BACKGROUND
oligodendrocyte lineage genes olig <dig> and olig <dig> encode basic helix-loop-helix  transcription factors.
olig <dig> is a master regulator of oligodendrocyte  lineage development  <cit> .
olig <dig> is also required for generation of some neurons, notably spinal motor neurons   <cit> .
mns are generated from neural stem/progenitor cells in a specialized region of the ventral ventricular zone  of the spinal cord known as pmn.
around embryonic day  <dig>  in mice, the same group of progenitors stops producing mns and switches to production of ol precursors , which proliferate and migrate away from the vz in all directions before associating with axons and differentiating into myelin-forming ols .
olig <dig> and olig <dig>  are involved at multiple stages of this developmental sequence.
olig <dig> is also required for specifying oligodendrocytes and some types of neurons in the brain – some ventrally-derived interneurons and cholinergic projection neurons in the forebrain, for example  <cit> .
olig <dig> can compensate for olig <dig> in some regions including the hindbrain and parts of the forebrain, because ops still form there in olig <dig> null mice but not in olig1/olig <dig> double nulls  <cit> .
olig <dig> also plays a later role in the differentiation of ops into myelinating ols, although there is disagreement about whether there is an absolute requirement for olig <dig> during normal development  <cit> .
the original olig <dig> null allele, made by inserting a cre-frt-pgk-neo-frt cassette into the mouse olig <dig> locus  <cit>  caused a delay in the appearance of differentiated ols but no long-term myelin deficit.
however, a subsequent study by xin et al.
<cit> , who crossed the original line with flp-expressing mice to remove the pgk-neo selection cassette , found a severe myelination defect leading to early postnatal lethality.
apart from this contested role in ol lineage development, olig <dig> is known to be required for remyelination of experimentally-induced demyelinated lesions in the mouse spinal cord  <cit> .
given the central role of the oligs in ol lineage development, it is important to try to settle the controversy over the developmental requirement for olig <dig>  this might have added significance because the olig <dig> null locus  <cit>  contains an expressed cre cassette under olig <dig> transcriptional control and these olig <dig> mice are being used to delete floxed genes specifically in ol lineage cells.
for example, conditional deletion of dicer <dig>  using olig <dig> <cit>  caused severe impairment of myelination and death around p <dig>  <cit> , whereas analogous experiments using olig <dig> or cnp resulted in only slightly delayed myelination with full recovery by p <dig>  <cit> .
in another example, constitutively activating the wnt signaling pathway by conditional deletion of exon  <dig> of β-catenin <cit>  using olig <dig> completely prevented ol lineage specification, judging by the complete absence of op markers such as pdgfra  <cit> , whereas similar experiments using olig <dig> did not affect op specification but only their subsequent differentiation into ols  <cit> .
while there might be a simple explanation for these differences, such as earlier or more complete recombination by olig <dig> than by olig <dig>  the possibility remains that the olig <dig> null allele generated by xin et al.
<cit>  might carry some additional, unidentified defect that can amplify the phenotype of other deleterious mutations.
to attempt to throw some light on these matters we undertook a study of two independent olig <dig> null lines generated in our own laboratory.
we found that loss of olig <dig> causes a transient delay in ol development and myelination.
we quantified olig <dig> mrna in our olig <dig> mutant mice and found no increase relative to wild type controls.
the mild phenotype we observe is therefore likely to be a genuine consequence of olig <dig> loss, not moderated by cis regulatory effects on olig <dig>
methods
mice
mice were maintained on a  <dig> hour light–dark cycle.
for timed mating, male and female mice were caged together overnight  and  <dig> noon the following day was designated embryonic day  <dig>  .
all mouse work was approved by the home office of the uk government, and conformed to the animals  act  <dig>  new olig <dig> null lines, olig <dig> and olig , olig <dig> were generated as described previously  <cit>  .
embryonic stem  cell targeting
we generated a new olig <dig> line by es cell targeting.
briefly, olig <dig> targeting vector  was linearized and electroporated into r <dig> es cells   <cit> .
after  <dig> days’ selection in 150 μg/ml g <dig> ,  <dig> colonies were picked and expanded in 96-well plates.
targeted es clones were identified by southern blotting using a 700 bp ncoi—ecori fragment as probe .
positive es clones were confirmed by southern blotting using a 200 bp psti—ncoi probe .
five correctly targeted es cell clones were expanded for karyotyping; two clones with normal karyotype were used for c57/b <dig> blastocyst injection to produce chimeric mice.
male chimeras were bred to c57/b <dig> females to produce olig <dig> heterozygotes.
tissue preparation and histochemistry
embryonic and postnatal spinal cords or brains were immersion-fixed in 4%  paraformaldehyde in phosphate-buffered saline  overnight at 4°c.
the tissue was cryo-protected overnight at 4°c in 20%  sucrose in pbs.
tissue was embedded in oct compound , rapidly frozen on dry ice/isopentane and stored at -80°c.
tissue was cryo-sectioned  in a bright cryotome and sections collected on superfrost plus slides.
sections were treated with blocking solution  at 20-25°c for one hour before immuno-labeling.
primary antibodies were anti-sox <dig>  and anti-olig <dig> .
secondary antibodies were alexa fluor  <dig> conjugated goat anti-rabbit and cy3-conjugated goat anti-guinea pig igg .
sections were counterstained with hoechst  <dig> dye , for  <dig> minutes at 20-25°c after the secondary antibody and mounted under coverslips in fluorescence mounting medium .
our fluorescence in situ hybridization procedure has been described before; detailed protocols are available at http://www.ucl.ac.uk/~ucbzwdr/richardson.htm.
briefly, digoxigenin -labelled rna probes were transcribed in vitro from cloned cdnas of mbp or plp.
after hybridization, the dig signal was detected using horseradish peroxidase -conjugated anti-dig  followed by developing in fluorescein-tyramide reagent .
quantitative pcr
quantitative pcr  was performed using forebrain and spinal cord tissue collected from olig <dig> null mice and control littermates that carried either one or two endogenous copies of olig <dig> at embryonic day  <dig>   and/or e <dig> .
the tissue was homogenized in the presence of trizol reagent , and total rna was purified and used for cdna synthesis following the manufacturer’s instructions.
oligonucleotides 5′ att gta caa aac ggc cac aa 3′ and 5′ agt gct ctg cgt ctc gtc ta 3′ were used for olig <dig> cdna amplification.
oligonucleotides 5′ aca act ttg gca ttg tgg aa 3′ and 5′ gat gca ggg atg atg ttc tg 3′ were used to amplify gapdh as an internal control.
qpcr values were calculated using the relative standard curve method.
at least three embryos of each genotype were analyzed at each age.
mouse embryonic fibroblast  culture and western blotting
mouse embryos  were placed in pbs  and the head, vertebral column, dorsal root ganglia, and inner organs were removed.
the remaining tissue was digested in  <dig> %  trypsin, finely minced with a razor blade and incubated at 37°c for  <dig> minutes to make a single-cell suspension.
cells were then plated in 35 mm dishes coated with  <dig> %  gelatin and grown at 37°c in 5%  co <dig> in mef medium .
a plasmid encoding cre under the control of the pgk promoter  was used for transfection with fugene  <dig> .
proteins from transfected mefs and mouse spinal cord tissue were separated by sds-page and transferred to polyvinylidene difluoride membranes.
rabbit anti-myc antibody was purchased from abcam and used at a 1: <dig>  dilution.
protein bands were visualized by chemi-luminescence .
RESULTS
generation of new olig <dig> null mouse lines
to try to resolve the discrepancy between the reported phenotypes of two different olig <dig> null mouse lines  <cit>  we generated two new olig <dig> null strains, using different approaches.
for one, we replaced the olig <dig> open reading frame  with a dna fragment including an inverted phosphoglycerate kinase promoter -neomycin resistance cassette  flanked by loxp sites in mouse embryonic stem  cells, line r <dig>  <cit>  .
we refer to this line as olig <dig>  for purposes unrelated to the work described here, the modified locus also included a myc-tagged notch intracellular domain  coding sequence downstream of the floxed pgk-neo cassette; in the absence of cre recombinase this nicd cassette is not expressed  and is phenotypically neutral.
targeted clones were identified by southern blot analysis of genomic dna using a 700 bp ncoi–ecori fragment as a 5′ probe .
correct targeting was confirmed using a 200 bp psti-ncoi fragment as a 3′ probe .
one karyotypically normal es cell line was selected for blastocyst injection and germline transmission.
our second olig <dig> ko was generated by crossing olig1/olig <dig> double-null mice  <cit>  with a phage artificial chromosome  transgenic line that contains a single copy of mouse olig <dig> <cit>  .
we refer to this line as olig , olig <dig>  we confirmed that this line does not express olig <dig> protein .
lack of compensatory up-regulation of olig <dig> in olig1-null mice
the olig <dig> and olig <dig> genes are located about 40 kb apart on mouse chromosome  <dig> and there is a large degree of overlap in their expression patterns in vivo  <cit> .
xin et al.
<cit>  suggested that the pgk-neo cassette introduced by lu et al.
<cit>  to disrupt the olig <dig> orf might have exerted a cis-activating effect on the neighbouring olig <dig> locus, resulting in over-expression of olig <dig> which compensated for loss of olig <dig>  since our own olig <dig> mice also contain a pgk-neo cassette at the olig <dig> locus , we compared olig <dig> mrna levels in our olig <dig> mice and olig <dig> controls.
we collected forebrain tissue at two embryonic stages  and quantified olig <dig> transcripts by pcr, using total cellular rna as substrate.
we could not detect a significant difference in the brain or spinal cord between olig <dig> and olig <dig>  indicating that olig <dig> was not mis-regulated by the pgk-neo cassette at the olig <dig> locus in our mice .
note that although two pgk-neo cassettes are present at the disrupted olig1/olig <dig> locus in our olig, olig <dig>  mice  they are almost certainly physically remote from the randomly-integrated olig <dig> pac transgene and therefore are not expected to impose cis-regulation on olig <dig>
oligodendrocyte precursors are specified normally in olig <dig> null cns
we analyzed the expression of both platelet-derived growth factor receptor-alpha , a marker of ops, and sox <dig>  which marks all stages of the ol lineage, by immunofluorescence microscopy of e <dig>  spinal cord and p <dig> forebrain sections.
neither pdgfra nor sox <dig> expression were noticeably altered in our two olig <dig> null lines, relative to olig <dig> controls .
this is as expected, given that olig <dig> protein does not appear until after op specification  <cit> , and is consistent with the phenotypes of the two previously-described olig <dig> null lines  <cit> .
ol differentiation is delayed in olig <dig> null spinal cord
to investigate ol differentiation in our two new olig <dig> null lines, we visualized mrnas encoding mature ol markers myelin basic protein  and myelin proteolipid protein  by in situ hybridization.
at e <dig> , mbp and plp transcripts were absent from spinal cord in both olig <dig> null lines, in contrast to littermate controls that carried one good copy of endogenous olig <dig> .
at e <dig> , mbp and plp transcripts were present but in lower numbers of cells relative to olig <dig> 0heterozygotes ; by postnatal day  <dig> , there were normal numbers of mbp and plp-positive cells in the olig <dig> null spinal cord .
these results indicate that olig <dig> is involved in, but is not critically important for ol differentiation in the developing spinal cord, consistent with the original study by lu et al.
<cit> .
ol differentiation in mouse forebrain does not begin until after birth  <cit> .
on forebrain sections, few mbp and/or plp positive cells were detectable by fluorescence in situ hybridization at p <dig> .
at p <dig>  both olig <dig> null lines appeared to have normal numbers of mbp and plp positive cells in both the corpus callosum and cortex compared to control mice .
discussion
we generated two new olig <dig> null mouse lines by different routes -one by homologous recombination in es cells followed by blastocyst injection, and the other by transgenic rescue of a previously generated olig1/olig <dig> double-null line  <cit>  by pronuclear injection of an olig <dig> pac.
neither of the olig <dig> null lines showed any evidence of prenatal lethality and both lines lived and reproduced normally.
there was a transient delay in the production of differentiated ols in the spinal cords of both our olig <dig> null lines, as originally reported by lu et al.
<cit>  but in contrast to xin et al.
<cit> , who reported a severe myelination block that resulted in death around the third postnatal week.
xin et al.
<cit>  put the discrepancy down to the fact that the original olig <dig> null allele retained a pgk-neo cassette, speculating that the presence of this highly-transcribed element might have caused compensatory up-regulation of the neighbouring olig <dig> gene.
xin et al.
<cit>  removed the pgk-neo cassette  by crossing the original lu et al.
<cit>  line with flp-expressing mice.
however, they did not quantify olig <dig> expression in either of the olig <dig> mutants.
a cis-acting regulatory effect of pgk-neo has been implied in previous studies.
for example, the initially reported lethal phenotype of a germ line surf <dig> deletion  <cit>  was later attributed to the effect of pgk-neo on expression of unidentified genes near the surf <dig> locus, after a second surf <dig> knockout line lacking the pgk-neo cassette was found to be unusually long-lived  <cit> .
another example is the germ line knockout of the zinc finger transcription factor zfp <dig>  which was initially reported to be embryonic-lethal  <cit> .
subsequently, an independent line was found to survive after birth, developing a severe dysmyelinating phenotype and dying around p <dig>  <cit> .
one potential explanation for the difference was that the embryonic-lethal allele contained an expressed neo selection cassette.
we tested the hypothesis that the mild phenotype of our olig <dig> mice might have been due to compensatory up-regulation of the adjacent olig <dig> gene by pgk-neo, but found no evidence for this.
our data are consistent with a previous study by samanta et al.
<cit>  who found no evidence for up-regulation of olig <dig> when they used the olig <dig> line of lu et al.
<cit>   for conditional deletion of bone morphogenetic protein receptor-1a .
taken together, the data indicate that the presence or absence of pgk-neo cannot easily explain the dramatically different developmental phenotypes of different olig <dig> null mice.
different phenotypic outcomes for the same gene deletion can sometimes result from differences in the genetic backgrounds of the mice.
for example, the effect of knocking out nogo-a, a membrane protein of the adult myelin sheath and an inhibitor of neurite growth and axon regeneration, has a much larger effect on neurite regeneration ability in the 129x1/svj background than in the c57bl/6 j  background  <cit> .
our olig <dig> line was generated using r <dig> es cells .
homozygous nulls were maintained in a 129/c <dig> mixed background for many  generations with no sign of lethality.
they are now maintained on a 129/c57/cba background, also with no sign of lethality.
the olig <dig> null of lu et al.
<cit>  was made using j <dig> es cells  and crossed onto c <dig> for analysis.
the background of our olig,olig <dig> line is mixed c57/cba and these mice also display a mild phenotype.
the line displaying the contradictory lethal phenotype made by xin et al.
<cit>  was a modification of lu et al.’s  <cit>  line, maintained in a mixed 129/c <dig> background.
altogether, there is no compelling reason to think that genetic background underlies the differing severity of olig <dig> disruption in different lines.
another possible reason for the divergent phenotypes reported by lu et al.
<cit>  and xin et al.
<cit>  might lie in the way in which their mouse lines were generated.
xin et al.
<cit>  made their line by crossing the mice made previously by lu et al.
<cit>  with a line that expresses flp recombinase ubiquitously, in order to effect germ line excision of the frt-flanked pgk-neo cassette.
given that olig <dig> and olig <dig> lie close to each other on the chromosome  and share significant sequence homologies  <cit> , it is conceivable that an unintended recombination event might have taken place, altering the olig locus in some way that affects olig <dig> expression or structure in addition to disrupting olig <dig>
arnett et al.
<cit>  previously showed that the olig <dig> null line of lu et al.
<cit>  inefficiently remyelinates demyelinated lesions produced either by focal injection of lysolecithin or by systemic administration of cuprizone, despite the nearly normal developmental time course of myelination of these mice  <cit> .
this implied that olig <dig> and olig <dig> have complementary roles in myelin development and repair, respectively.
we have no reason to question this conclusion and have not tested the remyelination abilities of our new olig <dig> null mice.
ol differentiation is subject to two-tier transcriptional regulation: 1) epigenetic repression of transcriptional inhibitors and 2) direct transcriptional activation of myelin genes  <cit> .
transcription factors olig <dig>  <cit> , sox <dig>  <cit> , mrf  <cit>  and zfp <dig>  <cit>  are critical for ol differentiation and/or myelination.
ascl <dig> and nkx <dig>  also play important roles; germ line knockout of either nkx <dig>  or ascl <dig> leads to decreased expression of myelin genes in neonatal mice, suggesting that both genes can promote ol maturation  <cit> .
in the present study, we have confirmed that olig <dig> deletion delays myelin gene expression.
in addition, our previous work has shown that olig <dig> can synergize with sox <dig> to activate mbp gene transcription  <cit> .
taken together, we believe that ol development is controlled by indispensible core factors  in conjunction with other factors  that are not crucial but serve to adjust the timing of ol differentiation.
CONCLUSIONS
using two newly-generated olig <dig> null lines we show that loss of olig <dig> causes a transient delay in ol development and myelination.
our data confirm the original description of a mild phenotypic effect of olig <dig> loss  <cit> , but run counter to the subsequent report of a complete myelination block  <cit> .
we have shown that the mild phenotype is unlikely to result from compensatory up-regulation of olig <dig>  as suggested  <cit> .
we conclude that olig <dig> is non-essential for ol development.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
wdr obtained funding.
wdr, hl, ntk designed the experiments and interpreted the results.
jpdf, hl and pa carried out the experiments.
hl drafted the manuscript and wdr helped revise it.
all authors read and approved the final manuscript.
authors’ information
william d richardson and huiliang li are joint senior authors.
supplementary material
additional file 1: figure s1
no nicd expression in olig <dig> or olig <dig> mice.
proteins from e <dig>  spinal cord were subjected to sds-page, followed by western blotting with rabbit anti-myc antibody.
ppgkcresv40-transfected mefs derived from olig <dig> embryos were used as positive control.
the nicd band is indicated by an arrow.
click here for file
additional file 2: figure s2
our new olig <dig> null mice do not express olig <dig> protein.
co-immunolabeling for olig <dig>  and sox <dig>  was performed on sections of e <dig>  mouse spinal cords.
no olig1-positive cells were detected in either olig <dig>  or olig,olig <dig> spinal cords  in contrast to in olig <dig>  or olig,olig <dig> controls .
scale bar: 80 μm for a-d and 20 μm for a’-d’.
click here for file
additional file 3: figure s3
no up-regulation of olig <dig> expression in olig <dig> spinal cord.
quantitative pcr using cdna templates prepared from e <dig>  spinal cord tissue revealed that there was no appreciable difference in the expression of olig <dig> mrna between olig <dig> and olig <dig> lines or between olig,olig <dig> and olig,olig <dig> lines.
click here for file
additional file 4: figure s4
no change in ol numbers in olig <dig> null forebrain at p <dig>  in the developing forebrain, ol differentiation starts in the first postnatal week.
at p <dig>  coronal sections showed that the numbers of mbp- and plp-expressing cells in olig <dig> null forebrain  were similar to those in controls .
cc, corpus callosum; ctx, cortex.
scale bar: 80 μm.
click here for file
acknowledgements
we thank ulla dennehy, palma iannarelli, sabrina pacheco and matthew grist for excellent technical help, michael wegner  for the gift of anti-sox <dig> antibodies, and charles d stiles  and richard lu  for helpful comments on the manuscript.
our work was funded by the wellcome trust, the uk medical research council, a european research council starting grant  to nk and a fellowship from the portuguese fundação para a ciência e a tecnologia  to jpdf.

 oligodendrocyte lineage genes olig and olig encode basic helix-loop-helix transcription factors. olig is a master regulator of oligodendrocyte lineage development. olig is also required for generation of some neurons, notably spinal motor neurons. mns are generated from neural stem/progenitor cells in a specialized region of the ventral ventricular zone of the spinal cord known as pmn. around embryonic day in mice, the same group of progenitors stops producing mns and switches to production of ol precursors, which proliferate and migrate away from the vz in all directions before associating with axons and differentiating into myelin-forming ols. olig and olig are involved at multiple stages of this developmental sequence. olig is also required for specifying oligodendrocytes and some types of neurons in the brain – some ventrally-derived interneurons and cholinergic projection neurons in the forebrain, for example. olig can compensate for olig in some regions including the hindbrain and parts of the forebrain, because ops still form there in olig null mice but not in olig1/olig double nulls. olig also plays a later role in the differentiation of ops into myelinating ols, although there is disagreement about whether there is an absolute requirement for olig during normal development. the original olig null allele, made by inserting a cre-frt-pgk-neo-frt cassette into the mouse olig locus caused a delay in the appearance of differentiated ols but no long-term myelin deficit. however, a subsequent study by xin et al., who crossed the original line with flp-expressing mice to remove the pgk-neo selection cassette, found a severe myelination defect leading to early postnatal lethality. apart from this contested role in ol lineage development, olig is known to be required for remyelination of experimentally-induced demyelinated lesions in the mouse spinal cord. given the central role of the oligs in ol lineage development, it is important to try to settle the controversy over the developmental requirement for olig this might have added significance because the olig null locus contains an expressed cre cassette under olig transcriptional control and these olig mice are being used to delete floxed genes specifically in ol lineage cells. for example, conditional deletion of dicer using olig caused severe impairment of myelination and death around p, whereas analogous experiments using olig or cnp resulted in only slightly delayed myelination with full recovery by p. in another example, constitutively activating the wnt signaling pathway by conditional deletion of exon of β-catenin using olig completely prevented ol lineage specification, judging by the complete absence of op markers such as pdgfra, whereas similar experiments using olig did not affect op specification but only their subsequent differentiation into ols. while there might be a simple explanation for these differences, such as earlier or more complete recombination by olig than by olig the possibility remains that the olig null allele generated by xin et al. might carry some additional, unidentified defect that can amplify the phenotype of other deleterious mutations. to attempt to throw some light on these matters we undertook a study of two independent olig null lines generated in our own laboratory. we found that loss of olig causes a transient delay in ol development and myelination. we quantified olig mrna in our olig mutant mice and found no increase relative to wild type controls. the mild phenotype we observe is therefore likely to be a genuine consequence of olig loss, not moderated by cis regulatory effects on olig methods mice mice were maintained on a hour light–dark cycle. for timed mating, male and female mice were caged together overnight and noon the following day was designated embryonic day. all mouse work was approved by the home office of the uk government, and conformed to the animals act new olig null lines, olig and olig, olig were generated as described previously. embryonic stem cell targeting we generated a new olig line by es cell targeting. briefly, olig targeting vector was linearized and electroporated into r es cells. after days’ selection in 150 μg/ml g, colonies were picked and expanded in 96-well plates. targeted es clones were identified by southern blotting using a 700 bp ncoi—ecori fragment as probe. positive es clones were confirmed by southern blotting using a 200 bp psti—ncoi probe. five correctly targeted es cell clones were expanded for karyotyping; two clones with normal karyotype were used for c57/b blastocyst injection to produce chimeric mice. male chimeras were bred to c57/b females to produce olig heterozygotes. tissue preparation and histochemistry embryonic and postnatal spinal cords or brains were immersion-fixed in 4% paraformaldehyde in phosphate-buffered saline overnight at 4°c. the tissue was cryo-protected overnight at 4°c in 20% sucrose in pbs. tissue was embedded in oct compound, rapidly frozen on dry ice/isopentane and stored at -80°c. tissue was cryo-sectioned in a bright cryotome and sections collected on superfrost plus slides. sections were treated with blocking solution at 20-25°c for one hour before immuno-labeling. primary antibodies were anti-sox and anti-olig. secondary antibodies were alexa fluor conjugated goat anti-rabbit and cy3-conjugated goat anti-guinea pig igg. sections were counterstained with hoechst dye, for minutes at 20-25°c after the secondary antibody and mounted under coverslips in fluorescence mounting medium. our fluorescence in situ hybridization procedure has been described before; detailed protocols are available at http://www.ucl.ac.uk/~ucbzwdr/richardson.htm. briefly, digoxigenin -labelled rna probes were transcribed in vitro from cloned cdnas of mbp or plp. after hybridization, the dig signal was detected using horseradish peroxidase -conjugated anti-dig followed by developing in fluorescein-tyramide reagent. quantitative pcr quantitative pcr was performed using forebrain and spinal cord tissue collected from olig null mice and control littermates that carried either one or two endogenous copies of olig at embryonic day and/or e. the tissue was homogenized in the presence of trizol reagent, and total rna was purified and used for cdna synthesis following the manufacturer’s instructions. oligonucleotides 5′ att gta caa aac ggc cac aa 3′ and 5′ agt gct ctg cgt ctc gtc ta 3′ were used for olig cdna amplification. oligonucleotides 5′ aca act ttg gca ttg tgg aa 3′ and 5′ gat gca ggg atg atg ttc tg 3′ were used to amplify gapdh as an internal control. qpcr values were calculated using the relative standard curve method. at least three embryos of each genotype were analyzed at each age. mouse embryonic fibroblast culture and western blotting mouse embryos were placed in pbs and the head, vertebral column, dorsal root ganglia, and inner organs were removed. the remaining tissue was digested in % trypsin, finely minced with a razor blade and incubated at 37°c for minutes to make a single-cell suspension. cells were then plated in 35 mm dishes coated with % gelatin and grown at 37°c in 5% co in mef medium. a plasmid encoding cre under the control of the pgk promoter was used for transfection with fugene. proteins from transfected mefs and mouse spinal cord tissue were separated by sds-page and transferred to polyvinylidene difluoride membranes. rabbit anti-myc antibody was purchased from abcam and used at a 1: dilution. protein bands were visualized by chemi-luminescence.  generation of new olig null mouse lines to try to resolve the discrepancy between the reported phenotypes of two different olig null mouse lines we generated two new olig null strains, using different approaches. for one, we replaced the olig open reading frame with a dna fragment including an inverted phosphoglycerate kinase promoter -neomycin resistance cassette flanked by loxp sites in mouse embryonic stem cells, line r. we refer to this line as olig for purposes unrelated to the work described here, the modified locus also included a myc-tagged notch intracellular domain coding sequence downstream of the floxed pgk-neo cassette; in the absence of cre recombinase this nicd cassette is not expressed and is phenotypically neutral. targeted clones were identified by southern blot analysis of genomic dna using a 700 bp ncoi–ecori fragment as a 5′ probe. correct targeting was confirmed using a 200 bp psti-ncoi fragment as a 3′ probe. one karyotypically normal es cell line was selected for blastocyst injection and germline transmission. our second olig ko was generated by crossing olig1/olig double-null mice with a phage artificial chromosome transgenic line that contains a single copy of mouse olig. we refer to this line as olig, olig we confirmed that this line does not express olig protein. lack of compensatory up-regulation of olig in olig1-null mice the olig and olig genes are located about 40 kb apart on mouse chromosome and there is a large degree of overlap in their expression patterns in vivo. xin et al. suggested that the pgk-neo cassette introduced by lu et al. to disrupt the olig orf might have exerted a cis-activating effect on the neighbouring olig locus, resulting in over-expression of olig which compensated for loss of olig since our own olig mice also contain a pgk-neo cassette at the olig locus, we compared olig mrna levels in our olig mice and olig controls. we collected forebrain tissue at two embryonic stages and quantified olig transcripts by pcr, using total cellular rna as substrate. we could not detect a significant difference in the brain or spinal cord between olig and olig indicating that olig was not mis-regulated by the pgk-neo cassette at the olig locus in our mice. note that although two pgk-neo cassettes are present at the disrupted olig1/olig locus in our olig, olig mice they are almost certainly physically remote from the randomly-integrated olig pac transgene and therefore are not expected to impose cis-regulation on olig oligodendrocyte precursors are specified normally in olig null cns we analyzed the expression of both platelet-derived growth factor receptor-alpha, a marker of ops, and sox which marks all stages of the ol lineage, by immunofluorescence microscopy of e spinal cord and p forebrain sections. neither pdgfra nor sox expression were noticeably altered in our two olig null lines, relative to olig controls. this is as expected, given that olig protein does not appear until after op specification, and is consistent with the phenotypes of the two previously-described olig null lines. ol differentiation is delayed in olig null spinal cord to investigate ol differentiation in our two new olig null lines, we visualized mrnas encoding mature ol markers myelin basic protein and myelin proteolipid protein by in situ hybridization. at e, mbp and plp transcripts were absent from spinal cord in both olig null lines, in contrast to littermate controls that carried one good copy of endogenous olig. at e, mbp and plp transcripts were present but in lower numbers of cells relative to olig 0heterozygotes; by postnatal day, there were normal numbers of mbp and plp-positive cells in the olig null spinal cord. these results indicate that olig is involved in, but is not critically important for ol differentiation in the developing spinal cord, consistent with the original study by lu et al.. ol differentiation in mouse forebrain does not begin until after birth. on forebrain sections, few mbp and/or plp positive cells were detectable by fluorescence in situ hybridization at p. at p both olig null lines appeared to have normal numbers of mbp and plp positive cells in both the corpus callosum and cortex compared to control mice. discussion we generated two new olig null mouse lines by different routes -one by homologous recombination in es cells followed by blastocyst injection, and the other by transgenic rescue of a previously generated olig1/olig double-null line by pronuclear injection of an olig pac. neither of the olig null lines showed any evidence of prenatal lethality and both lines lived and reproduced normally. there was a transient delay in the production of differentiated ols in the spinal cords of both our olig null lines, as originally reported by lu et al. but in contrast to xin et al., who reported a severe myelination block that resulted in death around the third postnatal week. xin et al. put the discrepancy down to the fact that the original olig null allele retained a pgk-neo cassette, speculating that the presence of this highly-transcribed element might have caused compensatory up-regulation of the neighbouring olig gene. xin et al. removed the pgk-neo cassette by crossing the original lu et al. line with flp-expressing mice. however, they did not quantify olig expression in either of the olig mutants. a cis-acting regulatory effect of pgk-neo has been implied in previous studies. for example, the initially reported lethal phenotype of a germ line surf deletion was later attributed to the effect of pgk-neo on expression of unidentified genes near the surf locus, after a second surf knockout line lacking the pgk-neo cassette was found to be unusually long-lived. another example is the germ line knockout of the zinc finger transcription factor zfp which was initially reported to be embryonic-lethal. subsequently, an independent line was found to survive after birth, developing a severe dysmyelinating phenotype and dying around p. one potential explanation for the difference was that the embryonic-lethal allele contained an expressed neo selection cassette. we tested the hypothesis that the mild phenotype of our olig mice might have been due to compensatory up-regulation of the adjacent olig gene by pgk-neo, but found no evidence for this. our data are consistent with a previous study by samanta et al. who found no evidence for up-regulation of olig when they used the olig line of lu et al. for conditional deletion of bone morphogenetic protein receptor-1a. taken together, the data indicate that the presence or absence of pgk-neo cannot easily explain the dramatically different developmental phenotypes of different olig null mice. different phenotypic outcomes for the same gene deletion can sometimes result from differences in the genetic backgrounds of the mice. for example, the effect of knocking out nogo-a, a membrane protein of the adult myelin sheath and an inhibitor of neurite growth and axon regeneration, has a much larger effect on neurite regeneration ability in the 129x1/svj background than in the c57bl/6 j background. our olig line was generated using r es cells. homozygous nulls were maintained in a 129/c mixed background for many generations with no sign of lethality. they are now maintained on a 129/c57/cba background, also with no sign of lethality. the olig null of lu et al. was made using j es cells and crossed onto c for analysis. the background of our olig,olig line is mixed c57/cba and these mice also display a mild phenotype. the line displaying the contradictory lethal phenotype made by xin et al. was a modification of lu et al.’s line, maintained in a mixed 129/c background. altogether, there is no compelling reason to think that genetic background underlies the differing severity of olig disruption in different lines. another possible reason for the divergent phenotypes reported by lu et al. and xin et al. might lie in the way in which their mouse lines were generated. xin et al. made their line by crossing the mice made previously by lu et al. with a line that expresses flp recombinase ubiquitously, in order to effect germ line excision of the frt-flanked pgk-neo cassette. given that olig and olig lie close to each other on the chromosome and share significant sequence homologies, it is conceivable that an unintended recombination event might have taken place, altering the olig locus in some way that affects olig expression or structure in addition to disrupting olig arnett et al. previously showed that the olig null line of lu et al. inefficiently remyelinates demyelinated lesions produced either by focal injection of lysolecithin or by systemic administration of cuprizone, despite the nearly normal developmental time course of myelination of these mice. this implied that olig and olig have complementary roles in myelin development and repair, respectively. we have no reason to question this conclusion and have not tested the remyelination abilities of our new olig null mice. ol differentiation is subject to two-tier transcriptional regulation: 1) epigenetic repression of transcriptional inhibitors and 2) direct transcriptional activation of myelin genes. transcription factors olig, sox, mrf and zfp are critical for ol differentiation and/or myelination. ascl and nkx also play important roles; germ line knockout of either nkx or ascl leads to decreased expression of myelin genes in neonatal mice, suggesting that both genes can promote ol maturation. in the present study, we have confirmed that olig deletion delays myelin gene expression. in addition, our previous work has shown that olig can synergize with sox to activate mbp gene transcription. taken together, we believe that ol development is controlled by indispensible core factors in conjunction with other factors that are not crucial but serve to adjust the timing of ol differentiation.  using two newly-generated olig null lines we show that loss of olig causes a transient delay in ol development and myelination. our data confirm the original description of a mild phenotypic effect of olig loss, but run counter to the subsequent report of a complete myelination block. we have shown that the mild phenotype is unlikely to result from compensatory up-regulation of olig as suggested. we conclude that olig is non-essential for ol development. competing interests the authors declare that they have no competing interests. authors’ contributions wdr obtained funding. wdr, hl, ntk designed the experiments and interpreted the results. jpdf, hl and pa carried out the experiments. hl drafted the manuscript and wdr helped revise it. all authors read and approved the final manuscript. authors’ information william d richardson and huiliang li are joint senior authors. supplementary material additional file 1: figure s1 no nicd expression in olig or olig mice. proteins from e spinal cord were subjected to sds-page, followed by western blotting with rabbit anti-myc antibody. ppgkcresv40-transfected mefs derived from olig embryos were used as positive control. the nicd band is indicated by an arrow. click here for file additional file 2: figure s2 our new olig null mice do not express olig protein. co-immunolabeling for olig and sox was performed on sections of e mouse spinal cords. no olig1-positive cells were detected in either olig or olig,olig spinal cords in contrast to in olig or olig,olig controls. scale bar: 80 μm for a-d and 20 μm for a’-d’. click here for file additional file 3: figure s3 no up-regulation of olig expression in olig spinal cord. quantitative pcr using cdna templates prepared from e spinal cord tissue revealed that there was no appreciable difference in the expression of olig mrna between olig and olig lines or between olig,olig and olig,olig lines. click here for file additional file 4: figure s4 no change in ol numbers in olig null forebrain at p in the developing forebrain, ol differentiation starts in the first postnatal week. at p coronal sections showed that the numbers of mbp- and plp-expressing cells in olig null forebrain were similar to those in controls. cc, corpus callosum; ctx, cortex. scale bar: 80 μm. click here for file acknowledgements we thank ulla dennehy, palma iannarelli, sabrina pacheco and matthew grist for excellent technical help, michael wegner for the gift of anti-sox antibodies, and charles d stiles and richard lu for helpful comments on the manuscript. our work was funded by the wellcome trust, the uk medical research council, a european research council starting grant to nk and a fellowship from the portuguese fundação para a ciência e a tecnologia to jpdf.
a plausible explanation was proposed, that the highly expressed pgk-neo cassette in the original line might have up-regulated the neighbouring olig gene, compensating for loss of olig however, this was not tested, so the importance of olig for oligodendrocyte development has remained unclear. however, a subsequent study of the same line following removal of pgk-neo found severe disruption of oligodendrocyte production, myelination failure and early postnatal lethality. both lines had a mild phenotype featuring slightly delayed oligodendrocyte differentiation and maturation but no long-term effect. olig and olig encoding closely related basic helix-loop-helix transcription factors, were originally identified in screens for glial-specific genes. olig and olig are both expressed in restricted parts of the neuroepithelium of the embryonic spinal cord and telencephalon and subsequently in oligodendrocyte lineage cells throughout life. in the spinal cord, olig plays a crucial role in the development of oligodendrocytes and motor neurons, and both cell types are lost from olig null mutant mice.
 olig and olig encoding closely related basic helix-loop-helix transcription factors, were originally identified in screens for glial-specific genes. olig and olig are both expressed in restricted parts of the neuroepithelium of the embryonic spinal cord and telencephalon and subsequently in oligodendrocyte lineage cells throughout life. in the spinal cord, olig plays a crucial role in the development of oligodendrocytes and motor neurons, and both cell types are lost from olig null mutant mice. the role of olig has been more cryptic. it was initially reported that olig null mice have a mild developmental phenotype characterized by a slight delay in oligodendrocyte differentiation. however, a subsequent study of the same line following removal of pgk-neo found severe disruption of oligodendrocyte production, myelination failure and early postnatal lethality. a plausible explanation was proposed, that the highly expressed pgk-neo cassette in the original line might have up-regulated the neighbouring olig gene, compensating for loss of olig however, this was not tested, so the importance of olig for oligodendrocyte development has remained unclear.  we generated two independent lines of olig null mice. both lines had a mild phenotype featuring slightly delayed oligodendrocyte differentiation and maturation but no long-term effect. in addition, we found that olig transcripts were not up-regulated in our olig null mice.  our findings support the original conclusion that olig plays a minor and non-essential role in oligodendrocyte development and have implications for the interpretation of studies based on olig deficient mice from different sources. oligodendrocyteolig1olig2myelinknockout micespinal cordforebrain 