Transcriptional regulation of mouse alpha A-crystallin gene in a 148kb Cryaa BAC and its derivates

Abstract

Background: alphaA-crystallin is highly expressed in the embryonic, neonatal and adult mouse lens. Previously, we identified two novel distal control regions, DCR1 and DCR3. DCR1 was required for transgenic expression of enhanced green fluorescent protein, EGFP, in lens epithelium, whereas DCR3 was active during "late" stages of lens primary fiber cell differentiation. However, the onset of transgenic EGFP expression was delayed by 12-24 hours, compared to the expression of the endogenous Cryaa gene.

Results: Here, we used bacterial artificial chromosome (BAC) and standard transgenic approaches to examine temporal and spatial regulation of the mouse Cryaa gene. Two BAC transgenes, with EGFP insertions into the third coding exon of Cryaa gene, were created: the intact alphaA-crystallin 148 kb BAC (alphaA-BAC) and alphaA-BAC(DeltaDCR3), which lacks approximately 1.0 kb of genomic DNA including DCR3. Expression of EGFP in the majority of both BAC transgenics nearly recapitulated the endogenous expression pattern of the Cryaa gene in lens, but not outside of the lens. The number of cells expressing alphaA-crystallin in the lens pit was higher compared to the number of cells expressing EGFP. Next, we generated additional lines using a 15 kb fragment of alphaA-crystallin locus derived from alphaA-BAC(DeltaDCR3), 15 kb Cryaa/EGFP. A 15 kb region of Cryaa/EGFP supported the expression pattern of EGFP also in the lens pit. However, co-localization studies of alphaA-crystallin and EGFP indicated that the number of cells that showed transgenic expression was higher compared to cells expressing alphaA-crystallin in the lens pit.

Conclusion: We conclude that a 148 kb alphaA-BAC likely contains all of the regulatory regions required for alphaA-crystallin expression in the lens, but not in retina, spleen and thymus. In addition, while the 15 kb Cryaa/EGFP region also supported the expression of EGFP in the lens pit, expression in regions such as the hindbrain, indicate that additional genomic regions may play modulatory functions in regulating extralenticular alphaA-crystallin expression. Finally, deletion of DCR3 in either alphaA-BAC(DeltaDCR3) or Cryaa (15 kb) transgenic mice result in EGFP expression patterns that are consistent with DCR's previously established role as a distal enhancer active in "late" primary lens fiber cells.

Figure 1

αA-crystallin protein expression during lens morphogenesis. Nuclear DAPI staining is blue and αA-crystallin immunolabeling is red. αA-crystallin is not expressed at E9.5 in the lens placode (A) but becomes visible in the lens pit at E10.5 (B). Expression intensifies in the developing lens vesicle at E11.5 (C), and is evident in the newly differentiated fiber cells at E14.5 (D), as well as within the lens epithelium. αA-crystallin expression continues in the fiber (E) and epithelial cells of the P1 lens (F). Lens epithelial cells, e; lens fiber cells, f; lens vesicle, lv; lens pit, lp. Scale bar = 100 μm.

Figure 2

Schematic representation of three αA-crystallin transgene constructs. (A) A schematic diagram of the mouse Cryaa locus (chromosome 17) and its adjacent loci, U2af1 and Snf1lk. The 148 kb BAC Clone RP-23-465G4 is shown in red. The XmaI-SpeI sites delineate 15 kb of the Cryaa locus. Exons in the Cryaa gene (black box), DCR1 and DCR3 (orange box), centromere, cen; telomere, tel; Exon, ex; rodent specific Cryaa exon, Ins. (B) Modification of αA-BAC using the λ prophage system for homologous recombination [25]. (C) Generation of αA-BAC(ΔDCR3) using a shuttle vector [26]. (D) Diagrammatic representation of the 15 kb portion of the mouse αA-crystallin locus with an EGFP insert (see panel (C)) marked by unique XmaI and SpeI restriction sites.

Figure 3

Expression analysis of αA-BAC transgenic mice. EGFP expression at E10.5 in the lens pit (A, F) and within the E11.5 lens vesicle (B, G). Expression is upregulated in lens fiber cells at E12.5, and very small expression is also apparent in a few cells of the lens epithelium (arrow) (C, H). Strong EGFP expression continues in the fiber cells at E14.5 (D, I) and in the PND1 lens (E, J). Nuclei are stained blue with DAPI, and the cytoskeletal staining is red. Lens fiber cells, f; lens pit, lp; lens vesicle, lv. Scale bar = 100 μm.

Figure 4

αA-BAC(ΔDCR3) transgene expression in the lens. Expression of EGFP from αA-BAC(ΔDCR3) is first observed at E10.5 in the lens pit (A, F). EGFP is expressed in the lens vesicle at E11.5 (Fig. B, G) and within the differentiating primary fiber cells at E12.5 (C, H), E14.5 (D, I) and in PND 1 lens (E, J). Fluorescent nuclear DAPI staining is blue and cytoskeletal staining is red. Lens fiber cells, f; lens pit, lp; lens vesicle, lv. Scale bar = 100 μm.

Figure 5

Expression of EGFP from the 15 kb Cryaa/EGFP fragment in lens. EGFP expression is first evident at E10.5 in the lens pit (A, F) and becomes expressed throughout the lens vesicle at E11.5 (B, G). As fiber cell differentiation commences at E12.5 (C, H), intense EGFP expression is observed. Prominent expression is also evident in the overlaying lens epithelium (C, H). EGFP expression continues to be highly expressed both in the lens fiber and epithelial cells of the developing E14.5 (D, I) and PND1 lens (E, J). Nuclear DAPI staining is blue, and the cytoskeletal phalloidin staining is red. Lens epithelial cells, e; lens fiber cells, f; lens pit, lp; lens vesicle, lv. Scale bar = 100 μm.

Figure 6

Extralenticular expression of EGFP in 15 kb Cryaa/EGFP transgenics. EGFP expression was observed at E9.5 (A), E10.5 (B), E11.5 (C) and (D) (higher magnification of (C)) (red arrows) in the hindbrain. The dashed white line illustrates the embryo and red arrowhead indicates EGFP expression in the lens. Scale bar = 1 mm.

Figure 7

Transgene expression coincides with αA-crystallin protein. Panels A-E are sections from αA-BAC mice; F-J are from αA-BAC(ΔDCR3) mice; and K-O are from the 15 kb Cryaa/EGFP mice. Red staining is αA-crystallin immunofluorescence, green is EGFP fluorescence and yellow represents co-localization of these two signals. Scale bar = 100 μm.