Congress Abstracts - Society for Developmental Biology

normally form, hence producing proximal-distal axis duplications (PDd). The molecular mechanism behind these observations and the
natural role of RA during limb regeneration are still unknown. RA exerts its effects through several nuclear receptors (RARs) which
are associated with specific functions, furthermore, RARs dimerization with the retinoid “X” receptors (RXRs) might contextually
affect RAR function. The present work aims to determine the possible specific functions of the RA effectors, RARs and RXRs, during
the axolotl limb regeneration. By using specific RAR agonists and antagonists we could determine that RARgamma is important for
autopod morphogenesis during normal regeneration and it is also responsible of the PDd and delay in regeneration when there is an
excess of RA. Besides, panagonist and panantagonist of RXRs did not have any major effects suggesting a meaningless function
during limb regeneration, although the antagonisms of RXRs increase the effects of the RARgamma agonist, suggesting that RXRs
might act by regulating the activity of RARs. This study was partially supported by CONACyT grants 53484 and 168642, DGAPA-
UNAM grants IN214511 and IN220808.
Program/Abstract # 337
Visualizing endogenous morphogen gradients and their modulation in vivo.
Sosnik, Julian; Zheng, Likun; Digman, Michelle; Nie, Qing; Gratton, Enrico; Schilling, Thomas (UC Irvine, USA)
During development, morphogens provides cells with critical information that determines their final fates. Retinoic Acid (RA), a
vitamin A derivative, functions as a morphogen to establish the anterior-posterior (A-P) axis in early embryogenesis. Defects in RA
signaling in animal models and humans lead to birth and developmental defects and cancer. In early development, RA is thought to
form a morphogen gradient, and although in the past we have been able to describe such distribution and formulate mathematical
models, we lack direct evidence of RA distribution. In this work we make use of the intrinsic fluorescence of RA and analyze RA
distribution by combining Fluorescence Lifetime Imaging Microscopy (FLIM) with a phasor approach to FLIM data analysis. Using
this approach we were able to detect and measure the wildtype endogenous distribution of RA in vivo without dyes or labels. The
observed distribution is consistent with models that predict RA forming a gradient that extends anteriorly during the second half of the
embryo’s gastrulation. In addition, using this technique, we can semi-quantitatively measure the endogenous fluctuations in the system
in the spatial and temporal domains. This is important in light of resent work suggesting that cells don’t respond immediately to
signals from morphogens, but rather temporally integrate this signals, with fluctuations playing an important role in accuracy of the
outcome. We were also able to generate mathematical models that predict these behaviors. In all, this work constitutes the first time
we can directly observe the graded distribution of RA and provides a novel mathematical model that allows us to make testable
predictions beyond the limitations of our measurements.
Program/Abstract # 338
Retinoic acid regulates musculoskeletal patterning in the zebrafish head
McGurk, Patrick; Swartz, Mary; Eberhart, Johann (University of Texas-Austin, USA)
Proper regulation of the morphogen all-trans retinoic acid (RA) is essential for head and craniofacial development. The distribution of
RA is mediated by its synthesis in Raldh-expressing cells and its degradation in Cyp26-expressing cells. In zebrafish, loss of the RAcatabolizing
enzyme Cyp26b1 causes reduction of the anterior neurocranium and midline fusions of Meckel’s cartilage and
ceratohyals, ventral first and second arch cartilage elements, respectively. These cartilages and the tendons joining them to head
muscles are cranial neural crest cell derivatives. The muscles themselves are mesoderm-derived, and in cyp26b1 mutants, muscle
fibers that attach to the neural crest-derived skeleton display disrupted bundling and terminate ectopically. Restoration of cyp26b1
function in neural crest rescues ventral cartilage and muscle defects in cyp26b1 mutant embryos. We predict that the specification of
neural crest derivatives is sensitive to RA signaling during craniofacial development. Consistent with this model, cyp26b1 mutant
zebrafish embryos express higher levels of the tendon specification transcription factor scxa in the head. Chemical inhibition of RA
synthesis during facial cartilage morphogenesis rescues muscle phenotypes and reduces scxa expression in cyp26b1 mutants. We are
currently investigating the differentiation of neural crest over time in cyp26b1 mutants. We are also developing transgenic models for
observing the coordination of growing muscle, tendon, and skeleton in the head. Further results will help shed light into the molecular
mechanisms that direct musculoskeletal development.
Program/Abstract # 339
Identifying the mechanism of action for Dispatched-mediated Hedgehog ligand release.
Bodeen, William (Univ of TN HSC - St. Jude Children's Res Hosp, USA), Ogden, Stacey (St. Jude Children's Research Hospital, USA)
The Hedgehog (Hh) signal transduction pathway plays a conserved patterning role during metazoan development. In Hh ligandproducing
cells, Hh protein is synthesized as a ~45 kDa precursor that undergoes an auto-catalytic processing reaction upon its entry
into the ER. This results in formation of a ~20 kDa mature peptide ligand that is modified by palmitate on its amino-terminus and
cholesterol on its carboxyl-terminus. To function as a morphogen, lipid modified Hh must be solubilized and released from its site of
synthesis to travel multiple cell diameters across developing tissues. One protein that is required for this release is the 12-pass
transmembrane protein Dispatched (Disp), a member of the resistance-nodulation division superfamily. Despite much effort, the exact
mechanism by which Disp functions to facilitate Hh ligand release is not known. We have initiated biochemical, cell biological and
Drosophila genetic studies aimed at dissecting Disp recognition and solubilization of mature, lipid modified Hh. We describe an in
vitro assay system designed to dissect Disp activity, and provide evidence that Disp may function as part of a large molecular weight
complex. Future experiments will be aimed at identifying Disp-interacting partners and ascertaining their role in Hh release.
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