Abstracts - Society for Developmental Biology

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containing protein FBXO30, found in a two-hybrid screen for TH binding proteins. Using primers based on the sequence
we obtained, along with primers based on the 5’ and 3’ UTRs of the Xenopus tropicalis FBXO3O mRNA, we obtained
RT-PCR products with total RNA samples from eggs and embryos at early developmental stages. Using this approach, we
have uncovered the presence of two FBXO30 homolog genes in X. laevis, FBXO30-A and FBXO30-B. The predicted
FBXO30-A full length protein sequence is 91% and 63% identical to its counterparts from Xenopus tropicalis and Homo
sapiens, respectively. These proteins contain very conserved Traf-like zinc finger-containing domains at their N-terminus,
and F-Box domains at their C-terminus, while the internal part of the proteins diverge extensively. By RT-PCR, we have
found that FBXO30-A and FBXO30-B are maternal factors as their messages are present in the unfertilized egg. The
FBXO30-A mRNA persists during the cleavage stages, but decreases after the mid-blastula transition and is barely
detected once gastrulation starts. Our studies show the presence of two homologs of FBXO30 in X. laevis that are
maternally expressed, which could be key regulators of early development working with TH to promote cell proliferation.
Program/Abstract # 92
Transition between two types of oscillators during Xenopus laevis early embryonic cell cycle
Tsai, Tony; Theriot, Julie; Ferrell, James, Stanford University, Stanford, United States
Before mid-blastula transition, the Xenopus laevis embryonic cell cycle is driven by an autonomous biochemical oscillator
based on Cdk1 activation and inactivation. Cycle 2 to 12 have a period of 25 minutes and are highly accurate while the 1st
cycle takes ~85 minutes, raising the question of how an autonomous oscillator can be initially tunable yet precise
afterwards. We reconstruct the temporal dynamicsof cell cycle oscillation in vivo using individual Xenopus laevis embryos
collected in fine temporal resolution. We observe a higher threshold for cyclin to trigger mitotic entry in the first cycle due
to stronger inhibitory phosphorylation of Cdk1. A positive feedback involving the cyclinB1-Cdk1 complex, their inhibitory
kinases Wee1 and Myt1, as well as their activating phosphatase Cdc25, is shown invitro to be important for the robustness
of the cell cycle oscillations. Surprisingly,bypassing the positive feedback created a significant phenotype in the first cell
cycle, but minimal impact on the subsequent cycles. This implies a transition from a strong positive-feedback oscillator to
a weak positive-feedback oscillator. Several mechanisms contributed to this transition, such as the increase of phosphatase
abundances and the decrease of kinase activities. We identified that the negative feedback is highly ultrasensitive and
could improve the robustness of the oscillator in the absence of the positive feedback. We demonstrated computationally
that the presence of positive feedback in the first cycle allows the oscillator to be tunable, and turning down the positive
feedback in the subsequent cycles help increase the precision of the oscillatory period. The Xenopus laevis may turn down
a subset of the regulatory circuit during early embryonic development to match changing developmental objectives.
Program/Abstract # 94
Dynamic cell shape changes are required for mesenchymal condensation
Ray, Poulomi; Chapman, Susan, Clemson University, Clemson, United States
The physical mechanism of mesenchymal condensation during skeletal development is not well understood. Here, we show
that dynamic cell shape changes are required for mesenchymal condensation during chick middle ear morphogenesis. The
chick contains a single middle earbone – the columella. The chick columella arises from two separate condensations; the
cartilaginous extracolumella and an osseous columella. We demonstrate that the respective condensations arise at distinct
timepoints. Our modeling results show that the extracolumella undergoes condensation earlier than the columella. In
contrast, overt differentiation of chondrocytes occurs first in the columella condensation. The cellular characteristics
between the columella and the extracolumella condensation differ substantially. The extracolumella condensation
resembles the classical definition of condensation with tightly packed cells. Conversely, the columella condensation has a
novel appearance with a loosely organized web-like network of cells, with elongated cell-to-cell connections. However,
dynamic cytoskeletal reorganization is observed in both condensations over several days, indicating that cell shape changes
are important. Using Cytochalasin D, an inhibitor of actin polymerization, we disrupted the ability of the mesenchyme cells
to reorganize their cytoskeleton. Our results show that inhibition of cell shape changes disrupts mesenchymal condensation
during chick middle ear morphogenesis. Overall, our experiments will be helpful in understanding the general principles of
self-assembly of multi-potent progenitor cells to form a cartilage template of correct shape and size.
Program/Abstract # 95
Mechanism of cranial neural crest cell migration.
Alfandari, Dominique; Abbruzzese, Genevieve; Cousin, Helene, Univ of Massachusetts, Amherst, United States
Cranial Neural Crest (CNC) are pluripotent cells induced at the lateral edge of the neural plate. In Xenopus laevis, CNC
migrate as a cohesive sheet of cells initially and then as individual cells to produce the face of the embryo. We have
previously shown that cell surface metalloproteases from the ADAM family are essential for CNC induction (ADAM19)