Human FEV cis-regulatory elements direct expression to developing and adult 5HT neurons
Rodent 5HT neurons develop in two ventral hindbrain domains, a rostral domain extending from rhombomere (r) r1 to r3, and a caudal domain extending from r5-r7 (Lidov and Molliver, 1982
; Wallace and Lauder, 1983
; Hendricks et al., 1999
expression is initiated at E10.5-E11 in the mouse rostral domain followed about 1 day later by expression in the caudal domain (Pfaar et al., 2002
; Pattyn et al., 2003
). In the adult brain a large number of 5HT neurons are present in the midline raphe nuclei, although significant numbers are also found scattered in more lateral regions of the pontine reticular formation and ventrolateral medulla (Steinbusch, 1981
). Expression of the endogenous Pet-1
gene is maintained in what appears to be all 5HT neurons of the adult brain (Hendricks et al., 1999
Our previous studies showed that a 1.8 kb fragment immediately upstream of mouse Pet-1
was sufficient to direct transgene expression in embryonic and adult 5HT neurons (Scott et al., 2005
). Comparison of the Pet-1
upstream regions revealed two blocks of sequence conservation (). The proximal block includes the FEV
5'-untranslated exon and extends 310 bp into the FEV
upstream region. The distal block comprises three subregions, the first of which begins 895 bp upstream of the FEV
start site. To determine whether or not 5HT neuron-specific elements are located among these sequences we isolated a 2.2 kb FEV
genomic fragment whose 3' end is located in the FEV
5' untranslated region (). This fragment was ligated upstream of a β-globin TATA box/lacZ/poly(A) signal sequence cassette (Yee and Rigby, 1993
; Helms et al., 2000
) to prepare transgene reporter FEV2.2Z. The β-globin TATA box was included in this and other transgenes to ensure the presence of a start site as neither the Pet-1
promoters have been experimentally localized.
Seven FEV2.2Z lines were analyzed for expression at embryonic stages. Similar to endogenous Pet-1, FEV2.2Z expression was initiated in the rostral hindbrain between E10.5-E11 (data not shown) and then expanded into the caudal domain by E12.5 () in six of seven lines (). Co-immunostaining with anti-5HT and anti-β-galactosidase (βgal) revealed a 5HT neuron specific pattern of FEV2.2Z expression in the hindbrain (). Expression of FEV2.2Z, however, was weak in the posterior half of the caudal hindbrain near the cervical flexure () in all but one line.
FEV is controlled by a cis-regulatory region that directs expression to developing and adult 5HT neurons
In eleven of twelve lines, adult FEV2.2Z expression was reproducibly present in the DRN and median raphe nuclei (MRN) as well as in the reticular formation and ventrolateral medulla ( and data not shown). Coimmunostaining of FEV2.2Z mice indicated that the majority of βgal+ cells expressed tryptophan hydroxylase (TPH), the rate-limiting enzyme for 5HT biosynthesis, in each of the adult raphe nuclei (). However, a small number of βgal−/TPH+cells was observed in all FEV2.2Z lines (), and FEV2.2Z expression was missing from most 5HT neurons in the B2 raphe obscurus (data not shown) in all but one line. These cells likely correspond to cells derived from the posterior half of the embryonic caudal hindbrain, and therefore additional cis regulatory elements probalby direct expression to this subdomain. Nevertheless, these findings show that a conserved regulatory region upstream of FEV directs transgene expression to developing and adult 5HT neurons.
FEV promoter sequences are necessary and sufficient for embryonic serotonergic transgene expression
We next tested several additional FEV transgenes to determine the location of essential serotonergic cis regulatory elements within the FEV 2.2 kb fragment. To investigate the importance of distal conserved sequences, we generated two transgenes with 5' truncations of either 1.1 kb or 1.6 kb (FEV1.1Z or FEV0.6Z, respectively) (). Similar to FEV2.2Z, both FEV1.1Z and FEV0.6Z expression was restricted to the rostral and caudal hindbrain (). Further analysis of FEV0.6Z indicated that FEV0.6Z was expressed in a 5HT neuron specific pattern in the hindbrain (), but expression was again lacking in posterior caudal 5HT neurons (). Thus, important cis regulatory elements for embryonic expression are not likely to be associated with the distal conserved sequences. These findings additionally suggest that conserved sequences surrounding the FEV transcriptional start site (−275/+318) constitute a minimal FEV cis-regulatory region sufficient for 5HT neuron-specific transgene expression in the developing hindbrain. However, when four FEV0.6Z lines showing restricted serotonergic expression of LacZ in the embryonic hindbrain were evaluated at adult stages, only one of them continued to express LacZ, and this expression was very weak (data not shown). Thus, additional regulatory elements that are present in FEV2.2Z but missing in FEV0.6Z are likely to be required for maintenance of expression.
Proximal conserved sequences are necessary and sufficient for 5HT neuron-specific expression in the developing hindbrain
We next asked whether proximal conserved sequences were necessary for 5HT neuron specific expression by testing a transgene, FEV2.2ΔZ, in which sequences −214 to +25 of the proximal block were deleted in the context of the 2.2 kb upstream fragment (). Transgenic founders were analyzed at E12.5 using whole-mount Xgal staining. Deletion of these sequences led to a complete loss of serotonergic expression of FEV2.2ΔZ although ectopic expression was present (). Similar results were obtained for a second transgene, FEV1.1ΔZ, in which −214/+25 sequences were removed from FEV1.1Z ().
Our findings show that proximal conserved sequences are not only sufficient but also necessary for 5HT neuron expression of FEV in the developing rostral and anterior portion of the caudal hindbrain. Furthermore, they suggest that the FEV promoter comprises the 5' flanking proximal sequences that are deleted in FEV2.2ΔZ and FEV1.1ΔZ. Consistent with this hypothesis, a 2.2 kb FEV transgene lacking the β-globin TATA box, FEV2.2ZΔβg, was expressed in a 5HT neuron-specific pattern (). Therefore, the β-globin start site is not required, and likely does not contribute to, transgene expression. Since FEV sequences can initiate transcription in the absence of a heterologous start site, and deletion of proximal sequences results in loss of specific expression, we conclude that these conserved sequences immediately upstream of the FEV start site constitute the FEV promoter region.
Conserved GATA binding sites are present in the FEV promoter region
We next sought to identify putative transcription factor binding sites within the FEV
upstream region to begin to determine factors that directly control FEV
transcription. Upstream sequences were analyzed for candidate binding sites using the rVista (Loots and Ovcharenko, 2004
) and MatInspector (Cartharius et al., 2005
) tools. Interestingly, this analysis identified possible binding sites for NUDR/DEAF-1, Engrailed-1, and GATA transcription factors clustered in the promoter region (). NUDR/DEAF-1 has been shown to be a repressor of the 5HT1A
receptor gene (Htr1a
) through binding to an upstream polymorphic site (Lemonde et al., 2003
) and loss of function studies have implicated Engrailed-1/2 in the development of 5HT neurons (Simon et al., 2005
). Of particular interest was the presence of two consensus GATA binding motifs (). Multispecies comparisons of the upstream region revealed these sites were conserved even among distantly related mammals (). As Gata-2
is essential for Pet-1
induction in the hindbrain (Craven et al., 2004
), the presence of conserved tandem GATA sites in the FEV
promoter region suggested that Gata-2 might directly induce FEV
in serotonergic precursors. Therefore, we focused our next set of studies at testing this idea.
Predicted transcription factor binding sites in the FEV upstream region
We first determined with co-immunostaining that Gata-2 was expressed in βgal+
cells of the rostral hindbrain in FEV2.2Z transgenic embryos (). We then asked whether the consensus sites could bind GATA protein in vitro. Gata-1, Gata-2, and Gata-3 all recognize a consensus site, WGATAR (Ko and Engel, 1993
), and both sites in the FEV
upstream region perfectly match this sequence (). We tested the ability of purified recombinant Gata-1 protein to bind either the FEV
) or proximal (GATA2
) sites () using electrophoretic mobility shift assays (EMSAs). Recombinant Gata-1 protein formed a single complex with duplex oligonucleotides carrying either GATA site (), thus demonstrating that these are indeed bona fide in vitro GATA binding sites. To test whether these sites are also recognized by Gata-2, we carried out EMSAs with HeLa cell nuclear extracts, as these cells express endogenous Gata-2, but not other GATA factors (Dorfman et al., 1992
; Zon et al., 1993
; Siltanen et al., 1999
; Ho et al., 2005
). Oligonucleotides containing the distal conserved GATA site, GATA1
formed two complexes when incubated with HeLa nuclear extracts (arrow and arrowhead in ). These complexes were competed away with an excess of the unlabeled GATA1
site but not an equimolar mass of an oligonucleotide in which the GATA site had been altered (, respectively). When the proximal GATA site, GATA2
, was used as a competitor, wildtype but not mutant oligonucleotides specifically competed the upper complex formed with GATA1
oligonucleotides (). GATA2
oligonucleotides formed similar complexes with HeLa extract that were competed with wildtype but not mutated GATA1
oligonucleotides (data not shown). These cross-competition experiments demonstrate that common proteins bind specifically to the “GATA” motif found in both oligonucleotide sequences. Finally, the addition of anti-Gata-2 antibody, but not an equal amount of anti-GFP antibody, disrupted the upper complex, indicating that Gata-2 is indeed present in that complex (). Taken together, these results suggest that both sequences are functional in vitro binding sites for Gata-2.
In vitro and in vivo analysis of GATA sites in FEV/Pet-1 upstream region
Gata-2 directly interacts with the Pet-1 cis regulatory region
We next used chromatin immunoprecipitation (ChIP) to determine whether Gata-2 occupies the homologous Pet-1 cis regulatory region in vivo. To our knowledge there is no neuronal cell line that expresses high levels of Pet-1 or FEV together with Gata-2. Therefore, we developed a ChIP assay with E11.5 mouse hindbrain as a source of serotonergic chromatin. Chromatin was immunoprecipitated with an anti-Gata-2 antibody, and the immunoprecipitate was assessed by quantitative PCR. We tested for Gata-2 interaction with the Pet-1 upstream region, as well as sequences located in upstream genes, Cryba2 and Ccdc108, where consensus GATA motifs are located. Anti-Gata-2 antibody immunoprecipitation of sequences associated with the Cryba2 GATA site was slightly increased over background levels measured with the Untr17 control but not with the Untr8 control. No enrichment of Ccdc108 sequences was detected when compared to either of these controls (). In contrast, binding of Gata-2 to the Pet-1 upstream region was highly significantly enriched (p<0.001) compared to both negative control regions and to the Cryba2 or Ccdc108 regions (). These findings show that in the developing hindbrain Gata-2 directly binds to the FEV/Pet-1 upstream cis-regulatory region.
Conserved GATA binding sites in the FEV cis-regulatory region are required for serotonergic transgene expression
Having shown that conserved GATA binding sites are present in the FEV promoter region and that Gata-2 directly interacts with the homologous Pet-1 cis regulatory region, we next investigated the function of the GATA sites in vivo. Site-directed mutagenesis was carried out to destroy these GATA binding sites in FEV2.2Z, either singly (Gata1mut2.2Z or Gata2mut2.2Z) or in combination (Gata1,2mut2.2Z) (). Mutation of either GATA site alone resulted in little change in the transgene expression in 5HT neurons (compare 6C, D with 6A) or reproducibility of expression; however, we did observe an increase in ectopic expression as compared to FEV2.2Z or FEV2.2ZΔβg (data not shown). In contrast, nucleotide changes to both GATA sites (Gata1,2mut2.2Z, ), led to a complete loss of ventral hindbrain expression in 16 of 27 founders. The remaining eleven founders showed very weak expression in rostral 5HT neurons (), which was often accompanied by weak to strong ectopic expression. Little or no staining was observed in more median or caudal 5HT neurons (). Furthermore, robust staining, comparable to FEV2.2Z (, ), was never observed with Gata1,2mut2.2Z. Thus, mutation of both GATA sites greatly compromised the ability of the 2.2 kb fragment to direct expression to 5HT neurons, as evidenced both by the large numbers of founders showing no hindbrain expression and the dramatically reduced serotonergic expression in remaining founders.
GATA sites are functionally required for FEV transgene expression
FEV-directed transgene expression and fate of mutant 5HT neuron precursors in Pet-1−/− brain
We showed previously that a LacZ reporter controlled by the mouse Pet-1
upstream enhancer was weakly expressed in the Pet-1−/−
brain compared to its expression in wildtype mice (Scott et al., 2005
). To determine whether FEV
-directed transgene expression was also dependent on endogenous Pet-1
function we crossed the FEV2.2Z transgene into the Pet-1−/−
background. The number of βgal+
cells in the embryonic brain of FEV2.2Z, Pet-1−/−
mice was somewhat diminished compared to their numbers in Pet-1+/−
background (), but FEV2.2Z continued to be expressed in 5HT+
cells (). However, FEV2.2Z expression was greatly diminished in adult Pet-1−/−
mice (). When costained for TPH and βgal, only a few co-labeled cells were detected (). These findings show that maintenance of FEV2.2Z expression depends on endogenous Pet-1
FEV-directed transgenes mark Pet-1−/− cells in the adult brain
To determine whether a longer upstream fragment was less sensitive to loss of Pet-1
we made a new reporter, FEV60Z, with a 60 kb upstream FEV
fragment. Two independent FEV60Z transgenic lines were crossed into the Pet-1−/−
background. In wildtype mice, we observed strong FEV60Z expression in the embryonic hindbrain at E12.5 ( and data not shown) and in adult DRN ( and data not shown). In contrast to FEV2.2Z and the mouse Pet-1
enhancer-directed LacZ (Scott et al., 2005
), the number of LacZ+
cells in the DRN was similar in FEV60Z, Pet-1−/−
and FEV60Z, Pet-1+/−
mice at both embryonic () and adult () stages. Indeed, cell counts of adult B7 DRN from Pet-1+/−
animals (n=3 for each genotype, two sections per animal) revealed no differences between groups (means were 380 and 378 cells for Pet-1+/−
sections, respectively, t-test p=0.98).
In FEV60Z, Pet-1+/− mice all TPH+ cells were βgal+ and the majority of βgal+ cells were TPH+ (). However, in FEV60Z, Pet-1−/− mice, while all remaining TPH+ neurons in the B7 DRN expressed the transgene, a substantial number of βgal+ cells were TPH− (). In medullary raphe, FEV60Z was expressed in Pet-1−/− mice but not always in the remaining TPH+ cells ( and data not shown). Thus, FEV60Z does not require Pet-1 to maintain its B7 expression, but may be dependent on Pet-1 function in medullary 5HT neurons.
The comparable expression of FEV60Z in Pet-1+/−
B7 DRN suggested it could be used a novel fate marker of Pet-1−/−
5HT neuron precursors. We reported previously (Hendricks et al., 2003
) and confirmed here with FEV60Z () that normal numbers of B7 and B8 serotonergic precursors are generated in Pet-1−/−
mice but only about 30% of them can be detected in the adult brain with serotonergic markers. These observations suggested that cell death, misfating, or mismigration might account for the failure to detect these cells in adults. However, the similar numbers of βgal+
cells in the adult B7 DRN of FEV60Z, Pet-1+/−
and FEV60Z, Pet-1−/−
mice indicated that these cells are not eliminated by a cell death program but may remain in an arrested precursor stage. The continued activity of FEV60Z is consistent with the maintenance of serotonergic character despite the lack of TPH expression in these cells (). Further studies showed that all FEV60Z+
DRN cells co-expressed NeuN (), a general neuronal marker. The expression of Gata-3, which marks several distinct neuronal populations in the brain including postmitotic 5HT neuron precursors (Pattyn et al., 2004
; Zhao et al., 2008
), was also present in βgal+
cells () further supporting a serotonergic character of FEV60Z+
cells in the Pet-1−/−
brain. Importantly, these cells did not coexpress markers of nearby neuronal cell types, such as tyrosine hydroxylase (TH) (), a marker of dopaminergic neurons, or choline acetyltransferase (ChAT) (), expressed by motor neurons.
The marking of mutant 5HT neuron precursors with the FEV60Z transgene also provided a means to visualize the organization of these cells in the adult B7 DRN. Interestingly, analysis of serial sections through the entire anterior-posterior length of the B7 DRN suggested an aberrant distribution of βgal+ cell bodies. The stereotypical “fountain” shape distribution of serotonergic cell bodies in the DRN was never observed in Pet-1 deficient mice and βgal+ cells in the nucleus appeared laterally extended (). Lateralization of the DRN in the Pet-1−/− brain was quantified by counting βgal+ cells in two pairs of regions on each side of the midline (). Significantly more βgal+ cells were located further from the midline in Pet-1−/− animals () than in controls. This lateralization appeared to be limited to DRN, as median and caudal βgal+ cells were appropriately positioned. These findings suggest that Pet-1 may also be required for the precise migration of 5HT neurons to their proper locations in the DRN.