High Levels of Cre Expression in Neuronal Progenitors Cause Defects in Brain Development Leading to Microencephaly and Hydrocephaly

Abstract

Hydrocephalus is a common and variegated pathology often emerging in newborn children after genotoxic insults during pregnancy (Hicks and D'Amato, 1980). Cre recombinase is known to have possible toxic effects that can compromise normal cell cycle and survival. Here we show, by using three independent nestin Cre transgenic lines, that high levels of Cre recombinase expression into the nucleus of neuronal progenitors can compromise normal brain development. The transgenics analyzed are the nestin Cre Balancer (Bal1) line, expressing the Cre recombinase with a nuclear localization signal, and two nestin CreER T2 (Cre recombinase fused with a truncated estrogen receptor) mice lines with different levels of expression of a hybrid CreER T2 recombinase that translocates into the nucleus after tamoxifen treatment. All homozygous Bal1 nestin Cre embryos displayed reduced neuronal proliferation, increased aneuploidy and cell death, as well as defects in ependymal lining and lamination of the cortex, leading to microencephaly and to a form of communicating hydrocephalus. An essentially overlapping phenotype was observed in the two nestin CreER T2 transgenic lines after tamoxifen mediatedCreER T2 translocation into the nucleus. Neither tamoxifen-treated wild-type nor nestin CreER T2 oil-treated control mice displayed these defects. These results indicate that some forms of hydrocephalus may derive from a defect in neuronal precursors proliferation. Furthermore, they underscore the potential risks for developmental studies of high levels of nuclear Cre in neurogenic cells.

Disruption of Foxg1 expression by knock-in of Cre recombinase: Effects on the development of the mouse telencephalon

“…In the latter study, the genotoxic effects required the levels of Cre recombinase to reach a critical, high threshold in the cell nucleus, as had been reported previously in vitro (Baba et al, 2005). Thus, hydrocephalus was observed in homozygous, but not hemizygous, mice in a transgenic line, in which multiple copies of the cre transgene had been inserted into unknown locations in the genome (Forni et al, 2006). In contrast, the present study utilized a Foxg1-cre line that has only a single copy of the cre gene and effects of Cre recombinase at low doses in vivo have not been reported previously.…”

“…More detailed analysis indicated that proliferating, rather than postmitotic, cells were vulnerable to the effects of Cre recombinase, as cryptic recombination events were only observed in mitotically-active cells (Loonstra et al, 2001). There have been two reports of overt pathology in cre-expressing transgenic mice, where high levels of Cre resulted in male infertility when expressed in postmeiotic spermatids (Schmidt et al, 2000) and hydrocephaly when expressed in neuronal progenitors (Forni et al, 2006). In the latter study, the genotoxic effects required the levels of Cre recombinase to reach a critical, high threshold in the cell nucleus, as had been reported previously in vitro (Baba et al, 2005).…”

“…In vivo and in vitro studies indicate that the adverse effects of Cre recombinase in the absence of exogenous loxP sites occurs predominantly in proliferating, rather than postmitotic, cells (Loonstra et al, 2001, Forni et al, 2006, suggesting that the disruption in the radial domain that we observe in the adult occurred earlier in development. We therefore performed in situ hybridization (E16.5) or immunocytochemistry (P4), using markers that identified specific layers in the developing cortex, to examine potential laminar defects of Foxg1-Cre/+ mice during late corticogenesis and early postnatal development.…”

“…An important difference between the two lines is the presence of a nuclear localization signal in the Foxg1-cre line (Hebert and McConnell, 2000), which is absent in the Emx1-cre line (Gorski et al, 2002), likely resulting in higher levels of Cre recombinase in the nucleus. As noted above, there is evidence in vivo and in vitro that the accumulation of Cre recombinase specifically in the nucleus, rather than the absolute levels in the cell, underlies the DNA damage and growth inhibition observed in the absence of exogenous loxP sites (Baba et al, 2005, Forni et al, 2006. Thus, because of the time required to achieve threshold levels of nuclear Cre recombinase, overt abnormalities may not be apparent until several days after cre is first expressed.…”

“…For example, several in vitro studies have described effects of Cre recombinase, including growth inhibition and death, in mammalian cells lacking exogenous loxP sites (Loonstra et al, 2001, Pfeifer et al, 2001, Silver and Livingston, 2001. A recent study extended these observations to the mouse central nervous system, where it was reported that high levels of Cre recombinase in the nucleus of neural progenitors lead to impaired proliferation and increased cell death, resulting in hydrocephaly (Forni et al, 2006). Similar effects of Cre recombinase at low doses, however, have not been reported in vivo, although it is unclear whether subtle phenotypes have gone unnoticed.…”