Page 78 - Mouse Molecular Genetics

Full Abstracts
Program number is above title. Author in bold is the presenter.
midbrain, and directs proliferation, survival, patterning and neurogenesis. We reveal an autoregulatory negative feedback loop
between the transcription factor Lmx1b and a newly characterized microRNA, miR135a2, that controls the extent of Wnt1/Wnt
signaling. Conditional gain of function studies reveal that Lmx1b promotes Wnt1/Wnt signaling, and thereby increases midbrain
size and increases dopamine progenitor allocation. Conversely, conditional removal of Lmx1b has the opposite effect. Next, we
provide evidence that microRNAs are involved in restricting dopamine progenitor allocation. Conditional loss of Dicer1 in ES
cells results in expanded Lmx1a/b+ progenitors. In contrast, forced elevation of microRNA135a2 during an early window in vivo
phenocopies the Lmx1b conditional knockout, in that the proportion of Lmx1a/b+ progenitors is selectively reduced. Midbrain
dimensions are also reduced in these mutants. We demonstrate that this mutant displays reductions in Wnt1/ canonical Wnt
signaling, which underpins these phenotypes. MicroRNA modulation of Lmx1b/Wnt1 dosage thus determines midbrain size and
allocation of dopamine progenitors. Since canonical Wnt activity has recently been recognized as a key ingredient for
programming ES cells towards a dopaminergic fate in vitro, and since microRNA135a2 modulates Wnt1/Wnt signaling, these
studies could impact the rational design of such protocols.
Dissection of the role of Fgf10 after limb bud initiation. Anne M. Boulet
Mario R. Capecchi. Human Genetics,
HHMI/University of Utah, Salt Lake City, UT.
Fgf10 is known to be required for limb bud initiation. Knockout of Fgf10 results in the complete absence of limb bud
development. Although loss of Fgf10 expression during limb bud growth has been proposed to account for the limb phenotype in
other mutants, the role of Fgf10 in further development of the limb has not been studied. It is assumed that continued Fgf10
expression is required to maintain AER function, including AER FGF expression. Expression of FGFs in the AER is in turn
thought to be required to maintain Shh expression in the posterior limb bud and Fgf10 expression in the mesenchyme. Using an
Fgf10 conditional allele, the role of Fgf10 in post-initiation limb bud development has been investigated. The RARcre driver was
used to knock out Fgf10 at approximately E9. It appears that limb buds are initiated in these mutants, but fail to grow, and an
increase in the number of apoptotic cells is seen. This result would suggests a requirement for Fgf10 in maintenance of cell
survival immediately after limb buds form, perhaps indirectly through an effect on formation of the AER and initiation of Fgf8
gene expression. The AP2cre driver is expressed in distal limb bud mesenchyme by E10.5. Mice in which Fgf10 was inactivated
using AP2cre show digit malformations, with 100% penetrance but variable expressivity. Digits do not separate properly, and
some digits are truncated or curved. When the Shhcre driver was used to inactivate Fgf10 in the posterior limb bud, a defect in
the development of digit 5 could be detected at least as early as E13.5. Newborn skeleton preps revealed subtle defects in the
condensations of digit 5 in both forelimbs and hindlimbs. Additional Cre drivers will be utilized to inactivate Fgf10 at different
developmental time points in order to elucidate the role of this gene in limb bud growth and patterning.
Early forebrain patterning of the mouse embryo: a role for the non-neural ectoderm. Marieke I. Cajal
Jerome Collignon,
Anne Camus. Université Paris Diderot, Sorbonne Paris Cité, Institut Jacques Monod, UMR 7592, CNRS, F-75013 Paris, France.
The central nervous system of the mouse develops from a single epithelial layer. Between the end of gastrulation and the
beginning of somitogenesis, this layer is specified and regionalized in three regions, the forebrain, midbrain, and hindbrain,
mostly under the influence of signals emanating from the axial mesendoderm. Our results highlight a critical role of another
embryonic region in the formation of the forebrain. When the anterior proximal region, that comprises the non-neural precursors
which give rise to surface and buccal ectoderm, is surgically removed at 7.5 days post-coitum, the development of the forebrain
is highly affected. In particular, after 30 hours of in vitro culture, the prospective telencephalon is missing. We are interested in
understanding the interactions between these non-neural ectoderm cells and neural precursors during neural plate formation. We
found that in ablated embryos, all the tissues composing the head, the neuroectoderm, the surface ectoderm, and the neural crest,
are specified. The axial mesendoderm, anterior neural ridge (ANR) and isthmus organizer are present. The neuroectoderm
appears correctly regionalized as assessed by the exhaustive analysis of anterior regional markers. The morphological defects of
ablated embryos are associated with an increase in cell death whereas no change in cell proliferation is detected. Several
signaling pathways involved in brain specification and development are disturbed and may account directly or indirectly for the
loss of the telencephalon. In particular, the Bone Morphogenetic Protein (BMP) signaling pathway is disrupted, and the Nodal
signaling pathway is ectopically activated in the neuroectoderm. We are currently pursuing our investigation of the role of non-
neural ectoderm in forebrain development and regionalization using reporter mice strains and pharmacological inhibitors of
major signaling pathways.
The Dimple Mutation Uncovers a Link Between Mouse Gastrulation and Mitochondrial Function.
Ivan Duran
Maria J.
Garcia-Garcia. Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY.
During mouse gastrulation, a group of cells in the primitive streak, an area located at the posterior side of the embryo,
delaminate from the embryonic epithelia and migrate to form mesodermal and endodermal lineages. To identify novel genes
regulating these processes, we performed a forward mutagenesis screen and found dimple, a recessive mutation that causes early
embryonic lethality and severe gastrulation defects. In dimple embryos, cells delaminate to form mesoderm, but are unable to
migrate and accumulate close to the primitive streak area. Analysis of molecular markers revealed that several signaling
pathways required for gastrulation are up-regulated in dimple mutants, including NODAL, WNT3 and FGF. Interestingly, Fgf8 is