Neural crest cell-derived VEGF promotes embryonic jaw extension

Jaw morphogenesis depends on the growth of Meckel’s cartilage during embryogenesis. However, the cell types and signals that promote chondrocyte proliferation for Meckel’s cartilage growth are poorly defined. Here we show that neural crest cells (NCCs) and their derivatives provide an essential source of the vascular endothelial growth factor (VEGF) to enhance jaw vascularization and stabilize the major mandibular artery. We further show in two independent mouse models that blood vessels promote Meckel’s cartilage extension. Coculture experiments of arterial tissue with NCCs or chondrocytes demonstrated that NCC-derived VEGF promotes blood vessel growth and that blood vessels secrete factors to instruct chondrocyte proliferation. Computed tomography and X-ray scans of patients with hemifacial microsomia also showed that jaw hypoplasia correlates with mandibular artery dysgenesis. We conclude that cranial NCCs and their derivatives provide an essential source of VEGF to support blood vessel growth in the developing jaw, which in turn is essential for normal chondrocyte proliferation, and therefore jaw extension

HuB (elavl2) mRNA Is Restricted to the Germ Cells by Post-Transcriptional Mechanisms including Stabilisation of the Message by DAZL

The ability of germ cells to carry out a gene regulatory program distinct from the surrounding somatic tissue, and their capacity to specify an entire new organism has made them a focus of many studies that seek to understand how specific regulatory mechanisms, particularly post-transcriptional mechanisms, contribute to cell fate. In zebrafish, germ cells are specified through the inheritance of cytoplasmic determinants, termed the germ plasm, which contains a number of maternal mRNAs and proteins. Investigation of several of these messages has revealed that the restricted localisation of these mRNAs to the germ plasm and subsequent germ cells is due to cis-acting sequence elements present in their 3′UTRs. Here we show that a member of the Hu family of RNA-binding proteins, HuB, is maternally provided in the zebrafish embryo and exhibits germ cell specific expression during embryogenesis. Restriction of HuB mRNA to the germ cells is dependent on a number of sequence elements in its 3′UTR, which act to degrade the mRNA in the soma and stabilise it in the germ cells. In addition, we show that the germ cell specific RNA-binding protein DAZL is able to promote HuB mRNA stability and translation in germ cells, and further demonstrate that these activities require a 30 nucleotide element in the 3′UTR. Our study suggests that DAZL specifically binds the HuB 3′UTR and protects the message from degradation and/or enhances HuB translation, leading to the germ cell specific expression of HuB protein

Investigating mechanisms of post-transcriptional gene regulation in the germ cells of Zebrafish.

In most organisms, the primordial germ cells are specified and set aside from the surrounding somatic tissues very early in development. Their ability to carry out a gene regulatory program quite distinct from the surrounding somatic cells, and their capacity to specify entire new organisms has made them a focus of many studies that seek to understand how specific transcriptional and translational programs contribute to cell fate. Zebrafish, a vertebrate with external development of the embryo, is currently one of the best animal models for understanding the molecular basis of germ cell specification. Briefly, germ cell specification is dependent on maternally provided cytoplasmic determinants, termed the germ plasm. The germ plasm, is localised to areas of the embryo that will become the germ cells later in development by inheritance the germ plasm through cleavage divisions. A number of mRNA components of the germ plasm have been identified; interestingly many of them encode RNA-binding proteins, and almost all of them have invertebrate and mammalian orthologues. Evidence suggests that these maternally provided mRNA determinants are specifically maintained in the germ cells throughout embryonic development, and at least some of these gene products are essential for germ cell specification. A number of studies have begun to elucidate the molecular mechanisms that allow germ cell specific maintenance of these mRNAs, and also to identify how maternally provided messages destined for the germ cells are destabilised and eliminated in the somatic tissues. For example, the germ cell specific mRNAs nanos and TDRD7 are destabilised in somatic cells through interactions of the 3´UTR sequences with the microRNA miR-430. This miR-430-mediated repression is overcome in germ cells through the binding of an RNA-binding protein Dead end (DND) to distinct sites within the nanos and TDRD7 3´UTRs. This thesis details a study of the zebrafish orthologue of HuB, a highly conserved RNAbinding protein with expression in neurons, testes and ovaries in adult vertebrates. In zebrafish, HuB mRNA is maternally provided, and is restricted to the germ cells by 24 hours of development; this is the first report to indicate expression of HuB in the germ cells of vertebrates, suggesting a possible role for HuB in germ cell development. Through detailed mutagenesis studies, the HuB 3´UTR has been found to contain a set of four destabilising elements, which bring about somatic degradation of the mRNA, and a separate, 30-nucleotide motif that is responsible for germ cell specific stabilisation of the message. None of these identified destabilising elements are targets for miR-430, and thus they represent novel sequence elements for somatic message degradation in zebrafish. Through a candidate screening approach, DAZL, a germ cell specific RNA-binding protein, was identified as being capable of stabilising HuB mRNA. Further-more, DAZL was shown to mediate this stabilisation of HuB mRNA by interacting, either directly or indirectly, with the 30-nucleotide stabilisation element that was indentified in the HuB 3´UTR. This elucidation of the mechanisms of germ cell specific expression of the HuB mRNA is an important finding, for it reveals mechanisms of post-transcriptional regulation that are distinct from that of other germ cell specific mRNAs. In summary, the identification of HuB as a germ cell specific mRNA, and the determination of the post-transcriptional mechanisms responsible for this specific expression is an important first step in understanding how HuB and other germ cell specific RNA-binding proteins contribute to germ cell development and function.Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 201

Exploring the Intracrine Functions of VEGF-A

Vascular endothelial growth factor A (VEGF-A or VEGF) is a highly conserved secreted signalling protein best known for its roles in vascular development and angiogenesis. Many non-endothelial roles for VEGF are now established, with the discovery that VEGF and its receptors VEGFR1 and VEGFR2 are expressed in many non-vascular cell-types, as well as various cancers. In addition to secreted VEGF binding to its receptors in the extracellular space at the cell membrane (i.e., in a paracrine or autocrine mode), intracellularly localised VEGF is emerging as an important signalling molecule regulating cell growth, survival, and metabolism. This intracellular mode of signalling has been termed “intracrine”, and refers to the direct action of a signalling molecule within the cell without being secreted. In this review, we describe examples of intracrine VEGF signalling in regulating cell growth, differentiation and survival, both in normal cell homeostasis and development, as well as in cancer. We further discuss emerging evidence for the molecular mechanisms underpinning VEGF intracrine function, as well as the implications this intracellular mode of VEGF signalling may have for use and design of anti-VEGF cancer therapeutics

Heterozygous expression of the oncogenic Pik3ca (H1047R) mutation during murine development results in fatal embryonic and extraembryonic defects

AbstractThe phosphoinositide 3-kinase (PI3K)/AKT signalling pathway regulates many cellular functions including proliferation, migration, survival and protein synthesis. Somatic mutations in PIK3CA, the gene encoding the p110α catalytic subunit of PI3K enzyme, are commonly associated with many human cancers as well as recently being implicated in human overgrowth syndromes. However, it is not clear if such mutations can be inherited through the germline. We have used a novel mouse model with Cre recombinase (Cre)-conditional knock-in of the common H1047R mutation into the endogenous Pik3ca gene. Heterozygous expression of the Pik3caH1047R mutation in the developing mouse embryo resulted in failed ‘turning’ of the embryo and disrupted vascular remodelling within the embryonic and extraembryonic tissues, leading to lethality prior to E10. As vascular endothelial growth factor A (VEGF-A) signalling was disrupted in these embryos, we used Cre under the control of the Tie2 promoter to target the Pik3caH1047R mutation specifically to endothelial cells. In these embryos turning occurred normally but the vascular remodelling defects and embryonic lethality remained, likely as a result of endothelial hyperproliferation. Our results confirm the lethality associated with heterozygous expression of the Pik3caH1047R mutation during development and likely explain the lack of inherited germline PIK3CA mutations in humans

Heterozygous expression of the oncogenic Pik3ca (H1047R) mutation during murine development results in fatal embryonic and extraembryonic defects

AbstractThe phosphoinositide 3-kinase (PI3K)/AKT signalling pathway regulates many cellular functions including proliferation, migration, survival and protein synthesis. Somatic mutations in PIK3CA, the gene encoding the p110α catalytic subunit of PI3K enzyme, are commonly associated with many human cancers as well as recently being implicated in human overgrowth syndromes. However, it is not clear if such mutations can be inherited through the germline. We have used a novel mouse model with Cre recombinase (Cre)-conditional knock-in of the common H1047R mutation into the endogenous Pik3ca gene. Heterozygous expression of the Pik3caH1047R mutation in the developing mouse embryo resulted in failed ‘turning’ of the embryo and disrupted vascular remodelling within the embryonic and extraembryonic tissues, leading to lethality prior to E10. As vascular endothelial growth factor A (VEGF-A) signalling was disrupted in these embryos, we used Cre under the control of the Tie2 promoter to target the Pik3caH1047R mutation specifically to endothelial cells. In these embryos turning occurred normally but the vascular remodelling defects and embryonic lethality remained, likely as a result of endothelial hyperproliferation. Our results confirm the lethality associated with heterozygous expression of the Pik3caH1047R mutation during development and likely explain the lack of inherited germline PIK3CA mutations in humans

The ubiquitin ligase Nedd4 regulates craniofacial development by promoting cranial neural crest cell survival and stem-cell like properties

The integration of multiple morphogenic signalling pathways and transcription factor networks is essential to mediate neural crest (NC) cell induction, delamination, survival, stem-cell properties, fate choice and differentiation. Although the transcriptional control of NC development is well documented in mammals, the role of post-transcriptional modifications, and in particular ubiquitination, has not been explored. Here we report an essential role for the ubiquitin ligase Nedd4 in cranial NC cell development. Our analysis of Nedd4(-/-) embryos identified profound deficiency of cranial NC cells in the absence of structural defects in the neural tube. Nedd4 is expressed in migrating cranial NC cells and was found to positively regulate expression of the NC transcription factors Sox9, Sox10 and FoxD3. We found that in the absence of these factors, a subset of cranial NC cells undergo apoptosis. In accordance with a lack of cranial NC cells, Nedd4(-/-) embryos have deficiency of the trigeminal ganglia, NC derived bone and malformation of the craniofacial skeleton. Our analyses therefore uncover an essential role for Nedd4 in a subset of cranial NC cells and highlight E3 ubiquitin ligases as a likely point of convergence for multiple NC signalling pathways and transcription factor networks.Sophie Wiszniak, Samuela Kabbara, Rachael Lumb, Michaela Scherer, Genevieve Secker, Natasha Harvey, Sharad Kumar, Quenten Schwar