Cold shock domain family members YB-1 and MSY4 share essential functions during murine embryogenesis

Three cold shock domain (CSD) family members (YB-1, MSY2, and MSY4) exist in vertebrate species ranging from frogs to humans. YB-1 is expressed throughout embryogenesis and is ubiquitously expressed in adult animals; it protects cells from senescence during periods of proliferative stress. YB-1-deficient embryos die unexpectedly late in embryogenesis (embryonic day 18.5 [E18.5] to postnatal day 1) with a runting phenotype. We have now determined that MSY4, but not MSY2, is also expressed during embryogenesis; its abundance declines substantially from E9.5 to E17.5 and is undetectable on postnatal day 1(adult mice express MSY4 in testes only). Whole-mount analysis revealed similar patterns of YB-1 and MSY4 RNA expression in E11.5 embryos. To determine whether MSY4 delays the death of YB-1-deficient embryos, we created and analyzed MSY4-deficient mice and then generated YB-1 and MSY4 double-knockout embryos. MSY4 is dispensable for normal development and survival, but the testes of adult mice have excessive spermatocyte apoptosis and seminiferous tubule degeneration. Embryos doubly deficient for YB-1 and MSY4 are severely runted and die much earlier (E8.5 to E11.5) than YB-1-deficient embryos, suggesting that MSY4 indeed shares critical cellular functions with YB-1 in the embryonic tissues where they are coexpressed.     

Embryonic lethality and fetal liver apoptosis in mice lacking all three small Maf proteins

Embryogenesis is a period during which cells are exposed to dynamic changes of various intracellular and extracellular stresses. Oxidative stress response genes are regulated by heterodimers composed of Cap'n'Collar (CNC) and small Maf proteins (small Mafs) that bind to antioxidant response elements (ARE). Whereas CNC factors have been shown to contribute to the expression of ARE-dependent cytoprotective genes during embryogenesis, the specific contribution of small Maf proteins to such gene regulation remains to be fully examined. To delineate the small Maf function in vivo, in this study we examined mice lacking all three small Mafs (MafF, MafG, and MafK). The small Maf triple-knockout mice developed normally until embryonic day 9.5 (E9.5). Thereafter, however, the triple-knockout embryos showed severe growth retardation and liver hypoplasia, and the embryos died around E13.5. ARE-dependent cytoprotective genes were expressed normally in E10.5 triple-knockout embryos, but the expression was significantly reduced in the livers of E13.5 mutant embryos. Importantly, the embryonic lethality could be completely rescued by transgenic expression of exogenous MafG under MafG gene regulatory control. These results thus demonstrate that small Maf proteins are indispensable for embryonic development after E9.5, especially for liver development, but early embryonic development does not require small Mafs.     

Hydrops fetalis, cardiovascular defects, and embryonic lethality in mice lacking the calcitonin receptor-like receptor gene

Adrenomedullin (AM) is a multifunctional peptide vasodilator that is essential for life. To date, numerous in vitro studies have suggested that AM can mediate its biological effects through at least three different receptors. To determine the in vivo importance of the most likely candidate receptor, calcitonin receptor-like receptor, a gene-targeted knockout model of the gene was generated. Mice heterozygous for the targeted Calcrl allele appear normal, survive to adulthood, and reproduce. However, heterozygote matings fail to produce viable Calcrl-/- pups, demonstrating that Calcrl is essential for survival. Timed matings confirmed that Calcrl-/- embryos die between embryonic day 13.5 (E13.5) and E14.5 of gestation. The Calcrl-/- embryos exhibit extreme hydrops fetalis and cardiovascular defects, including thin vascular smooth muscle walls and small, disorganized hearts remarkably similar to the previously characterized AM-/- phenotype. In vivo assays of cellular proliferation and apoptosis in the hearts and vasculature of Calcrl-/- and AM-/- embryos support the concept that AM signaling is a crucial mediator of cardiovascular development. The Calcrl gene targeted mice provide the first in vivo genetic evidence that CLR functions as an AM receptor during embryonic development.     

Secalonic acid D blocks embryonic palatal mesenchymal cell-cycle by altering the activity of CDK2 and the expression of p21 and cyclin E

BACKGROUND: The mycotoxin, secalonic acid D (SAD), a known animal and potential human cleft palate (CP)-inducing agent, is produced by Pencillium oxalicum in corn. SAD selectively inhibits proliferation of murine embryonic palatal mesenchymal (MEPM) cells leading to a reduction in cell numbers. These effects can explain the reduction in shelf size and the resulting CP seen in the offspring of SAD-exposed mice. Ability of SAD to inhibit proliferation as well as to block the progression of cells from G1- to S-phase of the cell-cycle were also shown in the human embryonic palatal mesenchymal (HEPM) cells suggesting the potential CP-inducing effect of SAD in human beings METHODS: Gestation day (GD) 12 mouse embryos and HEPM cells were used to test the hypothesis that the cell-cycle block induced by SAD results from a disruption of stage-specific regulatory components both in vivo and in vitro. The effects of SAD on the activity of various cyclin dependent kinases (CDK) and on the levels of various positive (cyclins and CDK) and negative (CDK inhibitors p15, 16, 18, 19, 21, 27, 57) cell-cycle regulators were assessed by performing kinase assays and immunoblots, respectively. RESULTS: In the murine embryonic palates, SAD specifically inhibited G1/S-phase-specific CDK2 activity, reduced the level of cyclin E and tended to increase the level of the CIP/kip CDK inhibitor, p21. In the HEPM cell cultures, exposure to IC50 of SAD significantly affected all of the above targets. In addition, a reduction in the levels/activity of CDK 4/6, a reduction in the levels of cyclins D1, D2, D3, E, A, and all INK4 family proteins, and an increase in the level of the CIP/kip CDK inhibitor, p57, were also seen. CONCLUSIONS: These results suggest that the S-phase-specific cell-cycle proteins CDK2, cyclin E and possibly p21 are the common targets of SAD in murine palatal shelves in vivo and in human embryonic palatal mesenchymal cells in vitro and may be relevant to the pathogenesis of SAD-induced CP.