Congress Abstracts - Society for Developmental Biology

Zhang, Haoran; Wong, Elaine Yee-man; Tsang, Sze Lan (The Univeristy of Hong Kong, China); Xu, Pin-Xian (Mount Sinai School of
Medicine, USA); Sham, Mai Har (The Univeristy of Hong Kong, China)
Branchio-Oto-Renal (BOR) syndrome patients exhibit craniofacial and renal anomalies as well as deafness. BOR syndrome is caused
by mutations in Six1 or Eya1, both of which regulate cell proliferation and differentiation. The molecular mechanism underlying the
craniofacial and branchial arch (BA) defects in BOR syndrome is unclear. We have found that Hoxb3 is up-regulated in the second
branchial arch (BA2) of Six1 -/- mutants. Moreover, Hoxb3 over-expression in transgenic mice leads to BA abnormalities which are
similar to the BA defects in Six1 -/- or Eya1 -/- mutants, suggesting a regulatory relationship among Six1, Eya1 and Hoxb3 genes. The
aim of this study is to investigate the molecular mechanism underlying abnormal BA development in BOR syndrome using Six1 and
Eya1 mutant mice. Two potential Six1 binding sites were identified on the Hoxb3 gene. In vitro and in vivo Chromatin IP assays
showed that Six1 could directly bind to one of the sites specifically. Furthermore, using a chick in ovo luciferase assay we showed that
Six1 could suppress gene expression through one of the specific binding sites. On the other hand, in Six1 -/- mutants, we found that the
Notch ligand Jag1 was up-regulated in BA2. Similarly, in Hoxb3 transgenic mice, ectopic expression of Jag1 could be also detected in
BA2. To investigate the activation of Notch signaling pathway, we found that Notch intracellular domain (NICD), a direct indicator of
Notch pathway activation, was up-regulated in BAs of Six1 -/- ; Eya1 -/- double mutants. Our results indicate that Hoxb3 and Notch
signaling pathway are involved in mediating the craniofacial defects of Six1/Eya1-associated Branchio-Oto-Renal Syndrome.
Program/Abstract # 206
A family of FOX genes determines precise spatial patterns of growth and differentiation within facial bone and cartilage
precursors
Balczerski, Bartosz; Louie, Kristin; Crump, Gage D. (Univ of Southern California-LA, USA)
FOX genes encode a large family of winged helix/forkhead transcription factors that have been shown to play multiple roles during
development. While mutations in a number of FOX genes are known to cause craniofacial defects, how members of this large family
coordinate development of the craniofacial skeleton remains unclear. In situ analyses in mice have led to a proposal that FOX genes
form a complex expression code, much like the Dlx or Hox genes, that pattern the craniofacial primordia, yet this model remains to be
tested at the functional level. In this study, we find that the homologous FOX genes of zebrafish ( foxc1a , foxc1b , foxd1 , foxd2 ,
foxf1 and foxl1 ) are also expressed in distinct patterns within the neural-crest-derived pharyngeal arches that are the precursors to the
facial skeleton. By manipulating major signaling pathways, we show that these distinct expression patterns result from differential
sensitivity of FOX enhancers to Hh, Fgf, Notch, Bmp and Edn signaling. This suggests that FOX genes act as integrators of multiple
signaling cascades in the cranial preskeletal mesenchyme. Next, we use morpholino knock-down and conditional transgenic
misexpression approaches to show that FOX genes have very specific requirements in controlling bone differentiation and cartilage
growth in distinct regions of the developing face. In particular, we find that FOX genes act in region-specific manners to prevent the
differentiation of dermal bone and increase the proliferation of cartilage precursors. In summary, our evidence in zebrafish supports
the existence of a FOX code that shapes the future facial skeleton by precisely regulating the balance between the self-renewal and
differentiation of skeletal progenitors.
Program/Abstract # 207
Alx-related frontonasal dysplasia: developmental mechanisms and evolutionary implications
Takahashi, Tokiharu; Mills, Peter; Dee, Chris (University of Manchester, UK)
The Alx gene family comprises three homeobox transcription factors in vertebrates, namely Alx1, Alx3, and Alx4. Interestingly,
mutations in all the three human genes have been identified in three related craniofacial disorders, which have been recently
established as Alx-related frontonasal dysplasia (FND). It encompasses a spectrum of severities but main characteristic features
include ocular hypertelorism, malformations of the nose and forehead, and clefting of the facial midline. Most notably, loss of Alx1
has severe orofacial clefting and extreme microphthalmia. In contrast, mutations of Alx3 or Alx4 cause milder forms of FND. Whilst
Alx1, Alx3 and Alx4 are all known to be expressed in the facial mesenchyme, little is known about the function of these proteins during
development.
Here, we report the establishment of zebrafish models of Alx-related FND. Morpholino knock-down of zebrafish alx1 expression
causes a profound craniofacial phenotype including loss of the facial cartilages and defective ocular development. In contrast,
suppression of alx3 produces no obvious phenotype. We demonstrate for the first time that Alx1 plays a crucial role in regulating the
migration of cranial neural crest cells into the frontonasal primordia. Abnormal neural crest migration is coincident with aberrant
expression of foxd3 and sox10, two key genes of neural crest development. This novel function is specific to Alx1, and likely explains
the marked clinical severity of Alx1 mutation within the spectrum of Alx-related FND. Alx1, Alx3 and Alx4 have been originated by
whole genome duplications at early vertebrate evolution, and we also discuss a possible link between Alx-related FND and the
molecular evolution of Alx gene family.
Program/Abstract # 208
Normalized Shape and Location of Perturbed Craniofacial Structures in the Xenopus Tadpole Reveal an Innate Ability to
Achieve Correct Morphology
Vandenberg, Laura Vandenberg; Adams, Dany; Levin, Michael (Tufts University, USA)
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