Isolation and characterization of mammalian homologs of the Drosophila gene glial cells missing

Abstract

The glial cells missing (gcm) gene in Drosophila encodes a transcription factor that determines the choice between glial and neuronal fates. We report here the isolation of two mammalian gcm homologs, Gcm1 and Gcm2, and the characterization of their expression patterns during embryonic development. Although Gcm2 is expressed in neural tissues at a low level, the major sites of expression for both of the mammalian genes are nonneural, suggesting that the functions of the mammalian homologs have diverged and diversified. However, when expressed ectopically, Gcm1 can substitute functionally for Drosophila gcm by transforming presumptive neurons into glia. Thus, certain biochemical properties, although not the specificity of the tissue in which the gene is expressed, have been conserved through the evolution of the Gcm gene family.

Figure 1

(A) Comparison of rGCM1 and mGCM1. The deduced amino acid sequence of rGCM1 is aligned with that of mGCM1 in the database (GenBank accession no. U59876). Conserved residues are boxed. The extent of the gcm motif is indicated by arrowheads. (B) Comparison of the strain-specific mGCM2 sequences. The sequence for 129SvEv GCM2 is based on the partial cDNA sequence proposed from the genomic DNA sequence. For C57BL/6, the sequence is based on a full-length cDNA clone. D88611 (GenBank accession no. D88611) represents the published BALB/c GCMb sequence. Missense alterations found in C57BL/6 and BALB/c sequences with respect to residues in 129SvEv are boxed. (C) Comparison of mGCM2 and human GCM2 (hGCM2; GenBank accession no. AA782779). Conserved residues are boxed.

Figure 2

(A and B) Expression of rGcm1 in rat E14.5 placenta. The section was hybridized with an antisense cDNA probe for rGcm1. A positive region in A is shown in enlarged form in B. (C) Sagittal section of the pharyngeal region of an E16.5 mouse showing mGcm2 expression in the parathyroid tissue. (D) The adjacent section was positive for parathyroid hormone gene transcript. (Scale bars: 500 μm for A and 100 μm for C.)

Figure 3

RT-PCR analyses of the expression of Gcm genes. Agarose-gel electrophoresis of the PCR products is shown. Oligo(dT) primed cDNA derived from mouse tissues were amplified with gene-specific primers. Actin cDNA was amplified as the control product. Oligonucleotide primers that span exon–intron junctions were used so as to be able to distinguish spliced messages from genomic DNA contamination. In addition, for each of the PCRs, a control reaction that used a cDNA preparation without reverse transcription was performed and shown to generate no product (data not shown).

Figure 4

Panneural expression of rGCM1 promotes glial-cell differentiation in Drosophila. Photomicrographs of the central nervous system (CNS) in stage 16 embryos showing four adjacent segmental neuromeres as stained with anti-REPO antisera. (A) Wild-type embryo. Anti-REPO stains the nuclei of all glial cells except midline glia. (B) gcm^ΔP1 loss-of-function mutant embryo. Virtually no cells express REPO. (C) Panneural expression of Drosophila GCM. In sca-Gal4/UAS-gcm; UAS-gcm/+ embryos (expressing two copies of UAS-gcm), panneural expression of GCM causes nearly all CNS cells to express REPO. (D) Panneural expression of rGCM1. In UAS-rGcm1/+; sca-Gal4/UAS-rGcm1 embryos (expressing two copies of UAS-rGcm1), panneural expression of rGCM1 also causes an increase in REPO expression in the CNS. Anterior is up. (Scale bar: 10 μm.)

Figure 5

rGCM1 rescues glial differentiation in gcm loss-of-function mutant embryos. Photomicrographs of the CNS in stage 16 embryos showing four adjacent segmental neuromeres as stained with anti-REPO antisera. (A) Panneural expression of Drosophila GCM in a gcm loss-of-function mutant embryo (UAS-gcm gcm^ΔP1/gcm^ΔP1 sca-Gal4) promotes REPO expression and glial-cell development. (B) Panneural expression of rGCM1 in a gcm loss-of-function mutant embryo (UAS-rGcm1 gcm^ΔP1/gcm^ΔP1 sca-Gal4) also promotes REPO expression and glial-cell development. Anterior is up. (Scale bar: 10 μm.)