|Molecular Vision 2004;
Received 23 March 2004 | Accepted 15 July 2004 | Published 30 August 2004
Expression of cytochrome P4501b1 (Cyp1b1) during early murine development
Ivaylo Stoilov,1 Tayebeh
Rezaie,2 Ingela Jansson,1 John B. Schenkman,1 Mansoor
1Department of Pharmacology and 2The Molecular Ophthalmic Genetics Laboratory, Surgical Research Center, Department of Surgery, University of Connecticut Health Center, Farmington, CT
Correspondence to: Ivaylo Stoilov, Department of Pharmacology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT, 06030-6125; Phone: (860) 679-4997; FAX: (860) 679-3693; email: firstname.lastname@example.org
Purpose: To examine the embryonic expression of cytochrome P4501b1 (Cyp1b1) gene by whole mount in situ hybridization.
Methods: FVB/NcrlBR mouse embryos staged at 9.5, 10.5, and 11.5 dpc were obtained by timed breeding experiments. Antisense and sense RNA probes labeled with digoxigenin UTP were generated by in vitro transcription of an 848 bp Cyp1b1 cDNA fragment that was subcloned into transcription vector pCR®II-TOPO®. The digoxigenin labeled RNA was localized using an alkaline phosphatase conjugated anti-digoxigenin Fab fragment. Colorimetric detection of the digoxigenin labeled probe was performed with substrate solution containing 4-nitro-blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP).
Results: During early stages of murine development Cyp1b1 mRNA was detected in the developing eye, hindbrain, branchial arches, forelimb bud, ligaments supporting the liver primordium and developing kidney. In the eye and forelimb bud Cyp1b1 displayed restricted expression along the axes of development. In the developing eye Cyp1b1 exhibited dorsal expression with respect to the dorso-distal/proximo-ventral axis and anterior expression with respect to the anterior-nasal/posterior-temporal axis. In the forelimb bud Cyp1b1 expression was localized posteriorly. The polarity of Cyp1b1 expression was lost at 11.5 dpc, at which time expression was additionally seen in ventral (eye) and anterior (forelimb bud) areas.
Conclusions: The spatio-temporal expression patterns observed in this study suggest that during early stages of murine development, Cyp1b1 participates in establishment and/or maintenance of polarity along the axes of embryonic development. Expression of Cyp1b1 in the dorso-distal end of the optic cup, from which the ciliary body and iris are derived, correlates with the expression patterns seen in adult tissues and the abnormal development of these structures as part of the glaucoma phenotypes resulting from Cyp1b1 mutations.
Cytochrome P4501B1 (CYP1B1) gene [1,2] encodes a monooxygenase capable of metabolizing both xenobiotics  and endogenous compounds such as estrogen, testosterone and retinoids [4-7]. In humans, mutations affecting CYP1B1 are associated with abnormal development of the anterior chamber angle of the eye manifested as aggressive, early onset glaucoma, which most frequently is classified as primary congenital glaucoma [8-11]. Cyp1b1-/- mice have milder phenotype due to the presence of both normal and abnormal filtration structures [12-14]. In situ hybridization analysis of tissue sections detected Cyp1b1 mRNA in the developing and adult mouse eye at embryonic day 15 (E15) and postnatal days 4, 7, and 30 . PCR based assay of whole mouse embryo cDNA suggested that Cyp1b1 is expressed as early as E11 . The objective of this study was to examine Cyp1b1 expression domains during the early stages of murine development by whole mount in situ hybridization.
Male and female FVB/NCrlBR mice were purchased from Charles River Laboratories (Wilmington, MA). For timed mating experiments breeding units (one male and two females) were set at 3.30-4 PM. Inspection for vaginal plugs was performed before 9 AM on the following day. For staging purposes, noon of the day of vaginal plug was considered 0.5 days post-conception (dpc). On the desired day, animals were euthanized at noon by carbon dioxide inhalation. Embryos from two pregnant females were collected at 9.5 dpc. Embryos from one pregnant female were collected at 10.5 dpc and 11.5 dpc, respectively. The embryos were dissected from the uterus, fixed overnight with 4% paraformaldehyde in phosphate buffered saline (PBS) at 4 °C, dehydrated through a methanol series (25%, 50%, 75%, 100%, and 100%) and stored in 100% methanol at -20 °C. All animal manipulations were conducted in accordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals and were approved by The Animal Care Committee (ACC) of the University of Connecticut Health Center.
In situ hybridization
A 848 bp Cyp1b1 cDNA fragment was amplified by polymerase chain reaction (PCR) by using primer set of 5'-AGC TGA GCT CGC TGT CTA CC-3' and 5'-GTC CGT CAT GTC TCG AGG AG-3' (GenBank accession number U03283, nt 391-1238). This fragment was subcloned into transcription vector pCR®II-TOPO® (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Antisense and sense RNA probes labeled with digoxigenin-UTP were generated by in vitro transcription of linearized plasmids with SP6 and T7 RNA polymerase (Roche Molecular Biochemicals, Mannheim, Germany) according to manufacturer's protocol. In situ hybridization of mRNA with digoxigenin labeled probes was performed as described by Xu and Wilkinson . The digoxigenin labeled RNA was localized using an alkaline phosphatase conjugated Fab fragment from a sheep anti-digoxigenin antibody. Colorimetric detection of the digoxigenin labeled probe was performed with substrate solution containing 350 mg/ml of nitroblue tetrazolium (NBT) and 175 mg/ml 5-bromo-4-chloro-3-indolyl phosphate (BCIP). For sectioning, the whole-mounts were fixed with 4% paraformaldehyde at 4 °C overnight and equilibrated overnight in 30% sucrose in PBS. The embryos were mounted on cryostat chuck with Tissue Tek® O.C.T. (Sakura Finetek U.S.A., Inc., Torrance, CA). Sections of 20-50 μm were cut on cryostat and mounted under a coverslip with Gel/MountTM (Biomeda Corp., Foster City, CA).
The Cyp1b1 expression domains were described in accordance with the standard anatomical nomenclature (developed at the Department of Biomedical Sciences, University of Edinburgh, The MRC Human Genetics Unit, Edinburgh, Scotland) and The Gene Expression Database Project (The Jackson Laboratory, Bar Harbor, ME).
Results & Discussion
Cyp1b1 Expression at 9.5 dpc
Upon inspection of the whole-mounts, Cyp1b1 expression domains were seen in the developing branchial arches and future hindbrain (Figure 1, Figure 2). The hindbrain expression domain is located immediately above the level of the otic vesicle and is most likely localized to the fourth rhombomere. A less pronounced signal is seen in the forelimb bud and the optic eminence (Figure 1A). Inspection of frontal (coronal) sections of the optic eminence revealed that the neuroectoderm of the optic vesicle has come in close contact with the surface ectoderm (Figure 2B). Previous studies have suggested that the close contact permits initiation of inductive signals from the surface ectoderm essential for the development of the neural retina . At this critical stage Cyp1b1 expression was detected in neuroectoderm adherent to the surface ectoderm. With respect to the dorso-distal/proximo-ventral axis of the developing eye Cyp1b1 expression was restricted to the dorsal half of the future neural retina (Figure 2B). Cyp1b1 mRNA was detected in ectoderm and endoderm of the second and third branchial arches including branchial grooves, pouches and membranes (Figure 2C). The expression was limited to the cranial portions of these structures. Cyp1b1 mRNA was also detected in the mesenchyme of the second branchial arch (Figure 2D). The first branchial groove develops into the external auditory meatus while the corresponding pouch forms the middle ear cavity and the Eustachian tube. The primordium of palatine tonsil originates from the second branchial pouch. The stapes, styloid process, stylohyoid ligament and the lesser horn of the hyoid bone originate from the second branchial arch . While no developmental abnormalities in these structures have been reported in individuals with CYP1B1 mutations or theCyp1b1-/- mouse, the expression profiles described here invite a more detailed inspection of the structures derived from the branchial region.
Cyp1b1 expression at 10.5 dpc
In contrast to the 9.5 dpc embryo, demarcated and asymmetric expression domains could be seen in the optic eminence and forelimb bud (Figure 3A and Figure 4A). During this stage the thickened surface ectoderm is indented and forms the lens pit while the optic cup is formed by invagination of the optic vesicle (Figure 3C). The expression of Cyp1b1 in the distal portion of the optic cup (Figure 3C), from which the ciliary body and iris are derived , correlates with the expression patterns reported in adult eyes and the abnormal development of these structures as part of the glaucoma phenotypes associated with CYP1B1 mutations [8-14]. Bejjani et al., reported that Cyp1b1 is expressed in the neural retina from embryonic day 15 (E15) onward and in the ciliary body of the adult mouse eye . In the developing mouse eye (10.5 dpc) Cyp1b1 expression continues to be restricted along the dorso-distal/proximo-ventral axis. Its expression is limited to dorsal half of the inner layer of the optic cup which will become the neural layer (neural epithelium) of the retina Figure 3C. Along the anterior-nasal/posterior-temporal axis Cyp1b1 expression was limited to the anterior (nasal) half of the retina (Figure 3A, Figure 4A, Figure 5D). Therefore, in relation to both axes, Cyp1b1 expression is limited to the dorso-distal/anterior-nasal quadrant of the future neural retina. The expression domains in the branchial arches are consolidated to relatively small caudal areas on the tip of each arch (Figure 3C, Figure 4B). Cyp1b1 expression is no longer seen in the endoderm and the branchial pouches. In the forelimb bud Cyp1b1 expression is localized posteriorly (Figure 3A). There are no apparent changes in the hindbrain expression domain, where Cyp1b1 mRNA is detected in the ventricular layer of the lateral walls of hindbrain (Figure 4C,F,G).
Cyp1b1 expression at 11.5 dpc
This stage is marked by alterations in the polarity of Cyp1b1 expression (Figure 5). In the developing eye, Cyp1b1 expression is also seen in the ventral portion of the neural retina (Figure 5A,B and Figure 6E). Another novel element is the detection of fine rim of Cyp1b1 positive tissue around the optic eminence (Figure 5A,B and Figure 6J). This layer is detected after 15 min of proteinase K digestion (10μg/ml at room temperature), but it was lost after 25 min digestion. In this area Cyp1b1 expression is limited to the ectoderm (Figure 6). Symmetric anterior and posterior domains limited to the mesenchyme underlying the apical ectodermal ridge are observed in the forelimb (Figure 5A,B and Figure 5F). No Cyp1b1 expression is detected in the hindlimb bud. In the developing pharyngeal area, Cyp1b1 expression is detected in area of the second branchial arch representing the future palatine tonsils (Figure 5E). The hindbrain expression is essentially unchanged (Figure 5G). Cyp1b1 expression was detected in ligaments supporting the hepatic primordium but not in the liver parenchyma itself (Figure 5H). Cyp1b1 expression was detected also in the metanephric diverticulum (Figure 5J).
Theoretical considerations based on the expression pattern and metabolic activity of Cyp1b1
Studies on the early patterning of the optic vesicle have identified genes that display dorsal-ventral (Pax2, Vax2, Xbr-1, Tbx5) and anterior-posterior (Foxg1, Foxd1 SOHo1, and GH6) polarity . Interestingly, according to our results, Cyp1b1 displays both dorsal-ventral and anterior-posterior polarity. Better understanding of the substrate specificity of Cyp1b1 should provide basis for proper interpretation of the importance such highly restricted expression may have for the regional specification of the optic vesicle. Cyp1b1 encodes a monooxygenase [1,2]. The substrate specificities of human, rat, and mouse P4501B1 have been the subject of numerous studies. One consistent observation is that the 1B1 family of enzymes is capable of metabolizing ligands for the nuclear receptor family such as steroids and retinoids [4-7]. Due to their relatively small size and lipophilicity, these molecules can readily diffuse from the site of their synthesis forming either gradient or sharply demarcated patterns. These patterns might then be translated into spatial patterns of cellular differentiation as a result of steroid and retinoid abilities to modulate gene transcription via binding to various nuclear receptors [21,22]. It is reasonable to expect that the restricted expression of Cyp1b1 along the axes of development in the eye and forelimb bud, revealed by this study, would result in asymmetric distribution of Cyp1b1 metabolites. Any alterations in the topography of Cyp1b1 expression, as a result of DNA mutations or environmental influences, may alter the normal distribution of Cyp1b1 metabolites and interfere with the normal development of the affected areas .
IS gratefully acknowledges Professor Christo Chouchkov for his comments and Ms. Marissa Caudill for her assistance in the preparation of the manuscript. This work was supported by grants from the Glaucoma Research Program of The American Health Assistance Foundation G2001-021 (IS) and by the National Eye Institute R03 EY014654 (IS) and R01 EY11095 (MS).
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