C 2014 Wiley Periodicals, Inc. V
genesis 52:864–869 (2014)
LETTER
Characterization of Missense Alleles of the glial cells missing Gene of Drosophila Bradley W. Jones* Department of Biology, The University of Mississippi, 122 Shoemaker Hall, University, Mississippi Received 21 March 2014; Revised 2 July 2014; Accepted 3 July 2014
Summary: Glial cells missing (Gcm) is the primary regulator of glial cell fate in Drosophila. Gcm belongs to a small family of transcriptional regulators involved in fundamental developmental processes found in diverse animal phyla including vertebrates. Gcm proteins contain the highly conserved DNA-binding GCM domain, which recognizes an octamer DNA sequence. To date, studies in Drosophila have primarily relied on gcm alleles caused by P-element induced DNA deletions at the gcm locus, as well as a null allele caused by a single base pair substitution in the GCM domain that completely abolishes DNA binding. Here I characterize two hypomorphic missense alleles of gcm with intermediate glial cells missing phenotypes. In embryos homozygous for either of these gcm alleles the number of glial cells in the central nervous cystem (CNS) is reduced approximately in half. Both alleles have single amino acid changes in the GCM domain. These results suggest that Gcm protein activities in these mutant alleles have been attenuated such that they are operating at threshold levels, and trigger glial cell differentiation in neural precursors in the CNS in a stochastic fashion. These hypomorphic alleles provide additional genetic resources for understanding Gcm functions and structure in Drosophila and other species. genesis 52:864– C 2014 Wiley Periodicals, Inc. 869, 2014. V Key words: Drosophila; CNS; glia; gcm; missense mutations; hypomorphic allele
INTRODUCTION In Drosophila melanogaster the glial cells missing gene (gcm) is the primary regulator of glial cell determination (Hosoya et al., 1995; Jones, et al., 1995; Vincent et al., 1996). gcm encodes a transcription factor that is transiently expressed in all embryonic lateral glia
(excluding the midline glia). gcm mutant embryos lack nearly all lateral glial cells, and presumptive glia are transformed into neurons. Conversely, when gcm is ectopically expressed, presumptive neurons are transformed into glia. Thus, within the nervous system, gcm acts as a binary genetic switch, with Gcm-positive cells becoming glia and Gcm-negative cells becoming neurons (Hosoya et al., 1995; Jones, et al., 1995; Vincent et al., 1996). In addition to acting as a regulator of glial cell differentiation, gcm is also required for the proper differentiation of embryonic hemocytes (Alfonso and Jones, 2002; Bernardoni et al., 1997; Lebestky et al., 2000), tendon cells (Soustelle et al., 2004), and a subset of neurons in the adult brain (Chotard et al., 2005; Soustelle and Giangrande, 2007). Thus gcm controls different developmental programs according to cellular context. Gcm belongs to a small family of transcriptional regulators found in diverse animal phyla including vertebrates, and, where they have been studied, they are involved in fundamental developmental processes (reviewed in Wegner and Riethmacker, 2001). Significantly, mouse Gcm1/GCMa is required for the development of placental labyrinthine trophoblasts, (AnsonCartwright et al., 2000; Schrieber et al., 2000), mouse Gcm2/GCMb is a master regulator parathyroid gland development (Gunther et al., 2000), and both mouse Gcm1 and Gcm2 together regulate neural stem cell development (Hittoshi et al., 2011).
* Correspondence to: Dr. Bradley Jones, 122 Shoemaker Hall, University, MS 38677. E-mail: [email protected] Published online 12 July 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/dvg.22801
MISSENSE ALLELES OF glial cells missing
865
FIG. 1. Missense alleles of gcm show reduction in glial cell differentiation. (a–g) Dissected stage 16 embryos showing five adjacent segments of the CNS labeled with Repo monoclonal antibody with the following genotypes: (a) Wild type, (b) gcmDP1, (c) gcmG78, (d) gcm1308, (e) gcmG78/gcm1308, (f) gcmG78/gcmDP1, and (g) gcm1308/gcmDP1. Anterior is up. Scale bar, 20 mm. In the gcm null mutant embryo (b) glial cells are nearly absent as shown by Repo expression. In the missense alleles gcmG78 (c), gcm1308, (d) and in the transheterozygote gcmG78/ gcm1308 (f) glial cell differentiation is reduced by half. (h) A gcm gene diagram is shown with the regions corresponding to the Gcm DNA binding domain, the remainder of the coding region, and the noncoding sequence indicated in black, gray, and white, respectively. The encoded amino acids affected by the analyzed missense gcm mutations are indicated.
Gcm family proteins share a highly conserved N-terminal 150 amino acid DNA binding GCM domain with unique structure (Cohen et al., 2003). Drosophila Gcm binds with high affinity to the octameric consensus sequence AT(G/A)CGGG(T/C) found repeated in the regulatory regions of glial-specific genes (Akiyama et al., 1996; Freeman et al., 2003; Granderath et al., 2000; Schreiber et al., 1997). Its mammalian counterparts also bind to the same octamer sequence (Cohen
et al., 2003; Shchrieber et al., 1998), and expression of murine and rat Gcm1 in Drosophila promotes glial cell differentiation, indicating conserved biochemical properties (Kim et al., 1998; Reifegerste et al., 1999). In summary Gcm proteins act as essential tissue-specific transcriptional regulators in several tissues that share the common action of binding to a highly conserved octamer sequence motif, activating a variety of different genes in different contexts.
866
JONES
FIG. 2. Locations of three Drosophila missense mutations in the GCM domain. Alignment of the Gcm domains from Drosophila melanogaster (dGcm, dGcm2), mouse (mGcm1, mGcm2) and zebrafish (zfGcm2). Conserved amino acids are shaded. Secondary structural elements derived from the crystal structure mGcm1 as described in Cohen et al. 2003 are shown above the alignment. Blue lines S1 through S7 represent strands in the two ß-sheets, and red bars H1 through H3 represent three small helical segments. 1 symbols represent DNA contacting amino acids; star symbols represent cysteine and histidines involved in coordinating two Zn ions, with green stars coordinating the first Zn ion and black stars cording the second Zn ion.
To date, studies in Drosophila have relied on loss-offunction gcm P-element excisions alleles that cause DNA deletions at the gcm locus (Hosoya et al., 1995; Jones et al., 1995; Vincent et al., 1996). Only one identified allele gcmN7-4 represents a point mutation in the gcm gene, where a G to C substitution in codon 93 of gcm (Fig. 2) changes a highly conserved cysteine to a serine in the Gcm domain that abolishes DNA binding and leads to complete loss-of-function (Miller et al., 1998). In this article I characterize two hypomorphic missense alleles of gcm with intermediate glial cells missing phenotypes. In embryos homozygous for either of these gcm alleles the number of glial cells in the central nervous cystem (CNS) is reduced approximately in half. Both alleles have point mutations that cause amino acid changes in the highly conserved GCM domain. These results suggest that the biochemical activities of Gcm proteins in these mutants have been attenuated such that they are operating at threshold levels, and trigger
glial cell differentiation in only half of neural precursors in the CNS in a stochastic fashion. RESULTS AND DISCUSSION gcm1308 is an ethyl methanesulfonate (EMS) induced allele of gcm that was isolated in a systematic screen for mutations that disrupt the organization of CNS axon pathways in Drosophila (Jones et al., 1995; Seeger et al., 1993). gcmG78 was isolated in an EMS screen for genes that disturb the pattern of Reversed Polarity (Repo) protein. Repo is expressed constitutively in the nuclei of all lateral glial cells can therefore be used as a glial cell marker (Campbell et al., 1994; Halter et al., 1995; Xiong et al., 1994). The pattern of glial cells as revealed by anti-Repo antibody show that gcmG78 and gcm1308 mutant embryos have similar phenotypes; both have a reduced number of glial cells in the CNS (Fig. 1c,d) compared to wild type (Fig. 1a). The pattern of missing glial cells appears to be random in both
MISSENSE ALLELES OF glial cells missing
Table 1 Number of Glial Cells per Abdominal CNS Segment in Wild type and gcm Mutant Stage 16/17 Embryos Genotype wild type gcmDP1 gcmG78 gcm1308 gcmG78/gcm1308 gcmG78/gcmDP1 gcm1308/gcmDP1
Total glia Number of Average # glia % of counted segments per segment wild type 3195 56 57.05 100% 226 56 4.04 7% 1399 56 24.98 44% 1550 56 27.67 49% 1494 56 26.68 47% 981 56 17.51 31% 761 56 13.59 24%
mutants, with deficiencies in all CNS glial subtypes in each segment. By comparison embryos carrying the null allele gcmDP1 in which the gcm locus is deleted show a dramatic decrease in Repo-positive cells (Fig. 1b), with isolated single Repo-positive cells, as well as occasional small clusters in many segments. Transheterozygotes of the two alleles (gcmG78/ gcm1308, Fig. 1e) are similar in appearance to the homozygotes of each allele, and crossing each allele with the gcmDP1 null allele appears to achieve a further reduction in glial cell numbers with similar phenotypes (Fig. 1f,g). Interestingly, reducing the dosage by half for both gcmG78 and gcm1308 alleles in embryos transheterozygous with gcmDP1 affects the distribution of glial cells in a similar way, with few glial cells near the midline along the longitudinal axon tracts; instead, the glial cells are abnormally clustered along the lateral edges of the of the CNS (Fig. 1f,g), suggesting a change in behavior and/or identity of the glial cells. Taken together, these data suggest that that both alleles function in a similar manner. To assay the degree to which glial cells are missing in gcm1308, gcmG78, and in transheterozygous embryos I counted the number of Repo-positive cells in 7 adjacent abdominal CNS segments in eight individuals of each mutant phenotype (56 segments) at stages 16 and 17, as well as in wild type and gcmDP1 null mutant embryos, and obtained an average number of glial cells per abdominal segment (Table 1). In wild type embryos I counted an average of 57 Repo-positive glia per abdominal segment, which is in accordance with abdominal glial cell counts done by others when the midline glia are excluded (Ito et al., 1995). gcmG78 mutant embryos have an average of 25 Repo-positive glia per abdominal segment, which is 44% of wild type, or a 56% reduction in the number of glia. gcm1308 mutant embryos have an average of 28 Repo-positive glia per abdominal segment, which is 49% of wild type, or a 51% reduction in the number of glia. gcmG78/ gcm1308 transheterozygous embryos have an average of 27 Repo-positive glia per abdominal segment, which is 47% of wild type, or a 53% reduction in the number of glia. gcmG78/gcmDP1 transheterozygous embryos have an average of 16 Repo-positive glia per abdominal
867
segment, which is 31% of wild type, or a 69% reduction in the number of glia. gcm1308/gcmDP1 transheterozygous embryos have an average of 14 Repo-positive glia per abdominal segment, which is 24% of wild type, or a 76% reduction in the number of glia. By contrast in gcmDP1 null mutant embryos have an average of 4 Repo-positive glia per abdominal segment, which constitutes a 93% reduction in the number of glia. gcm1308 and gcmG78 alleles were induced by EMS treatment, suggesting the presence of point mutations. Genomic DNA from gcm1308 homozygous embryos and from homozygous embryos of the mutant strain l(2)GA1019 from the same EMS mutagenesis screen were sequenced at the gcm locus and compared. gcm1308 contains a single G to A nucleotide substitution within the 75th codon of the gcm open reading frame, changing a conserved glycine to aspartic acid (Figs. 1e and 2). Genomic DNA from gcmG78 homozygous embryos and from the parental strain of the EMS mutagenesis screen were sequenced at the gcm locus and compared. gcmG78 contains a single T to A nucleotide substitution within the 53rd codon of the gcm open reading frame, changing a conserved tryptophan to arginine (Figs. 1e and 2). Both of these mutations change conserved amino acid residues in the GCM domain (Fig 2). The crystal structure of the murine Gcm1 (also known as GCMa) GCM domain-DNA complex has been resolved (Cohen et al., 2003). The GCM domain is complicated, consisting of two subdomains, a large and a small, comprised of five- and three-stranded ß-sheets respectively, with three helical segments also incorporated (Cohen et al., 2003). These two subdomains are held together by two Zn ions bound to conserved cysteine and histidine residues. The importance of Zn ions in coordination in GCM domain structure is demonstrated by mutant allele gcmN7-4 in which a cysteine that binds Zn is changed to a serine; this change completely abolishes DNA binding activity and leads to a complete loss-of-function (Miller et al., 1998, Fig. 2). DNA recognition is largely accomplished by the large subdomain that consists of a five-stranded ß-sheet, comprised of strands S1, S2, S3, S6, and S7 shown in Fig. 2. This five-stranded ß-sheet protrudes into the major groove perpendicular to the DNA axis. The large subdomain also consists of the N terminal part of the GCM domain packed on one side of the ß-sheet and the helix H1 packed on the other. Both mutations described here, gcm1308 and gcmG78, change hydrophobic amino acids into charged amino acids within this large subdomain and are likely to subtly affect its structure and perhaps the affinity of DNA binding. While both alleles have significant changes in the amino acid sequence in the conserved GCM domain, one cannot rule out the possibility that additional EMS induced mutations exist within gcm cis-regulatory
868
JONES
sequences that could contribute to the observed phenotypes by attenuating patterns or levels of gcm expression. However, I have looked at the expression pattern of Gcm protein using anti-Gcm antibodies in homozygous embryos of both alleles and find the early expression patterns to be normal at Stage 11 in the glial precursor cells that give rise to the longitudinal glia. I also find Gcm expressed at normal levels in both alleles in neural and glial cells through Stage 14, although in a disorganized fashion (data not shown). Both gcm alleles reduce glial cell differentiation by approximately half. The glial cells that do develop do so in an apparent random pattern. The results presented here suggest that the biochemical activities of Gcm proteins in these mutants have been attenuated such that they are operating at threshold levels, and trigger glial cell differentiation in a stochastic fashion. These mutants are the first hypomorphic alleles of gcm to be characterized that are likely to be caused by point mutations in the GCM domain; they provide additional genetic resources for understanding Gcm functions and structure in Drosophila and other species.
contrast and sharpness with Adobe Photoshop CS4 (Adobe). Molecular Biology Genomic DNA was prepared from homozygous mutant embryos or from parental strain adults by the method of Gloor et al., 1993. Homozygous mutant embryos balanced over CyO, P{Ubi-GFP.S65T}PAD1 were recognized by the absence of GFP expression. gcm gene regions were PCR amplified using the following oligonucleotides flanking the coding region: 50 - AGT GACCGCGTGTGAACTTG-30 and 50 - TGGGAAATGTGAC TTAAAACTCTA-30 . PCR products were prepared for sequencing by treatment with Exonuclease I (New England Biolabs) and Shrimp Alkaline Phosphatase (USB), and both strands were sequenced (MacrogenUSA, Rockville, MD) using the above two oligonucleotides and the following oligonucleotides: 50 -TCTTCCTCCTGCCAAAA GTC-30 ; 50 -GACAGAAATTGCAATGGTCG-30 ; 50 -GTACTC CTACGATATGCTCC-30 ; 50 -GAGTTGTCCACTCCCGTATT30 ; and, 50 -TAATCCTCGGAACAGTAGCC-30 . Sequence assembly and alignments were performed using Lasergene software (DNASTAR).
MATERIALS AND METHODS ACKNOWLEDGMENTS Fly Stocks Wild type embryos were y1w67c23. CyO, P{UbiGFP.S65T}PAD1 balancer was used to recognize homozygous mutant embryos for sequencing. An isogenized b pr cn wxwxt bw was the parental strain from which gcmG78 had been induced was used as a reference strain to compare gcm sequences from the same genomic background. l(2)GA1019 is a mutant strain generated in the same EMS mutagenesis as gcm1308 (Seeger et al., 1993) and was used as a reference strain to compare gcm sequences from the same genomic background. Mutant lines described in this paper are freely available to the research community on request. Immunohistochemistry and Microscopy Horseradish peroxidase (HRP) immunohistochemistry and embryo dissection were done as previously described (Patel, 1994). Anti-Repo Monoclonal antibody MAb 8D12 (Alfonso and Jones, 2002) was used at a 1:5 dilution. Secondary goat anti-mouse IgG antibody conjugated to HRP (Jackson Immunoresearch) was used at a1 : 300 dilution. Secondary antibodies were detected using HRP/Diaminobenzidine (DAB) reaction. DAB reactions were enhanced to give a black color by addition of 0.067% NiCl2. Rat anti-Gcm antisera was used as previously described (Jones et al., 1995). Digital images were taken with an Axioskop 2 microscope and Axiocam HR camera (Carl Zeiss) with x40 Nomarski optics and x1.6 Optovar. Images were adjusted for brightness,
The author thanks Miyuki Yussa for her contribution in screening for mutations affecting glial cell patterning, Matthew Abeysekera and Denisha Parker for technical help, Guy Tear, and the Bloomington Stock Center for fly stocks. LITERATURE CITED Akiyama Y, Hosoya T, Poole AM, Hotta Y. 1996. The gcm-motif: A novel DNA-binding motif conserved in Drosophila and mammals. Proc Natl Acad Sci USA 93:14912–14916. Alfonso TB, Jones BW. 2002. Gcm2 promotes glial cell differentiation and is required with glial cells missing for macrophage development in Drosophila. Dev Biol 248:369–383. Anson-Cartwright L, Dawson K, Holmyard, D, Fisher SJ, Lazzarini RA, Cross, JC. 2000. The glial cells missing-1 protein is essential for branching morphogenesis in the chorioallantoic placenta. Nat Genet 25:311–314. Bernardoni R, Vivancos V, Giangrande A. 1997. glide/ gcm is expressed and required in the scavenger cell lineage. Dev Biol 191:118–130. Campbell G, Goring H, Lin T, Spana E, Andersson S, Doe CQ, Tomlinson A. 1994. RK2, a glial-specific homeodomain protein required for embryonic nerve cord condensation and viability in Drosophila. Development 120:2957–2966.
MISSENSE ALLELES OF glial cells missing
Chotard C, Leung W, Salecker I. 2005. glial cells missing and gcm2 cell autonomously regulate both glial and neuronal development in the visual system of Drosophila. Neuron 48:237–251. Cohen SX, Moulin M, Hashemolhosseini S, Kilian K, Wegner M, M€ uller CW. 2003. Structure of the GCM domain-DNA complex: A DNA-binding domain with a novel fold and mode of target site recognition. EMBO J 22:1835–1845. Freeman MR, Delrow J, Kim J, Johnson E, Doe CQ. 2003. Unwrapping glial biology: Gcm target genes regulating glial development, diversification, and function. Neuron 38:567–580. Granderath S, Bunse I, Klambt C. 2000. gcm and pointed synergistically control glial transcription of the Drosophila gene loco. Mech Dev 91:197–208. Gunther T, Chen ZF, Kim J, Priemel M, Rueger JM, Amling M, Moseley JM, Martin TJ, Anderson DJ and Karsenty G. 2000. Genetic ablation of parathyroid glands reveals another source of parathyroid hormone. Nature 406:199–203. Halter DA, Urban J, Rickert C, Ner, SS, Ito K, Travers AA, Technau GM. 1995. The homeobox gene repo is required for the differentiation and maintenance of glia function in the embryonic nervous system of Drosophila melanogaster. Development 121:317–332. Hitoshi S, Ishino Y, Kumar A, Jasmine S, Tanaka KF, Kondo T, Kato S, Hosoya T, Hotta Y, Ikenaka K. 2011. Mammalian Gcm genes induce Hes5 expression by active DNA demethylation and induce neural stem cells. Nat Neurosci 14:957–964. Hosoya T, Takizawa K, Nitta K. Hotta Y. 1995. glial cells missing: A binary switch between neuronal and glial determination in Drosophila. Cell 82:1025–1036. Ito K, Urban J, Technau GM. 1995. Distribution, classification and development of Drosophila glial cells during late embryogenesis. Roux Arch Dev Biol 204:284–307. Jones BW, Fetter RD, Tear G, Goodman CS. 1995. glial cells missing: A genetic switch that controls glial versus neuronal fate. Cell 82:1013–1023. Kim J, Jones BW, Zock C, Chen Z, Wang H, Goodman CS, Anderson DJ. 1998. Isolation and characterization of mammalian homologs of the Drosophila gene glial cells missing. Proc Natl Acad Sci USA 95: 12364–12369. Lebestky T, Chang T, Hartenstein V, Banerjee U. 2000. Specification of Drosophila hematopoietic lineage
869
by conserved transcription factors. Science 288: 146–149. Miller AA, Bernardoni R, Giangrande A. 1998. Positive autoregulation of the glial promoting factor glide/ gcm. EMBO J 17:6316–6326. Patel NH. 1994. Imaging neuronal subsets and other cell types in whole-mount Drosophila embryos and larvae using antibody probes. In: Goldstein LSB, Fyrberg EA, editors. Methods in cell biology, Vol. 44. San Diego: Academic Press. pp 445–487. Reifegerste R, Schreiber J, Gulland S, Ludemann A, Wegner M. 1999. mGCMa is a murine transcription factor that overrides cell fate decisions in Drosophila. Mech Dev 82:141–150. Schreiber J, Sock E. Wegner M. 1997. The regulator of early gliogenesis glial cells missing is a transcription factor with a novel type of DNA-binding domain. Proc Natl Acad Sci USA 94:4739–4744. Schreiber J, Enderich J, Wegner M. 1998. Structural requirements for DNA binding of GCM proteins. Nucleic Acids Res 26:2337–2343. Schreiber J, Riethmacher-Sonnenberg E, Riethmacher D, Tuerk EE, Enderich J, B€ osl MR, Wegner M. 2000. Placental failure in mice lacking the mammalian homolog of glial cells missing, GCMa. Mol Cell Biol 20: 2466–2474. Seeger M, Tear G, Ferres-Marco D, Goodman CS. 1993. Mutations affecting growth cone guidance in Drosophila: Genes necessary for guidance toward or away from the midline. Neuron 10:409–426. Soustelle L, Giangrande A. 2007. Novel gcm-dependent lineages in the postembryonic nervous system of Drosophila melanogaster. Dev Dyn 236:2101–2108. Soustelle L, Jacques C, Altenhein B, Technau GM, Volk T, Giangrande A. 2004. Terminal tendon cell differentiation requires the glide/gcm complex. Development 131:4521–4532. Vincent S, Vonesch JL, Giangrande A. 1996. Glide directs glial fate commitment and cell fate switch between neurones and glia. Development 122:131– 139. Wegner M, Riethmacher D. 2001 Chronicles of a switch hunt: gcm genes in development. Trends Genet 17: 286–290. Xiong WC, Okano H, Patel NH, Blendy JA, Montell C. 1994. repo encodes a glial-specific homeo domain protein required in the Drosophila nervous system. Genes Dev 8:981–994.
genesis 52:864–869 (2014)
LETTER
Characterization of Missense Alleles of the glial cells missing Gene of Drosophila Bradley W. Jones* Department of Biology, The University of Mississippi, 122 Shoemaker Hall, University, Mississippi Received 21 March 2014; Revised 2 July 2014; Accepted 3 July 2014
Summary: Glial cells missing (Gcm) is the primary regulator of glial cell fate in Drosophila. Gcm belongs to a small family of transcriptional regulators involved in fundamental developmental processes found in diverse animal phyla including vertebrates. Gcm proteins contain the highly conserved DNA-binding GCM domain, which recognizes an octamer DNA sequence. To date, studies in Drosophila have primarily relied on gcm alleles caused by P-element induced DNA deletions at the gcm locus, as well as a null allele caused by a single base pair substitution in the GCM domain that completely abolishes DNA binding. Here I characterize two hypomorphic missense alleles of gcm with intermediate glial cells missing phenotypes. In embryos homozygous for either of these gcm alleles the number of glial cells in the central nervous cystem (CNS) is reduced approximately in half. Both alleles have single amino acid changes in the GCM domain. These results suggest that Gcm protein activities in these mutant alleles have been attenuated such that they are operating at threshold levels, and trigger glial cell differentiation in neural precursors in the CNS in a stochastic fashion. These hypomorphic alleles provide additional genetic resources for understanding Gcm functions and structure in Drosophila and other species. genesis 52:864– C 2014 Wiley Periodicals, Inc. 869, 2014. V Key words: Drosophila; CNS; glia; gcm; missense mutations; hypomorphic allele
INTRODUCTION In Drosophila melanogaster the glial cells missing gene (gcm) is the primary regulator of glial cell determination (Hosoya et al., 1995; Jones, et al., 1995; Vincent et al., 1996). gcm encodes a transcription factor that is transiently expressed in all embryonic lateral glia
(excluding the midline glia). gcm mutant embryos lack nearly all lateral glial cells, and presumptive glia are transformed into neurons. Conversely, when gcm is ectopically expressed, presumptive neurons are transformed into glia. Thus, within the nervous system, gcm acts as a binary genetic switch, with Gcm-positive cells becoming glia and Gcm-negative cells becoming neurons (Hosoya et al., 1995; Jones, et al., 1995; Vincent et al., 1996). In addition to acting as a regulator of glial cell differentiation, gcm is also required for the proper differentiation of embryonic hemocytes (Alfonso and Jones, 2002; Bernardoni et al., 1997; Lebestky et al., 2000), tendon cells (Soustelle et al., 2004), and a subset of neurons in the adult brain (Chotard et al., 2005; Soustelle and Giangrande, 2007). Thus gcm controls different developmental programs according to cellular context. Gcm belongs to a small family of transcriptional regulators found in diverse animal phyla including vertebrates, and, where they have been studied, they are involved in fundamental developmental processes (reviewed in Wegner and Riethmacker, 2001). Significantly, mouse Gcm1/GCMa is required for the development of placental labyrinthine trophoblasts, (AnsonCartwright et al., 2000; Schrieber et al., 2000), mouse Gcm2/GCMb is a master regulator parathyroid gland development (Gunther et al., 2000), and both mouse Gcm1 and Gcm2 together regulate neural stem cell development (Hittoshi et al., 2011).
* Correspondence to: Dr. Bradley Jones, 122 Shoemaker Hall, University, MS 38677. E-mail: [email protected] Published online 12 July 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/dvg.22801
MISSENSE ALLELES OF glial cells missing
865
FIG. 1. Missense alleles of gcm show reduction in glial cell differentiation. (a–g) Dissected stage 16 embryos showing five adjacent segments of the CNS labeled with Repo monoclonal antibody with the following genotypes: (a) Wild type, (b) gcmDP1, (c) gcmG78, (d) gcm1308, (e) gcmG78/gcm1308, (f) gcmG78/gcmDP1, and (g) gcm1308/gcmDP1. Anterior is up. Scale bar, 20 mm. In the gcm null mutant embryo (b) glial cells are nearly absent as shown by Repo expression. In the missense alleles gcmG78 (c), gcm1308, (d) and in the transheterozygote gcmG78/ gcm1308 (f) glial cell differentiation is reduced by half. (h) A gcm gene diagram is shown with the regions corresponding to the Gcm DNA binding domain, the remainder of the coding region, and the noncoding sequence indicated in black, gray, and white, respectively. The encoded amino acids affected by the analyzed missense gcm mutations are indicated.
Gcm family proteins share a highly conserved N-terminal 150 amino acid DNA binding GCM domain with unique structure (Cohen et al., 2003). Drosophila Gcm binds with high affinity to the octameric consensus sequence AT(G/A)CGGG(T/C) found repeated in the regulatory regions of glial-specific genes (Akiyama et al., 1996; Freeman et al., 2003; Granderath et al., 2000; Schreiber et al., 1997). Its mammalian counterparts also bind to the same octamer sequence (Cohen
et al., 2003; Shchrieber et al., 1998), and expression of murine and rat Gcm1 in Drosophila promotes glial cell differentiation, indicating conserved biochemical properties (Kim et al., 1998; Reifegerste et al., 1999). In summary Gcm proteins act as essential tissue-specific transcriptional regulators in several tissues that share the common action of binding to a highly conserved octamer sequence motif, activating a variety of different genes in different contexts.
866
JONES
FIG. 2. Locations of three Drosophila missense mutations in the GCM domain. Alignment of the Gcm domains from Drosophila melanogaster (dGcm, dGcm2), mouse (mGcm1, mGcm2) and zebrafish (zfGcm2). Conserved amino acids are shaded. Secondary structural elements derived from the crystal structure mGcm1 as described in Cohen et al. 2003 are shown above the alignment. Blue lines S1 through S7 represent strands in the two ß-sheets, and red bars H1 through H3 represent three small helical segments. 1 symbols represent DNA contacting amino acids; star symbols represent cysteine and histidines involved in coordinating two Zn ions, with green stars coordinating the first Zn ion and black stars cording the second Zn ion.
To date, studies in Drosophila have relied on loss-offunction gcm P-element excisions alleles that cause DNA deletions at the gcm locus (Hosoya et al., 1995; Jones et al., 1995; Vincent et al., 1996). Only one identified allele gcmN7-4 represents a point mutation in the gcm gene, where a G to C substitution in codon 93 of gcm (Fig. 2) changes a highly conserved cysteine to a serine in the Gcm domain that abolishes DNA binding and leads to complete loss-of-function (Miller et al., 1998). In this article I characterize two hypomorphic missense alleles of gcm with intermediate glial cells missing phenotypes. In embryos homozygous for either of these gcm alleles the number of glial cells in the central nervous cystem (CNS) is reduced approximately in half. Both alleles have point mutations that cause amino acid changes in the highly conserved GCM domain. These results suggest that the biochemical activities of Gcm proteins in these mutants have been attenuated such that they are operating at threshold levels, and trigger
glial cell differentiation in only half of neural precursors in the CNS in a stochastic fashion. RESULTS AND DISCUSSION gcm1308 is an ethyl methanesulfonate (EMS) induced allele of gcm that was isolated in a systematic screen for mutations that disrupt the organization of CNS axon pathways in Drosophila (Jones et al., 1995; Seeger et al., 1993). gcmG78 was isolated in an EMS screen for genes that disturb the pattern of Reversed Polarity (Repo) protein. Repo is expressed constitutively in the nuclei of all lateral glial cells can therefore be used as a glial cell marker (Campbell et al., 1994; Halter et al., 1995; Xiong et al., 1994). The pattern of glial cells as revealed by anti-Repo antibody show that gcmG78 and gcm1308 mutant embryos have similar phenotypes; both have a reduced number of glial cells in the CNS (Fig. 1c,d) compared to wild type (Fig. 1a). The pattern of missing glial cells appears to be random in both
MISSENSE ALLELES OF glial cells missing
Table 1 Number of Glial Cells per Abdominal CNS Segment in Wild type and gcm Mutant Stage 16/17 Embryos Genotype wild type gcmDP1 gcmG78 gcm1308 gcmG78/gcm1308 gcmG78/gcmDP1 gcm1308/gcmDP1
Total glia Number of Average # glia % of counted segments per segment wild type 3195 56 57.05 100% 226 56 4.04 7% 1399 56 24.98 44% 1550 56 27.67 49% 1494 56 26.68 47% 981 56 17.51 31% 761 56 13.59 24%
mutants, with deficiencies in all CNS glial subtypes in each segment. By comparison embryos carrying the null allele gcmDP1 in which the gcm locus is deleted show a dramatic decrease in Repo-positive cells (Fig. 1b), with isolated single Repo-positive cells, as well as occasional small clusters in many segments. Transheterozygotes of the two alleles (gcmG78/ gcm1308, Fig. 1e) are similar in appearance to the homozygotes of each allele, and crossing each allele with the gcmDP1 null allele appears to achieve a further reduction in glial cell numbers with similar phenotypes (Fig. 1f,g). Interestingly, reducing the dosage by half for both gcmG78 and gcm1308 alleles in embryos transheterozygous with gcmDP1 affects the distribution of glial cells in a similar way, with few glial cells near the midline along the longitudinal axon tracts; instead, the glial cells are abnormally clustered along the lateral edges of the of the CNS (Fig. 1f,g), suggesting a change in behavior and/or identity of the glial cells. Taken together, these data suggest that that both alleles function in a similar manner. To assay the degree to which glial cells are missing in gcm1308, gcmG78, and in transheterozygous embryos I counted the number of Repo-positive cells in 7 adjacent abdominal CNS segments in eight individuals of each mutant phenotype (56 segments) at stages 16 and 17, as well as in wild type and gcmDP1 null mutant embryos, and obtained an average number of glial cells per abdominal segment (Table 1). In wild type embryos I counted an average of 57 Repo-positive glia per abdominal segment, which is in accordance with abdominal glial cell counts done by others when the midline glia are excluded (Ito et al., 1995). gcmG78 mutant embryos have an average of 25 Repo-positive glia per abdominal segment, which is 44% of wild type, or a 56% reduction in the number of glia. gcm1308 mutant embryos have an average of 28 Repo-positive glia per abdominal segment, which is 49% of wild type, or a 51% reduction in the number of glia. gcmG78/ gcm1308 transheterozygous embryos have an average of 27 Repo-positive glia per abdominal segment, which is 47% of wild type, or a 53% reduction in the number of glia. gcmG78/gcmDP1 transheterozygous embryos have an average of 16 Repo-positive glia per abdominal
867
segment, which is 31% of wild type, or a 69% reduction in the number of glia. gcm1308/gcmDP1 transheterozygous embryos have an average of 14 Repo-positive glia per abdominal segment, which is 24% of wild type, or a 76% reduction in the number of glia. By contrast in gcmDP1 null mutant embryos have an average of 4 Repo-positive glia per abdominal segment, which constitutes a 93% reduction in the number of glia. gcm1308 and gcmG78 alleles were induced by EMS treatment, suggesting the presence of point mutations. Genomic DNA from gcm1308 homozygous embryos and from homozygous embryos of the mutant strain l(2)GA1019 from the same EMS mutagenesis screen were sequenced at the gcm locus and compared. gcm1308 contains a single G to A nucleotide substitution within the 75th codon of the gcm open reading frame, changing a conserved glycine to aspartic acid (Figs. 1e and 2). Genomic DNA from gcmG78 homozygous embryos and from the parental strain of the EMS mutagenesis screen were sequenced at the gcm locus and compared. gcmG78 contains a single T to A nucleotide substitution within the 53rd codon of the gcm open reading frame, changing a conserved tryptophan to arginine (Figs. 1e and 2). Both of these mutations change conserved amino acid residues in the GCM domain (Fig 2). The crystal structure of the murine Gcm1 (also known as GCMa) GCM domain-DNA complex has been resolved (Cohen et al., 2003). The GCM domain is complicated, consisting of two subdomains, a large and a small, comprised of five- and three-stranded ß-sheets respectively, with three helical segments also incorporated (Cohen et al., 2003). These two subdomains are held together by two Zn ions bound to conserved cysteine and histidine residues. The importance of Zn ions in coordination in GCM domain structure is demonstrated by mutant allele gcmN7-4 in which a cysteine that binds Zn is changed to a serine; this change completely abolishes DNA binding activity and leads to a complete loss-of-function (Miller et al., 1998, Fig. 2). DNA recognition is largely accomplished by the large subdomain that consists of a five-stranded ß-sheet, comprised of strands S1, S2, S3, S6, and S7 shown in Fig. 2. This five-stranded ß-sheet protrudes into the major groove perpendicular to the DNA axis. The large subdomain also consists of the N terminal part of the GCM domain packed on one side of the ß-sheet and the helix H1 packed on the other. Both mutations described here, gcm1308 and gcmG78, change hydrophobic amino acids into charged amino acids within this large subdomain and are likely to subtly affect its structure and perhaps the affinity of DNA binding. While both alleles have significant changes in the amino acid sequence in the conserved GCM domain, one cannot rule out the possibility that additional EMS induced mutations exist within gcm cis-regulatory
868
JONES
sequences that could contribute to the observed phenotypes by attenuating patterns or levels of gcm expression. However, I have looked at the expression pattern of Gcm protein using anti-Gcm antibodies in homozygous embryos of both alleles and find the early expression patterns to be normal at Stage 11 in the glial precursor cells that give rise to the longitudinal glia. I also find Gcm expressed at normal levels in both alleles in neural and glial cells through Stage 14, although in a disorganized fashion (data not shown). Both gcm alleles reduce glial cell differentiation by approximately half. The glial cells that do develop do so in an apparent random pattern. The results presented here suggest that the biochemical activities of Gcm proteins in these mutants have been attenuated such that they are operating at threshold levels, and trigger glial cell differentiation in a stochastic fashion. These mutants are the first hypomorphic alleles of gcm to be characterized that are likely to be caused by point mutations in the GCM domain; they provide additional genetic resources for understanding Gcm functions and structure in Drosophila and other species.
contrast and sharpness with Adobe Photoshop CS4 (Adobe). Molecular Biology Genomic DNA was prepared from homozygous mutant embryos or from parental strain adults by the method of Gloor et al., 1993. Homozygous mutant embryos balanced over CyO, P{Ubi-GFP.S65T}PAD1 were recognized by the absence of GFP expression. gcm gene regions were PCR amplified using the following oligonucleotides flanking the coding region: 50 - AGT GACCGCGTGTGAACTTG-30 and 50 - TGGGAAATGTGAC TTAAAACTCTA-30 . PCR products were prepared for sequencing by treatment with Exonuclease I (New England Biolabs) and Shrimp Alkaline Phosphatase (USB), and both strands were sequenced (MacrogenUSA, Rockville, MD) using the above two oligonucleotides and the following oligonucleotides: 50 -TCTTCCTCCTGCCAAAA GTC-30 ; 50 -GACAGAAATTGCAATGGTCG-30 ; 50 -GTACTC CTACGATATGCTCC-30 ; 50 -GAGTTGTCCACTCCCGTATT30 ; and, 50 -TAATCCTCGGAACAGTAGCC-30 . Sequence assembly and alignments were performed using Lasergene software (DNASTAR).
MATERIALS AND METHODS ACKNOWLEDGMENTS Fly Stocks Wild type embryos were y1w67c23. CyO, P{UbiGFP.S65T}PAD1 balancer was used to recognize homozygous mutant embryos for sequencing. An isogenized b pr cn wxwxt bw was the parental strain from which gcmG78 had been induced was used as a reference strain to compare gcm sequences from the same genomic background. l(2)GA1019 is a mutant strain generated in the same EMS mutagenesis as gcm1308 (Seeger et al., 1993) and was used as a reference strain to compare gcm sequences from the same genomic background. Mutant lines described in this paper are freely available to the research community on request. Immunohistochemistry and Microscopy Horseradish peroxidase (HRP) immunohistochemistry and embryo dissection were done as previously described (Patel, 1994). Anti-Repo Monoclonal antibody MAb 8D12 (Alfonso and Jones, 2002) was used at a 1:5 dilution. Secondary goat anti-mouse IgG antibody conjugated to HRP (Jackson Immunoresearch) was used at a1 : 300 dilution. Secondary antibodies were detected using HRP/Diaminobenzidine (DAB) reaction. DAB reactions were enhanced to give a black color by addition of 0.067% NiCl2. Rat anti-Gcm antisera was used as previously described (Jones et al., 1995). Digital images were taken with an Axioskop 2 microscope and Axiocam HR camera (Carl Zeiss) with x40 Nomarski optics and x1.6 Optovar. Images were adjusted for brightness,
The author thanks Miyuki Yussa for her contribution in screening for mutations affecting glial cell patterning, Matthew Abeysekera and Denisha Parker for technical help, Guy Tear, and the Bloomington Stock Center for fly stocks. LITERATURE CITED Akiyama Y, Hosoya T, Poole AM, Hotta Y. 1996. The gcm-motif: A novel DNA-binding motif conserved in Drosophila and mammals. Proc Natl Acad Sci USA 93:14912–14916. Alfonso TB, Jones BW. 2002. Gcm2 promotes glial cell differentiation and is required with glial cells missing for macrophage development in Drosophila. Dev Biol 248:369–383. Anson-Cartwright L, Dawson K, Holmyard, D, Fisher SJ, Lazzarini RA, Cross, JC. 2000. The glial cells missing-1 protein is essential for branching morphogenesis in the chorioallantoic placenta. Nat Genet 25:311–314. Bernardoni R, Vivancos V, Giangrande A. 1997. glide/ gcm is expressed and required in the scavenger cell lineage. Dev Biol 191:118–130. Campbell G, Goring H, Lin T, Spana E, Andersson S, Doe CQ, Tomlinson A. 1994. RK2, a glial-specific homeodomain protein required for embryonic nerve cord condensation and viability in Drosophila. Development 120:2957–2966.
MISSENSE ALLELES OF glial cells missing
Chotard C, Leung W, Salecker I. 2005. glial cells missing and gcm2 cell autonomously regulate both glial and neuronal development in the visual system of Drosophila. Neuron 48:237–251. Cohen SX, Moulin M, Hashemolhosseini S, Kilian K, Wegner M, M€ uller CW. 2003. Structure of the GCM domain-DNA complex: A DNA-binding domain with a novel fold and mode of target site recognition. EMBO J 22:1835–1845. Freeman MR, Delrow J, Kim J, Johnson E, Doe CQ. 2003. Unwrapping glial biology: Gcm target genes regulating glial development, diversification, and function. Neuron 38:567–580. Granderath S, Bunse I, Klambt C. 2000. gcm and pointed synergistically control glial transcription of the Drosophila gene loco. Mech Dev 91:197–208. Gunther T, Chen ZF, Kim J, Priemel M, Rueger JM, Amling M, Moseley JM, Martin TJ, Anderson DJ and Karsenty G. 2000. Genetic ablation of parathyroid glands reveals another source of parathyroid hormone. Nature 406:199–203. Halter DA, Urban J, Rickert C, Ner, SS, Ito K, Travers AA, Technau GM. 1995. The homeobox gene repo is required for the differentiation and maintenance of glia function in the embryonic nervous system of Drosophila melanogaster. Development 121:317–332. Hitoshi S, Ishino Y, Kumar A, Jasmine S, Tanaka KF, Kondo T, Kato S, Hosoya T, Hotta Y, Ikenaka K. 2011. Mammalian Gcm genes induce Hes5 expression by active DNA demethylation and induce neural stem cells. Nat Neurosci 14:957–964. Hosoya T, Takizawa K, Nitta K. Hotta Y. 1995. glial cells missing: A binary switch between neuronal and glial determination in Drosophila. Cell 82:1025–1036. Ito K, Urban J, Technau GM. 1995. Distribution, classification and development of Drosophila glial cells during late embryogenesis. Roux Arch Dev Biol 204:284–307. Jones BW, Fetter RD, Tear G, Goodman CS. 1995. glial cells missing: A genetic switch that controls glial versus neuronal fate. Cell 82:1013–1023. Kim J, Jones BW, Zock C, Chen Z, Wang H, Goodman CS, Anderson DJ. 1998. Isolation and characterization of mammalian homologs of the Drosophila gene glial cells missing. Proc Natl Acad Sci USA 95: 12364–12369. Lebestky T, Chang T, Hartenstein V, Banerjee U. 2000. Specification of Drosophila hematopoietic lineage
869
by conserved transcription factors. Science 288: 146–149. Miller AA, Bernardoni R, Giangrande A. 1998. Positive autoregulation of the glial promoting factor glide/ gcm. EMBO J 17:6316–6326. Patel NH. 1994. Imaging neuronal subsets and other cell types in whole-mount Drosophila embryos and larvae using antibody probes. In: Goldstein LSB, Fyrberg EA, editors. Methods in cell biology, Vol. 44. San Diego: Academic Press. pp 445–487. Reifegerste R, Schreiber J, Gulland S, Ludemann A, Wegner M. 1999. mGCMa is a murine transcription factor that overrides cell fate decisions in Drosophila. Mech Dev 82:141–150. Schreiber J, Sock E. Wegner M. 1997. The regulator of early gliogenesis glial cells missing is a transcription factor with a novel type of DNA-binding domain. Proc Natl Acad Sci USA 94:4739–4744. Schreiber J, Enderich J, Wegner M. 1998. Structural requirements for DNA binding of GCM proteins. Nucleic Acids Res 26:2337–2343. Schreiber J, Riethmacher-Sonnenberg E, Riethmacher D, Tuerk EE, Enderich J, B€ osl MR, Wegner M. 2000. Placental failure in mice lacking the mammalian homolog of glial cells missing, GCMa. Mol Cell Biol 20: 2466–2474. Seeger M, Tear G, Ferres-Marco D, Goodman CS. 1993. Mutations affecting growth cone guidance in Drosophila: Genes necessary for guidance toward or away from the midline. Neuron 10:409–426. Soustelle L, Giangrande A. 2007. Novel gcm-dependent lineages in the postembryonic nervous system of Drosophila melanogaster. Dev Dyn 236:2101–2108. Soustelle L, Jacques C, Altenhein B, Technau GM, Volk T, Giangrande A. 2004. Terminal tendon cell differentiation requires the glide/gcm complex. Development 131:4521–4532. Vincent S, Vonesch JL, Giangrande A. 1996. Glide directs glial fate commitment and cell fate switch between neurones and glia. Development 122:131– 139. Wegner M, Riethmacher D. 2001 Chronicles of a switch hunt: gcm genes in development. Trends Genet 17: 286–290. Xiong WC, Okano H, Patel NH, Blendy JA, Montell C. 1994. repo encodes a glial-specific homeo domain protein required in the Drosophila nervous system. Genes Dev 8:981–994.
