In the present study, we demonstrated that GATA-3 expression begins in the developing lens vesicle at mid-embryogenesis (around e11.5) and continues to be expressed in fiber cells throughout embryonic lens development. Its expression is specifically restricted to fiber cells during lens morphogenesis. Consistent with its spatiotemporal expression in the developing lens, the absence of GATA-3 led to interrupted differentiation of posterior lens fiber cells from e12.5 onward, as evidenced by the diminished γ-crystallin levels and prolonged E-cadherin expression in primary lens fiber cells. There was also an increase of mitotic (BrdU- or Ki67-immunopositive) and apoptotic fiber cells in the GATA-3–depleted lens.
Cell Cycle Regulation by GATA Factors Has Been Reported in a Variety of Different Tissues
Recently, it was reported that Gata2-deficient mouse embryonic neuroepithelial cells exhibited aberrant proliferation and that GATA-2 overexpression induced neural differentiation by inhibiting the proliferation of neuronal progenitors by means of activation of Cdkn1b/p27 expression (El Wakil et al.,2006). In erythroid cell differentiation, GATA-1 was reported to induce erythro-megakaryotic differentiation by suppressing the active cell cycle of hematopoietic progenitor cells by means of induction of Cdkn2a/p16 expression (Pan et al.,2005). Although it is still unclear if GATA-2 or GATA-1 directly regulates Cdkn1b/p27 or Cdkn2a/p16 expression, respectively, these reports as well as the present observations suggest that the potential cell cycle regulatory function for GATA factors in the normal differentiation process acts by activating expression of CKIs. GATA-3 has been reported to suppress abnormal proliferation of mesonephric cells as well as mammary epithelial cells, although the molecular basis for these phenomena remains elusive (Grote et al.,2006; Kouros-Mehr et al.,2006). More recently, transcriptome analysis of GATA-3 conditional deletion in hair follicles indicated that multiple cell cycle regulatory genes were altered in expression (Kurek et al.,2007). Further studies will be necessary to determine how GATA-3 functionally coordinates cell cycle regulation with normal differentiation in a variety of GATA-3–expressing tissues, including lens fiber cells.
Although the mechanistic details of how the loss of GATA-3 results in lens fiber differentiation failure remains to be elucidated, cell cycle regulators may be the potential key molecules underlying the abnormal increase of proliferating cells. In the wild-type lens, the epithelial cells near the equatorial zone exit the cell cycle to give rise to fiber cells, and in the process, they initiate the expression of Cdkn1b/p27 and Cdkn1c/p57. Cdkn1b/p27 and Cdkn1c/p57 cooperatively control the cell cycle exit and the subsequent differentiation of lens fiber cells. Cdkn1b/p27 is normally dispensable for lens development due to its redundancy with Cdkn1c/p57, whereas Cdkn1b/p27−/− and Cdkn1c/p57+/−m, like the Gata3-deficient mice, exhibit significant deficiencies in cell cycle withdrawal and in the subsequent differentiation of lens fiber cells (Zhang et al.,1998; Nagahama et al.,2001). In the Gata3-deficient lens, we demonstrated that Cdkn1b/p27 and Cdkn1c/p57 immunoreactivities were dramatically suppressed in the equatorial zone, and that their mRNA abundance was reduced in lens, suggesting that GATA-3 deficiency results in the suppression of these two CKIs at the transcription level. However, we have been unable to identify conserved GATA consensus binding sites around the Cdkn1b/p27 and Cdkn1c/p57 promoters or to observe GATA-3–dependent trans-activation of a reporter gene cis-linked to a 1.6-kbp Cdkn1b/p27 or a 2.0 kbp Cdkn1c/p57 promoter in several cell lines in co-transfection experiments (data not shown), suggesting that Cdkn1b/p27 and Cdkn1c/p57 are either not direct target genes of GATA-3 or that GATA-3 regulates those genes through enhancers that lie outside of the promoter boundaries.
We examined Sox1, Foxe3, Prox1, c-Maf, and Pax6 mRNA expression in e16.5 GATA-3–deficient lenses to examine potential genetic interactions between GATA-3 and each of those other known lens developmental regulators. Sox1 expression initiates in the lens vesicle at around e10 and continues to be expressed in lens fiber cells at e15.5 (Nishiguchi et al.,1998). Foxe3, Prox1, and c-Maf expression is first detected at around e9.0–e9.5 over the lens placode (Wigle et al.,1999; Kawauchi et al.,1999; Medina-Marinez et al.,2005). Foxe3 expression later becomes restricted to the anterior lens epithelium, while Prox1 and c-Maf expression are maintained in the lens fiber cells (Wigle et al.,1999; Kawauchi et al.,1999; Medina-Martinez et al.,2005). Pax6 expression is observed much earlier (in head neural ectoderm) including in the optic pit at e8.0, although from e13.5 onward, Pax6 expression is down-regulated in lens fiber cells (Grindley et al.,1995; Donner et al.,2007). Given those spaciotemporal expression patterns and the similarities in lens deficiencies encountered in various mutant mice, we initially expected to establish a genetic regulatory relationship between GATA-3 and Sox1 or Prox1 expression in the developing lens fiber cells. However, all of those transcriptional regulators are in general only modestly, if at all, changed in the GATA-3 deficient lens. Given the later appearance of GATA-3 expression in the e10.5 lens vesicle as well as the relatively mild lens deficiency in Gata3 mutant mice, we assumed that GATA-3 might be located at a lower position in the hierarchy, but upstream of γ-crystallin and both CKIs (Cdkn1b/p27 and Cdkn1c/p57) in the genetic program of lens development. Of interest, GATA-3 expression is strongly activated in the remnants of the c-Maf–deficient lens. This observation clearly demonstrates that GATA-3 expression is directly or indirectly negatively controlled by c-Maf in normal developing lens fiber cells, so that a c-Maf deficiency derepresses GATA-3 expression, possibly to compensate for the suppressed crystallin gene activation. Precise mapping of lens-specific Gata3 gene regulatory sequences, which are presumably located within a 2-kbp region lying 5′ to the gene (George et al.,1994; Lieuw et al.,1997), will provide additional insight into the identities of upstream regulators of GATA-3 expression in lens fiber cells.
During differentiation, mature lens fiber cells produce abundant β- and γ-crystallins (McAvoy,1978). Of the crystalline subtypes, α-crystallins are normally expressed in both lens epithelial and fiber cells, and are first expressed at the lens vesicle stage (McAvoy,1978; Murer-Orland et al.,1987; Goring et al.,1992; Horwitz,2003). β-Crystallin expression, which begins at e11 in the mouse embryo, serves as an early marker of fiber cell differentiation, whereas γ-crystallin gene activation initiates around e12.5 (Goring et al.,1992; Nishiguchi et al.,1998; Ring et al.,2000). We showed here that γA, γC and γD-crystallin expression, which are normally restricted in expression to terminally differentiated fiber cells, were more diminished than αA- and β1-crystallin in Gata3−/−:TghDBH-G3 lenses. In the Gata3 mutant lens, the fiber cell differentiation failure is associated with aberrant accumulation of mitotic posterior cells. There are two possible explanations for this observation. One is that GATA-3 primarily promotes fiber cell differentiation, i.e., activation of γ-crystallin genes as well as suppression of E-cadherin expression, so that a GATA-3 deficiency would primarily induce a fiber cell deficiency which in turn would lead to the accumulation of premature epithelial cell-like posterior cells. The other possibility is that the GATA-3 deficiency primarily but indirectly leads to transcriptional suppression of Cdkn1b/p27 and Cdkn1c/p57, which then cause the failure of cell cycle cessation during epithelial to fiber cell transition. Consequently, the posterior lens fiber cells do not fully differentiate and eventually apoptose. Although these two explanations could both be partially correct, cell differentiation and cell cycle cessation are probably tightly interwoven. It will, therefore, be of great interest to further define how GATA-3 functions during induction of cell differentiation as well as how it regulates cell cycle suppression during lens development.
In conclusion, we demonstrated here that GATA-3 is essential for terminal differentiation of lens fiber cells. It will be intriguing to clarify the underlying mechanisms by which the expression of CKIs and CDKs are controlled by GATA factors, and to identify other cell cycle/apoptosis-related factors which might be responsible for the increased cell death observed in the Gata3-deficient lens fiber cells. We conclude, from the data presented here, that the Gata3 mutant mouse lens may serve as another useful model for elucidating the general principles of cell cycle regulation by GATA transcription factors.