BACKGROUND
the ability to assay the transcriptional activity of every gene in the genome  has been facilitated by microarrays and now by next-generation high-throughput transcript sequencing   <cit> .
large-scale transcriptome profiling allows new questions to be asked about the roles of transcription factors in development and the cascades of genes that they influence to drive cell fate and differentiation.
understanding transcriptional programs is also central to understanding disease and pathological conditions resulting from failures in these fundamental processes.
multicellular organisms present challenges to understanding developmentally-relevant transcriptional programs, as cell-specific differences in gene expression can be masked by expression in multiple cell types.
isolation of specific cell types can solve this problem.
in c. elegans, embryonic neurons were isolated by fluorescence-activated cell sorting  and their transcriptional profile assayed by microarrays  <cit> .
in the same report, transcripts from larval neurons were isolated by expression of a tagged poly-a binding protein  in neurons and subsequent immunoprecipitation of flag-pab- <dig> and associated transcripts  <cit> .
recently, single migrating linker cells of the c. elegans male gonad were dissected from living animals and transcriptionally profiled using rna-seq  <cit> .
despite these advances, isolation of cell-specific transcripts from model organisms using these techniques is not trivial, and not feasible for every cell type at this time.
in the current study we test the efficacy of combining the advantageous genetics and well-understood development of the nematode c. elegans with whole organism rna-seq to identify genes with effects on the migration of the q neuroblasts.
by comparing transcript levels in wild type and various mab- <dig> mutant backgrounds defective in q cell migration, we test if genes with effects on q migration can be identified by rna-seq of whole animal lysates.
the q cells are neuroblasts that reside bilaterally in the posterior region of the worm .
the q cells, left and right, undergo an identical pattern of division coupled with cell death to produce three neurons  <cit>  .
while the q cells divide in a similar fashion, ql descendants migrate posteriorly and qr descendants migrate anteriorly.
ql on the left undergoes an initial migration towards the posterior of the worm and divides to give rise to the pvm, sdql, and pqr neurons  <cit> .
these ql descendants continue to migrate posteriorly, with pqr migrating the farthest to reside in the tail of the worm behind the anus .
qr on the right undergoes a similar pattern of migration and division, except the cells migrate to the anterior, generating the avm, sdqr, and aqr neurons  <cit> .
aqr migrates the farthest to reside in the head of the worm near the anterior deirid sensory organ .
q cell migration occurs in two stages.
the initial stage relies on q cell polarization regulated by the transmembrane molecules unc-40/dcc, ptp-3/lar, and mig- <dig>  <cit> .
when ql undergoes its initial migration towards the posterior, it encounters a graded egl-20/wnt signal, which is secreted from muscle and epidermal cells at the posterior of the animal  <cit>  .
in this second phase of migration, egl-20/wnt induces expression of the mab-5/hox transcription factor in ql, but not qr, via canonical wnt signaling  .
mab- <dig>  an antennapedia-like hox transcription factor, is essential for the posterior migration of the ql descendants and acts as a determinant for posterior migration of q descendants  <cit> .
loss of function of mab- <dig> results in anterior migration of the ql descendants, despite normal initial posterior migration of ql .
a gain-of-function mab- <dig> mutant that results in mab- <dig> expression in both ql and qr independent of wnt signaling causes both ql and qr descendants to migrate posteriorly despite normal initial q cell migrations   <cit> .
induced autonomous expression of mab- <dig> during anterior q descendant migration results in the cells changing direction to the posterior, and mab- <dig> is needed continuously during posterior migration  <cit> .
thus, mab- <dig> is both necessary and sufficient for autonomously directing posterior versus anterior migration of q descendants.
in addition to ql descendants, mab- <dig> is expressed in a handful of other cells in the posterior in the mid-l <dig> stage, including the hypodermal v <dig> and v <dig> cells, p7-p <dig>  the m mesoblast, and several posterior body wall muscles  <cit> .
mab- <dig> inhibits the expression of the transmembrane molecule mig- <dig> in posterior commissural motor neurons generated from p7-p <dig>  <cit> .
mig- <dig> is non-autonomously required for the anterior migration of qr descendants on the right  <cit> .
thus, mab- <dig> might control gene expression outside of the q cells that non-autonomously controls q descendant migration.
our whole organism rna-seq approach has the potential to identify both autonomous and non-autonomous targets of mab- <dig> in q descendant migration.
the results reported here do not distinguish between autonomy and non-autonomy, which will be the subject of future study.
it is important to note that ql cell descendants are still generated and still migrate in the absence of mab- <dig>  but in the opposite direction.
additionally, aqr and pqr are thought to have the same functions of sensing oxygen and regulating social feeding in the worm, even though mab- <dig> is expressed in pqr and not in aqr in wild type worms  <cit> .
these factors suggest that mab- <dig> is specifically involved in determining the direction of migration of the ql cell descendants and not in the specification of neuronal class identity or function or in their ability to migrate.
mab-5/hox represents an excellent opportunity to dissect the transcriptional programs that specify differentiation, in this case posterior versus anterior cell migration.
we take advantage of both loss-of-function and gain-of-function alleles of mab- <dig> to identify genes whose transcriptional levels are affected by each, and then functionally analyze their roles in q descendant migrations using rnai.
we find that while some genes are oppositely affected in loss versus gain-of-function backgrounds, the majority of genes are unique in each set, indicating redundancy and combinatorial function of mab- <dig> in regulating transcription.
we also find that mab- <dig> target genes are enriched for those encoding secreted proteins involved in extracellular matrix formation and remodeling, some of which have functional roles in q descendant migration.
this suggests that mab- <dig> might alter cell-extracellular matrix interaction to direct posterior migration.
finally, we functionally compare our rna-seq results with the mab- <dig> chip-seq results obtained by the modencode project  <cit>  and find that mutant rna-seq and chip-seq are both efficient methods to identify functional targets of mab- <dig> in posterior q descendant migration.
RESULTS
transcriptional profiling of wild-type and mab- <dig> mutant l <dig> animals using rna-seq
to identify genes whose expression is influenced by mab- <dig>  we conducted rna-seq analysis on wild-type worms, two mab- <dig> loss-of-function  alleles e <dig> and gk <dig> lof and mab-5lof, respectively), and the mab- <dig> gain-of-function  allele gof).
total rna was isolated from each genotype at the early l <dig> larval stage approximately  <dig> -5 h after hatching, after the q cells had undergone their initial migrations and divisions, and when mab- <dig> is activated by egl-20/wnt signaling in ql and ql descendants .
rna from multiple biological replicates of each genotype was then subjected to rna-seq using illumina single-end 60-bp reads.
reads were assigned to genes using tophat  <cit> , and pairwise expression differences between genotypes analyzed using the cuffdiff module in cufflinks  <cit> .
differential gene expression between genotypes was assessed using an fdr-adjusted q-value to account for multiple-testing, and only those genes with significant changes  are discussed.
cuffdiff files for each pairwise comparison can be found in additional files  <dig>   <dig>   <dig>   <dig> and  <dig>  and protein-coding genes with significant expression changes can be found in additional file  <dig>
due to our column-based total rna isolation procedure, and the poly-a selection step prior to rna-seq, few small, non-coding rna molecules should be represented in our sequencing data.
nevertheless, some small nuclear snrnas, small nucleolar snornas, and trans-spliced leader rnas  were found to be differentially expressed.
it is possible that certain high-abundance genes in these categories are inefficiently removed by poly-a selection.
no mirna genes were identified as differentially expressed, primarily because very few mirna reads were recovered by our protocol.
given the poor, and potentially inconsistent representation of reads associated small non-coding rna genes in our study, these genes are excluded from further analysis.
nevertheless, small non-coding rna genes are included in additional files  <dig>   <dig>   <dig>   <dig> and  <dig>  and those showing significant differential expression between genotypes are listed in additional file  <dig>
we first determined genes that were down- or upregulated in the two mab- <dig> lof backgrounds compared to wild-type.
ten genes were significantly downregulated in mab-5lof compared to wild-type, while  <dig> genes were downregulated in mab-5lof .
five genes were downregulated in both lof mutants, for a total of  <dig> genes downregulated in at least one comparison.
fewer genes were found to be upregulated in the lof backgrounds compared to wild-type: seven in mab-5lof and six in mab-5lof, with four in both, for a total of nine genes upregulated in at least one comparison .
1these categories also included the retired gene r07b <dig> , which was not included in further analysis.
we note that the disparity in the number of downregulated genes identified in the comparisons to the two different lof mutants is not explained by differences in overall rna-seq read count for the two mutant genotypes .
since mab- <dig> is a splice donor mutation in the second intron, whereas mab- <dig> is a deletion of a large region of the locus, it is possible that mab- <dig> retains some activity and thus more weakly affects gene expression than does mab- <dig>
interestingly, the mab- <dig> locus itself was not significantly underrepresented in the two lof backgrounds.
indeed, in the mab-5lof mutant, a 2-bp insertion at mab- <dig> was apparent in the rna-seq reads due to the use of a cryptic 5′ splice site 2 bp downstream from the wild-type site because of an altered splice donor site in the first mab- <dig> intron   <cit> .
in the mab-5lof mutant , the rna-seq data at mab- <dig> was consistent with the known deletion at this locus .
these data suggest that the mab- <dig> transcript might not be subject to nonsense-mediated mrna decay.
in the mab-5gof background,  <dig> genes were downregulated and  <dig> genes were upregulated compared to wild type, .
we expected that mab- <dig> itself would be upregulated in mab-5gof relative to wild-type, since this mutation causes upregulated mab- <dig> expression in multiple cells  <cit> .
while mab- <dig> gene expression is higher in the gof than wildtype  this change is not significant after correcting for multiple tests .
to identify other genes potentially regulated by mab- <dig>  we compared genes that were differentially expressed between each lof mutant and the gof, with the idea that this might be a more sensitive and powerful approach than comparing each mutant class separately with wild type .
seventeen genes were downregulated in mab-5gof compared to mab-5lof, and  <dig> were down in mab-5gof compared to mab-5lof, with  <dig> in both .
six of these genes were also downregulated in mab-5gof compared to wild-type .
twenty four genes were upregulated in mab-5gof relative to mab-5lof, and  <dig> upregulated relative to mab-5lof, with  <dig> of the  <dig> genes differentially expressed in the mab-5lof comparison also among those in the mab-5lof comparison.
differentially expressed gene sets for in silico evaluation and functional testing
our ultimate goal with this work was to identify and characterize genes arising from our rna-seq screen that function with mab- <dig> in neuronal migration.
in this work, we focus solely on those genes that exhibit significant gene expression changes after accounting for multiple testing.
this will clearly exclude some true mab- <dig> targets, but should provide a gene set with a high probability of success in experimental functional validation.
the mab- <dig> gene itself showed significant differential expression only in the comparison between the mab-5lof and the mab-5gof .
however, the comparisons between the mab-5lof and mab-5gof  and between wild-type and the mab-5gof  were nominally significant and in the anticipated direction.
this indicates that bona fide mab- <dig> targets might appear significant in only one of the comparisons of wild-type and the two lof backgrounds to the gof.
thus, for further characterization we combined genes from all the different comparisons described above into four groups :  <dig> genes total were downregulated in lof backgrounds compared to wildtype, while  <dig> were upregulated.
in combined gof comparisons to wild-type and the two lof backgrounds,  <dig> genes were downregulated in gof and  <dig> were upregulated.
opposing regulation in loss- versus gain-of-function
some transcriptional targets of mab- <dig> may be differentially-regulated in opposite directions in the lof and gof conditions  might be upregulated in mab-5).
of the  <dig> genes downregulated in either lof mutant compared to wild-type,  <dig> were upregulated in the mab-5gof combined gene set , while  <dig> of the  <dig> genes upregulated in the lof mutants was downregulated in gof combined  .
thus, there was not a strict concordance between gene expression in the lof and gof backgrounds, but there was significant overlap.
simple statistical power concerns could explain the lack of precise concordance, but it is also possible that mab- <dig> acts redundantly and combinatorially with other transcription factors to influence gene expression.
genes encoding secreted and transmembrane molecules are enriched among mab-5-regulated genes
for each of the four pools of genes  or mab- <dig> backgrounds), we classified genes based on their predicted cellular location and molecular function as annotated in wormbase .
within each group, molecules with predicted transmembrane domains and/or a predicted signal sequence  were by far the most abundant class .
this enrichment for secreted and transmembrane proteins was statistically significant in three of the four groups using a binomial analysis based on the fraction of such genes in the worm genome .
we note that while the mab- <dig> upregulated gene set did not show a significant excess of tmhmm and signalp-annotated genes, at  <dig> genes this is the smallest of the four groups of genes.
the remaining genes in the four pooled sets are predicted to encode molecules acting in transcription and gene expression, cytoplasmic signaling, metabolism, and the cytoskeleton .
1this set includes only protein-encoding genes and not small rna genes.
from gene annotation files from wormbase, there were  <dig> genes with tmhmm and/or signalp annotation out of  <dig> total.
2binomial distribution test comparing fractions of transmembrane and secreted proteins in the gene sets compared to the total fraction in the genome.
function and gene ontology of genes affected by mab-5
we used the database for annotation, visualization and integrated discovery  to perform a gene ontology term  enrichment and functional clustering analysis on the groups of mab-5-regulated genes .
genes upregulated in mab- <dig> were enriched for go terms involving aryl phosphatase and had phosphatase activity .
however, genes in this class were few in number and did not return highly significant go term functional clustering.
genes that were downregulated in mab- <dig> returned go term enrichment for transmembrane proteins, as expected from the domain analysis above.
this group was significantly enriched for go terms involving secreted glycine-rich proteins .
terms and a functional cluster involving atpase activity and nucleotide binding were also identified .
this is due to the downregulation of two genes in mab- <dig>  pmp-5/t10h <dig>  and pgp-4/f42e <dig> , that encode abc transporter-family proteins.
mab- <dig> affects genes involved in extracellular matrix
among genes upregulated in mab- <dig>  the most significantly enriched domain is related to an extracellular domain encoded by genes induced by fungal infection.
three genes, fipr- <dig>  fipr- <dig>  and fipr- <dig> , harbor this domain .
two other extracellular domains were also significantly-enriched; the complement c1r/c1s/uegf/bmp <dig>  domain , and the thrombospondin type i  .
other enriched domains include aryl phosphatase pho13/phosphoglycolate phosphatase domain and the had-superfamily hydrolase subfamily iia domain, both of which have been implicated in carbohydrate metabolism.
as mentioned above, two genes  harboring these domains were also upregulated in the mab- <dig> background, so their functional relationship to mab- <dig> is unclear.
this table is a summary of the significant go terms and functional clusters found in additional file  <dig>
genes downregulated in mab- <dig> were enriched for go terms and functional clusters involving collagens, secreted metalloproteases and their inhibitors, cysteine-rich repeats found in a variety of secreted proteins, proteins that form disulfide bonds, the shkt/metridin toxin domain found in secreted proteins, and histones .
this gene group includes:  <dig> collagen genes; seven genes encoding secreted keratin-like or keratin-associated molecules; three genes encoding secreted astacin metalloproteases ; three genes encoding secreted kunitz serine protease inhibitors; four genes encoding secreted proteins containing the shk/metridin toxin domain ; and four histone genes.
three genes encoding secreted glycine-rich repeats were downregulated in mab- <dig>  but these were also downregulated in mab- <dig>  suggesting that they might not be relevant targets.
in sum, this go term enrichment analysis suggests that mab- <dig> predominantly regulates genes encoding secreted and transmembrane molecules.
based on the mab- <dig> results, genes upregulated by mab- <dig> tend to encode secreted and transmembrane molecules with domains involved in signaling, cell adhesion, and fungal defense .
genes downregulated by mab- <dig> tend to encode secreted molecules that are involved in the formation and remodeling of the extracellular matrix , suggesting mab- <dig> might alter cell interactions with the extracellular matrix.
indeed, two genes upregulated by mab- <dig>  spon-1/f-spondin and mig-6/papilin, encode secreted molecules containing both kunitz-type proteinase inhibitor domains and tsp <dig> domains, and are important for cell migration  <cit> .
comparison of mab- <dig> rna-seq data with modencode chip-seq data
regions of the genome with which mab- <dig> interacts physically were identified previously by chromatin immunoprecipitation and dna sequencing  as part of the modencode project  <cit> .
we compared a list of potential mab- <dig> chip seq targets  with our rna seq data set : 2/ <dig> genes downregulated in mab- <dig> were present in the annotated chip seq targets, and 1/ <dig> mab- <dig> upregulated genes were included.
in the mab- <dig> comparisons, 4/ <dig> downregulated genes and 8/ <dig> upregulated genes were included.
thus, while some rna-seq and chip-seq targets were in common, there was not strict concordance between targets identified by the two methodologies.
one caveat is that the chip-seq data were gathered from l <dig> larval animals whereas our rna-seq data were gathered from l <dig> larval animals.
it is also possible that mab- <dig> regulates many genes indirectly, or that mab- <dig> is redundant with other transcription factors in the regulation of many genes.
the mab- <dig> gene itself was one of the  <dig> genes included in both the rna-seq and chip-seq data sets.
mab- <dig> has been shown to regulate its own expression by a positive feedback mechanism to maintain mab- <dig> expression in the q neuroblast descendants during their posterior migrations  <cit> .
also identified by both methods was k08f <dig> , which encodes a map kinase activated protein kinase similar to vertebrate mapkap <dig>  k08f <dig>  also showed opposite regulation in mab- <dig> lof and gof backgrounds .
posterior aqr migration in mab-5gof as a sensitized background to identify mab- <dig> targets that function in posterior migration
the goal of this work is to identify genes regulated by mab- <dig> that function in q descendant migration.
mab- <dig> is required for posterior migration of the q neuroblast descendants, and can also drive posterior migration of the qr descendants, which normally migrate anteriorly gof results in posterior migration of qr descendants including aqr; figure 2c).
genes upregulated in mab-5gof and downregulated in mab- <dig> are good candidates for those that might mediate posterior q descendant migration downstream of mab- <dig>  loss of such a gene’s function might modify mab-5gof posterior aqr migration, resulting in more anterior aqr positioning.
since aqr normally migrates anteriorly, posteriorly-migrating aqr in mab-5gof might represent a sensitized background to identify effects on posterior migration that might be missed in posterior pqr migration.
thus, we tested genes identified by our rna-seq analysis for their ability to modify posterior migration of aqr in the mab-5gof mutant.
ceh- <dig> and unc- <dig> are epistatic to mab- <dig> in posterior aqr migration
as proof of principle of using posterior aqr migration in mab-5gof as a background to define mab- <dig> targets that affect q cell descendant migrations, we studied genetic interactions of mab-5gof with two genes previously shown to interact with the hox gene lin- <dig> in q descendant migration, ceh- <dig> and unc- <dig> <cit> .
ceh- <dig> encodes a homeodomain transcription factor of the extradenticle/pbx family, and unc- <dig> encodes a homeodomain transcription factor of the homothorax/meis <dig> family  <cit> , vertebrate counterparts of which can act as cofactors with other hox factors to regulate gene expression  <cit> .
both ceh- <dig> and unc- <dig> controlled anterior-posterior migrations of the q neuroblasts descendants, including aqr and pqr .
in ceh- <dig> and unc- <dig> mutants, a fraction of aqr and pqr neurons migrated in the wrong direction.
unc- <dig> and ceh- <dig> are both predicted mab- <dig> targets by chip-seq  <cit> , suggesting that they might be direct transcriptional targets of mab- <dig>  nevertheless, neither gene was identified in the rna-seq analysis presented here.
we speculate that this is due to mab-5-independent expression, as ceh- <dig> broadly expressed in many cells including those that express mab- <dig> and those that do not  <cit> .
1aqr and pqr position as described in methods and figure 2d.
2significantly different  compared to mab- <dig> alone .
we tested if ceh- <dig> and unc- <dig> loss of function modified the mab- <dig> gain-of-function phenotype using a combination of mutations and rnai.
indeed, in mab-5; unc- <dig> and mab-5; ceh- <dig> double mutants, significantly fewer aqr neurons migrated posteriorly than in mab-5gof alone .
thus, genes that interact with mab- <dig> can modify the mab-5gof posterior aqr phenotype, providing proof of principle that this screen can identify genes that functionally interact with mab- <dig> in posterior migration.
it is possible that unc- <dig> and ceh- <dig> act downstream of mab- <dig> as suggested by chip seq, but it is also possible that they act together with mab- <dig>  indeed, mab- <dig> and ceh- <dig> act together to control transcription of the egl- <dig> gene to initiate programmed cell death in the q lineage  <cit> .
rna-seq targets functionally interact with mab- <dig> in posterior aqr migration
we tested mab- <dig> rna-seq targets for function in q descendant migration using rnai by feeding   <cit> .
based on availability in the rnai feeding library , we tested rnai of  <dig> genes upregulated in mab- <dig>  and  <dig> genes downregulated in mab- <dig> for the ability to modify posterior aqr migration in mab-5gof .
genes were tested in groups, and each independent experimental grouping included ceh- <dig> rnai as a positive control to ensure rnai efficacy.
each gene was retested at least once to ensure consistency of results.
mab-5gof caused 92% of aqr neurons to migrate posteriorly behind the anus to the normal posterior position of pqr, with the remaining 8% just anterior to this position .
ceh- <dig> reduced posterior aqr in mab-5gof to 59%  .
we tested three gene classes by rnai: those upregulated in mab-5gof , those downregulated in mab-5lof , and those in both , for a total of  <dig> genes tested.
sixteen of the  <dig> mab-5-upregulated genes, 3/ <dig> mab-5lof genes, and 3/ <dig> genes in both showed significantly fewer aqr in the posterior compared to the mab-5gof alone  .
rnai of the remaining  <dig> genes had no effect on mab-5gof .
1also down in mab- <dig>
2also a mab- <dig> top  <dig> chip seq target.
3also a mab- <dig> chip seq target.
4aqr position as described in methods and figure 2d.
as expected, rnai of mab-5/c08c <dig>  itself, part of the upregulated in mab- <dig> category, significantly reduced posterior aqr migration .
two additional genes confirmed by rnai have previously been implicated in controlling cell migration, mig-6/papilin and spon-1/f-spondin <cit> , validating our genomics approach to identifying functional targets of mab- <dig>  in addition, four of our rnai hits are predicted mab- <dig> chip-seq targets, mab-5/c08c <dig>  itself, fkh-7/f26d <dig> , mig-6/c37c <dig> /papilin, and k08f <dig> .
none of these rnai treatments affected pqr migration in mab-5gof, nor did they cause aqr or pqr migration defects in a wild-type background .
this is surprising for mab- <dig>  since mutations in this gene typically cause nearly all pqr to migrate anteriorly.
this could be due to the low efficacy of rnai and the possible insensitivity of the mab- <dig> locus to rnai.
our data suggest that posterior aqr migration in mab-5gof is a sensitized system to detect effects on cell migration that might be masked in anterior aqr or posterior pqr migration because of genetic redundancy.
chip-seq targets functionally interact with mab-5
we tested the top  <dig> modencode chip-seq targets  <cit>  predicted to encode proteins and that were available in the rnai library for interaction with mab-5gof aqr migration .
rnai of  <dig> of these  <dig> significantly reduced posterior aqr migration in mab-5gof .
two of these  <dig> were also in the upregulated in mab-5gof set .
none affected aqr or pqr migration alone, nor did any affect pqr migration in mab-5gof .
we note that unc- <dig> is among the top  <dig> chip-seq targets, but unc- <dig> rnai did not modify mab-5gof posterior aqr migration as did unc- <dig> mutants .
this is likely due to inefficient rnai of the unc- <dig> locus and indicates that our rnai studies are likely not identifying all of the genes that modify mab-5gof posterior aqr migration.
in total,  <dig> genes identified by rna-seq  were tested by rnai, and  <dig> affected aqr migration in mab-5gof .
fifty chip-seq targets were tested, and  <dig> had an effect .
thus, both methods  were similarly efficacious in identifying potential functional targets of mab- <dig> in posterior q descendant migration.
discussion
cell-specific transcriptome analysis in multicellular organisms usually relies on isolating rna samples from isolated single cells or cell populations.
in invertebrate model organisms such as c. elegans, isolation of rna from specific cell populations can be challenging, but has been achieved through cell sorting, cell-specific expression of an immuno-tagged poly-a binding protein and subsequent immunoprecipitation of mrnas  <cit> , and single cell dissection  <cit> .
while powerful, these techniques are not applicable to all cell types at this time.
in this work, we use whole organism rna-seq combined with mutant analysis in c. elegans to identify genes involved in q neuroblast migration.
the mab-5/hox transcription factor is necessary and sufficient for posterior q descendant migration and likely controls the expression of genes that direct posterior versus anterior migration.
our idea is that by identifying transcripts in staged l <dig> animals whose expression changes in mab- <dig> loss and gain of function mutants, the problems with using whole animal rna-seq to identify genes involved in q migration  might be mitigated.
we then used rnai and the functional test of modifying posterior aqr migration in mab-5gof to validate our approach.
this approach will not distinguish between genes that act autonomously in the q cells or non-autonomously, as mab- <dig> can act both autonomously and non-autonomously in q descendant migration.
further downstream experimentation will be required to distinguish these possibilities.
mutant rna-seq revealed genes regulated by mab- <dig> that function in q descendant migration
we conducted rna-seq on staged l <dig> animals at a time when ql has just divided and mab- <dig> expression was activated in the q cells by egl-20/wnt.
by staging animals in this manner, temporal variability in gene expression was minimized.
for example, a gene might be expressed in many cells in embryogenesis but in fewer cells at the l <dig> time point.
to focus on genes that might be important in q descendant migration, we compared rna-seq profiles from wild-type, two loss-of-function mab- <dig> mutants, and a gain-of-function mab- <dig> mutant.
we focused on genes that were upregulated in the mab-5gof and genes that were downregulated in mab-5lof as the best candidates for mab- <dig> targets that drive posterior q migration.
rnai was used to test if these genes were required for posterior aqr migration in the mab-5gof mutant, which is a sensitized background for posterior migration.
in this manner we identified  <dig> genes that modified posterior aqr in mab-5gof when subject to rnai.
these genes include known players in cell migration  as well as genes with no previously known role in cell migration.
mab- <dig> is expressed in a handful of cells in the posterior in addition to ql.
our studies do not address autonomy of function of genes in ql, and these genes might act autonomously with mab- <dig> in ql, or might non-autonomously affect q migrations along with mab- <dig> in other mab-5-expressing cells in the posterior.
in any event, whole animal rna-seq combined with mutational analysis and functional validation succeeded in identifying mab- <dig> transcriptional targets involved in q migrations.
we envision caveats with our whole-organism mutant rna-seq approach limiting its effectiveness in identifying particular subsets of the relevant genes.
our approach should readily identify genes expressed only in the q cells whose expression is dependent on mab- <dig>  as well as mab-5-dependent genes expressed in multiple cells, assuming the change in expression is sufficient to be detectable in our study.
more difficult to identify by our approach are genes expressed in multiple cells whose expression is dependent upon mab- <dig> in only a subset of cells.
this is the case with the known mab- <dig> transcriptional target mig- <dig> <cit> .
mab- <dig> inhibits expression of the transmembrane molecule mig- <dig> in posterior commissural motor neurons generated from p7-p <dig>  <cit> .
mig- <dig> is also expressed in cells independently of mab- <dig>  including the intestinal valve cells and anterior commissural motor neurons  <cit> .
in our rna-seq results, mig- <dig> expression was not significantly altered  in any comparison.
this was likely due to expression of mig- <dig> in cells not controlled by mab- <dig> increasing background noise, preventing us from detecting the expected mab-5-dependent expression change in this bona fide mab- <dig> target.
further functional validation of the candidate genes coming out of our rna-seq study may yield additional relevant loci, since mig- <dig> expression was consistently reduced in the mab-5gof genotype in comparison to wild-type and both loss-of-function mutants , consistent with mab- <dig> repression of mig- <dig> <cit> .
in addition, in comparison with wild-type, both loss-off-function alleles had increased mig- <dig> expression as expected.
the observed expression changes in these five comparisons were small, and only two were nominally significant .
functional validation of genes showing the mig- <dig> pattern might identify more relevant candidates, even if the changes are slight and not formally significant.
however, the success rate would likely be lower given the expected increase in false positives when considering genes that did not pass a stringent statistical threshold controlling for multiple tests.
the use of posterior aqr migration in mab-5gof as a sensitized background was illustrated, as none of the genes we tested by rnai had effects on pqr or aqr migration in a wild-type background but did reduce posterior aqr migration in mab-5gof.
this could in part be due to the variable efficacy of rnai as evidenced by mab- <dig> rnai having no effect on pqr migration in wild type.
it is also likely that transcriptional cascades downstream of mab- <dig> involve redundancy of function that is unmasked in the mab-5gof background.
in sum,  <dig> genes were identified that were regulated by mab- <dig> in our expression study, and modified posterior aqr migration in mab-5gof via rnai.
nineteen were upregulated in mab-5gof, six were downregulated in mab-5lof and three genes showed both patterns  .
these three genes showing opposing patterns of expression in the mab- <dig> loss- and gain-of-function genotypes are excellent candidates for targets of mab- <dig> that control q migrations.
c35d <dig>  encodes a conserved transmembrane protein of the duf <dig> family, k08f <dig>  encodes a conserved serine/threonine kinase of the mapkap <dig> family, and c53a <dig>  encodes a potential nitrophenylphosphatase that regulates phosphoglycolate metabolism  <cit> .
in addition, k08f <dig>  is a chip-seq target of mab- <dig>   <cit> , making it the only gene in this analysis that satisfies each of the criteria described here gof, and a mab- <dig> chip-seq target).
mab- <dig> targets are enriched for genes encoding molecules involved in extracellular matrix
overall, a large fraction of the genes we identified as differentially-expressed in response to mab- <dig> encoded secreted and transmembrane proteins .
of these, go terms for genes involved in extracellular matrix formation and rearrangement were enriched .
genes upregulated by mab- <dig> were enriched for those encoding cub domains and thrombospondin type i domains.
those downregulated by mab-5gof were enriched for genes encoding collagens, zinc metalloproteases, and kunitz protease inhibitors, which form and modify extracellular matrices.
genes encoding the shk/metridin domain, found in sea anemone toxins that are inhibitors of voltage gated potassium channels  <cit> , were enriched, as well as a glycine-rich domain of unknown function.
relatively fewer genes were found that encode molecules involved in gene expression, such as transcription factors, although the bath- <dig>  a potential transcriptional regulator, and fhk- <dig>  a forkhead family transcription factor, both modified mab- <dig> posterior aqr.
mab- <dig> predominantly affected genes encoding secreted and transmembrane molecules, suggesting that mab- <dig> influences cell-extracellular matrix interactions.
this is consistent with the mab- <dig> phenotype in ql descendants where only direction of migration is affected but not cell division, cell death, or cell fate.
migrating cells must modify interactions with basements membranes, including remodeling during normal development as well as invasion across basement membranes in normal development and tumor metastasis gof could be detected despite broad background expression).
expression of spon- <dig> and mig- <dig> in the q cells has not been demonstrated, but different mig- <dig> isoforms can act autonomously and non-autonomously in gonadal distal tip cell migration  <cit> , indicating that a secreted molecule can act autonomously on the cell producing it.
future studies will be aimed at determining the autonomy of function of mab- <dig> targets and their mechanistic roles in driving posterior migration of q descendants in response to the mab-5/hox transcription factor.
CONCLUSIONS
we used whole organism rna seq combined with mutant analysis to identify genes regulated by the mab-5/hox transcription factor in q neuroblast descendant anterior-posterior migrations in c. elegans.
wild-type, mab- <dig> loss-of-function and mab- <dig> gain-of-function strains were subjected to rna seq, and genes upregulated or downregulated in the different mutant lines were identified.
genes regulated by mab- <dig> were enriched for those encoding secreted and transmembrane molecules that affect extracellular matrix, indicating that mab- <dig> might mediate cell interactions with the extracellular matrix.
31% of genes upregulated in the mab- <dig> gain-of-function and downregulated in mab- <dig> loss-of-function had an effect on q descendant migration when perturbed by rnai, similar to the 38% of genes identified previously by chip seq.
our results indicate that whole organism rna seq combined with mutant analysis can identify genes involved in a specific developmental process, in this case q neuroblast descendant anterior-posterior migration.

 the ability to assay the transcriptional activity of every gene in the genome has been facilitated by microarrays and now by next-generation high-throughput transcript sequencing. large-scale transcriptome profiling allows new questions to be asked about the roles of transcription factors in development and the cascades of genes that they influence to drive cell fate and differentiation. understanding transcriptional programs is also central to understanding disease and pathological conditions resulting from failures in these fundamental processes. multicellular organisms present challenges to understanding developmentally-relevant transcriptional programs, as cell-specific differences in gene expression can be masked by expression in multiple cell types. isolation of specific cell types can solve this problem. in c. elegans, embryonic neurons were isolated by fluorescence-activated cell sorting and their transcriptional profile assayed by microarrays. in the same report, transcripts from larval neurons were isolated by expression of a tagged poly-a binding protein in neurons and subsequent immunoprecipitation of flag-pab- and associated transcripts. recently, single migrating linker cells of the c. elegans male gonad were dissected from living animals and transcriptionally profiled using rna-seq. despite these advances, isolation of cell-specific transcripts from model organisms using these techniques is not trivial, and not feasible for every cell type at this time. in the current study we test the efficacy of combining the advantageous genetics and well-understood development of the nematode c. elegans with whole organism rna-seq to identify genes with effects on the migration of the q neuroblasts. by comparing transcript levels in wild type and various mab- mutant backgrounds defective in q cell migration, we test if genes with effects on q migration can be identified by rna-seq of whole animal lysates. the q cells are neuroblasts that reside bilaterally in the posterior region of the worm. the q cells, left and right, undergo an identical pattern of division coupled with cell death to produce three neurons. while the q cells divide in a similar fashion, ql descendants migrate posteriorly and qr descendants migrate anteriorly. ql on the left undergoes an initial migration towards the posterior of the worm and divides to give rise to the pvm, sdql, and pqr neurons. these ql descendants continue to migrate posteriorly, with pqr migrating the farthest to reside in the tail of the worm behind the anus. qr on the right undergoes a similar pattern of migration and division, except the cells migrate to the anterior, generating the avm, sdqr, and aqr neurons. aqr migrates the farthest to reside in the head of the worm near the anterior deirid sensory organ. q cell migration occurs in two stages. the initial stage relies on q cell polarization regulated by the transmembrane molecules unc-40/dcc, ptp-3/lar, and mig-. when ql undergoes its initial migration towards the posterior, it encounters a graded egl-20/wnt signal, which is secreted from muscle and epidermal cells at the posterior of the animal. in this second phase of migration, egl-20/wnt induces expression of the mab-5/hox transcription factor in ql, but not qr, via canonical wnt signaling. mab- an antennapedia-like hox transcription factor, is essential for the posterior migration of the ql descendants and acts as a determinant for posterior migration of q descendants. loss of function of mab- results in anterior migration of the ql descendants, despite normal initial posterior migration of ql. a gain-of-function mab- mutant that results in mab- expression in both ql and qr independent of wnt signaling causes both ql and qr descendants to migrate posteriorly despite normal initial q cell migrations. induced autonomous expression of mab- during anterior q descendant migration results in the cells changing direction to the posterior, and mab- is needed continuously during posterior migration. thus, mab- is both necessary and sufficient for autonomously directing posterior versus anterior migration of q descendants. in addition to ql descendants, mab- is expressed in a handful of other cells in the posterior in the mid-l stage, including the hypodermal v and v cells, p7-p the m mesoblast, and several posterior body wall muscles. mab- inhibits the expression of the transmembrane molecule mig- in posterior commissural motor neurons generated from p7-p. mig- is non-autonomously required for the anterior migration of qr descendants on the right. thus, mab- might control gene expression outside of the q cells that non-autonomously controls q descendant migration. our whole organism rna-seq approach has the potential to identify both autonomous and non-autonomous targets of mab- in q descendant migration. the results reported here do not distinguish between autonomy and non-autonomy, which will be the subject of future study. it is important to note that ql cell descendants are still generated and still migrate in the absence of mab- but in the opposite direction. additionally, aqr and pqr are thought to have the same functions of sensing oxygen and regulating social feeding in the worm, even though mab- is expressed in pqr and not in aqr in wild type worms. these factors suggest that mab- is specifically involved in determining the direction of migration of the ql cell descendants and not in the specification of neuronal class identity or function or in their ability to migrate. mab-5/hox represents an excellent opportunity to dissect the transcriptional programs that specify differentiation, in this case posterior versus anterior cell migration. we take advantage of both loss-of-function and gain-of-function alleles of mab- to identify genes whose transcriptional levels are affected by each, and then functionally analyze their roles in q descendant migrations using rnai. we find that while some genes are oppositely affected in loss versus gain-of-function backgrounds, the majority of genes are unique in each set, indicating redundancy and combinatorial function of mab- in regulating transcription. we also find that mab- target genes are enriched for those encoding secreted proteins involved in extracellular matrix formation and remodeling, some of which have functional roles in q descendant migration. this suggests that mab- might alter cell-extracellular matrix interaction to direct posterior migration. finally, we functionally compare our rna-seq results with the mab- chip-seq results obtained by the modencode project and find that mutant rna-seq and chip-seq are both efficient methods to identify functional targets of mab- in posterior q descendant migration.  transcriptional profiling of wild-type and mab- mutant l animals using rna-seq to identify genes whose expression is influenced by mab- we conducted rna-seq analysis on wild-type worms, two mab- loss-of-function alleles e and gk lof and mab-5lof, respectively), and the mab- gain-of-function allele gof). total rna was isolated from each genotype at the early l larval stage approximately -5 h after hatching, after the q cells had undergone their initial migrations and divisions, and when mab- is activated by egl-20/wnt signaling in ql and ql descendants. rna from multiple biological replicates of each genotype was then subjected to rna-seq using illumina single-end 60-bp reads. reads were assigned to genes using tophat, and pairwise expression differences between genotypes analyzed using the cuffdiff module in cufflinks. differential gene expression between genotypes was assessed using an fdr-adjusted q-value to account for multiple-testing, and only those genes with significant changes are discussed. cuffdiff files for each pairwise comparison can be found in additional files and and protein-coding genes with significant expression changes can be found in additional file due to our column-based total rna isolation procedure, and the poly-a selection step prior to rna-seq, few small, non-coding rna molecules should be represented in our sequencing data. nevertheless, some small nuclear snrnas, small nucleolar snornas, and trans-spliced leader rnas were found to be differentially expressed. it is possible that certain high-abundance genes in these categories are inefficiently removed by poly-a selection. no mirna genes were identified as differentially expressed, primarily because very few mirna reads were recovered by our protocol. given the poor, and potentially inconsistent representation of reads associated small non-coding rna genes in our study, these genes are excluded from further analysis. nevertheless, small non-coding rna genes are included in additional files and and those showing significant differential expression between genotypes are listed in additional file we first determined genes that were down- or upregulated in the two mab- lof backgrounds compared to wild-type. ten genes were significantly downregulated in mab-5lof compared to wild-type, while genes were downregulated in mab-5lof. five genes were downregulated in both lof mutants, for a total of genes downregulated in at least one comparison. fewer genes were found to be upregulated in the lof backgrounds compared to wild-type: seven in mab-5lof and six in mab-5lof, with four in both, for a total of nine genes upregulated in at least one comparison. 1these categories also included the retired gene r07b, which was not included in further analysis. we note that the disparity in the number of downregulated genes identified in the comparisons to the two different lof mutants is not explained by differences in overall rna-seq read count for the two mutant genotypes. since mab- is a splice donor mutation in the second intron, whereas mab- is a deletion of a large region of the locus, it is possible that mab- retains some activity and thus more weakly affects gene expression than does mab- interestingly, the mab- locus itself was not significantly underrepresented in the two lof backgrounds. indeed, in the mab-5lof mutant, a 2-bp insertion at mab- was apparent in the rna-seq reads due to the use of a cryptic 5′ splice site 2 bp downstream from the wild-type site because of an altered splice donor site in the first mab- intron. in the mab-5lof mutant, the rna-seq data at mab- was consistent with the known deletion at this locus. these data suggest that the mab- transcript might not be subject to nonsense-mediated mrna decay. in the mab-5gof background, genes were downregulated and genes were upregulated compared to wild type,. we expected that mab- itself would be upregulated in mab-5gof relative to wild-type, since this mutation causes upregulated mab- expression in multiple cells. while mab- gene expression is higher in the gof than wildtype this change is not significant after correcting for multiple tests. to identify other genes potentially regulated by mab- we compared genes that were differentially expressed between each lof mutant and the gof, with the idea that this might be a more sensitive and powerful approach than comparing each mutant class separately with wild type. seventeen genes were downregulated in mab-5gof compared to mab-5lof, and were down in mab-5gof compared to mab-5lof, with in both. six of these genes were also downregulated in mab-5gof compared to wild-type. twenty four genes were upregulated in mab-5gof relative to mab-5lof, and upregulated relative to mab-5lof, with of the genes differentially expressed in the mab-5lof comparison also among those in the mab-5lof comparison. differentially expressed gene sets for in silico evaluation and functional testing our ultimate goal with this work was to identify and characterize genes arising from our rna-seq screen that function with mab- in neuronal migration. in this work, we focus solely on those genes that exhibit significant gene expression changes after accounting for multiple testing. this will clearly exclude some true mab- targets, but should provide a gene set with a high probability of success in experimental functional validation. the mab- gene itself showed significant differential expression only in the comparison between the mab-5lof and the mab-5gof. however, the comparisons between the mab-5lof and mab-5gof and between wild-type and the mab-5gof were nominally significant and in the anticipated direction. this indicates that bona fide mab- targets might appear significant in only one of the comparisons of wild-type and the two lof backgrounds to the gof. thus, for further characterization we combined genes from all the different comparisons described above into four groups: genes total were downregulated in lof backgrounds compared to wildtype, while were upregulated. in combined gof comparisons to wild-type and the two lof backgrounds, genes were downregulated in gof and were upregulated. opposing regulation in loss- versus gain-of-function some transcriptional targets of mab- may be differentially-regulated in opposite directions in the lof and gof conditions might be upregulated in mab-5). of the genes downregulated in either lof mutant compared to wild-type, were upregulated in the mab-5gof combined gene set, while of the genes upregulated in the lof mutants was downregulated in gof combined. thus, there was not a strict concordance between gene expression in the lof and gof backgrounds, but there was significant overlap. simple statistical power concerns could explain the lack of precise concordance, but it is also possible that mab- acts redundantly and combinatorially with other transcription factors to influence gene expression. genes encoding secreted and transmembrane molecules are enriched among mab-5-regulated genes for each of the four pools of genes or mab- backgrounds), we classified genes based on their predicted cellular location and molecular function as annotated in wormbase. within each group, molecules with predicted transmembrane domains and/or a predicted signal sequence were by far the most abundant class. this enrichment for secreted and transmembrane proteins was statistically significant in three of the four groups using a binomial analysis based on the fraction of such genes in the worm genome. we note that while the mab- upregulated gene set did not show a significant excess of tmhmm and signalp-annotated genes, at genes this is the smallest of the four groups of genes. the remaining genes in the four pooled sets are predicted to encode molecules acting in transcription and gene expression, cytoplasmic signaling, metabolism, and the cytoskeleton. 1this set includes only protein-encoding genes and not small rna genes. from gene annotation files from wormbase, there were genes with tmhmm and/or signalp annotation out of total. 2binomial distribution test comparing fractions of transmembrane and secreted proteins in the gene sets compared to the total fraction in the genome. function and gene ontology of genes affected by mab-5 we used the database for annotation, visualization and integrated discovery to perform a gene ontology term enrichment and functional clustering analysis on the groups of mab-5-regulated genes. genes upregulated in mab- were enriched for go terms involving aryl phosphatase and had phosphatase activity. however, genes in this class were few in number and did not return highly significant go term functional clustering. genes that were downregulated in mab- returned go term enrichment for transmembrane proteins, as expected from the domain analysis above. this group was significantly enriched for go terms involving secreted glycine-rich proteins. terms and a functional cluster involving atpase activity and nucleotide binding were also identified. this is due to the downregulation of two genes in mab- pmp-5/t10h and pgp-4/f42e, that encode abc transporter-family proteins. mab- affects genes involved in extracellular matrix among genes upregulated in mab- the most significantly enriched domain is related to an extracellular domain encoded by genes induced by fungal infection. three genes, fipr- fipr- and fipr-, harbor this domain. two other extracellular domains were also significantly-enriched; the complement c1r/c1s/uegf/bmp domain, and the thrombospondin type i. other enriched domains include aryl phosphatase pho13/phosphoglycolate phosphatase domain and the had-superfamily hydrolase subfamily iia domain, both of which have been implicated in carbohydrate metabolism. as mentioned above, two genes harboring these domains were also upregulated in the mab- background, so their functional relationship to mab- is unclear. this table is a summary of the significant go terms and functional clusters found in additional file genes downregulated in mab- were enriched for go terms and functional clusters involving collagens, secreted metalloproteases and their inhibitors, cysteine-rich repeats found in a variety of secreted proteins, proteins that form disulfide bonds, the shkt/metridin toxin domain found in secreted proteins, and histones. this gene group includes: collagen genes; seven genes encoding secreted keratin-like or keratin-associated molecules; three genes encoding secreted astacin metalloproteases; three genes encoding secreted kunitz serine protease inhibitors; four genes encoding secreted proteins containing the shk/metridin toxin domain; and four histone genes. three genes encoding secreted glycine-rich repeats were downregulated in mab- but these were also downregulated in mab- suggesting that they might not be relevant targets. in sum, this go term enrichment analysis suggests that mab- predominantly regulates genes encoding secreted and transmembrane molecules. based on the mab- results, genes upregulated by mab- tend to encode secreted and transmembrane molecules with domains involved in signaling, cell adhesion, and fungal defense. genes downregulated by mab- tend to encode secreted molecules that are involved in the formation and remodeling of the extracellular matrix, suggesting mab- might alter cell interactions with the extracellular matrix. indeed, two genes upregulated by mab- spon-1/f-spondin and mig-6/papilin, encode secreted molecules containing both kunitz-type proteinase inhibitor domains and tsp domains, and are important for cell migration. comparison of mab- rna-seq data with modencode chip-seq data regions of the genome with which mab- interacts physically were identified previously by chromatin immunoprecipitation and dna sequencing as part of the modencode project. we compared a list of potential mab- chip seq targets with our rna seq data set: 2/ genes downregulated in mab- were present in the annotated chip seq targets, and 1/ mab- upregulated genes were included. in the mab- comparisons, 4/ downregulated genes and 8/ upregulated genes were included. thus, while some rna-seq and chip-seq targets were in common, there was not strict concordance between targets identified by the two methodologies. one caveat is that the chip-seq data were gathered from l larval animals whereas our rna-seq data were gathered from l larval animals. it is also possible that mab- regulates many genes indirectly, or that mab- is redundant with other transcription factors in the regulation of many genes. the mab- gene itself was one of the genes included in both the rna-seq and chip-seq data sets. mab- has been shown to regulate its own expression by a positive feedback mechanism to maintain mab- expression in the q neuroblast descendants during their posterior migrations. also identified by both methods was k08f, which encodes a map kinase activated protein kinase similar to vertebrate mapkap k08f also showed opposite regulation in mab- lof and gof backgrounds. posterior aqr migration in mab-5gof as a sensitized background to identify mab- targets that function in posterior migration the goal of this work is to identify genes regulated by mab- that function in q descendant migration. mab- is required for posterior migration of the q neuroblast descendants, and can also drive posterior migration of the qr descendants, which normally migrate anteriorly gof results in posterior migration of qr descendants including aqr; figure 2c). genes upregulated in mab-5gof and downregulated in mab- are good candidates for those that might mediate posterior q descendant migration downstream of mab- loss of such a gene’s function might modify mab-5gof posterior aqr migration, resulting in more anterior aqr positioning. since aqr normally migrates anteriorly, posteriorly-migrating aqr in mab-5gof might represent a sensitized background to identify effects on posterior migration that might be missed in posterior pqr migration. thus, we tested genes identified by our rna-seq analysis for their ability to modify posterior migration of aqr in the mab-5gof mutant. ceh- and unc- are epistatic to mab- in posterior aqr migration as proof of principle of using posterior aqr migration in mab-5gof as a background to define mab- targets that affect q cell descendant migrations, we studied genetic interactions of mab-5gof with two genes previously shown to interact with the hox gene lin- in q descendant migration, ceh- and unc-. ceh- encodes a homeodomain transcription factor of the extradenticle/pbx family, and unc- encodes a homeodomain transcription factor of the homothorax/meis family, vertebrate counterparts of which can act as cofactors with other hox factors to regulate gene expression. both ceh- and unc- controlled anterior-posterior migrations of the q neuroblasts descendants, including aqr and pqr. in ceh- and unc- mutants, a fraction of aqr and pqr neurons migrated in the wrong direction. unc- and ceh- are both predicted mab- targets by chip-seq, suggesting that they might be direct transcriptional targets of mab- nevertheless, neither gene was identified in the rna-seq analysis presented here. we speculate that this is due to mab-5-independent expression, as ceh- broadly expressed in many cells including those that express mab- and those that do not. 1aqr and pqr position as described in methods and figure 2d. 2significantly different compared to mab- alone. we tested if ceh- and unc- loss of function modified the mab- gain-of-function phenotype using a combination of mutations and rnai. indeed, in mab-5; unc- and mab-5; ceh- double mutants, significantly fewer aqr neurons migrated posteriorly than in mab-5gof alone. thus, genes that interact with mab- can modify the mab-5gof posterior aqr phenotype, providing proof of principle that this screen can identify genes that functionally interact with mab- in posterior migration. it is possible that unc- and ceh- act downstream of mab- as suggested by chip seq, but it is also possible that they act together with mab- indeed, mab- and ceh- act together to control transcription of the egl- gene to initiate programmed cell death in the q lineage. rna-seq targets functionally interact with mab- in posterior aqr migration we tested mab- rna-seq targets for function in q descendant migration using rnai by feeding. based on availability in the rnai feeding library, we tested rnai of genes upregulated in mab- and genes downregulated in mab- for the ability to modify posterior aqr migration in mab-5gof. genes were tested in groups, and each independent experimental grouping included ceh- rnai as a positive control to ensure rnai efficacy. each gene was retested at least once to ensure consistency of results. mab-5gof caused 92% of aqr neurons to migrate posteriorly behind the anus to the normal posterior position of pqr, with the remaining 8% just anterior to this position. ceh- reduced posterior aqr in mab-5gof to 59%. we tested three gene classes by rnai: those upregulated in mab-5gof, those downregulated in mab-5lof, and those in both, for a total of genes tested. sixteen of the mab-5-upregulated genes, 3/ mab-5lof genes, and 3/ genes in both showed significantly fewer aqr in the posterior compared to the mab-5gof alone. rnai of the remaining genes had no effect on mab-5gof. 1also down in mab- 2also a mab- top chip seq target. 3also a mab- chip seq target. 4aqr position as described in methods and figure 2d. as expected, rnai of mab-5/c08c itself, part of the upregulated in mab- category, significantly reduced posterior aqr migration. two additional genes confirmed by rnai have previously been implicated in controlling cell migration, mig-6/papilin and spon-1/f-spondin, validating our genomics approach to identifying functional targets of mab- in addition, four of our rnai hits are predicted mab- chip-seq targets, mab-5/c08c itself, fkh-7/f26d, mig-6/c37c /papilin, and k08f. none of these rnai treatments affected pqr migration in mab-5gof, nor did they cause aqr or pqr migration defects in a wild-type background. this is surprising for mab- since mutations in this gene typically cause nearly all pqr to migrate anteriorly. this could be due to the low efficacy of rnai and the possible insensitivity of the mab- locus to rnai. our data suggest that posterior aqr migration in mab-5gof is a sensitized system to detect effects on cell migration that might be masked in anterior aqr or posterior pqr migration because of genetic redundancy. chip-seq targets functionally interact with mab-5 we tested the top modencode chip-seq targets predicted to encode proteins and that were available in the rnai library for interaction with mab-5gof aqr migration. rnai of of these significantly reduced posterior aqr migration in mab-5gof. two of these were also in the upregulated in mab-5gof set. none affected aqr or pqr migration alone, nor did any affect pqr migration in mab-5gof. we note that unc- is among the top chip-seq targets, but unc- rnai did not modify mab-5gof posterior aqr migration as did unc- mutants. this is likely due to inefficient rnai of the unc- locus and indicates that our rnai studies are likely not identifying all of the genes that modify mab-5gof posterior aqr migration. in total, genes identified by rna-seq were tested by rnai, and affected aqr migration in mab-5gof. fifty chip-seq targets were tested, and had an effect. thus, both methods were similarly efficacious in identifying potential functional targets of mab- in posterior q descendant migration. discussion cell-specific transcriptome analysis in multicellular organisms usually relies on isolating rna samples from isolated single cells or cell populations. in invertebrate model organisms such as c. elegans, isolation of rna from specific cell populations can be challenging, but has been achieved through cell sorting, cell-specific expression of an immuno-tagged poly-a binding protein and subsequent immunoprecipitation of mrnas, and single cell dissection. while powerful, these techniques are not applicable to all cell types at this time. in this work, we use whole organism rna-seq combined with mutant analysis in c. elegans to identify genes involved in q neuroblast migration. the mab-5/hox transcription factor is necessary and sufficient for posterior q descendant migration and likely controls the expression of genes that direct posterior versus anterior migration. our idea is that by identifying transcripts in staged l animals whose expression changes in mab- loss and gain of function mutants, the problems with using whole animal rna-seq to identify genes involved in q migration might be mitigated. we then used rnai and the functional test of modifying posterior aqr migration in mab-5gof to validate our approach. this approach will not distinguish between genes that act autonomously in the q cells or non-autonomously, as mab- can act both autonomously and non-autonomously in q descendant migration. further downstream experimentation will be required to distinguish these possibilities. mutant rna-seq revealed genes regulated by mab- that function in q descendant migration we conducted rna-seq on staged l animals at a time when ql has just divided and mab- expression was activated in the q cells by egl-20/wnt. by staging animals in this manner, temporal variability in gene expression was minimized. for example, a gene might be expressed in many cells in embryogenesis but in fewer cells at the l time point. to focus on genes that might be important in q descendant migration, we compared rna-seq profiles from wild-type, two loss-of-function mab- mutants, and a gain-of-function mab- mutant. we focused on genes that were upregulated in the mab-5gof and genes that were downregulated in mab-5lof as the best candidates for mab- targets that drive posterior q migration. rnai was used to test if these genes were required for posterior aqr migration in the mab-5gof mutant, which is a sensitized background for posterior migration. in this manner we identified genes that modified posterior aqr in mab-5gof when subject to rnai. these genes include known players in cell migration as well as genes with no previously known role in cell migration. mab- is expressed in a handful of cells in the posterior in addition to ql. our studies do not address autonomy of function of genes in ql, and these genes might act autonomously with mab- in ql, or might non-autonomously affect q migrations along with mab- in other mab-5-expressing cells in the posterior. in any event, whole animal rna-seq combined with mutational analysis and functional validation succeeded in identifying mab- transcriptional targets involved in q migrations. we envision caveats with our whole-organism mutant rna-seq approach limiting its effectiveness in identifying particular subsets of the relevant genes. our approach should readily identify genes expressed only in the q cells whose expression is dependent on mab- as well as mab-5-dependent genes expressed in multiple cells, assuming the change in expression is sufficient to be detectable in our study. more difficult to identify by our approach are genes expressed in multiple cells whose expression is dependent upon mab- in only a subset of cells. this is the case with the known mab- transcriptional target mig-. mab- inhibits expression of the transmembrane molecule mig- in posterior commissural motor neurons generated from p7-p. mig- is also expressed in cells independently of mab- including the intestinal valve cells and anterior commissural motor neurons. in our rna-seq results, mig- expression was not significantly altered in any comparison. this was likely due to expression of mig- in cells not controlled by mab- increasing background noise, preventing us from detecting the expected mab-5-dependent expression change in this bona fide mab- target. further functional validation of the candidate genes coming out of our rna-seq study may yield additional relevant loci, since mig- expression was consistently reduced in the mab-5gof genotype in comparison to wild-type and both loss-of-function mutants, consistent with mab- repression of mig-. in addition, in comparison with wild-type, both loss-off-function alleles had increased mig- expression as expected. the observed expression changes in these five comparisons were small, and only two were nominally significant. functional validation of genes showing the mig- pattern might identify more relevant candidates, even if the changes are slight and not formally significant. however, the success rate would likely be lower given the expected increase in false positives when considering genes that did not pass a stringent statistical threshold controlling for multiple tests. the use of posterior aqr migration in mab-5gof as a sensitized background was illustrated, as none of the genes we tested by rnai had effects on pqr or aqr migration in a wild-type background but did reduce posterior aqr migration in mab-5gof. this could in part be due to the variable efficacy of rnai as evidenced by mab- rnai having no effect on pqr migration in wild type. it is also likely that transcriptional cascades downstream of mab- involve redundancy of function that is unmasked in the mab-5gof background. in sum, genes were identified that were regulated by mab- in our expression study, and modified posterior aqr migration in mab-5gof via rnai. nineteen were upregulated in mab-5gof, six were downregulated in mab-5lof and three genes showed both patterns. these three genes showing opposing patterns of expression in the mab- loss- and gain-of-function genotypes are excellent candidates for targets of mab- that control q migrations. c35d encodes a conserved transmembrane protein of the duf family, k08f encodes a conserved serine/threonine kinase of the mapkap family, and c53a encodes a potential nitrophenylphosphatase that regulates phosphoglycolate metabolism. in addition, k08f is a chip-seq target of mab-, making it the only gene in this analysis that satisfies each of the criteria described here gof, and a mab- chip-seq target). mab- targets are enriched for genes encoding molecules involved in extracellular matrix overall, a large fraction of the genes we identified as differentially-expressed in response to mab- encoded secreted and transmembrane proteins. of these, go terms for genes involved in extracellular matrix formation and rearrangement were enriched. genes upregulated by mab- were enriched for those encoding cub domains and thrombospondin type i domains. those downregulated by mab-5gof were enriched for genes encoding collagens, zinc metalloproteases, and kunitz protease inhibitors, which form and modify extracellular matrices. genes encoding the shk/metridin domain, found in sea anemone toxins that are inhibitors of voltage gated potassium channels, were enriched, as well as a glycine-rich domain of unknown function. relatively fewer genes were found that encode molecules involved in gene expression, such as transcription factors, although the bath- a potential transcriptional regulator, and fhk- a forkhead family transcription factor, both modified mab- posterior aqr. mab- predominantly affected genes encoding secreted and transmembrane molecules, suggesting that mab- influences cell-extracellular matrix interactions. this is consistent with the mab- phenotype in ql descendants where only direction of migration is affected but not cell division, cell death, or cell fate. migrating cells must modify interactions with basements membranes, including remodeling during normal development as well as invasion across basement membranes in normal development and tumor metastasis gof could be detected despite broad background expression). expression of spon- and mig- in the q cells has not been demonstrated, but different mig- isoforms can act autonomously and non-autonomously in gonadal distal tip cell migration, indicating that a secreted molecule can act autonomously on the cell producing it. future studies will be aimed at determining the autonomy of function of mab- targets and their mechanistic roles in driving posterior migration of q descendants in response to the mab-5/hox transcription factor.  we used whole organism rna seq combined with mutant analysis to identify genes regulated by the mab-5/hox transcription factor in q neuroblast descendant anterior-posterior migrations in c. elegans. wild-type, mab- loss-of-function and mab- gain-of-function strains were subjected to rna seq, and genes upregulated or downregulated in the different mutant lines were identified. genes regulated by mab- were enriched for those encoding secreted and transmembrane molecules that affect extracellular matrix, indicating that mab- might mediate cell interactions with the extracellular matrix. 31% of genes upregulated in the mab- gain-of-function and downregulated in mab- loss-of-function had an effect on q descendant migration when perturbed by rnai, similar to the 38% of genes identified previously by chip seq. our results indicate that whole organism rna seq combined with mutant analysis can identify genes involved in a specific developmental process, in this case q neuroblast descendant anterior-posterior migration. 
here we test the utility of combining a whole organism rna-seq approach with mab- loss and gain-of-function mutants and functional validation using rnai to identify genes regulated by mab- to control q descendant migration. in c. elegans the mab-5/hox transcription factor is a determinant of posterior migration of the q neuroblast descendants. our results indicate that a whole-organism rna-seq approach, when combined with mutant analysis and functional validation, can be a powerful method to identify genes involved in a specific developmental process, in this case q descendant posterior migration. cell-specific rna samples can be difficult to obtain from invertebrate model organisms such as drosophila and c. elegans. in this work, mab- transcriptional targets that control q descendant migration are identified by comparing rna-seq profiles in wild type and mab- mutant backgrounds. transcriptome profiling is a widely-used and potent tool to identify genes involved in developmental and pathological processes, and is most informative when rna can be isolated from individual cell or tissue types. directed cell migration is a fundamental process in normal development and in tumor metastasis. we identified genes whose expression was controlled by mab- and that controlled q descendant migration. genes regulated by mab- were enriched for secreted and transmembrane molecules involved in basement membrane interaction and modification, and some affected q descendant migration. these genes could act either autonomously in the q cells, or non-autonomously in other cells that express mab- the identities of the genes regulated by mab- indicate that mab- acts by modifying interactions with the basement membrane, resulting in posterior versus anterior migration.
 directed cell migration is a fundamental process in normal development and in tumor metastasis. in c. elegans the mab-5/hox transcription factor is a determinant of posterior migration of the q neuroblast descendants. in this work, mab- transcriptional targets that control q descendant migration are identified by comparing rna-seq profiles in wild type and mab- mutant backgrounds.  transcriptome profiling is a widely-used and potent tool to identify genes involved in developmental and pathological processes, and is most informative when rna can be isolated from individual cell or tissue types. cell-specific rna samples can be difficult to obtain from invertebrate model organisms such as drosophila and c. elegans. here we test the utility of combining a whole organism rna-seq approach with mab- loss and gain-of-function mutants and functional validation using rnai to identify genes regulated by mab- to control q descendant migration. we identified genes whose expression was controlled by mab- and that controlled q descendant migration. genes regulated by mab- were enriched for secreted and transmembrane molecules involved in basement membrane interaction and modification, and some affected q descendant migration.  our results indicate that a whole-organism rna-seq approach, when combined with mutant analysis and functional validation, can be a powerful method to identify genes involved in a specific developmental process, in this case q descendant posterior migration. these genes could act either autonomously in the q cells, or non-autonomously in other cells that express mab- the identities of the genes regulated by mab- indicate that mab- acts by modifying interactions with the basement membrane, resulting in posterior versus anterior migration. 