During vertebrate lens development, a group of head ectoderm cells thickens and forms the lens placode in response to inductive signals from the underlying optic vesicle at embryonic day (e) 9.5 of mouse embryogenesis (Muthukkaruppan,1965; McAvoy,1980; Lovicu and McAvoy,2005; Medina-Martinez and Jamerich,2007). By e11.5, the lens placode, through invagination, develops into a lens vesicle in which the primary lens fiber cells in the posterior region eventually exit the cell cycle to elongate toward the anterior wall. Three days later, this elongation is complete, and the fully differentiated fiber cells come into contact with the monolayer of cuboidal lens epithelial cells at the anterior of the eye. Throughout most of life, cell proliferation occurs preferentially in a subset of the epithelial cells located near the equatorial zone. After undergoing cell division, they withdraw from the cell cycle and move posteriorly to differentiate into secondary lens fiber cells (McAvoy,1980; Piatigorsky,1981; Lovicu and McAvoy,2005; Medina-Martinez and Jamerich,2007). As fiber cells differentiate, they rapidly increase in length and volume and accumulate high levels of crystallins, the proteins that account for the transparency and high refractive index of the lens (Piatigorsky,1981; Lovicu and McAvoy,2005; Andley,2007). After completing elongation, fiber cells partially fuse with their neighbors and degrade all membrane bound organelles, including the nuclei.
The cessation of cell proliferation requires the expression of cyclin-dependent kinase (CDK) inhibitors (CKIs). Two families of CKIs have been identified. The Cip/Kip family contains Cdkn1a/p21, Cdkn1b/p27, and Cdkn1c/p57, which inhibit all kinases involved in the G1/S transition. The INK4a family, composed of Cdkn2b/p15, Cdkn2a/p16, Cdkn2c/p18, and Cdkn2d/p19, specifically inhibit Cdk4 and Cdk6, blocking entry into the cell cycle (Harper and Elledge,1996; Sherr and Roberts,1995; Nakayama and Nakayama,1998). Cdkn1b/p27 and Cdkn1c/p57 are coexpressed during murine lens development, especially in the equatorial zone of the fetal lens (Zhang et al.,1998; Nagahama et al.,2001). The withdrawal of lens fiber cells from the cell cycle largely depends on the expression of Cdkn1b/p27 and Cdkn1c/p57 because in mice that lack these genes, fiber cells continue to proliferate and cause incomplete lens fiber elongation. Eventually, these fiber cells undergo apoptosis in Cdkn1b/p27−/− and Cdkn1c/p57+/−m (m, denoting maternal active Cdkn1c/p57 allele) compound mutant mice (Zhang et al.,1998). The other cell cycle regulators involved in lens differentiation are the D-type cyclins. All three D-type cyclins are expressed during lens differentiation, with Ccnd2/Cyclin D2 being the most highly expressed cyclin in the posterior region (Zhang et al.,1998). Down-regulation of Ccnd2/Cyclin D2 in the postmitotic lens fiber cell is required for the maintenance of the postmitotic state (Gomez et al.,1999).
Several genes have been identified that play important roles in the development of the lens. Gata3 encodes a transcription factor containing two steroid hormone receptor-like zinc fingers that serve as a DNA binding domain, a motif that is highly conserved amongst all six members (GATA-1 to -6) (Patient and McGhee,2002). These zinc fingers bind most avidly to the consensus motif AGATCTTA (Ko and Engel,1993). The physiological roles of GATA-3 has been revealed through the analysis of GATA-3–deficient ES cells or various germ line mutant mice: GATA-3 plays a critical role in the differentiation of T lymphocytes, hair follicles, mammary gland, renal, and central nervous systems (Pandolfi et al.,1995; Ting et al.,1996; van Doorninck et al.,1999; Kaufman et al.,2003; Grote et al.,2006; Kouros-Mehr et al.,2006; Kurek et al.,2007; Hasegawa et al.,2007; Asselin-Labat et al.,2007). Moreover, GATA-3 is prominently expressed in the primary sympathetic chain and persists during the development of all sympathoadrenal (SA) lineages, including sympathetic neurons, adrenal chromaffin cells, and para-aortic chromaffin cells (the Zuckerkandl organ; George et al.,1994; Lakshmanan et al.,1999; Lim et al.,2000; Moriguchi et al.,2006).
Gata3 null mutants die around e11 as a consequence of primary noradrenalin biosynthetic defect and secondary cardiac failure (Gata3−/−; Pandolfi et al.,1995; Lim et al.,2000). But they can be rescued by feeding Gata3 heterozygous intercrossed dams with synthetic catecholamine intermediates, or by restoring GATA-3 function specifically in SA lineages using the human dopamine β-hydroxylase (hDBH) promoter to direct GATA-3 transgenic expression (TghDBH-G3; Lim et al.,2000; Moriguchi et al.,2006). We and others have previously reported that GATA-3 is expressed in lens fiber cells of murine embryos (Oosterwegel et al.,1992; Lakshmanan et al.,1999), although the physiological significance of this observation is unknown. In the present study, we examined the consequences of a GATA-3 loss-of-function mutation in lens development of TghDBH-G3-rescued Gata3 null mutants. We demonstrate that Gata3 inactivation lead to abnormal development of the posterior lens fiber cells, which exhibit reduced levels of the differentiation marker γ-crystallin, sustained expression of lens vesicle marker E-cadherin and the increased signal of proliferation markers, i.e., BrdU incorporation and Ki67 immunoreactivity. The abnormal proliferation of the lens fiber cells in TghDBH-G3-rescued Gata3 null mutant lenses correlates with reduced levels of Cdkn1b/p27 and Cdkn1c/p57 CKIs as well as increased Ccnd2/Cyclin D2 abundance. Subsequently, these cells succumbed to apoptotic cell death. The molecular pathway that regulates lens differentiation is intimately intertwined with normal cell cycle control, and GATA-3 plays an important role in cellular differentiation of lens fiber cells by inducing cell cycle exit as a part of its regulatory functions.