Developmental regulation of heparan sulfate proteoglycan synthesis during Drosophila  embryogenesis.

Abstract 2005 MHE Conference

Douglas Bornemann, Sangbin Park and Rahul Warrior.
Department of Developmental and Cell Biology and   
Developmental Biology Center, University of California, Irvine, Irvine CA 92617.

Signaling by the BMP4 homolog Decapentaplegic (Dpp) is critical for cell fate specification in a wide variety of tissues and at
several stages during Drosophila development. The most extensively studied roles for Dpp are in patterning the dorsal region of
the early embryo and in regulating cell proliferation and patterning in the wing imaginal discs. Dpp activity in the wing disc
requires heparan sulfate proteoglycans (HSPGs), and mutations in tout velou (ttv) and sister of tout velou (sotv) that encode
the GAG chain polymerases, impair Dpp signaling.

Ttv and Sotv activities are also essential for Hedgehog (Hh) and Wingless (Wg) signaling, both in  the imaginal discs as well as in
the embryo.

Surprisingly however, embryos  lacking ttv, sotv and other HSPG biosynthetic enzymes do not show any  alterations in dorsal
patterning, a phenotype characteristic of disruptions in the Dpp pathway. Thus our data suggested that the Dpp pathway, in
contrast to the Hh and Wg pathways, appears to be differentially sensitive to loss of HSPGs at different developmental stages.

In order to understand the basis for these observations we examined the  temporal regulation of GAG chain addition to HSPG
core proteins. We found  that GAG chain synthesis is under tight developmental control. Essentially no synthetic activity is
detectable in the first three hours of  embryogenesis with a rapid onset between three and four hours following  fertilization.The
early time period when the biosynthetic process is  inactive correlates precisely with the interval during which a Dpp/BMP activity
gradient is established, while the onset of GAG chain addition coincides with the time when Hh and Wg signaling first become
active in patterning the embryonic epidermis. We find that the timing of GAG chain addition is not controlled by the regulated
expression or activity of a pathway component. Instead our data argue that GAG chain synthesis is controlled at a
post-transcriptional level through regulated translation of at least one of the GAG chain polymerases.

Interestingly, this regulatory mechanism appears to be phylogenetically conserved, suggesting  that stage or tissue-specific
regulation of GAG chain addition could  represent an important strategy in altering the sensitivity of specific signaling pathways
at unique stages during development.  
Research authored by Dr. Warrior
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2009 Conference abstract
Developmental Regulation of Heparan Sulfate Proteoglycan Synthesis

Rahul Warrior, Ph.D.
Developmental Biology Center, and Department of Developmental and Cell Biology, University of California, Irvine.

email:
rwarrior@uci.edu

In the fruitfly Drosophila, the genes toutvelou (ttv) and sister of toutvelou (sotv) encode GAG polymerase subunits
orthologous to human EXT1 and EXT2 that are affected in Hereditary Multiple Exostoses. We isolated mutations in sotv, and
showed that both Sotv and its partner co-polymerase Ttv, are essential for enzymatic activity. Importantly we found that
mutations in sotv and ttv result in wide ranging developmental defects and impair the activity of 3 major signaling pathways –
Hedgehog (Hh), Wnt and Bone Morphogenetic Protein (BMP) - in larval imaginal discs that give rise to adult tissues (Bornemann
et al., 2004, Development 131, 1927-1938). This work highlighted the fact that consequences of mutations in the EXT genes
are likely to reflect dysregulation of multiple growth factor signaling pathways, in contrast to previous studies that argued for a
more restricted effect on Hedgehog signaling alone.

More recently we investigated how loss of sotv and ttv activity affects early embryonic patterning. Surprisingly, in the embryo
the absence of GAG chains perturbs only Wnt/Wg and Hh but not BMP signaling. We found that GAG chain synthesis is under
tight temporal regulation and HSPG core proteins are not glycosylated in the early embryo during the first 3 hours post-
fertilization, when BMP activity specifies cell fates along the dorsal ventral axis. GAG chain synthesis is initiated an hour later
when Wg and Hh signaling play critical roles. The absence of GAG modifications in the early embryo is unexpected since HSPGs
are required for BMP signaling at other stages and argues that the mechanism by which a BMP activity gradient is established in
the embryo is significantly different from that utilized in imaginal discs. The absence of detectable GAG chain polymerase activity
in the early embryo was surprising because high levels of ttv and sotv transcripts are present at these stages. Examination of
the genomic organization, expression data and cDNA clones revealed that these maternally provided transcripts have complex
structures typically associated with mRNAs that are translationally regulated. Our data show that the mRNA structure of ttv is
critical for regulating protein levels, suggesting a novel mechanism for regulating GAG chain synthetic activity. Importantly, this
mechanism is likely to be evolutionarily conserved since we have identified similar complex 5’ UTR in several human enzymes
that play critical roles in GAG synthesis and modification (Bornemann et al., 2008, Development 135, 1039-1047).

Current studies indicate that the 5’ UTR from human Ext1 can function as an IRES and direct regulated translation in
Drosophila. This exciting finding argues that these regulatory mechanisms may be evolutionarily conserved, and that cis-
elements and trans-acting factors identified in Drosophila could be relevant to understanding the basis of the human disease.
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