Expression Profile of Endogenous Lycat Examined by In Situ Hybridization
Figure 1 shows the expression patterns of mouse Lycat on paraffin sections of embryos examined by a digoxigenin or 35S-labeled antisense Lycat probe. When examined at embryonic day 9.5 (E9.5), Lycat has already been turned on in the developing heart (H), epithelial lining in the main bronchus within lung bud (L) and junctions between somites (Fig. 1A). Lycat expression in these organs persists throughout the embryonic stages. In the developing heart, Lycat is expressed in cells of both atria and ventricles (Fig. 1B,C). At E14.5 when the septation of each chamber is complete, a strong expression of Lycat can be seen in the mesenchymal cells (MCs) (morphologically, MCs are not tightly packed compared with neighboring cardiomyocytes) in the cardiac septa and valves (arrow in Fig. 1D), which originate from endocardial cells within the atrioventricular canal region through endothelial-to-mesenchymal transformation (EMT) (Zeisberg et al.2007; Niessen and Karsan,2008). Impaired EMT fails to generate precursor cells that populate the endocardial cushions, resulting in many congenital heart defects (reviewed by Markwald and Butcher,2007). In the developing lung, Lycat is strongly expressed in the epithelial cells of the main bronchus (Fig. 1A–E). Interestingly, the bronchial epithelial linings are of endodermal origin. In addition, notochord (NC in Fig. 1D) and cartilage primordium of the developing vertebrate are positive for Lycat (CP in Fig. 1D). In adult heart, Lycat is present in the entire atria (data not shown) and a subset of cells in the ventricles including the mesenchymal cells at the base of tricuspid valves (arrowheads in Fig. 1F). A small portion of cardiomyocytes underlying the endocardium in the ventricle also express Lycat (arrow in Fig. 1F). Cells in the smooth muscle layer of the thoracic aorta and the epithelial layer of trachea are also Lycat-positive (Fig. 1G,H). In the female reproductive organs, Lycat is strongly expressed in oocytes in the growing secondary follicles (Fig. 1I and J). Weak signals can be seen in the corpus luteum (data not shown).
Figure 1. Lycat mRNA is expressed in the heart, aorta, lung and cartilage primordium, and maturing oocytes. Paraffin sections of wild-type mouse embryos and tissues were used for in situ hybridization with either digoxigenin-labeled or 35S-labeled full-length cDNA. Lycat signal was visualized by alkaline phosphatase staining (A-H; blue or purple signal) or autoradiography (I and J), respectively. Developmental stages are indicated at the bottom right of each panel. Lycat is expressed in the heart, and lung in sagittal sections of embryos at E9.5 (A), at E10.5 (B), and at E11.5 (C). A transverse section (D) and a sagittal section (E) show Lycat in the developing cardiomyocytes, lung epithelium, notochord (NC), and cartilage primordium of vertebrates (CP) at E14.5. Lycat signal is detectable in mesenchyme-like cells near valves and septum (arrowheads in F) in heart, smooth muscle cells in aorta (Ao in G), and epithelial cells in trachea (Tr in H) at 30 days postpartum. I, J: Bright-field view and the corresponding dark-field view of Lycat mRNA in a maturing oocyte after autoradiography. Ao, aorta; At, atrium; CP, cartilage primordium; dpp, day(s) after postpartum; H, heart; L, lung; NC, notochord; Tr, trachea; Vt, ventricle.
Download figure to PowerPoint
In summary, Lycat is specifically expressed in oocyte, heart, lung, the vascular structures associated to these organs, as well as the cartilage primordium of the vertebrate.
A 135-kb Genomic Fragment From the Lycat Locus Can Drive the Reporter Gene to Recapitulate the Endogenous Lycat Expression
The targeting strategy of this transgenic study is shown in Figure 2. The BAC clones RP23-294D3 and RP23-299H17 contain 170 and 135 kb of Lycat genomic DNA, respectively, but neither covers the entire Lycat gene (Fig. 2A). Mouse Lycat contains 6 exons that spread over 134 kb in length on chromosome 17. RP23-294D3 contains exons 2–6 and RP23-299H17 harbors exons 1–5 of Lycat. To map the Lycat regulatory elements, we first modified both BAC clones by inserting the reporter ires.lacZ into the second exon of Lycat by ET cloning to generate pW209 from RP23-299H7 and pW199 from RP23-294D3 (Fig. 2A). These transgenes were microinjected into one-cell embryos by pronuclear microinjection.
Figure 2. Identification of the Lycat regulatory elements by ET cloning and transgenesis. A: The size and position of RP23-299H17 and RP23-294D3 Lycat BACs are indicated. Linearized targeting vector, pW207, was used to modify the BACs by ET-mediated homologous recombination in DH10B E. coli cells. Correct integration of the targeting vector results in the insertion of the reporter cassette into the 2nd exon of Lycat. Transgenic mice were generated by pronuclear injection of PI-SceI linearized BAC DNAs (pW199 and pW209). B: Erratic expression pattern of a pW199 transgenic embryo. a: A whole mount embryo stained for β-galactosidase activity; b: a sagittal section of the same embryo shown in a. Embryonic stage of the embryo is listed at the bottom right of each panel. C: Stable pW209 transgenic lines were verified by Southern blot analysis using a 32P-labeled neo probe. Approximately 5 μg of tail-tip genomic DNA from each stable transgenic line was digested with restriction enzyme EcoRI. The neo probe detected a 2.9-kb DNA fragment comprising the entire neo coding region and part of the Lycat BAC insert. Endogenous Umodl1 gene was included as the internal control. Copy number of pW209 in each transgenic line was estimated by TaqMan real-time PCR method (see Table in C).
Download figure to PowerPoint
First, transient transgenic assays were performed to examine whether the entire or part of the Lycat regulatory elements are included in these BAC clones. Embryos were collected from embryonic stage E8.5 to E12.5. Each embryo was genotyped by PCR using genomic DNA extracted from the attached yolk sac membranes. Expression of the Lycat BAC-ires.lacZ-A+ transgenes (Lycat:lacZ) was examined by β-galactosidase activity (lacZ-staining).
Totally, 13 pW199 transgenic embryos verified by PCR were collected for lacZ staining. These embryos showed no or erratic lacZ expression that is inconsistent with the endogenous Lycat (Fig. 2B). Lack of consistent expression of the pW199 transgenic embryos indicates that the 170-kb genomic fragment carried by the RP23-294D3 BAC clone contains no functional Lycat regulatory elements. However, when examined at E11.5, six out of eight pW209 transgenic embryos, as genotyped by PCR, showed identical expression patterns to those of the endogenous Lycat with minor variations in expression intensity, confirming that all of the Lycat regulatory elements are well preserved in RP23-299H17. Furthermore, absence of regulatory elements in pW199 suggests that the Lycat promoter/enhancers are primarily located in the 53-kb genomic fragment of RP23-299H17 that is not overlapped with RP23-294D3 (Fig. 2A).
Encouraged by these remarkable results from the above transient transgenic assays, we decided to establish stable pW209 transgenic lines so that we can use them to examine Lycat:lacZ expression patterns at any developmental stages. Additionally, these transgenic mice will serve as control animals for phenotypic analysis of both transgenic mice carrying extra copies of Lycat (over-expression) and the Lycat knockout mice in our future studies. After two rounds of pronuclear injection, 42 pups were born and 4 of them were transgenic as confirmed by PCR (data not shown) and Southern blot analysis (left panel in Fig. 2C). Transgene copy numbers were estimated by 2ΔΔCt TaqMan method using tail-tip genomic DNA samples from both the founders and their F3 offspring generated by crossing BAC transgene founder (F0) with FVB males/females (right panel in Fig. 2C). Absence of deviation in transgene copy number after several generations of breeding suggests a single site integration of the transgenic construct in all of these stable transgenic lines.
When their F1 embryos were tested by lacZ-staining, colonies pW209-17, 23, and 42 showed identical lacZ expression patterns to those of the endogenous Lycat. Transgenic line number 8 showed an irrelevant lacZ expression, which might be interpreted by positional effects or inter-genomic rearrangements of the BAC. Figures 3 and 4 show Lycat:lacZ expression of transgenic embryos and adult organs collected from Line pW209-17.
Figure 3. Embryonic expression of the pW209 BAC transgene visualized by β-galactosidase activity. A–E: Whole-mount embryos showing dynamic expression profile of the BAC transgene from E7.5 (A) to E 11.5 (E). F–J: Paraffin sections of lacZ-stained embryos showing transgene expression in embryonic heart cells (F–I), epithelial cells of developing lung and midgut (G), and the vessel walls of umbilical artery (J). Sections were counter-stained with Nuclear Red. AER, apical ectodermal ridge; LA, left atrium; MG, midgut; RA, right atrium; So, somite; Tg, transgenic; UA, umbilical artery; UC, umbilical cord; WT, wild type. The rest of the abbreviations are as in Figure1.
Download figure to PowerPoint
Figure 4. Expression of pW209 transgene in adult organs. A–E: lacZ-stained whole mounts of heart (B), lung (C), uterus (D), ovary (E), and associated vessels (A, D). F–T: Paraffin sections showing lacZ-positive cells. LacZ is detected in the trachea (A and F), pulmonary artery (Pa in A), aorta (Ao in A, K, and P), coronary vessels (arrow in B and arrowheads in Q), atrial myocardium (At in L), pericardium-derived mesenchymal cells at the base of the tricuspid valve (arrowheads in G and L), terminal bronchi or bronchioles of lung (C, H, and M), as well as a subset of nucleated blood cells (K and R). P: Higher-power view of the aortic smooth muscle layer. R: The same view of the boxed region in K at a higher magnification. In female reproductive organs, the transgene is seen in some of the maturing oocytes (E, J, and arrow in S) in the hemizygote ovary, as well as cells of smooth muscle lineage in the transgenic oviduct (S) and uterus (D, I, and N). In lactating mammary glands, the smooth muscle cells in the lactiferous ducts (arrowheads in O) and the epithelial cells in the extracellular matrix of the lactiferous lobes (arrows in O) are actively expressing the transgene. The lacZ-cells can also be found in bone marrow (T). Magnification of each section is indicated at the bottom right of each panel. AD, alveolar duct; AL, alveolus; Bn, bone; Bv, blood vessel; TriV, tricuspid valve; Pa, pulmonary artery. The rest of the abbreviations are as in previous figures.
Download figure to PowerPoint
In fact, Lycat:lacZ (pW209) transgene is detectable in all developmental stages examined from oocytes and fertilized eggs to adult mice; however, its expression domains are progressively restricted. At E7.5, Lycat:lacZ is expressed in the extraembryonic mesoderm (top arrow in Fig. 3F) and the cardiogenic plate (bottom arrow in Fig. 3F) and primitive streak, but not the extraembryonic endoderm (arrowheads in Fig. 3F). This supports the supposition that Lycat is expressed in the mesodermal precursors for generating hematopoietic and endothelial lineages, which is consistent with our previous findings in zebrafish and mouse embryonic stem cell–derived embryoid bodies (Wang et al.,2007; Xiong et al.,2008). At E8.5, no cells in the extraembryonic tissues including yolk sacs are positive for Lycat:lacZ (Fig. 3B). In addition to the heart, the lung bud, AER, and somites become positive for the transgene from E9.5 (Fig. 3C,D,E, and G). Interestingly, more cells in the atria are lacZ-positive than in the ventricles (Fig. 3G–I). β-galactosidase (lacZ) is also expressed in the epithelial layer of the third bronchial arch artery (arrow in Fig. 3H). At E11.5, lacZ is strongly expressed in the atrioventricular canal (arrow in Fig. 3I). These mesenchymal cells are critical for the development of septa and valves. The smooth muscle cells of umbilical artery are also Lycat-positive (Fig. 3J). When adult pW209 transgenic organs were examined, we found that Lycat:lacZ is restricted to the atrial myocardium (Fig. 4B and L), myocytes adjacent to ventricular endocardium (Fig. 4G and L), epicardium-derived mesenchymal cells at the base of the tricuspid valves and septa between chambers (arrows in Fig. 4G and L), and the smooth muscle cells of the coronary arteries (Fig. 4B and Q). In mature lungs, Lycat:lacZ is present in the periendothelial cells surrounding pulmonary arteries and veins (Fig. 4C,H, and M). In the arteries, veins, and airways associated with the heart or lung, LacZ is expressed in the epithelial layer of trachea and the smooth muscle layer of aorta (Fig. 4A,F,K, and P). Most strikingly, we found approximately 2% of the nucleated blood cells in the aorta and <1% of bone marrow cells are lacZ-positive (Fig. 4K, arrowhead in R and arrow in T). Previous studies demonstrate that Lycat is actively transcribed in hematopoietic and endothelial cell lineages, including the Flk1+ cells in embryoid bodies (EBs) derived from mouse ES cells and the Lin− Sca+C-Kit+/CD31+ CD45− bone marrow cells (Wang et al.,2007). Whether these lacZ-positive cells are of hematopoietic and endothelial cell lineages remains to be investigated in the future.
In female reproductive organs, Lycat:lacZ is expressed in the smooth muscle layer of uterine blood vessels (Bv in Fig. 4I), myometrial cells (arrows in Fig. 4I and N), and the epithelial layer of uterine glands (arrowheads in Fig. 4I and N). At the embryo implantation sites, uterine expression of Lycat:lacZ is up-regulated in the myometrium and glandular epithelium (Fig. 4N). In the oviduct, Lycat:lacZ expression is only discernible in the smooth muscle layer (arrowhead in Fig. 4S). In the mature ovaries, approximately half of the growing secondary follicles are positive for the Lycat:lacZ transgene (arrows in Fig. 4E,J, and S), whereas the primordial follicles are Lycat:lacZ-negative (arrowheads in Fig. 4J). Sections of these ovaries indicate that the Lycat:lacZ signals are located in the cytoplasm of maturing oocytes (Fig. 4J and S). In the lactating mammary glands, the smooth muscle cells of the lactiferous ducts (arrowheads in Fig. 4O) and the epithelial cells in the extracellular matrix of the lactiferous lobes (arrows in Fig. 4O) are actively expressing Lycat:lacZ.
Immunofluorescence labeling was also performed to determine the identity of Lycat-expressing cells. Overlapped expression of Lycat-FITC and SM-MHC-TRITC reiterates the vascular smooth muscle lineage of Lycat-expressing cells (Fig. 5A and B). Furthermore, the pW209 transgene, which co-localizes with the endogenous Lycat, is excluded from the CD31+ vascular endothelial layer (Fig. 5C and D).
Figure 5. Immunofluorescence micrographs of Lycat:lacZ transgenic (pW209) cardiac vessels labeled with the rabbit anti-mouse Lycat polyclonal (A), rabbit anti-bovine smooth muscle myosin heavy chain (B), and FITC-conjugated rat anti-mouse CD31 antibodies (C). Primary antibodies against Lycat and SM-MHC were visualized with FITC-conjugated goat anti-rabbit IgG and TRITC-conjugated goat anti-rabbit IgG secondary antibodies, respectively. Nuclei were stained with DAPI. D: Expression of Lycat:lacZ transgene in the vascular smooth muscle cells. Optical magnification is indicated at the bottom right of each panel.
Download figure to PowerPoint
In conclusion, our transgenic assay demonstrates that the RP23-299H17 BAC clone contains all of the Lycat regulatory elements, which are capable of driving the reporter gene to recapitulate the full spectrum of endogenous Lycat expression (Figs. 1, 3, 4, and 5).
Identification of a Cardiac-Specific Regulatory Region of Lycat
To identify the cis-regulatory element(s) responsible for cardiac/vascular smooth muscle/oocyte-specific expression of the Lycat gene, we first aligned the putative promoter regions of the human, mouse, and zebrafish Lycat genes, hoping that these cis-regulatory elements might have been well preserved during evolution. Unfortunately, no conserved binding motifs have been found among Lycat genes from different species. The Lycat genomic sequence was subjected for promoter scanning using databases including TransFAC (Heinemeyer et al.,1999) and ProScan (Bioinformation and Molecualr Analysis Section, NIH). Interestingly, as indicated in Figure 6A, the consensus binding motifs for various transcription factors are clustered in two genomic regions, named Cluster 1 and Cluster 2, which are located 30 and 38 kb from the 5′ end of RP23-299H17, respectively. To test the promoter activity of this proximal domain, a 9.2-kb genomic fragment containing Cluster 1, Cluster 2, and part of the putative first exon were amplified by PCR and fused to a reporter cassette, pW196a (Wang and Lufkin,2000). The resulting vector pW220 was used for transient transgenic assay (Fig. 5A). Figure 6B and C shows that this 9.2-kb proximal region of Lycat can only confer cardiac-specific transcription to the reporter. Embryonic expression of endogenous Lycat in somite, AER, lung bud, and bronchial arch artery was not observed, demonstrating that the 9.2 kb upstream sequence contains genetic information only for cardiac-specific expression, while other regulatory elements are likely located in the more 5′ distal region or the intragenic domain(s) of the Lycat gene.
Figure 6. Mapping of the cardiac-specific promoter of mouse Lycat by transient transgenesis. A: Promoter scanning demonstrates that the cis-regulatory elements are clustered in two genomic regions at the 5′ end of Lycat. These consensus-binding motifs for various transcription factors are indicated. A 9.2-kb genomic fragment containing part of the 1st exon of Lycat was tested to drive reporter gene, ires.lacZ. After pronuclear injection, embryos were collected for promoter activity by lacZ-staining. B: This 9.2-kb genomic fragment is capable of directing the reporter gene specifically in the developing heart. The white line in B indicates the level and orientation of the section in C. C: A paraffin section shows cardiac-specific expression of the reporter gene, β-galactosidase. A, anterior; D. dorsal; P, posterior; and V, ventral. The rest of the abbreviations are as in previous figures.
Download figure to PowerPoint