Unique Requirement for Rb/E2F3 in Neuronal Migration: Evidence for Cell Cycle-Independent Functions

Abstract

The cell cycle regulatory retinoblastoma (Rb) protein is a key regulator of neural precursor proliferation; however, its role has been expanded to include a novel cell-autonomous role in mediating neuronal migration. We sought to determine the Rb-interacting factors that mediate both the cell cycle and migration defects. E2F1 and E2F3 are likely Rb-interacting candidates that we have shown to be deregulated in the absence of Rb. Using mice with compound null mutations of Rb and E2F1 or E2F3, we asked to what extent either E2F1 or E2F3 interacts with Rb in neurogenesis. Here, we report that E2F1 and E2F3 are both functionally relevant targets in neural precursor proliferation, cell cycle exit, and laminar patterning. Each also partially mediates the Rb requirement for neuronal survival. Neuronal migration, however, is specifically mediated through E2F3, beyond its role in cell cycle regulation. This study not only outlines overlapping and distinct functions for E2Fs in neurogenesis but also is the first to establish a physiologically relevant role for the Rb/E2F pathway beyond cell cycle regulation in vivo.Neurogenesis is a highly regulated process by which neural precursors divide and differentiate, giving rise to the cells that make up the nervous system (reviewed in references 24 and 25). While the role of cell cycle genes in regulating proliferation of neural precursor cells is well appreciated, accumulating data point convincingly to their unique roles in regulating diverse cellular processes, independent of cell cycle regulation (reviewed in reference 63). The retinoblastoma (Rb) tumor suppressor is a key cell cycle regulator that we along with others have shown to play a number of roles in neurodevelopment including proliferation, survival, and, more recently, neuronal migration (7,8,10,18,19,33,40,52). Differentiating Rb-deficient neural precursor cells exhibit delayed cell cycle exit, while the absence of Rb in the telencephalon leads to ectopic proliferation of neural precursor cells and enhanced brain size at midgestation (7,19,52). In a recent study we described a role for Rb in regulating the survival of discrete neuronal subpopulations and a novel cell autonomous role for Rb in regulating neuronal migration (18).The mechanism by which Rb regulates neurogenesis and the extent to which defects in migration and survival are the result of cell cycle deregulation remain unknown. While Rb is known to interact with numerous proteins (reviewed in reference 67), many of which are expressed in quiescent cells or have cell cycle-independent functions, members of the cell cycle regulatory E2F family are likely targets in neurogenesis. The E2F family of transcription factors is comprised of E2Fs 1 to 8; however, E2F1, E2F2, and E2F3, the so-called activating E2Fs, are key Rb-interacting targets best known for their role in promoting cell cycle progression (9, 14, 17, 48, 49, 54; reviewed in reference78). Both E2F1 and E2F3 are likely candidates involved in Rb-mediated regulation of neurogenesis. Deficiency of e...

Tissue-specific targeting of cell fate regulatory genes by E2f factors

“…5,6,8,9,17,18,[41][42][43] Thus through our identification of E2f3&4-bound promoters in NPCs, we have significantly expanded our understanding of how cell cycle regulators can direct cell fate control. While this study provides a number of important insights, we offer two key conceptual findings: E2f transcription factors are poised as widespread regulators of cell fate-associated genes in NPCs, establishing a pervasive direct role for the cell cycle machinery Figure 4 E2f3 and Ctcf are co-enriched at nervous system-related genes.…”

“…Electrophoretic mobility shift assay was performed as previously described. 9 The material was neurospheres at stage E14.5 cultured from either littermate WT and E2f3 − / − embryos, or littermate WT and E2f3a − / − embryos. The antibodies used were sc-878, to detect E2f3, and sc-193 and sc-633 to detect E2f1 and E2f2.…”

“…Cell cycle dynamics strongly influence neural precursor cell (NPC) maintenance and neurogenesis, [1][2][3][4] and gain-or loss-of-function studies have demonstrated key roles for cell cycle proteins, including the E2f family, in NPC fate decisions. [3][4][5][6][7][8][9][10][11][12][13][14][15][16] E2f3 is required for proper cortical migration of neurons and to maintain the balance between NPC self-renewal, proliferation and differentiation, and its loss disrupts long-term neurogenesis and cortical function; E2f1 deficiency impairs NPC proliferation, and E2f4 deficiency leads to inhibition of NPC self-renewal and severe defects in telencephalic development. 6,[8][9][10]17 A pivotal question is whether cell fate control by the pRb/E2f pathway is largely a consequence of cell cycle regulation, or due to direct regulation of cell fate-associated genes.…”

Specific In Vivo Roles for E2Fs in Differentiation and Development

“…Expression of E2F3 alone induces S-phase entry in fibroblasts but not in cultured myeloid cells 31,105 Expression of E2F3a induces cell cycle re-entry in lens fibre cells 45 Transgenic expression of E2F3a in the epidermis leads to hyperproliferation (similar to E2F4 and 1) 114 Positive regulator of neural precursor proliferation as loss of E2F3 leads to reduction of neural precursor cells 77 Oncogenic in bladder cancer as it is frequently amplified and knockdown of E2F3 in bladder cells reduces proliferation [115][116][117] E2F4 HYPOTHESIZED UNIVERSAL FUNCTION Alone loss of E2F4 does not result in cell cycle or proliferation defects in MEFs 118,119 Expression of E2F4&5 together with DP-1 induces quiescent fibroblasts into S-phase 31 Together with E2F5, required for pocket protein mediated G 1 arrest in response to p16 INK4a120…”

“…E2F3a& b Required for normal embryonic development as lethality begins at E13.5 during development and likely required Unique for proper heart development as surviving animals die from congestive heart failure hypothesized to be a result of a primary heart defect 19,20 Mediates neuronal migration and differentiation through Rb 76,77 Unique E2F4 Involved in regulation of gut epithelium development and craniofacial development as E2F4 -/-exhibit shortening Unique and broadening of the snout and reduced intestinal crypts and villi 118,119 Required to repress adipocyte differentiation, in part through regulation of PPARg expression. Repression of Opposing role with adipogenesis appears to be separable from any role of E2F4 in cell cycle regulation or pocket protein binding 72,73 E2F1 and 3…”