Barrott, Jared J.

Barrow, Jeffery R.

Dahl, Tiffany M.

Kendall, Jed J.

Kmetzsch, Kate E.

Low, Keri L.

Smith, Aaron P.

Elsevier - Publisher Connector 

The Limb Mesenchyme Recruitment Model for Patterning the Vertebrate Limb 

(Dlx5/6). Dlx5/6 induce expression of Hand2, a basic helix-loop-helix
transcription factor. This pathway places Hand2 at the center of a
complex signaling cascade, but little is known of its function in
mammalian craniofacial development since Hand2-/- embryos die
around embryonic day (E) 10.5 from vascular failure. To bypass these
defects, we created a conditional deletion of Hand2 using a traditional
Cre-loxP system. Using the Wnt1-Cre mouse line, we delete Hand2
within all migrating NCCs. Mutant mice exhibit severe craniofacial
defects including mandibular hypoplasia, a single incisor, aglossia,
and loss of tympanic rings and Meckel's cartilage. These changes are
preceded by aberrant maintenance of Dlx5/6 expression in the distal
mandibular arch and subsequent upregulation of Runx2 expression.
In vitro studies show that Hand2 is able to repress the Dlx5/6
enhancer, I56i. This suggests that Hand2 functions by repressing
Dlx5/6 expression within the distal midline. In its absence, Dlx5/6
expression is maintained and results in expression of Runx2 followed
by the repatterning of distal tongue mesenchyme to bone.
doi:10.1016/j.ydbio.2010.05.129
Program/Abstract # 91
Gain-of-function in Ras signaling perturbs dental development in
mouse and human
Alice Goodwina, Snehlata Oberoia, Cyril Charlesa,
Jessica C. Grotha, Cecilia F. Fairleya, Xu Chenb,
James A. Fagina, Katherine A. Rauena, Ophir D. Kleina
aUCSF, San Francisco, CA
bMemorial Sloan-Kettering Cancer Center, New York, NY
Ras/MAPK signaling is critical in animal development, and RTK
signaling, which activates Ras signaling, is known to play an important
role in tooth development. Our previous work has shown that
increasing Ras/MAPK signaling by inactivating Sprouty genes adversely
affects tooth morphogenesis. Here, we directly examined the effects of
activating Ras/MAPK signaling in both humans and mice. Costello
Syndrome (CS) is caused by a heterozygous de novo germline mutation
in HRAS that results in a constitutively active Ras protein. We examined
a cohort of CS patients and identified a number of craniofacial and
dental anomalies. We found that a large majority of patients presented
with pronounced enamel hypoplasia. Microcomputed tomography of
exfoliated primary teeth from CS patients showed a significant
decrease in enamel thickness compared to controls. We next examined
the CS mouse model and found that the mice also had an enamel
defect. Further inspection revealed disorganization of the ameloblasts
in the mouse incisor. We are currently studying cell proliferation and
polarity of the amelobasts in the mutant mouse incisors. In addition,
we are using an ameloblast-like cell line to determine the effects of
increased Ras/MAPK signaling on the behavior of the cells.
doi:10.1016/j.ydbio.2010.05.130
Program/Abstract # 92
The control of inner ear morphogenesis by Sprouty and Tbx1
genes in mouse models of 22q11.2 deletion syndrome
Yuichiro Yaguchia,b, Jennifer Gardinerb, Tian Yub, Katherine Shimc,
Bernice Morrowd, M. Albert Bassonb
aDept.of Otolaryngology, Saku Central Hospital, Japan
bDept.of Craniofacial Dev, KCL, UK
cDept.of Otolaryngology & Com Science at the MCW, USA
dDept.of Molecular Genetics, AECM, New York, USA
DiGeorge/Velo-caldio-facial/22q11 deletion syndrome (22q11DS)
is one of the most common microdeletion disorders, characterized by
severe malformations of many organ systems. Most of the patients
have some form of hearing loss and about 10% have defects in the
structure of their inner ears. However, while for some people the
condition is severe, for others it is not, and it is not yet known what
causes the differences between individuals. Haploinsufficiency for the
TBX1 gene has been linked to 22q11DS defects, including inner ear
defects. We have recently identified another group of genes, the
Sprouty (Spry) genes, that regulate inner ear development. Mice with
loss of function mutations in the Spry1 and Spry2 genes have
abnormally shaped cochleas and semi-circular canals. To determine
whether these genes interact with Tbx1 during ear development, we
generated Spry1-/-;Spry2-/-;Tbx1 embryos and examined their inner
ear structures by paint-fill metod. Spry1-/-;Spry2-/-;Tbx1 embryos
have a more severe phenotype than Spry1-/-;Spry2-/- or Tbx1
mutants. We found that the several signaling pathways implicated
in inner ear development are deregulated in the Spry1-/-;Spry2-/-
otic vesicle and that these effects are further enhanced by Tbx1
haploinsufficiency. These data suggest that Sprouty genes have the
potential to modify inner ear development in patients with 22q11DS.
doi:10.1016/j.ydbio.2010.05.131
Program/Abstract # 93
The Role of FGF Gradients in the Regulation of Early Limb Growth
Ying Zhanga,b, Nikodem J. Poplawskib, James A. Glazierb
aGenetics of Vertebrate Development Section,
Cancer and Developmental Biology Lab, National Cancer Institute,
Frederick Cancer and Developmental Center, Box B, Building 539,
Frederick, MD 21702, USA
bBiocomplexity Institute and Department of Physics, Indiana University,
Simon Hall 047, 212 South Hawthorne Drive, Bloomington,
IN 47405, USA
While molecular biology can identify the molecular components
regulating tissue growth, by itself it can not explain how embryos
determine their shape and size. FGFs, which control cell prolifera-
tion, differentiation, migration and survival, are key molecules in
embryonic morphogenesis. In this paper, we use a reaction-
diffusion model for morphogen diffusion and a Glazier-Graner-
Hogeweg multi-cell model to simulate numerically the role of FGF4
and FGF8 in regulating the early growth of the vertebrate limb. FGF
diffusion, decay and secretion, and cell growth in response to FGF
concentrations, determine the shape and size of limbs, and hence
more generally, of tissues and organs during embryonic develop-
ment. Physiologically reasonable values for FGF secretion, diffusion
and decay grow a simulated limb with correct shape, size and
antero-posterior asymmetry. We show that the limb mainly
expands by growth of the distal domain which has high FGF
concentrations and that the distalized expansion locks the region of
high FGF concentration into the distal tip. We conclude that the
interaction between growth and FGF gradients dominates regula-
tion of the proximo-distal and antero-posterior outgrowth of the
limb and the FGF distribution.
doi:10.1016/j.ydbio.2010.05.132
Program/Abstract # 94
The Limb Mesenchyme Recruitment Model for Patterning the
Vertebrate Limb
Jeffery R. Barrow, Tiffany M. Dahl, Aaron P. Smith, Kate E. Kmetzsch,
Jared J. Barrott, Jed J. Kendall, Keri L. Low
Dept. of Phys. and Developmental Biology, Brigham Young University,
Provo, UT, 84602
444 Abstracts
John Saunders’ classic apical ectodermal ridge (AER) removal
experiments performed over 60 years ago demonstrated that the AER
is required for proximodistal (PD) outgrowth and patterning of the
limb. Despite intense investigation, the mechanisms whereby the
AER regulates these processes remain poorly understood. We propose
that one of the primary roles of the AER is to regulate directional
growth of the adjacent limb mesenchyme. First, we show that Fgf
signaling is necessary and sufficient to activate Wnt5a expression in
gradient fashion in the limb mesenchyme. We also demonstrate that
Wnt5a/Ror2 signaling is necessary and sufficient for directional
growth of the limb mesenchyme. Taken together, we propose that the
AER directs polarized growth of the adjacent mesenchyme through
establishment of a Wnt5a gradient. Because the AER, through Wnt5a,
regulates directional growth of the mesenchyme, it follows that its
shape will in turn play a crucial role in shaping the mesenchyme it
recruits. We report that the AER is almost circular at the time of its
induction which would be predicted to recruit a cylindrical popula-
tion of mesenchyme which condenses to form the stylopod. Over
time, the AER extends along the anteroposterior (AP) and thins along
the dorsoventral (DV) axes in a manner consistent with the formation
of the wider (AP) and thinner (DV) zeugopod and autopod elements.
These results highlight a novel morphogenetic paradigm: the di-
mensions of recruitment signaling centers ultimately shape organs.
This model also provides mechanistic insight for evolutionary change.
doi:10.1016/j.ydbio.2010.05.133
Program/Abstract # 95
Twist Function in Limb Morphogenesis
Peter Farlie, Christine Wade, Inigo Brinas
Murdoch Childrens Research Institute
Twist1 has been demonstrated to play critical roles in the early
development of neural crest and mesodermally derived tissues. Twist2
has been lesswell characterised but its relatively late onset of expression
suggests specific roles in the development of a number of sites. We have
used RCAS-mediated overexpression to investigate the function of
Twist2 in limbdevelopment. Expressionof Twist2within the developing
limbs begins prior to formation of the limb bud and persists within the
peripheral mesenchyme until digital rays condense when Twist2
expression becomes restricted to the interdigital mesenchyme. Viral
misexpression following injection into the lateral plate mesoderm
results in a spectrum of hypoplastic limb phenotypes. These include
generalized shortening of the entire limb, fusion of the autopod skeletal
elements, loss of individual digits or distal truncation resulting in
complete loss of the autopod. These phenotypes appear to result from a
premature termination of limb outgrowth. In situ hybridisation analysis
demonstrates thatmanycomponents of theShh/Fgf/Gremlin regulatory
loop that controls early limb outgrowth are downregulated by Twist2
overexpression. However, despite loss of AER Fgf8 and other regulatory
loop factors such as Gre1, Shh expression is sustained at normal levels.
Twist2 is endogenously co-expressed with Shh but is complementary to
Gre1. This suggests that Shh and Gre may be transcriptional targets of
Twist2. These data indicate that Twist2 regulates limb morphogenesis
through control of the Shh/Fgf/Gre autoregulatory loop.
doi:10.1016/j.ydbio.2010.05.134
Program/Abstract # 96
Does Lunatic fringe play a distinct role in tail development?
Susan E. Cole, Dustin R. Williams
Molecular Genetics, The Ohio State University, Columbus, OH
During vertebrate segmentation, paired somites bud from the
presomitic mesoderm (PSM). The Notch pathway, and a key Notch
modulator Lunatic fringe (Lfng), play multiple roles during segmen-
tation. In the posterior PSM, cyclic Notch activity and Lfng expression
function in the segmentation clock, timing somitogenesis. In the
anterior PSM, stable Lfng expression is involved in pre-somite
patterning. Recent results from our lab indicate that oscillatory Lfng
expression in the clock is required during formation of the anterior
skeleton (primary body formation), but is largely dispensable during
tail development (secondary body formation). In contrast, we find
that Lfng expression in the anterior PSM, during R/C somite
patterning, is required during tail development. Specifically, we find
that mice that retain this expression form largely normal tails, while
tail truncation is observed in Lfng null animals. This suggests the
possibility that Lfng activity in the anterior PSM may regulate long-
range signals that are required for tail outgrowth. To address the
functional requirements for Lfng during tail development, we are
examining secondary body development in embryos that lack Lfng, as
well as embryos that retain Lfng expression only in the anterior PSM.
In addition, we are examining gene expression in embryos that retain
Lfng expression only in the anterior PSM to identify genes and
pathways that may be critical for tail extension. These studies will
elucidate the role of Lfng during tail outgrowth and further test the
hypothesis that the Notch pathway plays distinct roles during
primary and secondary body formation.
doi:10.1016/j.ydbio.2010.05.135
Program/Abstract # 97
SIX1 initiates branching morphogenesis by regulating gremlin 1
expression in the metanephric mesenchyme, which acts to locally
restrict BMP4 activity
Pin-Xian Xu, Jinshu Xu, Xuguang Nie
Dept. of Genetics and Genomic Sciences Mount Sinai School of Medicine
of NYU, New York, NY, USA
Urinary tract morphogenesis requires subdivision of the ureteric
bud (UB) into tip- and trunk-specific domains, which willdifferentiate
into intra-renal collecting system and the extra-renal ureter,
respectively. We report here that Six1-/- UB tip cells failed to form a
tip-specific domain. Failure of invasion into the metanephric
mesenchyme (MM), the mutant UB elongates within Tbx18- and
Bmp4-expressing mesenchyme for its differentiation into ureter. We
find that the expression of the extra-cellular BMP antagonist Grem1 in
the MM is Six1-dependent.Treatment of mutant kidney rudiments in
culture with recombinant GREM1 protein restores ampulla formation
and its subsequent branching morphogenesis. As GREM1 acts as an
antagonist of BMP4 to induce UB outgrowth and branching
morphogenesis, we tested whether genetically lowering BMP4
activity could restore kidney organogenesis. Indeed, genetic reduction
of BMP4 levels in Six1-/- embryos restores urinary tract morphogen-
esis and kidney formation. This study provides the first direct
evidence for the requirement of Six1 in spatially restricting BMP4
signaling in the mesenchyme during the initiation of UB patterning
and branching morphogenesis. Our results indicate that Six1 acts by
regulating Grem1 in the MM to locally antagonizing BMP4 activity to
ensure that the UB tip cells are induced for ampulla formation and its
subsequent branching. This finding uncovers an essential function for
SIX1 as acritical upstream regulator of Gremlin-mediated BMP4
signaling during urinary tract morphogenesis.
doi:10.1016/j.ydbio.2010.05.136
445Abstracts


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The Limb Mesenchyme Recruitment Model for Patterning the Vertebrate Limb

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