2012 Theses Doctoral

Complex Regulation of Pax6 Neuronal Progenitors By Rb Family Members During Corticogenesis

Naglieri, Benedetta

The retinoblastoma tumor suppressor (pRB) inhibits tumorigenesis by restraining cell cycle progression via repression of the E2F transcription factor family and by promoting cell differentiation via activation of lineage-specific transcription factors. In contrast, the closely related pRB homologues, p107 and p130, are known to inhibit cell cycle progression by repressing the E2F transcription factor family, but are not known to have roles in promoting cell differentiation. Interestingly, the Rb promoter contains a critical cassette of binding sites (Sp1/Ets, ATF and E2F) that is conserved between mice and humans. Previously, our lab developed a wild type Rb promoter-LacZ transgenic reporter line (T157) that displayed dynamic and neuronal-specific expression (Agromayor et al., 2006).

We generated mutant Rb promoter-LacZ transgenic lines and demonstrated that the conserved cassette controls Rb expression, positively through the Sp1/Ets site and negatively through the E2F site. Repression of the Rb promoter through this critical E2F site means that the E2F family lies both upstream and downstream of Rb, and suggests that Rb family members regulate the Rb promoter during neuronal development. To identify which Rb family member represses the Rb promoter during corticogenesis, we generated RbP-LacZ lines in genetic backgrounds deficient in various Rb family members and looked for deregulation of RbP-LacZ activity within the embryo (Aim 1).

Surprisingly, RbP-LacZ activity responds in opposing ways with either loss of Rb or dual loss of p107 and p130, demonstrating that regulation of the Rb promoter by Rb family members during corticogenesis is complex. To determine whether direct or indirect mechanisms are responsible for the opposing changes in RbP-LacZ expression with loss of Rb family members in the developing cortex, we evaluated occupancy at the Rb promoter (ChIP analysis), proliferation, cell death (BrdU incorporation and TUNEL analysis) and changes in gene expression (RT-PCR) in wild type vs. mutant cortices from embryos lacking various Rb family members (Aim 2). Interestingly, we found evidence for both direct and indirect action of Rb family member inactivation on the Rb promoter.

To determine if the opposing changes in RbP-LacZ activity with either loss of Rb or dual loss of p107 and p130 occurs in a cell autonomous or a non-cell autonomous manner, we optimized and analyzed primary cortical neuron cultures from wild type and mutant embryos to quantitate RbP-LacZ activity on a cell-by-cell basis (Aim 3). We compared changes in the frequency and intensity of RbP-LacZ activity, the distribution of neuronal subpopulations, identified the cells expressing RbP-LacZ activity and evaluated differences in these populations with loss of various Rb family members. Through these studies, we have discovered a complex relationship exists between Rb family members and Pax6 progenitors during corticogenesis, underscoring the intricate nature of the network connecting the Rb and E2f families in vivo.