Abstracts - Society for Developmental Biology

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Program/Abstract # 116
Structural changes of the nuclear envelope impact murine embryonic stem cell differentiation
Moore, Robert; Smith, Elizabeth; Rosario, Santas; Yeasky, Toni; Xu, Xiang-Xi, University of Miami Dept. of Medicine,
United States
Murine embryonic stem cells can be differentiated as embryoid bodies or by treatment with retinoic acid and
differentiation is associated with changes in chromatin conformation. It is thought that the nuclear lamina plays a role in
organizing chromatin, with a high affinity for heterochromatin. We have investigated the roles of the nuclear envelope
(NE), including the outer and inner nuclear membranes (ONM and INM), in ES cell differentiation. Using transmission
electron microscopy we found that the lumen between the ONM and INM is 60% larger in ES cells when compared to
differentiated cells, although actual nuclear pore density remained unaltered. Gene expression profiling revealed that the
synthesis of several nuclear lamina and envelope proteins, including Syne1, lamin A/C and emerin are upregulated during
differentiation. Differentiation was also associated with enrichment of heterochromatin attached to the nuclear lamina.
Surprisingly, mice deficient for Syne1, emerin and/or laminA/C are capable of completing embryonic development. We
have also found that ES cells deficient of NE proteins show only subtle changes in differentiation in vitro. However,
overexpression of exogenous laminA and other NE structural proteins in undifferentiated ES cells changed the propensity
of the cells to differentiate. We conclude that the NE and lamina undergo drastic structural changes during the
differentiation of embryonic stem cells and that these changes exert an impact upon gene expression and differentiation.
Program/Abstract # 117
Eya1 mice as models for understanding middle ear developmental defects
Joshi, Leena, King's College London, United Kingdom
The mammalian middle ear is composed of three bony ossicles, the malleus, incus and stapes which function to conduct
sound from the external ear via the tympanic membrane, to the inner ear through the oval window. Normal development of
these three ossicles and the formation of a tissue and liquid free middle ear space are integral for this transduction of
sound, defects resulting in conductive deafness. Branchio- oto- renal syndrome is a disorder of craniofacial development
which causes developmental defects in both the middle and inner ear structures and accounts for 2% of profoundly deaf
children worldwide. 40% of patients with this syndrome possess mutations in the Eya1 gene, one of several genetic factors
influencing the development of this syndrome. Eya1 heterozygous mice exhibit variable hearing loss and possess extra
ossicle like structures between the malleus and incus. We show that the extra ossicles originate from the incus as a result of
ectopic joint formation. This provides us with a tool to investigate the mechanisms of ossicle morphogenesis and cartilage
joint formation in the middle ear. Eya1 mice are further predisposed to developing otitis media (middle ear infections) in
adult life, implicating Eya1 as a regulator of both embryonic and postnatal middle ear development.
Program/Abstract # 118
In vivo knock down of Wnt signalling components via shRNA in the inner ear anlage
Funke, Constanze; Sienknecht, Ulrike J., Carl von Ossietzky University Oldenburg, Germany
Development of the vertebrate inner ear relies on elaborate morphogenesis. Finally, the resulting 3-dimensional apparatus
consists of the dorsal semicircular canals and underneath the vestibular macula organs. Amniotes (land vertebrates), in
addition, extend ventrally the cochlear duct housing a dedicated auditory organ. Involvement of Wnt signalling in both cell
proliferation and programmed cell death necessary for proper morphogenesis has been shown for other organ systems.
However, studies on the function of endogenous Wnts or Wnt inhibitors in the inner ear are still sparse. Our hypothesis is
that Wnt signalling is capable of maintaining cells in an undifferentiated proliferative state and it also regulates apoptosis
during chicken inner ear morphogenesis. Here we introduce a method for regional loss-of function of otherwise vital genes,
adapted for studies of inner ear development. Gene silencing is carried out via shRNA (short hairpin RNA). Briefly: Genes
such as Wnt9a or SFPR2 are targeted by custom-designed hairpins with a non-collapsing spacer and an additional
restriction site to facilitate sequencing. We utilize a primer extension method with two oligos of different length (one
hairpin containing template oligo and a short generic primer for PCR extension). Injection and electroporation of the
hairpin expression vector into the otocyst is performed at embryonic day (E)3 prior to inner ear morphogenesis. Embryos
are then harvested at desired stages of development. Gene knock down efficiency is evaluated on histological serial
sections via RNA in-situ hybridization and the ear morphology is analyzed with the aid of immunohistology. Supported by
CvO University Oldenburg
Program/Abstract # 119
Cardiac contractility and blood flow regulate cardiac form
Glickman Holtzman, Nathalia S., Queens College, CUNY Biology, United States; Estevez, Jaymie; Kigler, Gabriella