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Research Article
Computational and Experimental Approach for the Identification of Mesencephalon-Specific Cis- Element of Dbx1 Gene
Jennifer Kim#, Mohammed M. Islam#, Evangeline Tzatzalos, Hailing Hao, Ying Li, Haim Y. Tawil, Sungtae Doh and Li Cai*
Department of Biomedical Engineering, Rutgers University, USA
#Both the authors contribute equally to this work

Abstract
Gene regulation is central to the biological processes of differentiation, morphogenesis, and the creation of diversity of cell types. The identification of gene regulatory elements is thus essential for the understanding of the molecular mechanism underlying these processes. Here, we present an integrative approach for the identification and characterization of a cis-element of Dbx1 gene. Dbx1 (developing brain homeobox 1) plays an essential role during development of the central nervous system (CNS). Dbx1 expression is regionally restricted within the brain and spinal cord to define neuronal specific domains. Although the functional roles of Dbx1 have been established, the molecular mechanism that governs Dbx1 regional specific expression is not well understood. Using comparative genomics method, we revealed a highly evolutionarily conserved noncoding DNA fragment of 624 bp located approximately18 kb upstream of Dbx1 locus (CR5) and is predicted as a cis-element. Using experimental approaches, we show that this cis-element CR5 directs mesencephalon-specific gene expression during chick embryonic CNS development. Cells with CR5 activity contain a heterogeneous neuronal population suggesting that CR5 is involved in multiple neuronal lineage development. Together, these findings provide additional insight into the complex regulation of Dbx1 expression during CNS development. In addition, our study established an effective approach for the discovery and characterization of genomic regulatory elements.
Keywords
Gene regulation; cis-Element; Enhancer; Dbx1; Mesencephalon; Brain, and Chick embryo

INTRODUCTION
Regulation of gene expression is essential for cell development and growth; it controls the processes of morphogenesis and the generation of different cell types [1-3]. The identification and characterization of the gene regulatory elements is essential for the understanding of the molecular mechanism underlying these biological processes and for the annotation of the genome.
Previously, the discovery of cis-regulatory elements typically relied on extensive experimentation. Tissue-specific cis-regulatory elements were generally identified by promoter/ enhancer deletion studies in transgenic mice [4] or by DNase I hypersensitivity mapping in expressing tissues [5-7]. The DNase I hypersensitivity mapping technique is based on the observation that local disruptions of the regular nucleosomal array create preferential targets for DNase I. Binding of transcription factors to the DNA at cis-regulatory elements is thought to cause the removal or dispersal of a nucleosome. Thus, DNase I hypersensitive sites are often found at active cis-regulatory elements. Genomic regions close to the promoter are sometimes further explored by footprinting and by electrophoretic mobility-shift assays, which seek to identify regulatory protein-binding DNA regions [5,8]. The ability to evaluate the expression of thousands of genes across various experimental conditions has allowed bioinformatics approaches to these problems. Clusters of genes that show similar patterns of expression were searched within their upstream sequences for over-represented or conserved sequence motifs. This method has worked well in bacteria and yeast [8]; however, due to the highly complex nature of the genome, finding cisregulatory elements in vertebrates still remains as a challenge [9]. Completion of the human genome sequencing along with many other species has allowed comparative genomic approach to identify evolutionarily conserved DNA sequences and with a great success [10-15]. However, detailed characterization of the spatiotemporal gene regulatory activities is still lacking. In this study, we use developing brain homeobox 1 (Dbx1) gene as an example to demonstrate a relatively rapid and effective approach for this problem.
During neurogenesis, various genes have been found to be fundamental in defining the basic structure and organization of the central nervous system (CNS) [16]. These genes are expressed in a spatiotemporal pattern and provide specific cues that control tissue development. Dbx1 has been identified to have specific regional expression, and it plays an essential role in the patterning of the CNS during embryogenesis [17,18]. Dbx1 is expressed in both the brain and spinal cord of developing mouse embryos [17-20]; the expression initially starts in the analogs of diencephalon and spinal cord at embryonic day 9.5 (E9.5) mouse brain. From E10.5 to E12.5, Dbx1 is expressed in the dorsal mesencephalon. The expression is also detected in the telencephalon and hindbrain. At later gestation, however, Dbx1 expression discontinues in the spinal cord and becomes even more restricted within the diencephalon, dorsal mesencephalon, as well as the primitive cerebellum [17-19]. On a cellular level, Dbx1 expression has been observed to be limited to regions of active mitosis, indicating its role in early specification of Dbx1- expressing cells and their progenies [17]. Further evidence correlates Dbx1 expression strongly to progenitor cells in the ventricular zone at the boundaries between dorsal and ventral parts of the neural tube [18-21]. Within the spinal cord, Dbx1 has long been established to define distinct progenitor domains of V0 interneurons along the dorsoventral axis [17-20,22]. In more recent years, studies have demonstrated that Dbx1-derived cells have a high capacity to migrate from their sites of origin to populate different cortical regions [23-25]. The Dbx1-expressing neural progenitors are responsible for the generation of a subset of oligodendrocytes [26], interneurons [27,28] in the spinal cord, Reelin-positive Cajal-Retzius cells [25,29], and a subtype of inhibitory neuron in the medial amygdala [30], indicating that these Dbx1-expressing cells are a source of neuronal diversity. Although the specific roles and functions of Dbx1 in the CNS development have been established, the transcriptional mechanism that governs the complex expression of Dbx1 is still not well understood. One transgenic mouse study shows that a 5.7 kbp region located directly 5’ of Dbx1 gene can control Dbx1 expression similar to the expression pattern of the endogenous Dbx1 gene within both the brain and the spinal cord [31].
Here, we report the development of an integrative approach for the identification and characterization of an evolutionarily conserved DNA element of 624 bp (CR5) located approximately 18 kbp upstream of the Dbx1 transcription start site. Using our newly developed reporter assay system in chick embryos, we found that CR5 acts as a novel regulatory element for brain-specific gene expression. In particular, CR5 is capable of directing reporter GFP expression specifically in the developing mesencephalon, distinct from the previously reported 5.7 kbp sequence element of Dbx1 gene. Furthermore, several subregions of CR5 were also able to individually direct mesencephalonspecific GFP expression. Our findings indicate that CR5 is a novel cis-element for Dbx1 responsible for mesencephalon-specific gene expression during the chick brain development. In addition, CR5 activity was present in various neuronal cell types. Thus, we have established an integrative approach for the annotation and characterization of genomic regulatory elements.
MATERIALS AND METHODS
Computational prediction of Dbx1 cis-elements
Multiple genome sequence alignment methods were used to identify evolutionarily conserved non-coding DNA regions as putative gene regulatory elements. The genomic Dbx1 sequences and their orthologs from the various species including human, mouse, rat, and other vertebrate species were retrieved using non-coding sequence retrieval system, NCSRS [32]. These orthologous sequences were then aligned using multi-LAGAN [33] to identify regions with ≥70% identity and ≥ 100 bp. The percent identity and length of the conserved regions (CR) were used to calculate a ranking score for each CR (ranking score = percent identity + (length/60)). The minimum required length of 100 bp was to ensure significance in sequence conservation. Based on this scoring system, the percent identity was more heavily weighted to ensure that shorter (i.e., 200 bp) and very highly conserved sequences are not ranked below longer (i.e., 1000 bp) sequences with lower levels of conservation (Figure 1A).
Design of plasmid constructs for reporter assay
CR5 and its subregions (Figure 1B-C) were PCR amplified (Table 1) from mouse tail genomic DNA and individually subcloned into a GFP reporter construct containing a human basal promoter, β-globin promoter (BGP) [34]. Resulting constructs were verified by DNA sequencing.
Table 1 List of PCR primers for Dbx1CR5 and its subregions.

Conserved Region

PCR product length (bp)

Primer

Primer Sequence

CR5

1185

forward

CGA TAT ATA CTA GTA AGC ATG AAG CAT GAA GGG CTG TG

reverse

CGA TAT ATG GCC GGC CAG GCC ATG AAC AAC CTG AGA GGA A

CR5.1

318

forward

CGA TAT ATA CTA GTA AGC ATG AAG CAT GAA GGG CTG TG

reverse

CGA TAT ATG GCC GGC CAC ACA CAC ACA CAC ACA CAC ACA C

CR5.2

182

forward

CGA TAT ATA CTA GTA GCT TTG AAT GTG CAA CCC GAT CC

reverse

CGA TAT ATG GCC GGC CAG GAA CAA GGG ACA ACC TCT GCT T

CR5.3

258

forward

CGA TAT ATA CTA GTA AGC AGA GGT TGT CCC TTG TTC CT

reverse

CGA TAT ATG GCC GGC CTA ATC AAG CCA CGG TGC TGT GAG A

CR5.4

641

forward

CGA TAT ATA CTA GTA AGC ATG AAG CAT GAA GGG CTG TG

reverse

CGA TAT ATG GCC GGC CAG GAA CAA GGG ACA ACC TCT GCT T

CR5.5

417

forward

CGA TAT ATA CTA GTA GCT TTG AAT GTG CAA CCC GAT CC

reverse

CGA TAT ATG GCC GGC CTA ATC AAG CCA CGG TGC TGT GAG A

CR5.6

23

forward

CGA TAT ATA CTA GTA AGC AGA GGT TGT CCC TTG TTC CT

reverse

CGA TAT ATG GCC GGC CAG GAA CAA GGG ACA ACC TCT GCT T

Table 1 List of PCR primers for Dbx1CR5 and its subregions.

×
For control experiments, we designed two negative control constructs (one contains a random DNA sequence coupled with BGP-GFP and the other is BGP-GFP alone without a conserved sequence); and two transfect control constructs (CAG-DsRed and CAG-GFP) (Figure 1C); CAG promoter is chicken β-actin promoter with a CMV enhancer element that is able to drive high levels of reporter expression in all transfected cells [35].
Figure 1 Sequence analysis to predict Dbx1 cis-elements and experimental construct design for functional verification. (A). Multiple-species alignment of Dbx1 sequences from human, chicken, and zebrafish using mouse sequence as baseline. The sequence alignment reveals highly conserved regions (CR, pink peaks) of the noncoding Dbx1 locus as putative cis-elements. CR5 is the highest ranked region. Blue peaks represent exons of Dbx1. (B). Pair-wise alignment of the CR5 region from mouse and human. CR5.1-CR5.6 are six smaller fragments. They were individually cloned into the reporter construct. A DnaseI protected site is also depicted between the dotted lines, which overlaps with CR5.5. (C). Schematic of experimental and control reporter constructs for testing gene regulatory activity.

Figure 1 Sequence analysis to predict Dbx1 cis-elements and experimental construct design for functional verification. (A). Multiple-species alignment of Dbx1 sequences from human, chicken, and zebrafish using mouse sequence as baseline. The sequence alignment reveals highly conserved regions (CR, pink peaks) of the noncoding Dbx1 locus as putative cis-elements. CR5 is the highest ranked region. Blue peaks represent exons of Dbx1. (B). Pair-wise alignment of the CR5 region from mouse and human. CR5.1-CR5.6 are six smaller fragments. They were individually cloned into the reporter construct. A DnaseI protected site is also depicted between the dotted lines, which overlaps with CR5.5. (C). Schematic of experimental and control reporter constructs for testing gene regulatory activity.

×
Chicken embryos and in ovo electroporation
Fertilized pathogen-free (SPF) white leghorn chicken eggs (Sunshine Farms, Catskill, NY) were incubated (GQF manufacturing, Savannah, GA) at 38°C with 58-60% humidity and protected from light for approximately 40-45 hours to obtain embryos that were at the Hamburger Hamilton stage 10-12 (HH10-12; ~E2) [36]. All of the animal experiments were approved by the Animal Care and Facilities Committee at Rutgers University (approval ID 05-060).
Plasmid DNA (3-5µg/µl) was injected into the neural tube of chick embryos using a PCR micropipette glass-pulled needle. Fast green dye (0.0125%) was mixed with plasmid DNA for visualization purposes. Gold-plated electrodes were set approximately 4mm apart to electroporate embryos using a BTX ECM 830 system (Harvard Apparatus, Holliston, MA) to deliver 5 pulses at 18 volts, 50 ms duration, and with 950ms intervals. Electroporated eggs were incubated until appropriate harvest time points.
Tissue processing and immunostaining
Transfected tissues were harvested at various developmental stages. Tissue processing and immunostaining were performed as described previously [37] with the following antibodies: NeuN (1:1000, Millipore), GFAP (1:250, Accurate), GS (1:250, Santa Cruz), Pkcα (1:400, Santa Cruz), Chx10 (1:50, Santa Cruz); and the following antibodies were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology (Iowa City, IA 52242): Acetylcholine receptor (mab270), Hb9 (81.5C10), 3CB2, Vimentin (40E-C, and H5), Pax6, Lim1+2 (4F2), Evx1 (99.1-3A2).
Image analysis
Whole-mount samples were examined using a Leica MZ16FA fluorescent dissection microscope. Tissue sections were analyzed using a Zeiss Axio Imager A1 and an Olympus FluoView FV10i confocal microscope.
Statistical analysis
GFP expression in each sample was categorized based on its anatomical location within the CNS: telencephalon, diencephalon, mesencephalon, metencephalon, myencephalon, and spinal cord. Frequency of GFP occurrence within each CNS regions at each embryonic stage was determined by counting the number of GFP+ samples. Statistical analysis was performed using a paired two-tailed t-test, P<0.05 indicates a statistical significance.
RESULTS AND DISCUSSION
Prediction of evolutionarily conserved noncoding DNA of Dbx1 as cis-elements
To predict evolutionarily conserved noncoding sequence elements that may serve as transcription regulators for Dbx1 expression, we performed DNA sequence analysis. The intergenic sequences flanking the 5´ and 3´ regions of Dbx1 from various species, including human, mouse, chicken, and other vertebrate species, were retrieved using a sequence retrieval system NCSRS [32], and aligned using multi-LAGAN [33,38]. Resulting alignment revealed that many non-coding fragments highly conserved among different species (Figure 1A). The highest conserved region CR5 is located approximately 18 kbp upstream of Dbx1 transcription start site containing 624bp nucleotides (pink peak in Figure 1B), and it also contains a known DNaseI hypersensitivity site [39], indicating that CR5 is a strong candidate as a functional cis-element for Dbx1 gene.
CR5 directs tissue-specific GFP expression in the developing chick CNS
To examine the possibility that CR5, conserved among human, mouse, chick and other vertebrate species, might represent cisregulatory activity, we explored its ability to direct tissue-specific gene expression during chick CNS development with an in ovo reporter assay. Mouse CR5 was cloned upstream of a human beta-globulin minimal promoter (βGP) [34] coupled to a reporter gene, green fluorescent protein (GFP)(Figure 1C). Plasmid constructs CAG-GFP or CAG-DsRed were used as transfection controls. CAG (chicken β-actin promoter with CMV enhancer),a strong and ubiquitous promoter, can drive gene expression in all transfected cell types [35]. The experimental and transfection control constructs were injected into the developing chick neural tube at Hamilton-Hamburger stage 11-12 (~ embryonic day 2, E2), and followed by electroporation to facilitate the uptake of DNA constructs into the neural stem/progenitor cells lining the neural tube. The patterns of reporter expression from CR5- GFP and CAG-DsRed were compared at various stages from E3 to E18. The control CAG-DsRed+ cells (Figures 2A, D) and CAGGFP+ cells (Figure 2E) were observed in both CNS tissues (e.g., telencephalon, mesencephalon, spinal cord, eye) and non-CNS tissues (e.g., skin, limb, and heart), while CR5-GFP+ cells were restricted to the developing CNS (Figures 2B, F; n=22).
Figure 2 CR5 directs mesencephalon-specific GFP expression in developing chick CNS. Expression of GFP was examined in the developing brain of E3-E4 embryos after in ovo electroporation at E2 (i.e., HH stage10-12). The control CAG-DsRed+ cells (A and D) and CAG-GFP+ cells (E) were found in the eye, telencephalon, diencephalon, mesencephalon, spinal cord of the CNS and also in the non-CNS tissues, e.g., skin (arrowheads) and heart (arrow). Expression of CR5-GFP was restricted in the mesencephalon (B, C and F). T, telencephalon; D, diencephalon; M, mesencephalon; SC, spinal cord.

Figure 2 CR5 directs mesencephalon-specific GFP expression in developing chick CNS. Expression of GFP was examined in the developing brain of E3-E4 embryos after in ovo electroporation at E2 (i.e., HH stage10-12). The control CAG-DsRed+ cells (A and D) and CAG-GFP+ cells (E) were found in the eye, telencephalon, diencephalon, mesencephalon, spinal cord of the CNS and also in the non-CNS tissues, e.g., skin (arrowheads) and heart (arrow). Expression of CR5-GFP was restricted in the mesencephalon (B, C and F). T, telencephalon; D, diencephalon; M, mesencephalon; SC, spinal cord.

×
CR5 is preferentially active in the developing mesencephalon
To determine temporal specificity of CR5 in directing gene expression during chick CNS development. GFP expression was examined in transfected chick embryos at various developmental stages from E3 to E18. The differences in reporter expression between the control (CAG-GFP or CAG-DsRed) and experimental group (CR5-GFP) were determined by categorizing reporter expression pattern into different CNS regions with respect to time (Figure 3). CR5-GFP expression was found predominantly within the mesencephalon and occasionally the diencephalon or other regions (Figure 3B). No CR5-GFP+ cells were observed within the spinal cord (data not shown). The pattern of CR5- GFP expression was largely consistent within the developing mesencephalon (Figure 3A-B). This result indicates that CR5 activity exists primarily in the mesencephalon of the developing chick brain and not in the spinal cord.
Figure 3 CR5 and its subregions direct GFP expression at various stages during chick embryonic development. (A) GFP expression mediated by CR5 in chick developing brain at E3, E6, E13, and E18 after in ovo electroporation at E2 (HH stage 10-12). (B) Quantification shows that CR5 directs GFP expression predominantly in the mesencephalon (For each stage, n ≥ 4. p < 0.01). (C) GFP expression derived by CR5 subregions (CR5.2-CR5.6) in chick developing brain at E3 after in ovo electroporation at E2 (HH stage 10-12). The number of samples with reporter GFP expression in various developing subregions of the CNS is listed in Tables 3 and 4.

Figure 3 CR5 and its subregions direct GFP expression at various stages during chick embryonic development. (A) GFP expression mediated by CR5 in chick developing brain at E3, E6, E13, and E18 after in ovo electroporation at E2 (HH stage 10-12). (B) Quantification shows that CR5 directs GFP expression predominantly in the mesencephalon (For each stage, n ≥ 4. p < 0.01). (C) GFP expression derived by CR5 subregions (CR5.2-CR5.6) in chick developing brain at E3 after in ovo electroporation at E2 (HH stage 10-12). The number of samples with reporter GFP expression in various developing subregions of the CNS is listed in Tables 3 and 4.

×
To determine minimum active region, CR5 subregions (CR5.1-5.6 in Figure 1B) were tested for their gene regulatory activity. Except CR5.1, all other five subregions, CR5.2-CR5.6, were shown to drive GFP expression in E3 chick embryos (Figure 3C). GFP expression pattern resulted from each of CR5 subregions was analyzed at E3 (Table 3) and at E6 (Table 4). The observation revealed a predominant GFP expression in the mesencephalon (all 4 samples at E3 and all 3 samples at E6) resulted from CR5.5 construct. While CR5-GFP expression was never observed anywhere but the mesencephalon in E3 samples, each of the subregional constructs (CR5.2-CR5.6) had varying amounts of GFP expression in different regions of the brain (e.g., telencephalon, diencephalon, spinal cord, etc.) in addition to mesencephalon. These results indicate each or combination of CR5.2 - 5.6 subregions may be important in restricting GFP expression specifically in the mesencephalon.
CR5 is active in various neuronal subtypes
To determine the cellular identity of CR5-GFP+ cells, cryosections of E13 and E18 brain tissues were immunostained using various antibodies marking a specific neuronal cell type (Figure 4). E13 samples were analyzed, because the chick neuronogenesis is near completion at this stage [40], and many differentiated neuron cell types can be determined by immunostaining [37,40-42]. Immunostaining showed that
Figure 4 CR5-GFP+ cells in late chick embryos contain various neuronal types. Transverse sections of optic tectum (mesencephalon) from E18 (A) and E13 (B) embryos after in ovo electroporation at E2. CR5-GFP+ cells were co-labeled with Evx1 (A) and Lim1+2 (B). Scale bar = 50μm.

Figure 4 CR5-GFP+ cells in late chick embryos contain various neuronal types. Transverse sections of optic tectum (mesencephalon) from E18 (A) and E13 (B) embryos after in ovo electroporation at E2. CR5-GFP+ cells were co-labeled with Evx1 (A) and Lim1+2 (B). Scale bar = 50μm.

×
CR5-GFP+ cells were labeled with neuronal markers, e.g., Evx1 (21±0.07%; Figure 4A) and Lim1+2 (15±0.06%; Figure 4B). Confocal microscopic analysis further confirmed co-labeling of CR5-GFP+ cells in E18 samples with Evx1 labeling (Figure 5). Lim1+2 [43] and Evx1 [44] are markers for interneurons. However, no CR5-GFP+ cells were co-labeled with the glial marker GFAP (data not shown). To our surprise, markers for neural stem/progenitor cells and radial glia (e.g., Vimentin and Pax6) did not co-label with GFP+ cells, indicating CR5 is not active in neural stem/progenitor cells. Staining with other neuronal markers (e.g., GABA, Tyrosine Hydroxylase, Brn3a, Engrailed-1; see summary in Table 2) failed to co-label with CR5-GFP+ cells at any time points examined. Other regionally expressed molecules were also examined on CR5-GFP+ cells, including Islet1 on E6 and E13 samples, and Hb9, Lim3, and Chx10 on E6 samples, glutamine synthetase on E13 samples, acetylcholine receptor, and PKCα on E18 tissues, which all failed to co-label with CR5- GFP+ cells (Table 2). These results indicate that CR5 activity exists in different neuronal cell types, but not in glial cells at the stages we examined.
Figure 5 CR5-GFP+ cells co-labeled with a neuronal marker Evx1. (A) Side view of transfected E18 brain sample in grey scale. (B) Transverse section as indicated by the white bar in grey scale. (C-F) Confocal images of stained tissue sections. Brain region of white box inset of (B) is shown in (C-D). Brain region in white boxes inset of (C-D) are shown in (E-F). Evx1 antibody staining co-localization with GFP+ cells is indicated by white arrowheads. SC, spinal cord, Cbm, cerebellum, OT, optic tectum, D, dorsal, V, ventral. Scale bar = 50 μm.

Figure 5 CR5-GFP+ cells co-labeled with a neuronal marker Evx1. (A) Side view of transfected E18 brain sample in grey scale. (B) Transverse section as indicated by the white bar in grey scale. (C-F) Confocal images of stained tissue sections. Brain region of white box inset of (B) is shown in (C-D). Brain region in white boxes inset of (C-D) are shown in (E-F). Evx1 antibody staining co-localization with GFP+ cells is indicated by white arrowheads. SC, spinal cord, Cbm, cerebellum, OT, optic tectum, D, dorsal, V, ventral. Scale bar = 50 μm.

×
Table 2 Summary of antibody staining with CR5-GFP transfected tissue samples.

 

NeuN

Lim1/2

Evx1

GFAP

Vimentin

Pax6

En1

Isl1

Hb9

Lim3

Chx10

GS

Ach-receptor

PKCα

E3

 

 

 

 

-

 

 

 

 

 

 

 

 

 

E4

-

-

 

 

-

-

 

 

 

 

 

 

 

 

E6

-

+

+

 

-

-

-

-

-

-

-

 

 

 

E13

+

+

+

-

-

-

-

-

 

 

 

-

 

 

E18

+

+

+

-

-

-

-

-

 

 

 

 

-

-

Table 2 Summary of antibody staining with CR5-GFP transfected tissue samples.

×
Table 3 Number of samples with reporter GFP expression in various developing subregions of the CNS at E3.

Telencephalon

Diencephalon

Mesencephalon

Metencephalon

Myencephalon

Spinal Cord

CR5 (n=3)

0/3

0/3

3/3

0/3

0/3

0/3

CR5.2 (n=2)

1/2

1/2

2/2

1/2

1/2

1/2

CR5.3 (n=4)

0/4

2/4

2/4

1/4

2/4

2/4

CR5.5 (n=4)

0/4

3/4

4/4

1/4

1/4

2/4

CR5.6 (n=9)

1/9

3/9

4/9

5/9

6/9

5/9

Table 3 Number of samples with reporter GFP expression in various developing subregions of the CNS at E3.

×
Table 4 Number of samples with reporter GFP expression in various developing subregions of the CNS at E6.

Telencephalon

Diencephalon

Mesencephalon

Metencephalon

Myencephalon

Spinal Cord

CR5 (n=7)

0/7

0/7

7/7

1/7

1/7

0/7

CR5.2 (n=7)

0/7

0/7

3/7

7/7

7/7

0/7

CR5.3 (n=5)

0/5

0/5

0/5

5/5

5/5

0/5

CR5.5 (n=3)

0/3

0/3

3/3

1/3

1/3

0/3

CR5.6 (n=6)

0/6

0/6

0/6

0/6

0/6

0/6

Table 4 Number of samples with reporter GFP expression in various developing subregions of the CNS at E6.

×
Conclusion
In this study, we demonstrate that CR5, a 624bp noncoding DNA fragment, acts as a cis-element that regulates gene expression during chick brain development using an integrative approach combining computational prediction and experimental verification. CR5 is able to direct CNS-specific reporter GFP expression in the developing mesencephalon during embryonic days 3 (E3) through E18. CR5-GFP+ cells contain a heterogeneous neuronal population indicating that CR5 may be involved in regulating gene expression for the development of multiple neuronal lineages. The capability of several subregions within CR5 (CR5.2-CR5.6) in directing GFP expression indicates that multiple cis-regulatory modules are involved in restricting mesencephalon-specific gene expression in the developing chick CNS.
In previous studies, an enhancer of Dbx1 of 5.7 kb DNA fragment that contains the transcriptional start site has been reported [31]. The 5.7kb enhancer showed reporter gene expression in the forebrain, midbrain, and spinal cord [17]. However, CR5-GFP expression is highly restricted in the mesencephalon, which indicates that 1) cis-element CR5 plays a specific role in the midbrain (particularly the mesencephalon) development, instead of having a global effect on overall Dbx1 gene expression (e.g., 5.7 kb enhancer) [31]; 2) the regulation of Dbx1 expression is a complex process involves multiple cis-regulatory elements for various patterns of Dbx1 expression in the CNS. The fact that GFP expression is not detected in the spinal cord suggests that CR5 is not involved in the regulation of gene expression in the developing spinal cord.
The previously characterized 5.7 kb Dbx1 enhancer should be a control for this study, however, the corresponding regions (i.e., CR3 and CR4 in Figure 1A) are not highly conserved in chick, and and their ability to regulate gene expression in chick was not demonstrated (data not shown). This indicates that 1) there is a difference in regulating Dbx1 gene expression between the two species of mouse and chicken; 2) not all conserved noncoding DNA sequence can function as a gene expression regulator.
Our study identified that CR5 is a functional cis-regulatory element outside of 5.7 kb promoter/enhancer region located directly upstream of the transcription start site. It provides an additional piece of information in the complex transcription regulation of Dbx1 expression. There are several other conserved regions, and their gene regulatory activity needs to be determined in order to gain a full understanding the complex Dbx1 expression. In addition, the mechanism of CR5 in regulating gene expression is likely via its interactions with specific protein factors. Future research should include the determination of such specific CR5 binding factors and their role in regulating Dbx1 expression. Finally, our integrative approach used in this study has proven to be effective. Using this approach, we have successfully identified and characterized cis-elements in the Notch1 [45], CD44 [46], and Foxn4 [47] genes.
Acknowledgements
We thank Drs. Richard S. Nowakowski and Mladen- Roko Rasin for discussion and comments on the manuscript. Antibodies against En1, Evx1, Lim1/2, Lim3, Vimentin, and Pax6 were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242. This work was supported in part by grants from the NIH (EY018738), the New Jersey Commission on Spinal Cord Research (08-3074-SCR-E-0 and 10-3091-SCR-E-0), Busch Biomedical Research Award, and The Aresty Center for Undergraduate Research.

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Cite this article: Kim J, Islam MM, Tzatzalos E, Hao H, Li Y, Tawil HY, et al. (2013) Computational and Experimental Approach for the Identification of Mesencephalon-Specific Cis-Element of Dbx1 Gene. JSM Biotechnol Bioeng 1(3): 1017.
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