Target Discovery and Validation Reviews and Protocols

Keratin Transgenics and Knockouts 215
demonstrated by coimmunoprecipitation (61). TRADD, the adaptor protein linking
FADD and RIP to the TNFR1, is thought to be sequestered by K8 and K18
IFs, thereby precluding TRADD to operate as a mediator of apoptosis and thus
modulating TNFR1 signaling. This type of explanation also was used as a paradigm
for the interpretation of results after targeted deletion of keratins suspected
to be essential for embryonic development. The K8 deficiency and subsequent
embryonic lethality at ED12.5 in the sensitive C57BL/6 and 129Sv background
is caused by trophoblast giant cell layer failure, suggested to result from TNFsensitive
failure of trophoblast giant cell barrier function (55). Maternal TNFα
was thought to trigger an apoptotic response in extraembryonic trophoblast giant
cells becasue after the loss of K8/K18 IF network, cells can no longer moderate
apoptosis by sequestering TNFR1 and TRADD (55).
Targeted deletion of K18 in C57BL/6 mice was less dramatic than that of K8.
Keratin K18(–/–) animals are viable, fertile, and show a normal life-span. Old
K18 null mice, however, developed a distinctive liver pathology with abnormal
hepatocytes containing K8-positive aggregates, identified as Mallory bodies, a
hallmark of human alcoholic hepatitis (34). Absence of K8 and K18 in hepatocytes
furthermore disturbs the function of at least some cell cycle regulators,
such as the signaling integrator 14-3-3, and causes disturbances in cell cycle
regulation and aberrant cytokinesis, which might drive cells into the S-to-G 2
transition (62). Except for hepatocytes, in which K19 is not expressed, perfect
IFs between K8 and K19 have been found in these mice, underlining that functional
redundancy is common in keratin biology (34). The obvious lack of
defects in K19 deficient mice also falls into this category (63).
To identify the role of keratins during embryogenesis, double-deficient animals
for K8 and K19 (63) as well as K18 and K19 (64) have been generated (Tables 1
and 2) to get rid of nearly all K8/K19 or all K18/K19 IFs [Note that in
K8(–/–)/K19(–/–) mice, a compensatory IF network composed of K7 and K18 is
formed.] Mice from both lines die concomitantly between ED9.5 and 10 but from
different phenotypes. In K8(–/–)/K19(–/–)-embryos, a decreased number of disorganized
labyrinthine trophoblast and spongiotrophoblast cells will lead to the penetration
of maternal as well as embryonic blood into lesions of the placenta (63).
In K18(–/–)/K19(–/–)-embryos, a rupture of giant trophoblast cells because of
mechanical fragility, provoked by the maternal blood pressure, lead to hematomas
and deformations of the yolk sac, which might disturb the nutrition of the embryo
(64). Very recently, this defect could be rescued by aggregation of double-deficient
embryos with tetraploid wild-type embryos up to ED11.5 that underscored the
necessity of keratin filaments for the proper function of extraembryonic (like trophoblastic
and placental tissue) rather than embryonic epithelia (65).
A possible explanation for the early embryonic death of K8 (–/–)/K19(–/–)
embryos was in line with that of the single K8 knockout (55). An increment