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

Wolf L, Yang Y, Wawrousek E, Cvekl A - BMC Dev. Biol. (2008)

Bottom Line: The number of cells expressing alphaA-crystallin in the lens pit was higher compared to the number of cells expressing EGFP.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Departments of Ophthalmology and Visual Sciences, Bronx, NY 10461, USA. lwolf@aecom.yu.edu

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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2567317&req=5

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.

Mentions: All four remaining lines expressed EGFP at E10.5 in the invaginating lens pit (Fig. 5A, F). As development progressed, strong EGFP was found throughout the lens vesicle at E11.5 (Fig. 5B, G). In addition to the characteristic upregulation of EGFP in the differentiating fiber cells at E12.5, all transgenic lines also displayed intense EGFP expression in the lens epithelium (Fig. 5C, H), which was less evident in the αA-crystallin BAC transgenics (compare with Fig. 3C, H and Fig. 4C, H). Similarly, the lens epithelial cells at E14.5 (Fig. 5D, I; Additional file 1E) and at postnatal day 1 (Fig. 5E, J; Additional file 1F) also displayed stronger EGFP expression in the lens epithelium compared to their BAC counterparts. However, while high levels of EGFP expression were observed in lens, numerous embryos from different lines also exhibited extralenticular expression in regions such as the hindbrain, and other areas of the head (Fig. 6). From these results we conclude that expression of αA-crystallin/EGFP in the lens pit is regulated by one or more enhancers present presumably within the 15 kb XmaI-SpeI genomic fragment, functionally distinct from DCR3.


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

Wolf L, Yang Y, Wawrousek E, Cvekl A - BMC Dev. Biol. (2008)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2567317&req=5

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.
Mentions: All four remaining lines expressed EGFP at E10.5 in the invaginating lens pit (Fig. 5A, F). As development progressed, strong EGFP was found throughout the lens vesicle at E11.5 (Fig. 5B, G). In addition to the characteristic upregulation of EGFP in the differentiating fiber cells at E12.5, all transgenic lines also displayed intense EGFP expression in the lens epithelium (Fig. 5C, H), which was less evident in the αA-crystallin BAC transgenics (compare with Fig. 3C, H and Fig. 4C, H). Similarly, the lens epithelial cells at E14.5 (Fig. 5D, I; Additional file 1E) and at postnatal day 1 (Fig. 5E, J; Additional file 1F) also displayed stronger EGFP expression in the lens epithelium compared to their BAC counterparts. However, while high levels of EGFP expression were observed in lens, numerous embryos from different lines also exhibited extralenticular expression in regions such as the hindbrain, and other areas of the head (Fig. 6). From these results we conclude that expression of αA-crystallin/EGFP in the lens pit is regulated by one or more enhancers present presumably within the 15 kb XmaI-SpeI genomic fragment, functionally distinct from DCR3.

Bottom Line: The number of cells expressing alphaA-crystallin in the lens pit was higher compared to the number of cells expressing EGFP.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Departments of Ophthalmology and Visual Sciences, Bronx, NY 10461, USA. lwolf@aecom.yu.edu

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.

Show MeSH
Related in: MedlinePlus