of one of nine cell sublineages leading to nine sensory rays. In
 mab-21 mutants, two cells, one neuronal and one glial-like, in one of the
 nine rays assume characteristics of the same cells found in a more
 anterior ray. In addition, a hypodermal cell generated by the same cell
 sublineage makes a different choice of fusion partner. The fourth cell
 affected is a hypodermal cell, which in mab-21 mutants is transformed into
 a neuroblast. All these cells lie together in the lateral tail epidermis,
 suggesting that mab-21 acts as part of a localized pattern formation
 mechanism. mab-21 mutant males and hermaphrodites have additional
 pleiotropic phenotypes affecting movement, body shape, and fecundity,
 suggesting that mab-21 has functions outside the tail region of males.
 Applicant shows that the three known alleles of mab-21 are hypomorphs of a
 new essential gene with an embryonic function. The mab-21 gene encodes a
 novel protein of 386 amino acids. In further studies of the action of
 mab-21 in the male tail epidermis, applicant shows by mosaic analysis that
 mab-21 acts cell autonomously to specify the properties of one sensory
 ray, but non-autonomously in the hypodermal versus neuroblast cell fate
 choice. Presence of cell signaling in the choice of the neuroblast fate
 was confirmed by cell ablation experiments. Mutations in mab-21 were shown
 previously to be genetic modifiers of the effects of HOM-C/Hox gene
 mutations on ray identity specification. The results presented here
 support the conclusion that mab-21 acts as part of a mechanism required
 for correct cell fate choice, possibly involving the function of HOM-C/Hox
 genes in several body regions.
 During animal development, pattern formation mechanisms guide the
 generation of variant forms of a variety of serially repeated structures,
 such as segments, rhombomeres, digits, or cell sublineages. One way in
 which these pattern formation mechanisms exert their influence is by
 determining unique states of expression of HOM-C/Hox transcription factors
 within cells (McGinnis and Krumlauf, 1992). HOM-C/Hox transcription
 factors, in turn, dictate transcription of characteristic patterns of
 downstream target genes that determine the individual properties of
 separate units (Andrew and Scott, 1992; Botas, 1993).
 In C. elegans, HOM-C/Hox genes determine variant forms of epidermal and
 neuronal cell sublineages that are serially repeated along the
 anteroposterior body axis (Sulston and Horvitz, 1977; Wang et al. 1993).
 As one example of this process, applicant has studied development of a set
 of nine bilateral pairs of peripheral sensory organs known as rays present
 in the posterior region of males. Each ray develops from an identical ray
 cell sublineage, yet each develops at a different epidermal site, can have
 a distinct morphology and pattern of neurotransmitter expression, and can
 mediate distinct behavioral responses during mating (Suiston et al., 1980;
 Loer and Kenyon, 1993; Liu and Sternberg, 1995). In addition, the cells of
 each ray appear to express a unique combination of cell recognition
 functions necessary for their assembly as a separate organelle. Evidence
 for such distinct ray morphogenetic identities came from studies of
 mutations in several genes required for independent development of subsets
 of rays (Baird et al., 1991). In males mutant for these genes, neighboring
 rays fuse together during development, suggesting that they have lost
 their distinct identities and instead express common assembly functions.
 It was shown that HOM- C/Hox genes play a role in endowing separate
 developmental and morphological identities to the rays (Chow and Emmons,
 1994). Levels of expression of these genes within the terminal cells of
 the ray lineages dictate distinct morphological identities that allow the
 rays to assemble independently. Several of the ray fusion genes applicant
 identified were shown to be genetic modifiers of HOM-CiHox effects on ray
 development, suggesting that ray fusion genes act in a common pathway with
 HOM-CoHox genes (Chow and Emmons, 1994). In the present paper applicant
 characterizes further one of these HOM-C/Hox gene modifier loci.
 Mutations in the gene mab-21 resulted in the specific transformation of the
 identity of one of the rays into that of a more anterior ray (Baird et
 al., 1991). Here applicant characterizes this transformation in greater
 detail, and document effects of mnab-21 mutations on the cell fate choices
 and/or differentiation of neural and hypodermal cells. mab-21 also appears
 to function outside of the tail region of males, and applicant shows that
 his amab-21 mutations are hypomorphic alleles of a new essential gene with
 an embryonic function. Applicant cloned the gene and show that it encodes
 a novel protein of 386 amino acids. In further studies of its action in
 the male tail, applicant shows that mab-21 acts cell autonomously in
 specifying a ray identity, and thus could function together with the
 autonomously-acting HOM-C/Hox genes. Its action in the choice of epidermal
 or neuroblast cell fate by a neighboring cell was non-autonomous, however.
 The results are consistent with a model in which mab-21 acts as part of a
 localized pattern formation mechanism that dictates the fates of cells in
 the male tail lateral epidermis. It could as well have a wider function in
 specification by HOM-C/Hox genes of specialized structures or cell
 identities elsewhere in the body.
 SUMMARY OF THE INVENTION
 This invention provides an isolated polynucleotide comprising a member
 selected from the group consisting of: (a) a polynucleotide encoding
 MAB-21 protein; (b) a polynucleotide capable of hybridizing to and which
 is at least 60% identical to the polynucleotide of (a); and (c) a
 polynucleotide fragment of the polynucleotide of (a) or (b). This
 invention also provides a vector comprising the above-described DNA.
 This invention also provides a host cell comprising the above-described
 vector. This invention further provides a host vector system for the
 production of MAB-21 protein comprising the above vector and a suitable
 host. In an embodiment, the suitable host is selected from a group
 consisting of a bacterial cell, plant and animal cell.
 This invention provides a nucleic acid molecule of at least 15 nucleotides
 capable of specifically hybridizing with a unique sequence of a nucleic
 acid molecule which is complementary to the above-described nucleic acid
 molecule.
 This invention also provides methods of detecting expression of nmab-21 or
 its homologs in a sample which comprises steps of: a) obtaining total mRNA
 from the sample; b) contacting the mRNA so obtained with a labelled
 nucleic acid probe molecules which are described above under hybridizing
 conditions; and c) determining the presence of mRNA hybridized to the
 molecule, and thereby detecting the expression of the mab-21 or its
 homologs in the sample.
 This invention provides methods of detecting expression of protein products
 of mab-21 or mab-21 homologs in a sample which comprises steps of: a)
 obtaining protein extracts from the sample; b) contacting the obtained
 protein extract with an antibody capable of specifically recognizing the
 protein products of mab-21 or mab-21 homologs under conditions permitting
 the formation of complexes between the antibody and the protein products,
 measuring the amount of the formed complexes, thereby detecting the
 expression of the protein product of mab-21 and mab-21 homologs.
 This invention also provides purified MAB-21 proteins or fragments thereof.
 This invention also provides methods for production of an antibody capable
 of binding to protein products of mab-21 or mab-21 homologs comprising: a)
 administering an amount of purified protein products of mab-21 or mab-21
 homologs to a suitable animal effective to produce an antibody against the
 protein in the animal; and b) testing the produced antibody for capability
 to bind the administered protein. In an embodiment, the antibody is
 produced by in vitro immunization.
 This invention also provides methods for production of an antibody capable
 of binding to protein products of mab-21 or mab-21 homologs comprising: a)
 determining conserved regions revealed by alignment of MAB-21 protein and
 the protein product of mab-21 homologs; b) synthesizing peptides
 corresponding to the revealed conserved regions; c) administering an
 amount of the synthesized peptides to a suitable animal effective to
 produce an antibody against the peptides in the animal; and d) testing the
 produced antibody for capability to bind the administered protein. In an
 embodiment, the antibodies are produced by in vitro immunization.
 This invention further provides antibodies produced by the above methods.
 In an embodiment, these antibodies are monoclonal.
 This invention further provides antibodies capable of binding specifically
 to MAB-21 protein. In an embodiment, the antibodies are monoclonal.
 This invention also provides assays for measuring the amount of protein
 product of mab-21 or mab-21 homologs comprising steps of: a) contacting
 the sample with at least one of the antibody capable of binding
 specifically to MAB-21 protein to form a complex with said antibody and
 the protein, and b) measuring the amount of the protein in said biological
 sample by measuring the amount of said complex.
 This invention also provides methods to purify MAB-21 protein or the
 protein product of mab-21 homologs comprising steps of: a) coupling at
 least one antibody capable of binding specifically to the protein products
 of mab-21 or its homologs to a solid matrix; b) incubating the coupled
 antibody of a) with a cell lysate containing MAB-21 protein or the protein
 product of mab-21 homologs under the condition permitting binding of the
 coupled antibody and protein; c) washing the solid matrix to eliminate
 impurities and d) eluting the protein from the coupled antibody.
 This invention also provides transgenic animal comprising DNA molecule
 comprising a member selected from the group consisting of: (a) a
 polynucleotide encoding MAB-21 protein;(b) a polynucleotide capable of
 hybridizing to and which is at least 60% identical to the polynucleotide
 of (a); and (c) a polynucleotide fragment of the polynucleotide of (a) or
 (b). In an embodiment, the transgenic animal is a Caenorhabditis elegans.
 This invention further provides a transgenic Caenorhabditis elegans animal
 comprising a human homologous mab-21 gene.
 This invention provides methods for identifying a mutant mab-21 gene in
 animals which have reduced mab-21 function comprising steps of: a)
 treating wild type Caenorhabditis elegans hermaphrodite animals with
 effective amount of a mutagen; b) screening in F1, F2 or F3 generations
 for animals with Mab-21 mutant phenotype; and c) identifying, isolating
 and segregating the mutant animals in subsequent generations to
 homozygosity.
 This invention also provides a mutant mab-21 animal identified by the above
 method.
 This invention also provides methods for identification of a mutant gene
 which reduce mab-21 comprising performing DNA sequence analysis of the
 mutant mab-21 animal identified by the above method. This invention also
 provides mutant gene identified by the above method.
 This invention provides methods for identifying mutations in mab-21
 homologs in animals comprising steps of: a) obtaining DNA from the mutated
 and wild-type animals; b) amplifying on both wild-type and the mutant DNAs
 with appropriate mab-21 primers by polymerase chain reactions; c)
 hybridizing the amplified DNA under conditions permitting the formation of
 mismatched hybrid molecules a nd complementary hybrid molecules; d)
 separating the mismatched and complementary hybrid amolecules by
 electrophoresis; e) isolating the mismatched hybrid molecule; and f)
 determining the mismatched sequences, thereby identifying mutations in
 mab-21 homologs.
 Finally, this invention also provides methods for identifying various
 suppressor, enhancer and modifier mutations in animals carrying mab-21
 mutation. This invention provides animals carrying these suppressor,
 enhancer and modifier mutations. This invention also provides the
 suppressor, enhancer and modifier genes comprising steps of genetic
 mapping, physical mapping and DNA sequence analysis of these genes.

DETAILED DESCRIPTION OF THE INVENTION
 This invention provides an isolated polynucleotide comprising a member
 selected from the group consisting of: (a) a polynucleotide encoding
 MAB-21 protein; (b) a polynucleotide capable of hybridizing to and which
 is at least 60% identical to the polynucleotide of (a); and (c) a
 polynucleotide fragment of the polynucleotide of (a) or (b). In an
 embodiment, the polynucleotide is DNA.
 The isolated polynucleotide of the subject invention also include DNA
 molecules coding for polypeptide analogs, fragments or derivatives of
 antigenic polypeptides which differ from naturally-occurring forms in
 terms of the identity or location of one or more amino acid residues
 (deletion analogs containing less than all of the residues specified for
 the protein, substitution analogs wherein one or more residues specified
 are replaced by other residues and addition analogs where in one or more
 amino acid residues is added to a terminal or medial portion of the
 polypeptides) and which share some or all properties of
 naturally-occurring forms. These molecules include: the incorporation of
 codons "preferred" for expression by selected hosts; the provision of
 sites for cleavage by restriction endonuclease enzymes; and the provision
 of additional initial, terminal or intermediate DNA sequences that
 facilitate construction of readily expressed vectors.
 Mab-21 homologs in other species can be obtained by screening genomic
 library or cDNA libraries of different species using polynucleotide
 fragment of mab-21 gene as labelled nucleic acid molecule, in conditions
 permitting DNA hybridization of DNA molecules. Clones forming stable
 hybrid with the labelled molecules can be purified, and amplified for
 characterization. mab-21 homologs in other species can be obtained by
 polymerase chain reaction performed with genomic DNA from various species
 and a pair of oligonucleotides complementing to specific conserved regions
 of mab-21 gene. The amplified fragment will be used as probe for
 subsequent screening of genomic or cDNA libraries. mab-21 homologs in
 other species can also be obtained screening expression library with
 antibody capable of binding specifically to MAB-21 protein, provided that
 antigenic epitopes are conserved in these homologs. However, other methods
 for performing these screening steps are well known to those skilled in
 the art, and the discussion above is merely an example.
 This invention also provides a vector comprising the above-described DNA.
 In an embodiment, the vector is a plasmid.
 This invention also provides a host cell comprising the above-described
 vector. This invention further provides a host vector system for the
 production of MAB-21 protein comprising the above vector and a suitable
 host. In an embodiment, the suitable host is selected from a group
 consisting of a bacterial cell, plant and animal cell. In another
 embodiment, the suitable host cells are insect cells.
 As an example to obtain these vectors, insert and vector DNA can both be
 exposed to a restriction enzyme to create complementary ends on both
 molecules which base pair with each other and are then ligated together
 with DNA ligase. Alternatively, linkers can be ligated to the insert DNA
 which correspond to a restriction site in the vector DNA, which is then
 digested with the restriction enzyme which cuts at that site. Other means
 are also available and known to an ordinary skilled practitioner.
 Regulatory elements required for expression include promoter sequences to
 bind RNA polymerase and transcription initiation sequences for ribosome
 binding. For example, a bacterial expression vector includes a promoter
 such as the lac promoter and for transcription initiation the
 Shine-Dalgarno sequence and the start codon AUG. Similarly, a eukaryotic
 expression vector includes a heterologous or homologous promoter for RNA
 polymerase II, a downstream polyadenylation signal, the start codon AUG,
 and a termination codon for detachment of the ribosome. Such vectors may
 be obtained commercially or assembled from the sequences described by
 methods well known in the art, for example the methods described above for
 constructing vectors in general. Expression vectors are useful to produce
 cells that express the MAB-21 protein.
 This invention further provides an isolated DNA or cDNA molecule described
 hereinabove wherein the host cell is selected from the group consisting of
 bacterial cells (such as E. coli), yeast cells, fungal cells, insect cells
 and animal cells. Suitable animal cells include, but are not limited to
 Vero cells, HeLa cells, Cos cells, CV1 cells and various primary mammalian
 cells.
 This invention provides a nucleic acid molecule of at least 15 nucleotides
 capable of specifically hybridizing with a unique sequence of a nucleic
 acid molecule which is complementary to the above-described nucleic acid
 molecule. In an embodiment, this probe molecule is a DNA molecule. In
 another embodiment, this probe molecule is an RNA molecule.
 This nucleic acid molecule of at least 15 nucleotides capable of
 specifically hybridizing with a sequence of a nucleic acid molecule
 encoding MAB-21 protein can be used as a probe. Nucleic acid probe
 technology is well known to those skilled in the art who will readily
 appreciate that such probes may vary greatly in length and may be labeled
 with a detectable label, such as a radioisotope or fluorescent dye, to
 facilitate detection of the probe. DNA probe molecules may be produced by
 insertion of a DNA molecule which encodes MAB-21 protein into suitable
 vectors, such as plasmids or bacteriophages, followed by transforming into
 suitable bacterial host cells, replication in the transformed bacterial
 host cells and harvesting of the DNA probes, using methods well known in
 the art. Alternatively, probes may be generated chemically from DNA
 synthesizers.
 RNA probes may be generated by inserting a selected nucleic acid molecule
 downstream of a bacteriophage promoter such as T3, T7 or SP6. Large
 amounts of RNA probe may be produced by incubating the labeled nucleotides
 with the linearized mab-21 fragment where it contains an upstream promoter
 in the presence of the appropriate RNA polymerase.
 This invention also provides methods of detecting expression of mab-21 or
 its homologs in a sample which comprises steps of: a) obtaining total mRNA
 from the sample; b) contacting the mRNA so obtained with a labelled
 nucleic acid probe molecules which are described above under hybridizing
 conditions; and c) determining the presence of mRNA hybridized to the
 molecule, and thereby detecting the expression of the mab-21 or its
 homologs in the sample.
 In one embodiment of this invention, nucleic acids are extracted by
 precipitation from lysed cells and the mRNA is isolated from the extract
 using an oligo-dT column which binds the poly-A tails of the mRNA
 molecules. The mRNA is then exposed to radioactively labelled probe on a
 nitrocellulose membrane, and the probe hybridizes to and thereby labels
 complementary mRNA sequences. Binding may be detected by luminescence
 autoradiography or scintillation counting. However, other methods for
 performing these steps are well known to those skilled in the art, and the
 discussion above is merely an example.
 This invention provides methods of detecting expression of protein products
 of mab-21 or mab-21 homologs in a sample which comprises steps of: a)
 obtaining protein extracts from the sample; b) contacting the obtained
 protein extract with an antibody capable of specifically recognizing the
 protein products of mab-21 or mab-21 homologs under conditions permitting
 the formation of complexes between the antibody and the protein products,
 measuring the amount of the formed complexes, thereby detecting the
 expression of the protein product of mab-21 and mab-21 homologs.
 As used herein, the sample may include any biological samples obtained from
 an living organism. The sample may contain animal tissues or animal cells.
 Animals tissue or clinical samples can be obtained by biopsy, or sample
 can be cell lines generated from tissue from animals or patients. These
 samples will be processed to obtain purified protein or RNA for the
 detection assays, based on nucleic acid hybridization or on
 antibody-antigen complex formation.
 This invention also provides purified MAB-21 proteins or fragments thereof.
 This invention also provides purified MAB-21 proteins or fragments
 comprising steps of: a) inserting full length or truncated mab-21 cDNAs
 into a bacterial, yeast or mammalian expression vector with appropriate
 promoter regulatory elements, fusion protein cassettes (tag), and
 termination signals, b) expressing the fusion genes in the corresponding
 host cells, c) obtaining cell lysate containing the fusion protein, d)
 employing affinity chromatographic procedure using matrix coupled with
 antibody specific for the protein tag to permit binding of the fusion
 protein to the matrix, e) washing the matrix to eliminate impurities, f)
 eluting the protein from the coupled antibody, g) cleaving the tag from
 the fusion protein with appropriate protease, h) removing the tag in the
 solution by affinity chromotograpy, i) eluting the purified protein
 products of mab-21 or mab-21 homolog.
 This invention also provides methods for production of an antibody capable
 of binding to protein products of mab-21 or mab-21 homologs comprising: a)
 administering an amount of purified protein products of mab-21 or mab-21
 homologs to a suitable animal effective to produce an antibody against the
 protein in the animal; and b) testing the produced antibody for capability
 to bind the administered protein. In an embodiment, the antibody is
 produced by in vitro immunization.
 This invention also provides methods for production of an antibody capable
 of binding to protein products of mab-21 or mab-21 homologs comprising: a)
 determining conserved regions revealed by alignment of MAB-21 protein and
 the protein product of mab-21 homologs; b) synthesizing peptides
 corresponding to the revealed conserved regions; c) administering an
 amount of the synthesized peptides to a suitable animal effective to
 produce an antibody against the peptides in the animal; and d) testing the
 produced antibody for capability to bind the administered protein. In an
 embodiment, the antibodies are produced by in vitro immunization.
 This invention further provides antibodies produced by the above methods.
 In an embodiment, these antibodies are monoclonal.
 This invention further provides antibodies capable of binding specifically
 to protein product of mab-21 or mab-21 homologs.
 In an embodiment, the antibodies are monoclonal.
 This invention provides a method to select specific regions on the MAB-21
 or protein products of mab-21 homologs to generate antibodies. The protein
 sequence may be determined from the mab-21 sequence. Amino acid sequences
 may be analyzed by methods well known to those skilled in the art to
 determine whether they produce hydrophobic or hydrophilic regions in the
 proteins which they build. In the case of cell membrane proteins,
 hydrophobic regions are well known to form the part of the protein that is
 inserted into the lipid bilayer of the cell membrane, while hydrophilic
 regions are located on the cell surface, in an aqueous environment.
 Usually, the hydrophilic regions will be more immunogenic than the
 hydrophobic regions. Therefore the hydrophilic amino acid sequences may be
 selected and used to generate antibodies specific to MAB-21 protein. The
 selected peptides may be prepared using commercially available machines.
 As an alternative, DNA, such as a cDNA or a fragment thereof, may be
 cloned and expressed and the resulting polypeptide recovered and used as
 an immunogen.
 Polyclonal antibodies against these peptides may be produced by immunizing
 animals using the selected peptides. Monoclonal antibodies are prepared
 using hybridoma technology by fusing antibody producing B cells from
 immunized animals with myeloma cells and selecting the resulting hybridoma
 cell line producing the desired antibody. Alternatively, monoclonal
 antibodies may be produced by in vitro techniques known to a person of
 ordinary skill in the art.
 This invention further provides a method for production of an antibody
 capable of binding to protein products of mab-21 and mab-21 homolog
 comprising steps of: a) stimulating antibody response in a mouse by
 non-specific antigen; b) isolating mRNA from the stimulated mouse; c)
 performing polymerase chain reaction on the isolated mRNA with appropriate
 primers to produce cDNAs of the antibodies; d)cloning the cDNAs into an
 expression library; e) screening the expression library resulting from
 step d), which contains clones of cDNAs, with the protein product of
 mab-21 or mab-21 homolog; f) identifying and isolate the clone capable of
 binding to the protein product. The above-described in vitro immunization
 procedure is known in art (See e.g. McCafferty, J. et al. (1990) Phage
 antibodies: Filamentous phage display antibody variable domains. Nature
 348:552-554.). The expression library used may be a phage library. In a
 preferred embodiment, the library is a phage display library. The
 amplified cDNAs may be linked to a phage coat protein gene of a
 filamentous phage vector. Appropriate cells may be transformed by the
 vector to generate a library of phages expressing various immunoglobulin
 proteins on the phage surface. This library may then be screened with the
 antigen of interest. The identified clone will be the antibody capable of
 binding to the antigen of interest.
 These antibodies are useful to detect the expression of protein product of
 mab-21 or mab-21 homologs in living animals, in humans, or in biological
 tissues or fluids isolated from animals or humans.
 This invention also provides assays for measuring the amount of a protein
 product of mab-21 or mab-21 homologs comprising steps of: a) contacting
 the sample with at least one of the antibody capable of binding
 specifically to MAB-21 protein or protein product of mab-21 homologs to
 form a complex with said antibody and the protein, and b) measuring the
 amount of the protein in said biological sample by measuring the amount of
 said complex.
 This invention also provides methods to purify MAB-21 protein or the
 protein product of mab-21 homologs comprising steps of:
 a) coupling at least one antibody capable of binding specifically to MAB-21
 protein or protein product of mab-21 homologs to a solid matrix; b)
 incubating the coupled antibody of a) with a cell lysate containing MAB-21
 protein or the protein product of mab-21 homologs under the condition
 permitting binding of the coupled antibody and protein; c) washing the
 solid matrix to eliminate impurities and d) eluting the protein from the
 coupled antibody.
 This invention also provides transgenic animal comprising DNA molecule
 comprising a member selected from the group consisting of: (a) a
 polynucleotide encoding MAB-21 protein;(b) a polynucleotide capable of
 hybridizing to and which is at least 60% identical to the polynucleotide
 of (a); and (c) a polynucleotide fragment of the polynucleotide of (a) or
 (b). In an embodiment, the transgenic animal a Caenorhabditis elegans.
 This invention further provides a transgenic Caenorhabditis elegans animal
 comprising a human homologous mab-21 gene.
 This invention provides methods for identifying a mutant mab-21 animal
 which reduces mab-21 function comprising steps of: a) treating wild type
 Caenorhabditis elegans hermaphrodite animals with effective amount of a
 mutagen; b) screening in F1, F2 or F3 generations for animals with Mab-21
 mutant phenotype; and c) identifying, isolating and segregating the mutant
 animals in subsequent generations to homozygosity.
 This invention also provides a mutant mab-21 animal identified by the above
 method.
 This invention also provides methods for identification of a mutant mab-21
 gene which reduces mab-21 function comprising performing DNA sequence
 analysis of the mutant mab-21 animal identified by the above method.
 This invention also provides mutant genes identified by the above method.
 This invention provides methods for identifying mutations in mab-21
 homologs in animals comprising steps of: a) obtaining DNA from the mutant
 and wild-type animals; b) amplifying both wild-type and the mutant DNAs
 with a pair of mab-21 homolog-specific oligonucleotide primers by
 polymerase chain reactions; c) hybridizing the amplified DNA under
 conditions permitting the formation of mismatched hybrid molecules and
 complementary hybrid molecules; d) separating the mismatched and
 complementary hybrid molecules by electrophoresis; e) isolating the
 mismatched hybrid molecule; and f) determining the mismatched sequences,
 thereby identifying mutations in mab-21 homologs.
 This invention provides methods for producing an animal carrying an
 extragenic suppressor gene mutation of a mab-21 allele comprising: a)
 mutagenizing mab-21 mutant hermaphrodites with an effective amount of a
 mutagen; b) screening for revertants in the F1, F2 and F3 generations; and
 c) identifying, isolating and segregating the animals carrying the
 suppressor gene mutation in subsequent generations to homozygosity. This
 invention further provides an animal carrying a mutation of a mab-21
 suppressor gene produced by the above method. This invention also provides
 methods for identification of a suppressor gene comprising performing
 genetic mapping, physical mapping and DNA sequence analysis of the
 suppressor mutation of the above isolated animal to identify the
 suppressor gene. This invention also provides a suppressor gene identified
 by the above method.
 This invention provides methods for producing an animal carrying an
 extragenic enhancer mutation of a mab-21 allele comprising: a)
 mutagenizing mab-21 mutant hermaphrodites with an effective amount of a
 mutagen; b) screening for enhancement of Mab-21 mutant phenotype in the
 F1, F2 and F3 generations; and c) identifying, isolating and segregating
 the animals carrying the enhancer mutation in subsequent generations to
 homozygosity. This invention also provides an animal carrying a mutation
 of a mab-21 enhancer gene produced by the above method. This invention
 also provides a method for identification of an enhancer gene comprising
 performing genetic mapping, physical mapping and DNA sequence analysis of
 the enhancer mutation of the above isolated animal to identify the
 enhancer gene. This invention further provides an enhancer gene identified
 by above method.
 This invention provides methods for producing an animal carrying an
 extragenic modifier mutation of a mab-21 allele comprising: a)
 mutagenizing mab-21 mutant hermaphrodites with an effective amount of a
 mutagen; b) screening for alteration of Mab-21 mutant phenotype in the F1,
 F2 and F3 generations; and c) identifying, isolating and segregating the
 animals carrying the modifier mutation in subsequent generations to
 homozygosity. This invention also provides an animal carrying a mutation
 of a mab-21 modifier gene produced by the above method. This invention
 also provides methods for identification of a modifier gene comprising
 performing genetic mapping, physical mapping and DNA sequence analysis of
 the modifier mutation of the isolated animal to identify the modifier
 gene. This invention further provides a modifier gene identified by above
 method.
 The mutagen includes but is not limited to ethyl methanesulfonate,
 diethylsulfate, ethyl nitrosy-urea, diepoxybutane, diepoxyoctane,
 acetaldehyde, formaldehyde, porsalein, UV irradiation, ionizing radiation
 and activated transposons.
 This invention will be better understood from the examples which follow.
 However, one skilled in the art will readily appreciate that the specific
 methods and results discussed are merely illustrative of the invention as
 described more fully in the claims which follow thereafter.
 EXPERIMENTAL DETAILS
 First Series of Experiments
 Materials and Methods
 General Methods and Mapping
 Nematodes were reared at 20.degree. C. following standard procedures
 (Brenner, 1974). Most strains carried the him- 5(e1490) mutation, which
 results in a high frequency of spontaneous males in populations of selfing
 hermaphrodites (Hodgkin et al., 1979). Isolation after EMS mutagenesis of
 two alleles of mab-21 (b.times.41 and b.times.53) has been described
 previously (Baird et al., 1991); a third allele (sy155) was isolated from
 a mutant strain kindly provided by H. Chamberlin and P. Sternberg. mab-21
 mutations were mapped to the left arm of linkage group III between unc-79
 and pal-1 by four factor cross in which the following recombination events
 were scored: unc-79(12/18) mab-21(3/18) pal-1(3/18) dpy-17 (see FIG. 4A) .
 mab-21 was placed between two restriction fragment length polymorphisms,
 MJ#NEC2 and MH#NEC1, by crossing a mab-21 strain to strain EM375 of
 genotype unc-79 MJ#NEC2 MJ#NEC1 dyp-17 with the following recombination
 results: unc-79(29/59) MJ#NEC2(11/59) mab-21(9/59)MJ#NEC1
 (10/59)dpy-17(FIG. 4B). EM375 was generated from strain MJ569 provided by
 J. Miwa.
 Cell Lineage Analysis and Cell Ablation Experiments
 Cell lineages of mab-21(b.times.41) (3 sides) and mab-21(b.times.53) (5
 sides) were followed in living animals by Nomarski microscopy following
 methods described by Sulston and Horvitz (1977). Lineages were followed
 from late L1 through the L3 molt and division of Rn.a cells. Division of
 Rn.a cells was taken as an indication of the expression of the ray
 sublineage.
 Cells were ablated with a laser microbeam following procedures described in
 Chow and Emmons (1994).
 Electron Microscopy
 Young adult males were rinsed in M9 buffer and place into buffered aldehyde
 fixative at room temperature. While in fix, the tails were quickly cut off
 with a scalpel blade. After 1-3 hours in aldehyde fix, samples were
 transferred through several buffer rinses and refixed in 1% osmium
 tetroxide. Samples were then dehydrated and embedded in Medcast resion
 (Ted Pella) and serially sectioned on a diamond knife (cf. Hall, 1995).
 Some tails were sectioned transverse to the body axis, others lengthwise,
 either sagittal or frontal, in order to obtain different perspmicrograph
 the curving rays. Electron micrographs were collected with a Philips CM10
 at 2,000-12,000.sup..times.. Several rays that showed especially good
 fixation were reconstructed using the Eutectics Electronics 3D-SSRS
 computer program, after manual tracing of every 3-5th section on a
 digitizing table. In mab-21 males, normal rays, an ectopic ray, and a
 fused (4+6) ray could thus be compared from any perspective, and used ans
 models with which to compare sample sections from additional animals. In
 all, nine mab-21 adults and four wild type adults were compared in serial
 thin sections. In particular, effort was made to compare the details of
 ray openings, ciliary tip specializations, the relative sizes of cellular
 elements along the length of the rays, and the number and type of cell
 processes invading the base of each ray. Not all features could be
 assessed in each specimen, depending upon section angle and varying
 quality of preservation.
 Complementation of Emb Genes
 The following embryonic lethal mutations, all temperature sensitive and
 closely linked to mab-21, were tested for complementation with mab-21:
 emb-1(hc57), emb-2(hc58), emb-5(hc61), emb-7(hc66), errb-8(hc69),
 emb-13(g6), and emb-32(g58). Crosses were set up at 16.degree. C. between
 mab-21(bx53); him-5(e1490) males and emb-n hermaphrodites. After mating
 took place for two days, the parents were removed, and plates with mostly
 eggs and a few L1 larvae were transferred to 25.degree. C. mab-21/emb-n
 male cross progeny were scored for ray fusion phenotype.
 Indirect Immunofluorescence Staining of Cell Boundaries
 Animals were fixed with 1% paraformaldehyde and permeabilized by reduction
 and oxidation of the cuticle (Finney and Ruvkun, 1990). Permeabilized
 animals were incubated at 37.degree. C. for 8 hr with a hundred fold
 dilution of monoclonal antibody MH27 (provided by J. Preiss), and then for
 12 hr with a hundred fold dilution of secondary antibody (rhodamine
 isothiocyanate conjugate goat anti-mouse, Boehringer Mannheim Biochemical,
 Indianapolis Ind.). Stained animals were mounted in a solution containing
 30mM Tris pH7.5, 70% glycerol, and 2% N-propylgalate. Observation and
 photography were performed with a Zeiss Axioplan microscope equipped for
 epifluorescence.
 Visualization of Hypodermal Cell Boundaries
 To visualize the SET compartment in the male tail, late L4 larval males
 were mounted for Nomarski microscopy (Sulston and Hodgkin, 1988) in 0.25%
 sodium dodecyl-sulfate (Austin and Kenyon, 1994). Animals were immediately
 observed at 1000.times.. The boundary of hypodermal cells, including the
 SET, gradually became visible and then started to deteriorate. When the
 boundaries were clear, SET nuclei were counted and photographed.
 Transformation Rescue of mab-21
 Cosmids for transformation rescue of mab-21 were obtained from R. Shownkeen
 and A. Coulson (MRC Laboratory of Molecular Biology, Cambridge, UK).
 Microinjection of cloned DNA into hermaphrodite gonads was performed
 following the procedure of Mello et al. (1991). The number of cosmids
 including those listed in FIG. 4B were individually injected at a
 concentration of 15 .mu.g/ml together with the dominant rol-6(su1006)
 marker carried on plasmid pRF4 (Kramer et al., 1990) at a concentration of
 20 .mu.g/ml. mab-21 (b.times.53); him-5 (e1490) (EM128) was the host of
 microinjection. Rol males appearing among the progeny of injected
 hermaphrodites were scored for ray 6 to 4 transformation phenotype. A
 minimal rescuing genomic BamHI-SacI fragment was identified by deletion
 analysis (EM#227) (FIG. 4B), and its sequence determined (Sequenase Kit,
 US Biochemicals).
 To confirm the identification of the mab-21 locus, a frame shift mutation
 was introduced into the open reading frame present in EM#219 (FIG. 4B). A
 unique EcoRI site in the 3' end of the open reading frame was filled in
 with Klenow polymerase and religated. This resulted in generation of a
 premature translation termination codon at position 311, and consequently
 a truncated MAB-21 conceptual protein lacking 79 amino acids from the
 carboxyl terminus. Transformation with DNA carrying this frame shift
 mutation failed to result in rescue of -mab-21(b.times.53) (0/54 Rol males
 showed a wild type ray 6 phenotype).
 Isolation of cDNA Clones and Heat Shock-Induced Expression
 A C. elegans lamda cDNA library (Palazzolo et al., 1990) was screened with
 EM#219 (FIG. 4B). Plasmid derivatives of the positive phage clones were
 obtained following the procedure described by Palazzolo et al. (1990).
 Inserts were sequenced with Sequenase Kit (US Biochemicals). DNA sequences
 were analyzed with GCG sequence analysis software (Sequence Analysis
 Software Package by Genetics Computer Group, Inc., [Devereux et al.,
 1984]). The Genbank Accession Number for the mab-21 cDNA sequence is
 U19861.
 cDNA inserts A, B, and C (FIG. 4B) were released by digestion with ApaI and
 SacI and subcloned into pPD49.78 (Fire et al., 1990), which allows their
 expression from the heat shock promoter hsp16.2 (generating respectively
 plasmids EM#230, EM#231, and EM#220). After cotransformation with pRF4,
 stable integrated lines were isolated by gamma ray radiation (95 rads per
 minute for 40 minutes). For heat shock of synchronized populations, eggs
 were isolated with sodium hypochlorite and allowed to hatch into buffer
 (Sulston and Hodgkin, 1988). Synchronized, arrested L1 larvae were
 transferred after 24 hours to 50 mm plates seeded with bacteria and
 allowed to develop at 20.degree. C. At various times during development,
 plates were shifted to 32.degree. C. for 3 hours and returned to
 20.degree. C. Adult Rol males were scored for ray fusion phenotype. For
 determining the effective heat shock interval with individual animals,
 single animals were randomly picked and staged by observation with
 Nomarski microscopy. They were then heat shocked for 3 hours at 32.degree.
 C., allowed to recover and develop at 20.degree. C., and their adult
 phenotype recorded (FIG. 5).
 Mosaic Analysis
 Mosaic analysis (Herman, 1984, 1989) for determination of the focus of
 mab-21 gene activity was carried out in strain EM289, genotype mab-21
 (b.times.53) ncl-1 (e1865) unc-36 (e251) III;him-5 (e1490)V;sDp3(III;f).
 The free duplication sDp3(III;f) (Rosenbluth et al., 1985) carries wild
 type copies of the LGIII genes and hence EM289 is generally nonMab nonUnc
 and nonNcl in phenotype. Genetic mosaics arise by spontaneous loss of the
 free duplication. ncl-1(e1865) results in enlarged nucleoli; its activity
 is cell-autonomous (Herman, 1989). The unc-36(e251) mutation results in
 very slow movement, probably due to loss of the gene in motor neurons; its
 focus of activity is among descendants of AB or AB.p (Kenyon, 1986;
 Herman, 1989). Mosaic males were identified as Unc animals with wild type
 rays, or as nonUnc or semiUnc animals with Mab rays. Because displacement
 of ray cell bodies during L4 morphogenesis of the male tail prevents
 identification of ray cells in the adult, the point of duplication loss
 was determined by examining the nuclei of lineally related cells (FIG. 6).
 The Ncl phenotype of PLM(L/R), V5.pa postdeirid neurons, and Q-derived
 neurons were scored to infer duplication loss in the AB.p branch; the Ncl
 phenotype in ALM(L/R) and BDU(L/R) were scored to infer loss in the AB.a
 branch. Loss of the duplication in the P1 lineage was scored by the Ncl
 phenotype of body muscle cells, pharyngeal neurons, and coelomocytes. In 4
 putative mosaic animals, no Ncl nucleoli could be found in the cells
 examined. The observed mab-21 phenotype observed on one side in these
 animals could be accounted for by loss of the duplication in a subset of
 cells within the V6 lineage (in two cases within AB.apppap or one of its
 descendants, in two cases within AB.arpppp or one of its descendants);
 these are not shown in FIG. 6.
 Experimental Results
 mab-21 is Required for the Choice of Alternate Fates by Several Cells in
 the Male Tail
 Mutations in the gene mab-21 were identified because of their effects on
 differentiation of a single pair of sensory rays in the C. elegans adult
 male tail (Baird et al., 1991). It is found that mab-21 mutations affected
 the differentiation of two cells in ray 6, as well as the fusion
 properties of the hypodermal cell generated by the ray 6 sublineage.
 mab-21 mutations also transform a nearby hypodermal cell into a neuroblast
 cell that expresses the ray sublineage and generates a ray. The C. elegans
 male has several specialized posterior structures and organs necessary for
 copulation with the hermaphrodite. Among these are a set of nine
 bilaterally symmetrical pairs of sensory rays, which project outward from
 the body within an acellular fan (FIG. 1A). The ultrastructure of the rays
 has been described by Sulston et al. (1980), and is illustrated in FIG. 2.
 Each ray consists of a cone or cylindar of hypodermis containing the
 dendritic processes of two ultrastructurally distinguishable neurons (RnA,
 RnB, n=1-9), together with the process of a support cell (called the
 structural cell, Rnst) (FIGS. 2J,N). The support cell is joined to the
 hypodermis at the tip of the ray and surrounds and holds the dendritic
 endings of the neurons, one of which, RnB, generally faces an opening to
 the exterior (FIGS. 2A,D). In wild type males, the rays are of identical
 morphology with one exception: the sixth ray counting from anterior to
 posterior, ray 6, is fatter and more conical in shape than the others rays
 (FIG. 1A). Ray 6 also fails to open to the exterior or is open only
 through a thin channel, and has a B neuron that differs slightly in
 ultrastructure from the B neurons of the other rays (Sulston et al., 1980)
 (FIG. 2C).
 In mab-21 mutants, the most obvious phenotype is that the distinctive
 conical ray 6, is absent, and a large ray of uniform diameter replaces ray
 4 (FIG. 1B). The large new ray consists of a fusion of rays 6 and 4, and
 contains the cells normally found in these two rays, as demonstrated by
 several lines of evidence. First, in mab-21 mutants the cell lineages
 leading to rays 4 and 6 are unaffected. In wild type, the three cells of
 each ray plus one hypodermal cell are generated as products of the ray
 sublineage. The ray sublineage is expressed by ray precursor cells (Rn
 cells) (FIGS. 1C,D) (Sulston and Horvitz, 1977, Sulston et al., 1980). In
 mab-21 mutants, both R4 and R6 express the ray sublineage at the normal
 time (8/8 sides lineaged). Secondly, the large ray at the position of ray
 4 extends from two adjacent papillae (not shown) and has two ray tips
 (FIG. 2F). Ray papillae and tips are structures formed by the ray
 structural cell and have a distinctive "ring-and-dot" morphology visible
 by Nomarski microscopy (Sulston et al., 1980). The presence of two ray
 papillae during L4 and two tips in the large ray suggests that the large
 ray contains two structural cells. These cells are apparently fused, as no
 cell boundaries separating them are visible in electron micrographs of the
 fused ray (FIGS. 2F-I,K). Thirdly, electron microscopy reveals that the
 large ray contains the processes of three or four neurons (FIGS. 2F-I,K).
 Two of the processes terminate respectively at each of the two openings
 and have the ultrastructure of B-type neurons, while two terminate within
 the ray and have the ultastructure of A-type neurons.
 Fusion of ray 6 to ray 4 in mab-21 mutants appears to be the result of
 altered properties of ray 6, and in particular, of the ray 6 structural
 cell, R6st. Mutational changes affecting ray 6 alone can occasionally be
 seen when fusion of ray 6 with ray 4 does not occur. In 5% (n&gt;600) of
 mab-21 mutant animals rays 4 and 6 do not fuse. In these animals, ray 4
 appears to be unaffected, while ray 6 lies between rays 4 and 5 anterior
 of its normal position. In such animals, ray 6 does not have a conical
 morphology, but instead has a uniformly thin morphology similar to the
 other rays. The unique conical morphology of ray 6 in wild type is
 determined by R6st (Y. Zhang and S. W. Emmons, in preparation). Altered
 ray 6 position is likely to result from altered interactions between R6st
 and surrounding hypodermal cells (Baird et al., 1991). Fusion of ray 4 and
 ray 6 in mab-21 mutants could be a consequence of misplacement of ray 6 to
 a position adjacent to ray 4, or could result because cell recognition
 functions expressed by cells of ray 6, ray 4, or both, are altered.
 In addition to affecting properties of R6st, mab-21 mutations affect the
 ultrastructure of R6B. In wild type, the B neurons of most rays are
 characterized by having a thin lip that is exposed to the exterior, and by
 the presence of an extracellular dense matrix material where the tip
 narrows (FIGS. 2A, B). In ray 7 in wild type, in which there is either no
 opening or only a narrow channel opening to the exterior (data not shown),
 the B neuron lacks this dense material (FIG. 2C). In mab-21 mutants, fused
 rays have two openings, each containing the tip of a B-type neuron. Both
 of these neurons are surrounded by the dense material normally
 characteristic of all the rays except ray 6 (FIGS. 2G, H). This in mab-21
 mutants, the ultrastructure of the ending of R6B has been transformed to
 that characteristic of the B neurons of the other rays. A second unique
 ultrastructural property of R6B in wild type, lack of dilated cisternae in
 the cell body (Sulston et al., 1980), was not examined.
 A third cell affected in mab-21 mutants is the hypodermal cell generated by
 the ray 6 sublineage (R6.p). At the end of the L4 larval stage, R6.p
 normally fuses with the large hypodermal syncytium, hyp7, covering most of
 the body (Sulston et al., 1980). In mab-21 mutants, R6.p instead fuses
 with R1.p-R5.p (n=15/15), thus becoming part of the tail seam (SET) (FIG.
 3).
 Finally, in mab-21 mutants, a hypodermal cell lying adjacent to ray 6 is
 transformed into a ray neuroblast. In wild type, the rays are generated by
 nine pairs of ray precursor cells. These are descended from three
 embryonic hypodermal blast cells denoted V5 (ray 1), V6 (rays 2-6), and T
 (rays 7-9). In mab-21 mutants, an additional descendant cell of T,
 T.apapa, the anterior sister of R7, is often (45%, n&gt;600) transformed from
 a hypodermal cell into a ray precursor cell and expresses the ray
 sublineage. Thus, mab-21 mutant animals can have 10 instead of 9 rays
 (FIG. 1B). The T.apapa-derived ray will sometimes be referred to as the
 "ectopic" ray.
 Altogether, the mab-21 male tail phenotype can be accounted for by the
 affects on the fates of the four cells, R6st, R6B, R6.p and T.apapa. These
 affected cells are of diverse types, and therefore nab-21 does not appear
 to be required for expression of any particular cell fate. In each case,
 the affected cells choose alternate cell fates normally assumed by
 neighboring cells and appear to execute those alternate fates correctly.
 The cells affected by mab-21 mutations have in common that they lie
 adjacent to one another in a restricted region of the lateral epidermis of
 the male tail, and differentiate during the late L3 and L4 larval stages.
 Therefore it appears most likely that mab-21 is required for a localized
 pattern formation mechanism that causes these cells to choose their wild
 type fates.
 All of the changes affecting cells of ray 6, together with the change in
 fusion partner of R6.p, can be parsimoniously interpreted as a change in
 the identity of the ray 6 precursor cell R6 to that of R4. T.apapa, like
 R6, is the anterior sister of a ray precursor cell (R7 in the case of
 T.apapa, R5 in the case of R6 [FIG. 1C]). R6 and T.apapa lie adjacent to
 each other ventral of the seam, and in mab-21 mutants each assumes the
 fate. mab-21 thus appears to act as one component of a pattern formation
 mechanism that assigns fates to at least two seam cells during the late L3
 larval stage.
 Additional mab-21 Mutant Phenotypes Suggest mab-21 Has Functions Outside
 the Tail Region of Males
 In addition to having ray defects in the male tail, srab-21 males and
 hermaphrodites are somewhat short and fat, are slightly uncoordinated, and
 have decreased fecundity. These pleiotropic defects imply that the action
 of mab-21 is not restricted to the tail hypodermis of males. The body
 length of mab-21 mutant adult hermaphrodites (856 mm.+-.55 mm [n=30]), was
 intermediate between that of wild type (1024 mm.+-.72 mm [n=30]) and
 dpy-17 (524 mm.+-.56 mm [n=30]). The latter have short and fat bodies
 typical of a large number of dpy (dumpy) and sma (small) mutants. The
 shorter body length of mab-21 animals was not due to a growth rate defect,
 nor was the timing of developmental stages abnormal (data not shown). As
 the hypodermis and overlying cuticle play a key role in determining body
 shape in C. elegans (Priess and Hirsh, 1986; Levy et al., 1993) these
 results suggested that mab-21 might play a role in differentiation of the
 hypodermis.
 mab-21 animals, particularly males, have an uncoordinated movement in
 backward locomotion. Upon being touched on the head, mab-21 males made a
 ventral bend at the tail during backward movement that was more severe
 than that of wild type males, resulting in a deep curving movement. A
 similar though less pronounced bend could be discerned in hermaphrodites.
 This backward Unc phenotype suggests a defect in the nervous system or
 possibly the posterior muscles in mab-21.
 mab-21 hermaphrodites had a reduced brood size due to a reduced brood size
 due to a decreased number of laid eggs, suggesting the presence of a
 gonadal defect (average number of self progeny at 20.degree. C. was 126
 (18 for mab-21(b.times.53);him-5(e1490), n=10, average number for
 him-5(e1490) was 221 ( 18, n=10; results were similar with the other two
 mab-21 alleles). The eggs laid by mab-21 hermaphrodites failed to hatch in
 numbers (several percent) significantly higher than mab-21(+). Dead
 embryos were arrested from morphogenesis stage up to the end of
 embryogenesis, and had an apparently disrupted hypodermis. A few percent
 of hatched animals had a deformed Vab (variable abnormal) phenotype with
 deformities mostly in the region of the pharynx. mab-21 males sired fewer
 progeny in mating tests (data not shown), but it was difficult to
 determined whether this was due to a gonad defect or was because the
 uncoordinated movement described above disrupted mating.
 mab-21 has an Essential Embryonic Function
 In order to gain information about the possible null phenotype of mab-21,
 the phenotype that resulted when alleles of mab-21 were placed over a
 deficiency was determined. Mapping experiments (Materials and Methods)
 placed mab-21 on the left arm of linkage group III between unc-79 and
 pal-1 (FIG. 4A). It was attempted to construct the heterozygote carrying
 mab-21(b.times.53) over the deficiency yDf10, but found that progeny of
 the expected phenotype were absent from the cross (Table 1).
 TABLE 1
 Result of crossing mab-21 to a deficiency
 Hermaphrodite gametes
 Male gametes
 Result unc-93 dpy-17 + yDf10 + unc - 32
 Cross 1
 mab-21 + nonUnc nonDpy Mab nonUnc 305 nonUnc
 mab-21 unc-32 nonUnc nonDpy Mab Unc 0 Unc
 Cross 2
 + + nonUnc nonDpy nonUnc 426 nonUnc
 + unc-32 nonUnc nonDpy Unc 124 Unc
 For map positions of genetic markers, see FIG. 4A.
 Male heterozygous for unc-32 were used because homozygous unc-32 males
 cannot mate.
 The underlined gives the phenotypes of expected cross progeny if
 mab-21/yDf10 were viable.
 Viable self progeny are Dpy or have a distinctive rubberband phenotype due
 to unc-93.
 In corresponding crosses lacking the mab-21 mutation, yDf10 heterozygotes
 were present in the expected proportion. mab-21/yDf10 is concluded to be a
 lethal combination, and that the null phenotype of mab-21 is lethality
 prior to hatching. Since the phenotype of the existing mab-21(b.times.53)
 is more severe over a deficiency, and because the mutant phenotype of the
 two mab-21 alleles is similar to that of b.times.53, All these mutations
 are concluded to be most likely hypomorphs.
 Alleles of known essential genes in the vicinity of mab-21 were tested for
 complementation with mab-21 (Schierenberg et al., 1980; Cassada et al.,
 1981) (Materials and Methods). Alleles of emb-1, emb-2, emb-5, emb-7,
 emb-8, emb-13, and emb-32 all complemented the ray 6 defect of
 mab-21(b.times.53) males. Therefore none of these mutations is likely to
 be a strong allele of mab-21, and mab-21 appears to define a new essential
 gene.
 In order to gain information about the stage of embryonic arrest in a
 mab-21 near-null background, the dead embryos arising from a cross between
 a mab-21 carrying strain and a yDf10 carrying strain were examined and
 compared these to dead embryos that segregated from a yDf10/+ strain
 during self propagation. 47% (36/76) of dead eggs resulting from the cross
 had completed the proliferation stage of embryogenesis and were arrested
 during the morphogenesis stage, whereas only 2% (1/56) of the dead eggs
 segregating from the yDf10/+ heterozygote progressed this far. Therefore,
 it was concluded that mab-21/yDf10 heterozygous embryos can complete
 cellular proliferation and that decreased level of mab-21 function causes
 arrest during morphogenesis.
 mab-21 Encodes a Novel Protein
 The mab-21 gene was cloned by identifying cosmids (kindly supplied by the
 C. elegans sequencing consortium) that rescued the mab-21 male tail
 phenotype. Candidate cosmids for testing were identified by mapping mab-21
 between two restriction fragment length polymorphisms, MJ#NEC2 and MH#NEC1
 (FIG. 4B, see Materials and Methods). A 5.6 kb rescuing genomic fragment
 (EM#219) was used to screen a cDNA library (Palazzolo et al., 1990),
 resulting in the recovery of 8 cDNA clones from 250,000 plaques screened.
 Seven of the cDNA clones were of 1.3 kb and differed only by having 5'
 ends located at different positions within a 26 bp genomic region. The
 eighth cDNA was of 0.7 kb and was colinear with the 3' portion of the
 other cDNA's. It was showed that these cDNA's represented transcripts of
 the mab-21 locus in two ways. First, expression of all these cDNA's
 (except the 0.7 kb cDNA) under the control of a heat shock promoter
 resulted in rescue of the mab-21 mutant phenotype, as described more fully
 below. Second, introduction of a mutation into the coding region of EM#219
 abolished the ability of this fragment to rescue a mab-21 mutation
 (Materials and Methods). Third, a genomic fragment (EM#228) covering all
 of the genomic region covered by EM#227 with the exception of the region
 encoding the 3' end of the cDNAs failed to rescue a mab-21 mutation (FIG.
 4B).
 DNA sequence analysis revealed that the cDNA clones contained an open
 reading frame encoding a protein of 386 amino acids (FIG. 4C). Search of
 Genebank and EMBL databases with GCG FASTA and TFASTA identified no
 previously described proteins with significant similarity to this
 conceptual protein. Thus the mab-21 locus encodes a novel protein.
 mab-21 Function is Required During Late L3 or L4 Larval Stages
 As discussed above, the male tail mutant phenotype of mab-21 suggested that
 mab-21 was required for a pattern formation mechanism that directed cell
 fate choices of seam cells during the late L3 larval stage. To determine
 whether mab-21 gene function was required at a time consistent with this
 model, applicant expressed mab-21 from a heat shock promoter at various
 times in a mab-21 mutant background (FIG. 5). It was found that the
 interval during which heat shock resulted in the highest frequency of
 rescue of the mab-21 mutant phenotype (both the ray 6 defect and the
 ectopic ray defect) included the time when ray precursor cells were
 present. Therefore mab-21 function could be required by one or more ray
 precursor cells. Alternatively, heat shock starting at this time might be
 necessary in order to accumulate enough mab-21 protein for function at a
 later time.
 mab-21 Functions Cell Autonomously for Choice of Ray Identity by R6, but
 Non-Autonomously for Choice of Hypodermal Versus Neuroblast Cell Fate by
 T.apapa
 Results of our previous cell ablation experiments suggested that genes
 required for specification of ray morphological identities acted
 autonomously within the terminal branches of the ray lineages (Chow and
 Emmons, 1994). (Terminal branches are defined as those branches leading to
 or contributing to single rays.) Therefore it was expected that mab-21(+)
 was required in R6 or a descendant of R6 for correct specification of ray
 6 morphology, ultrastructure, and position. In order to test this
 prediction, as well as to determine whether the choice of hypodermal
 versus neuroblast cell fate by T.apapa was cell autonomous, carried out a
 mosaic analysis of mab-21 function, as well as a laser ablation study in a
 mab-21 mutant background. It was found that expression of mab-21(+) within
 the V6 lineage was both necessary and sufficient for wild type ray 6
 position and morphology, whereas expression of mab-21(+) within T.apapa
 was not necessary for preventing expression of the neuroblast fate by this
 cell. Expression within either the T or the V6 lineage was sufficient for
 preventing expression of the neuroblast cell fate by T.apapa. The laser
 ablation study confirmed that the presence or absence of R6 can affect the
 fate of T.apapa.
 For mosaic analysis, a strain carrying the three linked mutations mab-21
 (b.times.53) ncl -1 (e1865) unc-36(e251) III as well as a free
 duplication, sDp3 (III;f), that carries wild type alleles of each of these
 genes were used (FIG. 4A). sDp3 is spontaneously lost during the cell
 lineage with a frequency variously estimated to be 1 per 400 cell
 divisions (Kenyon, 1986) or 1 per 300 cell divisions (Herman, 1989).
 Mosaic animals in which duplication loss had occurred were identified as
 Unc animals with wild type male tails, or nonUnc animals with mutant male
 tails, and the probable point of duplication loss was determined for such
 animals by analysis of the Ncl phenotype of cells representing several
 lineages (FIG. 6; Materials and Methods). By this means, 36 mosaic animals
 were identified and could be placed into one of four classes. The cells
 scored and the points of duplication loss for each mosaic class are shown
 in FIG. 6; the phenotypes of the mosaics are summarized in Table 2.
 Class I represented probable loss of the duplication in AB.p (Ia) or in the
 AB.pa (Ib) or AB.pp (Ic) branches; these animals lacked a wild type copy
 of the gene in T-apapa. Animals of this class had wild type male tails.
 This indicated that mab-21(+) gene function was not necessary within
 descendants of AB.p, which included T and its descendants T.apapa and R7,
 for either wild type ray 6 or correct choice of the hypodermal cell fate
 by T.apapa. Class II animals had lost the duplication in AB.a (IIa), or in
 the AB.al(IIb) or AB.ar(IIc) branches; these animals most likely lacked a
 wild type gene copy in R6. Such animals were mutant for ray 6 on one (IIb
 and IIc) or both (IIa) sides, consistent with a requirement for mab-21(+)
 within R6 or one of its descendants. Expression of mab-21 function within
 the large hypodermal syncytium, hyp7, appeared unlikely, because Class II
 mosaics would be expected to have large numbers of syncytial nuclei
 carrying the mab-21(+) gene. Class II animals were wild type for choice of
 hypodermal cell fate by T.apapa, indicating expression of mab-21(+) in R6
 was not necessary for correct specification of T.apapa cell fate.
 In class III animals, the duplication was lost in AB, and as expected, such
 animals were bilaterally mutant for ray 6. Two of the three class III
 animals had ectopic rays unilaterally. Taken together with the previous
 results, this indicates that expression of mab-21(+) in either the AB.a or
 AB.p branches is sufficient for specification of wild type T.apapa cell
 fate, but that expression in at least one of these two lineages is
 necessary. Finally, the single animal of class IV, where the duplication
 was lost in P1, had a wild type male tail as expected.
 One possible interpretation of mosaics is that the presence of mab-21(+)
 activity in the V6 lineage prevents expression of the neuroblast cell fate
 by a T.apapa cell lacking mab-21 gene function (class I mosaics). This
 suggested an existence of an interaction between R6 and T.apapa that could
 affect the fate of T.apapa. Direct evidence for such an interaction was
 obtained by cell ablation experiments carried out on mab-21 mutant animals
 (Table 2).
 TABLE 2
 V6 and T lineage phenotypes of mosaic animals
 mab-21
 genotype Phenotype
 Class Dp loss R6 T ray 6 ectopic ray
 I AB.p + - wild type absent
 II AB.a - + fused absent
 III AB - - fused
 present (2/6)
 IV P1 + + wild type absent
 Consistent with our earlier results in a wild type background (Chow and
 Emmons, 1994), most ablations in a mab-21 mutant background had no effect
 on unablated cells. However, ablation of R6 or its mother reduced the
 frequency of expression of the neuroblast fate by T.apapa from around 45%
 to 2.3% (1/43). Therefore in a mab-21 mutant background presence of R6
 causes T.apapa to express the neuroblast cell fate with increased
 frequency.
 This interaction between R6 and T.apapa in a mab-21 mutant background might
 be direct or indirect. R6 itself might send an inductive signal or blocks
 an inhibitory signal received by T.apapa (direct interaction), or R6 might
 simply by its physical presence cause T.apapa to be exposed to an inducing
 signal or prevent exposure to a blocking signal from another source
 (indirect interaction). Only the first of these two alternatives is
 consistent with the results of the mosaic analysis. Expression of
 mab-21(+) within one or more cells of the AB.a lineage, presumably R6,
 prevents interactions between R6 and T.apapa, or makes it ineffective (as
 does expression of mab-21 (+) within T.apapa itself). Since gene
 expression within R6 alters the interaction between R6 and T.apapa, this
 interaction is likely to be occurring directly between these two cells
 (FIG. 7).
 TABLE 3
 Frequency of T. apapa-derived ray after ablation
 of seam cells
 Cell Ablated n Ray from T. apapa
 None &gt;600 (45%)
 T. appp (R8/9) 16 10 (63%)
 R7 11 5 (46%)
 T. apapa 11 0 (0%)
 R6 25 1 (4%)
 R5 17 7 (41%)
 R4 13 7 (54%)
 R3 13 7 (54%)
 R2 10 5 (50%)
 R1 10 4 (40%)
 V6. pppp (R5/R6) 18 0 (0%)
 T 27 0 (0%)
 n, the number of sides examined.
 Ablation did not affect the identities of rays derived from the remaining
 cells.
 A fused 4-6 ray was present except in the following cases: after ablation
 of R6 and V6. pppp a normal ray 4 was present: after ablation of R5 (35%)
 and R4 (100%) ray 6 was not fused and was present as a thin, cylindrical
 ray located near or at the normal position of ray 4.
 Experimental Discussion
 mab-21 mutants affect the differentiation of four cells present bilaterally
 in the posterior epidermis of the male tail. Two of these cells, R6st and
 R6B are components of ray 6, one of nine sensory rays, while a third
 (R6.p) is a hypodermal product of the same cell sublineage that generates
 the cells of ray 6. In mab-21 mutants, each of the three cells R6st, R6B,
 and R6.p adopts a fate or differentiates in a manner similar to that of
 the corresponding cell of the adjacent more anterior ray sublineage (ray
 4). It was shown that mab-21(+) activity is likely to be required within
 the ray 6 sublineage. However, it is not known whether its action is
 required cell autonomously within each ray 6 cell individually. It could
 be that the effects on some cells are secondary to effects on other cells.
 One possible explanation for the diverse effects of mab-21 mutations on
 these three cells is that mab-21(+) activity is required only by the ray
 precursor cell R6, which in a mab-21 mutant background appears to assume
 the identity of its anterior neighbor R4. This interpretation is
 consistent with the earliest possible time of action of mab-21, which was
 when R6 was present.
 mab-21 mutations also affect a fourth cell, a hypodermal seam cell similar
 to R6. This cell, T.apapa, is born at the same time as R6 and, like R6,
 moves posteriorly out of the seam after it is born and becomes the
 immediate posterior neighbor of R6 (FIGS. 1C, D). In mab-21 mutants,
 T.apapa was frequently transformed into a ray precursor cell and expressed
 the ray sublineage. Thus in mab-21 mutants both T. appa and R6 assume
 characteristics of more anterior seam cells. It seems most likely that
 mab-21 acts as part of a pattern formation mechanism that dictates the
 fates of seam cells during the late L3 and early L4 larval stages.
 It is demonstrated that in a mab-21 background, a signal form R6 causes
 T.apapa to express the ray neuroblast cell fate. Action of mab-21(+) in
 either R6 or T.apapa blocks the effect of this signal. This evidence for
 signaling between seam cells and its modulation by cell-autonomous
 functions is consistent with previous observations, both in C.elegans and
 in other organisms. A signal from the posterior seam cell T inhibits the
 expression of ray lineages by its anterior neighbor V6 (Waring and Kenyon,
 1990). In wild type, V6 overcomes this inhibition by the cell-autonomous
 action of the homeobox transcription factor pal-1 (Waring and Kenyon,
 1991). Signalling between seam cells is necessary for expression of the
 postdeirid neuroblast cell fate by V5.pa (Waring et al., 1992). In this
 case, effective signalling requires cell contact, and this must be
 maintained on both sides of V5.p in order for this cell to divide
 asymmetrically (Austin and Kenyon, 1994). Seam cells in C. elegans, like
 epithelial cells in other organisms, are joined by adherens junctions
 (Priess and Hirsh, 1986; Baird et al., 1991). Signals passing between
 epithelial cells that influence their fates are important in the
 development of many if not all animals (for reviews see Horvitz and
 Herskowitz, 1992; Greenwald and Rubin, 1992; Peifer et al., 1993).
 One possible consequence of signalling between epidermal cells is
 modulation of levels of expression of HOM-C/Hox genes, which give cells
 their regional identities. In C. elegans, the effect of the
 above-mentioned T inhibition on V6 is proposed to be to prevent expression
 of the HOM-C/Hox gene mab-5, which is required for the male-specific
 divisions of the V6 lineage, including the ray sublineage (Waring and
 Kenyon, 1990; 1991). Likewise, a likely consequence of contact between
 V5.p and its neighbors is modulation of the expression of mab-5 within the
 V5 lineage (Austin and Kenyon, 1994).
 It was shown previously that the HOM-C/Hox genes mab-5 and egl-5 function
 in specifying the identities of rays 1-6 (Chow and Emmons, 1994). mab-5 is
 most closely related to Drosophila Antennapedia, while egl-5 is most
 closely related to Drosophila Abdominal B (Wang et al., 1993). The
 relative levels of expression of these two genes within the terminal cells
 of the ray lineages help to specify the morphological identity assumed by
 each ray. In particular, egl-5 is weakly haplo insufficient for expression
 of the identity of ray 6: in egl-5(0)/+heterozygotes, ray 6 occasionally
 (7% assumes the identity of ray 4 and fuses with ray 4 (Chow and Emmons,
 1994).
 The function of mab-21 may be related to the role of egl-5 in specification
 of the development of ray 6. mab-21 mutants are fully penetrant for the
 same ray 6 to 4 transformation phenotype weakly expressed in egl-5
 heterozygotes. Furthermore, mab-21 mutations, themselves recessive, are
 dominant enhancers of the haploinsufficient phenotype of egl-5.
 Heterozygosity for a mab-21 mutation increased the frequency of
 transformation of ray 6 to ray 4 in a heterozygous egl-5 background from
 7% to over 30% (Chow and Emmons, 1994). Thus a decreased level of egl-5(+)
 gene function makes R6 sensitive to the level of mab-21 gene function.
 Because egl-5 modifies the cellular environment in which mab-21 acts, this
 argues that the two genes function in the same or related pathways in
 determination of the identity of ray 6. As mab-21 also acts in other body
 regions in addition to toe posterior seam of males, and indeed has an
 essential embryonic function, it is possible that mab-21 plays a much
 wider role in implementing the action of HOM-C/Hox genes.
 In spite of the fact that HOM-C/Hox genes have been recognized for some
 time as encoding transcription factors that play a key role in regional
 specialization within the metazoan body, large gaps remain in our
 understanding of their mode of action. Because the mab-21 gene encodes a
 putative protein of hitherto unreported amino acid sequence, it is not
 possible to predict whether it might play a role in regulation of
 transcription of egl-5 or in target gene selectivity of egl-5, possibly as
 a cofactor. Another possibility for the function of a HOM-C/Hox gene
 modifier such as mab-21 is that it acts in the upstream pathways that
 restrict the expression of HOM-C/Hox genes to certain body regions. Thus a
 further possibility for mab-21 action is as a component of the pattern
 formation mechanisms that set the expression levels of HOM-C/Hox genes in
 the seam cells.
 References
 Andrew, D. J. and Scott, M. P. (1992). Downstream of the homeotic genes.
 new biol. 4, 5-15.
 Austin, J., and C. Kenyon. (1994). Cell contact regulates neuroblast
 formation in the Caenorhabditis elegans lateral epidermis. Development
 120:313-324.
 Baird, S. E., D. H. A. Fitch, I. A. A. Kassem, and S. W. Emmons. (1991).
 Pattern formation in the nematode epidermis: determination of the
 arrangement of peripheral sense organs in the C. elegans male tail.
 Development 113:515-526.
 Botas, J. (1993) Control of morphogenesis and differentiation by HOM/hox
 genes. Curr. Opin. Cell Biol. 5, 1015-1022.
 Brenner, S. (1974) The genetics of Caenorhabditis elegans. Genetics.
 77:71-94
 Cassada, R., E. Isnenghi, M. Culotti, and G. Von Ehrenstein. (1981).
 Genetic analysis of temperature-sensitive embryogenesis mutants in
 Caenorhabditis elegans. Develop. Biol. 84:193-205.
 Chow, K. L., and S. W. Emmons. (1994). HOM-C genes and four interacting
 loci determine the morphogenetic properties of single cells in the
 nematode male tail. Development.
 Devereux, J., Haeberli, P., and Smithies, O. (1984). A comprehensive set of
 sequence analysis programs for the VAX. NAR 12, 387-395.
 Finney, M., and G. Ruvkun. (1990). The unc-86 gene product couples cell
 lineage and cell identity in C. elegans. Cell 63:895-905.
 Fire, A., S. White-Harrison, and D. Dixon. (1990). A modular set of lacZ
 fusion vectors for studying gene expression in Caenorhabditis elegans.
 Gene 93:189-198.
 Greenwald, I. S., P. W. Sternberg, and H. R. Horvitz. (1983). The lin-12
 locus specifies cell fates in Caenorhabditis elegans. Cell 34:435-444.
 Hall, D. H. (1995) Electron microscopy and 3D image reconstruction. In C.
 elegans: Modern Biological Analysis of an Organism. Methods in Cell
 Biology, Vol.48. (H. F. Epstein and D. C. Shakes, eds.) Academic Press,
 New York, in press.
 Herman, R. K., (1984) Analysis of genetic mosaics of the nematode
 Caenorhabditis elegans. Genetics 108, 165-180.
 Herman, R. K. (1989) Mosaic analysis in the nematode Caenorhabditis
 elegans. J. Neurogenetics 5, 1-24.
 Hodgkin, J., H. R. Horvitz, and S. Brenner. (1979). Nondisjunction mutants
 of the nematode Caenorhabditis elegans Genetics. 91:67-94.
 Horvitz, H. R., and Herskowitz, I. (1992). Mechanisms of asymmetric cell
 division: two Bs or not two Bs, that is the question. Cell 68, 237-255.
 Kenyon, C. (1986). A gene involved in the development of the posterior body
 region of C. elegans. Cell 46:477-487.
 Kramer, J. M., R. P. French, E. C. Park, and J. J. Jonhson. (1990). The
 Caenorhabditis elegans rol-6 gene, which interacts with the sqt-1 collagen
 gene to determine organismal morphology, encodes a collagen. Mol. Cell.
 Biol. 10:2081-2089.
 Levy, A. D., J. Yang, and J. M. Kramer. (1993). Molecular and genetics
 analyses of the Caenorhabditis elegans dpy-2 and dpy-10 collagen genes: a
 variety of molecular alterations affect organismal morphology. Mol. Biol.
 Cell. 4:803-817
 Liu, K. S., and Sternberg, P. W. (1995). Sensory regulation of male mating
 behavior in Caenorhabditis elegans. Neuron 14, 1-20.
 Loer, C. M., and Kenyon, C. J. (1993) Serotonin-deficient mutants and male
 mating behavior in the nematode Caenorhabditis elegans. J.Neurosci. 13,
 5407-5417.
 McGinnis, W., and Krumlauf, R. (1992) Homeobox genes and axial patterning.
 Cell 68,283-302.
 Mello, C. C., J. M. Kramer, D. Stinchcomb, and V. Ambros. (1991). Efficient
 gene transfer in C. elegans:Extrachromosomal maintenance and integration
 of transforming sequences. EMBO L. 10:3959-3970.
 Palazzolo, M. J., B. A. Hamilton, D. Ding, C. H. Martin, D. A. Mead, R. C.
 Mierendorf, K. V. Raghavan, E. M. Meyerowitz, and H. D. Lipshitz. (1990).
 Phage lambda cDNA cloning vectors for subtractive hybridization,
 fusion-protein synthesis and Cre-loxP automatic plasmid subcloning. Gene
 88:25-36.
 Peifer, M., Orsulic, S., Pai, L. M., and Loureiro, J. (1993). A model
 system for cell adhesion and signal transduction in Drosophila.
 Development 1993 Suppl., 163-176.
 Priess, J. R., and D. I. Hirsh. (1986). Caenorhabditis elegans
 morphogenesis: the role of the cytoskeleton in elongation of the embryo.
 Dev. Biol. 117:156-173.
 Rosenbluth, R. E., Cuddeford, C., and Baillie, D. L. (1985). Mutagenesis in
 Caenorhabditis elegans. II. A spectrum of mutational events induced with
 1500 R of gamma-radiation. Genetics 109, 493-511.
 Schierenberg, E., J. Miwa, and G. von Ehrenstein. (1980). Cell lineages and
 developmental defects of temperature-sensitive embryonic arrest mutants in
 Caenorhabditis elegans. Develop. Biol. 76, 141-159.
 Sulston, J. E. and Horvits, H. R. (1977). Post-embryonic cell lineages of
 the nematode Caenorhabditis elegans. Dev. Biol. 76, 141-159.
 Sulston, J. E., D. G. Albertson, and J. N. Thompson. (1980). The
 Caenorhabditis elegans male: postembryonic development of nongonadal
 structures. Dev. Biol. 78:542-576.
 Sulston, J., and Hodgkin, J. (1988) Methods. In Wood, W. B. (ed.) The
 Nematode Caenorhabditis elegans. Cold Spring Harbor Laboratory Press.
 pp587-606.
 Wang, B. B., M. M. Muller-Immergluck, J. Austin, N. T. Robinson,
 A.Chisholm, and C. Kenyon. (1993). A homeotic gene cluster patterns the
 anteroposterior body axis of C. elegans. Cell 74:29-42.
 Waring, D., and Kenyon, C. (1990). Selective silencing of cell
 communication influences anteroposterior pattern formation in C. elegans.
 Cell 60, 123-131.
 Waring, D. A., and Kenyon, C. (1991). Regulation of cellular responsiveness
 to inductive signals in the developing C. elegans nervous system. Nature
 350, 712-715.
 Waring, D. A., Wrischnik, L., and Kenyon, C. (1992). Cell signals allow the
 expression of a pre-existent neural patter in C. elegans. Development 116,
 457-466.
 Zhang, Y. and Emmons, S. W. (1995). Specification of sense-organ identity
 by a Caenorhabditis elegans Pax-6 homologue. Nature 377, 55-59.
 Second Series of Experiments
 1. mab-21 AND DISEASES
 a. Comparison of mab-21 and mab-18 phenotypes
 It was noted that the mab-21 and mab-18 shared their mutant phenotypes.
 Both mutations affect the differentiation of sensory ray 6, conical shape
 structure in wild type animal. Ray 6 is transformed into a thin ray
 resembling ray 4, and both of them fuse to form a thick fused ray.
 Although these two genes are mapped on different chromosome (linkage group
 III and linkage group X respectively) (Baird et al., 1990), the phenotype
 alone has indicated that they may play similar or related functional role
 in sensory organ differentiation. This idea has prompted us to analyze the
 genetic interaction between them.
 b. Genetic interaction
 Subsequent genetic experiments show that heterozygous mutation of either
 one of the mutations results in no phenotype. On the other hand, double
 heterozygous populations have a fraction of the animals with a phenotype
 resembling homozygous mutation of mab-21 or mab-18 gene (Chow and Emmons,
 1994). Such genetic interaction test is a common method in genetic
 analysis to infer genes participating in the same genetic pathway.
 In parallel experiments, mab-21 has been shown to interact with another
 group of homeodomain containing transcription factor genes belonging to
 the HOM-C/Hox class (Chow and Emmons, 1994). While HOM-C/Hox class genes
 are highly conserved in hydra, worm, fly, fish, mouse and human, their
 interaction with mab-21 substantiates the fact that mab-21 is part of a
 conserved mechanism present in invertebrate and vertebrate systems. It has
 been implicated to interact with these conserved genes in local pattern
 formation of the sensory tissues.
 c. mab-18 Sequence is Homologous to PAX-6 (Aniridia) in Human, pax-6 (small
 eye) in Mouse, Eyeless in Drosophila
 mab-18 gene has been molecularly analyzed and sequenced. It encodes a
 homeodomain containing transcription factor (Zhang and Emmons, 1995) with
 homologs in invertebrate and vertebrate systems (Quiring et al., 1994;
 Krauss et al., 1991; Puschel et al., 1992).
 The human and mouse homologs are called PAX-6 (Jordan et al., 1992; Glaser
 et al., 1992) and pax-6 (Walther and Gruss, 1991). They were identified by
 sequence homology to a group of Drosophila developmentally important
 genes. These genes have a homeodomain and an adjacent paired domain, both
 of which are required for DNA binding. Subsequent mutation analyses have
 linked the human PAX-6 gene mutation with a human disease called Aniridia
 (Glaser et al., 1992) affecting iris differentiation, and pax-6 gene
 mutation with a mouse mutation called small eye (Schmahl et al., 1993). In
 both cases, the eye development has been affected.
 Recently, a Drosophila homolog of mab-18 was isolated (Quiring et al.,
 1994). The gene was called eyeless, mutation of which results in no eye in
 the animal. Ectopic expression of the gene can induce formation of
 additional eyes in tissue normally not differentiating into eye (Halder et
 al., 1995a). In the same study, experiments have been conducted to prove
 that the human PAX-6 gene can functionally substitute the Drosophila gene
 and rescue the Drosophila mutation.
 These results suggest that pax-6 like genes are involved in a developmental
 pathway of eye formation which is highly conserved from invertebrate to
 vertebrate (Halder et al. 1995b). Based on the conservation of the
 components in this pathway, applicant postulates that eye development in
 Drosophila, mouse and human is molecularly equivalent to the sensory ray
 differentiation in nematode.
 d. mab-21 Acts Together With pax-6 in the Same Pathway
 The genetic characterization of mab-21 indicates that mab-21 interacts with
 mab-18, and is acting in the same evolutionary conserved genetic pathway.
 While mutation of mab-18 and its homologs result in a variety of
 abnormality and disease associated with sensory organ formation as
 described in (c), applicant believes that mutations of mab-21 homologs in
 vertebrate are also associated with abnormality which may have a disease
 state related to that of Aniridia caused by mutation in the human PAX-6
 gene, and may affect peripheral neural tissue differentiation.
 e. Homologs of mab-21 Identified
 The cellular and genetic components with that mab-21 interacts are highly
 conserved across phyla in the animal kingdom. Applicant believes early on
 that the homologs of mab-21 exist in other invertebrates as well as
 vertebrates. The homolog of a closely related species of Caenorhabditis
 elegans, C. briggsae was isolated. From a genomic library screen,
 applicant isolated four genomic clones using the C. elegans mab-21 cDNA as
 probe. From the sequence analyzed so far, the homology with C. elegans
 mab-21 is greater than 95%. [see section 2 for detail].
 From the search of Genebank sequence database, two human EST clones were
 identified in the Merck-St.Louis EST project. When compared with the
 genomic sequence from the cosmid containing mab-21 locus, both EST
 sequences share greater than 70% homology to the worm gene. While these
 clones are isolated from a brain library, it implies that the human mab-21
 gene is expressed in neural tissue.
 Subsequently, Margolis et al. reported identification of a number of cDNAs
 they called CAGRl which are also homologous to the nematode mab-21 gene
 with an overall greater than 60 percent homology on the amino acid
 sequence level (Margolis et al., pers. comm.) (FIG. 8). Recent experiments
 have shown that expression of the human cDNA can substitute the mab-21
 function in mutant C. elegans worms. This result proves the functional
 homology of these genes. While these CAGR1 cDNAs were isolated from human
 retina and cerebral cortex cDNA libraries, this fact strengthens the
 association of mab-21 with neural function, and further hints its role in
 sensory organ development.
 f. Human mab-21 is Mapped to Human Chromosome 13q13
 Human mab-21 has been localized onto human chromosome 13 band q13 (Margolis
 et al., pers. comm.). Applicant believes that loci corresponding to the
 human mab-21 may have been described by mutations previously associated
 with that chromosomal region. Search in Genome Database at University of
 Pennsylvania revealed that there are a number of loci in the same region.
 However, as the above disclosure makes clear that mab-21 affects sensory
 organ and mab-18 homolog mutation phenotype affects eye development, a
 candidate locus stands out by its own. Moebius Syndrome mapped to 13q13
 (Slee et al., 1991). The phenotype described for Moebius include cranial
 facial palsy, congenital oculofacial paralysis, oculofacialbulbar palsy,
 hypoplasia of tongue, mask (expressionless) face, oculomotor nerve defect
 (failure of lateral eye movement), trochlear nerve defect, malformation of
 orofacial structures (swallowing and speech difficulties), and branchial
 muscle defect (Kumar, 1990). Based on the phenotypes which can be
 associated with cranial facial nerve function and development, applicant
 is currently testing that Moebius Syndrome is the manifestation of mab-21
 mutation.
 2. IDENTIFICATION OF mab-21 HOMOLOG IN OTHER NEMATODE SPECIES
 C. elegans mab-21 complete cDNA was used as the probe. The DNA fragment was
 labeled by random priming procedure to generate radioactively labeled DNA
 probe for screening C. briggsae genomic library kindly provided by Dr.
 David Baillie of Simon Fraser University, Canada. The phages were plated
 out at concentration of about 1000 pfu/plate. Out of about 30,000 pfu
 screened, i.e. greater than 5 genomic equivalents, four independent clones
 were isolated. Only Cb#17 was subcloned. Deletions were generated and
 sequenced. DNA sequence and the predicted protein amino acid sequence
 (FIG. 9) suggests that Cb#17 corresponds to the 3' end portion of the C.
 briggsae mab-21. While the other three clones corresponds to the 5'
 genomic region of the gene.
 Recently, an EST sequence deposited to the gene bank database was found to
 have high similarity to the 5' region of C. elegans mab-21 gene. It is
 representing the mab-21 homolog in a parasitic nematode Brugia malayi
 (FIG. 10).
 3. IDENTIFICATION OF HOMOLOGS OF mab-21
 a. F35G12.6 in C. elegans
 F35G12 and C56A8 are the cosmids that can rescue the mab-21 mutant
 phenotype by DNA transformation into mutant gonad (Chow and Emmons, 1995).
 Overlapping region of these two cosmids defines the mab-21 gene. In that
 region, a number of ORF has been predicted by the Genefinder program used
 in the A Caenorhabditis elegans Database (ACEDB), one of which is
 F35G12.6. The F35G12.6 ORF is at the corresponding position of a 5.6 Kb
 genomic fragment which has been shown by deletion/transformation rescue
 assay to contain mab-21 gene (Chow et al., 1995). However, due to errors
 in the submitted sequence of the cosmid F35G12, the ORF F35G12.6 encodes a
 protein slightly different from the protein product predicted from the
 mab-21 cDNA (FIG. 8 and FIG. 11).
 b. Human Homolog for mab-21 Search
 The human homolog reported by Margolis et al. can be obtained from NCBI's
 Genebank Database Query function using "mab-21" as the keyword. (FIG. 12)
 c. Human Homolog in EST Project
 In the University of Washington (St.Louis)-Merck EST project, two cDNA
 clones were deposited to the Genebank. They are ym36d10 (FIG. 13) and
 ym45dll (FIG. 14). Both have been identified as having sequence homology
 to F35G12.6. They were pulled out from search using F35G12 as the keyword
 (These two clones will not be identified using mab-21 as keyword.). Since
 F35G12.6 corresponds to the C.elegans mab-21 gene, the two EST clones
 should have homology to mab-21 cDNA sequence. Alignment test by SeqVu 1.0
 program does confirm this notion. The two EST clones corresponding to two
 truncated cDNAs with most of the 3' portion of the human mab-21
 transcript.
 4. EXPRESSION OF NEMATODE MAB-21 PROTEIN IN BACTERIAL CELLS AND YEAST
 a. mab-21 cDNA had been inserted into bacterial expression vector with a
 maltose binding protein tag (MBP). Fusion protein (MBP-MAB-21) was
 generated as cytosolic protein in bacterial cell. Cell lysate had been
 passed into affinity chromatographic column with maltose coupled with
 Sephadex resin. The impurities were eluted with the MBP-MAB-21 retained in
 the column. The fusion was eluted later with maltose solution, dialyzed
 and cleaved with factor Xa, a protease that could digest and separate the
 MBP tag from the MAB-21 protein (FIGS. 16, 17).
 b. mab-21 cDNA had also been inserted into yeast vector under the
 regulation of the GAL-1 promoter, and in front of a galactosidase gene.
 Induction by galactose in yeast culture medium generated fusion protein of
 MAB-21-Galactosidase. The fusion protein had enzymatic activity of
 galacosidase, by enzyme assay. The fusion protein could also be detected
 by antibody against galactosidase. In both enzymatic assay and
 immunostaining study, it was revealed that the fusion protein was
 expressed in the nuclei of yeast cells. Similar subcellular localization
 of fusion protein was observed in worm.
 5. PRODUCTION OF ANTIBODIES AGAINST MAB-21 PROTEIN
 The bacterial expressed MAB-21 protein had been used for immunization of
 mouse and rat, antisera from six mice and six rats are now available.
 Preliminary tests of the sera by both Western blot assay (FIG. 18) and
 ELISA assay (FIG. 19) show positive identification of the MAB-21 protein
 in bacteria and in nematode. This suggests that the rationale of
 generating immunoreagents in one the claims is sound, and valid, and
 experimental work is feasible.
 6. IDENTIFICATION AND ISOLATION OF MORE MAB-21 HOMOLOGS AND OTHER STUDIES
 Homologs of mab-21 in a variety of animals species, including hydra,
 Artemia, Drosophila, Xenopus (frog), zebrafish, mouse and human are being
 isolated. Preliminary data shown in FIG. 8 suggest that the gene products
 are highly conserved, and vertebrate homologs are present in human beings.
 The human homolog of mab-21 gene will be used to perform Northern analysis
 to examine the normal expression pattern of the gene, and to correlate its
 function in neural tissues during development.
 Isolation of the mouse mab-21 homolog by polymerase chain reaction and
 hybridization screening is also in progress. Preliminary data are shown in
 FIG. 15. The homologous sequences are currently used to localize the gene
 onto a specific chromosome by fluorescent in situ hybridization, and to
 correlate the gene with existing genetic mutation identified in mouse. In
 fact, by syntenic analysis of the human and mouse genome, there are
 candidate mutant loci which may correspond to the mouse mab-21 gene.
 Subsequently, knock out transgenic mouse will be used to establish a
 vertebrate animal model to examine the function of mab-21 gene, and the
 potential manifestation of the disease state with the absence of the gene
 function.
 Mutation(s) associated with the three existing allele of mab-21 mutation
 are being characterized to determine the functional significance of
 various domain of the protein.
 Genetic experiments are in the set up process for identification of
 suppressors, enhancers and modifiers of mab-21 mutation, in hope of
 identifying various components in the genetic pathway involving mab-21
 gene function.
 Experiments are in progress to overexpress the protein in various animal
 model systems to examine the role of mab-21 gene in normal development.
 Experiments using MAB-21 protein as starting material to looking for
 additional interacting proteins are in the preparation stage. It includes
 experiments using the antibodies specific for MAB-21 protein for
 co-immunoprecipitation of the interacting protein together with MAB-21
 protein. Genetic approach using yeast two hybrid system or interaction
 trap system to identify interacting proteins is also in progress.
 Specifically, demonstration of a direct interaction of MAB-21 protein with
 MAB-18 protein will strengthen the genetic support for the involvement of
 mab-21 gene or its homologs in developmental functions involving mab-18
 gene and its homologs, e.g. sensory organ differentiation and neural
 patterning.
 7. REFERENCES:
 Baird, S. E. Fitch, D. H. A., Kassem, I. A. A. and Emmons, S. W. (1991)
 Pattern formation in the nematode epidermis: determination of the
 arrangement of peripheral sense organs in the C. elegans male tail.
 Development 113, 515-526.
 Chow, K. L. and Emmons, S. W. (1994) HOM-C/Hox genes and four interacting
 loci determine the morphogenetic properties of single cells in nematode
 mail tail. Development 120, 2579-2593.
 Chow, K. L., Hall, D. and Emmons, S. W. (1995) The mab-21 gene of
 Caenorhabditis elegans encodes a novel protein required for choice of
 alternate cell fates. Development 121, 3615-3626.
 Glaser, T., Walton, D. S. and Maas, R. L. (1992) Genomic structure,
 evolutionary conservation and aniridia mutations in the human PAX 6 gene.
 Nature Genetics 2, 232-238.
 Grindley, J. C., Davidson, D. R. and Hill R. E. (1995) The role of Pax-6 in
 eye and nasal development. Development 121, 1433-1442.
 Halder, G., Gallaerts, P. and Gehring, W. J. (1995a) Induction of ectopic
 eyes by targeted expression of the eyeless gene in Drosoplila. Science
 267,1788-1792.
 Halder, G., Gallaerts, P. and Gehring, W. J. (1995b) New perspectives on
 eye evolution. Curr. Opin. Genet. Develop. 5,602-609.
 Hanson, I and Van Heyningen, V. (1995) Trends Genet. 11, 268-272.
 Jordan, T. Hanson, I., Zaletayev, D., Hodgson, S., Prosser, J., Seawright,
 A., Hastie, N. and Van Heyningen, V. (1992) Nature Genet. 1, 328-332.
 Krauss, S., Johansen, T., Korzh, V., Moens, U., Ericson, J. U. and Fjose,
 A., (1991) Zebrafish pax[zf-a]: a paired box containing gene expressed in
 the neural tube. EMBO J. 10,3609-3619.
 Kumar, D. (1990) Moebius syndrome. J. Med. Genet. 27, 122-126.
 Puschel, A. W., Gruss, P. and Westerfield, M. (1992) Sequence and
 expression pattern of pax-6 are highly conserved between zebrafish and
 mice. Development 114, 643-651.
 Quiring, R., Walldorf, U., Kloter, U. and Gehring, W. J. (1994) Homology of
 the eyeless gene of Drosophila to the small eye gene in mice and aniridia
 in humans. Science 265, 785-789.
 Schmahl, W., Knoedlseder, M., Favor, J. and Davidson, D. (1993) Defect of
 neuronal migration and the pathogenesis of cortical malformations are
 associated with Small eye (Sey) in the mouse, a point mutation at the
 Pax-6 locus. Acta Neuropathol. 86, 126-135.
 Slee, J. J., Smart, R. D. and Viljoen, D. L. (1991) Deletion of chromosome
 13 in Moebius syndrome. J. Med. Genet. 28, 413-414.
 Walther, C., Guenet, J. L., Simon, D., Deutsch, U., Jostes, B., Goulding,
 M. D., Plachov, D., Balling, R. and Gruss, P. (1991) Genomics 11, 424-434.
 Zhang, Y. and Emmons, S. W. (1995) Specification of sense organ identity by
 a Caenorhabditis elegans Pax-6 homologue. Nature 377, 55-59.