Subdividing the embryo: a role for Notch signaling during germ layer patterning in Xenopus laevis

The development of all vertebrate embryos requires the establishment of a three-dimensional coordinate system in order to pattern embryonic structures and create the complex shape of the adult organism. During the process of gastrulation, the three primary germ layers are created under the guidance of numerous signaling pathways, allowing cells to communicate during development. Cell-cell communication, mediated by receptors of the Notch family, has been shown to be involved in mediating diverse cellular behaviors during development and has been implicated in the regulation of cell fate decisions in both vertebrate and invertebrate organisms. In order to investigate a role for Notch signaling during boundary formation between the mesoderm and endoderm during gastrulation, we manipulated Notch signaling in gastrula stage embryos and examined gene expression in resultant tissues and organs. Our findings demonstrate a much broader role for Notch signaling during germ layer determination than previously reported in a vertebrate organism. Activation of the Notch pathway, specifically in gastrula stage embryos, results in a dramatic decrease in the expression of genes necessary to create many different types of mesodermal tissues while causing a dramatic expansion of endodermal tissue markers. Conversely, temporally controlled suppression of this pathway results in a loss of endodermal cell types and an expansion of molecular markers of mesoderm. Thus, our data are consistent with and significantly extend the implications of prior observations suggesting roles for Notch signaling during germ layer formation and establish an evolutionarily conserved role for Notch signaling in mediating mesoderm-endoderm boundaries during early vertebrate development.     

Notch signaling reveals developmental plasticity of Pax4(+) pancreatic endocrine progenitors and shunts them to a duct fate

Relatively little is known about the developmental signals that specify the types and numbers of pancreatic cells. Previous studies suggested that Notch signaling in the pancreas inhibits differentiation and promotes the maintenance of progenitor cells, but it remains unclear whether Notch also controls cell fate choices as it does in other tissues. To study the impact of Notch in progenitors of the beta cell lineage, we generated mice that express Cre-recombinase under control of the Pax4 promoter. Lineage analysis of Pax4(+) cells demonstrates they are specified endocrine progenitors that contribute equally to four islet cell fates, contrary to expectations raised by the dispensable role of Pax4 in the specification of the alpha and PP subtypes. In addition, we show that activation of Notch in Pax4(+) progenitors inhibits their differentiation into alpha and beta endocrine cells and shunts them instead toward a duct fate. These observations reveal an unappreciated degree of developmental plasticity among early endocrine progenitors and raise the possibility that a bipotent duct-endocrine progenitor exists during development. Furthermore, the redirection of Pax4(+) cells from alpha and beta endocrine fates toward a duct cell type suggests a positive role for Notch signaling in duct specification and is consistent with the more widely defined role for Notch in cell fate determination.     

Evidence that nervy, the Drosophila homolog of ETO/MTG8, promotes mechanosensory organ development by enhancing Notch signaling

In the imaginal tissue of developing fruit flies, achaete (ac) and scute (sc) expression defines a group of neurally-competent cells called the proneural cluster (PNC). From the PNC, a single cell, the sensory organ precursor (SOP), is selected as the adult mechanosensory organ precursor. The SOP expresses high levels of ac and sc and sends a strong Delta (Dl) signal, which activates the Notch (N) receptor in neighboring cells, preventing them from also adopting a neural fate. Previous work has determined how ac and sc expression in the PNC and SOP is regulated, but less is known about SOP-specific factors that promote SOP fate. Here, we describe the role of nervy (nvy), the Drosophila homolog of the mammalian proto-oncogene ETO, in mechanosensory organ formation. Nvy is specifically expressed in the SOP, where it interacts with the Ac and Sc DNA binding partner Daughterless (Da) and affects the expression of Ac and Sc targets. nvy loss- and gain-of-function experiments suggest that nvy reinforces, but is not absolutely required for, the SOP fate. We propose a model in which nvy acts downstream of ac and sc to promote the SOP fate by transiently strengthening the Dl signal emanating from the SOP.     

Cyclical expression of the Notch/Wnt regulator Nrarp requires modulation by Dll3 in somitogenesis

Delta-like 3 (Dll3) is a divergent ligand and modulator of the Notch signaling pathway only identified so far in mammals. Null mutations of Dll3 disrupt cycling expression of Notch targets Hes1, Hes5, and Lfng, but not of Hes7. Compared with Dll1 or Notch1, the effects of Dll3 mutations are less severe for gene expression in the presomitic mesoderm, yet severe segmentation phenotypes and vertebral defects result in both human and mouse. Reasoning that Dll3 specifically disrupts key regulators of somite cycling, we carried out functional analysis to identify targets accounting for the segmental phenotype. Using microdissected embryonic tissue from somitic and presomitic mesodermal tissue, we identified new genes enriched in these tissues, including Limch1, Rhpn2, and A130022J15Rik. Surprisingly, we only identified a small number of genes disrupted by the Dll3 mutation. These include Uncx, a somite gene required for rib and vertebral patterning, and Nrarp, a regulator of Notch/Wnt signaling in zebrafish and a cycling gene in mouse. To determine the effects of Dll3 mutation on Nrarp, we characterized the cycling expression of this gene from early (8.5 dpc) to late (10.5 dpc) somitogenesis. Nrarp displays a distinct pattern of cycling phases when compared to Lfng and Axin2 (a Wnt pathway gene) at 9.5 dpc but appears to be in phase with Lfng by 10.5 dpc. Nrarp cycling appears to require Dll3 but not Lfng modulation. In Dll3 null embryos, Nrarp displayed static patterns. However, in Lfng null embryos, Nrarp appeared static at 8.5 dpc but resumed cycling expression by 9.5 and dynamic expression at 10.5 dpc stages. By contrast, in Wnt3a null embryos, Nrarp expression was completely absent in the presomitic mesoderm. Towards identifying the role of Dll3 in regulating somitogenesis, Nrarp emerges as a potentially important regulator that requires Dll3 but not Lfng for normal function.     

Altered Notch signaling resulting from expression of a WAMTP1-MAML2 gene fusion in mucoepidermoid carcinomas and benign Warthin's tumors

Chromosome translocations in neoplasia commonly result in fusion genes that may encode either novel fusion proteins or normal, but ectopically expressed proteins. Here we report the cloning of a novel fusion gene in a common type of salivary and bronchial gland tumor, mucoepidermoid carcinomas (MEC), as well as in benign Warthin's tumors (WATs). The fusion, which results from a t(11;19)(q21-22;p13) translocation, creates a chimeric gene in which exon 1 of a novel gene of unknown function, designated WAMTP1, is linked to exons 2-5 of the recently identified Mastermind-like Notch coactivator MAML2. In the fusion protein, the N-terminal basic domain of MAML2, which is required for binding to intracellular Notch (Notch ICD), is replaced by an unrelated N-terminal sequence from WAMTP1. Mutation analysis of the N-terminus of WAMTP1-MAML2 identified two regions of importance for nuclear localization (amino acids 11-20) and for colocalization with MAML2 and Notch1 ICD in nuclear granules (amino acids 21-42). Analyses of the Notch target genes HES5 and MASH1 in MEC tumors with and without the WAMTP1-MAML2 fusion revealed upregulation of HES5 and downregulation of MASH1 in fusion positive MECs compared to normal salivary gland tissue and MECs lacking the fusion. These findings suggest that altered Notch signaling plays an important role in the genesis of benign and malignant neoplasms of salivary and bronchial gland origin.     

Mesonephric FGF signaling is associated with the development of sexually indifferent gonadal primordium in chick embryos

The gonad as well as the reproductive tracts, kidney, and adrenal cortex are derived from the intermediate mesoderm. In addition, the intermediate mesoderm forms the mesonephros. Although the mesonephros is the source of certain testicular cell types, its contribution to gonad formation through expression of growth factors is largely unknown. Here, we examined the expression profiles of FGF9 in the developing mesonephros of chick embryos at sexually indifferent stages, and found that the expression domain is adjacent to the gonadal primordium. Moreover, FGFR3 (FGF receptor 3) showed a strong expression in the gonadal primordium. Next, we examined the functions of FGF signal during gonadal development with misexpressed FGF9. Interestingly, misexpression of FGF9 led to gonadal expansion through stimulation of cell proliferation. In contrast, treatment with a chemical inhibitor for FGFR decreased cell proliferation and resulted in reduction of the gonadal size. Simultaneously, the treatment resulted in reduction of gonadal marker gene expression. Our study demonstrated that FGF expressed in the developing mesonephros is involved in the development of the gonad at the sexually indifferent stages through stimulation of gonadal cell proliferation and gonadal marker gene expression.     

Role of BMP signaling and the homeoprotein Iroquois in the specification of the cranial placodal field

Different types of placodes originate at the anterior border of the neural plate but it is still an unresolved question whether individual placodes arise as distinct ectodermal specializations in situ or whether all or a subset of the placodes originate from a common preplacodal field. We have analyzed the expression and function of the homeoprotein Iro1 in Xenopus and zebrafish embryos, and we have compared its expression with several preplacodal and placodal markers. Our results indicate that the iro1 genes are expressed in the preplacodal region, being one of the earliest markers for this area. We show that an interaction between the neural plate and the epidermis is able to induce the expression of several preplacodal markers, including Xiro1, by a similar mechanism to that previously shown for neural crest induction. In addition, we analyzed the role of BMP in the specification of the preplacodal field by studying the expression of the preplacodal markers Six1, Xiro1, and several specific placodal markers. We experimentally modified the level of BMP activity by three different methods. First, we implanted beads soaked with noggin in early neurula stage Xenopus embryos; second, we injected the mRNA that encodes a dominant negative of the BMP receptor into Xenopus and zebrafish embryos; and third, we grafted cells expressing chordin into zebrafish embryos. The results obtained using all three methods show that a reduction in the level of BMP activity leads to an expansion of the preplacodal and placodal region similar to what has been described for neural crest regions. By using conditional constructs of Xiro1, we performed gain and loss of function experiments. We show that Xiro1 play an important role in the specification of both the preplacodal field as well as individual placodes. We have also used inducible dominant negative and activator constructs of Notch signaling components to analyze the role of these factors on placodal development. Our results indicate that the a precise level of BMP activity is required to induce the neural plate border, including placodes and neural crest cells, that in this border the iro1 gene is activated, and that this activation is required for the specification of the placodes.     

A highly conserved Wnt-dependent TCF4 binding site within the proximal enhancer of the anti-myogenic Msx1 gene supports expression within Pax3-expressing limb bud muscle precursor cells

The product of the Msx1 gene is a potent inhibitor of muscle differentiation. Msx1 is expressed in muscle precursor cells of the limb bud that also express Pax3. It is thought that Msx1 may facilitate distal migration by delaying myogenesis in these cells. Despite the role played by Msx1 in inhibiting muscle differentiation, nothing is known of the mechanisms that support the expression of the Msx1 gene within limb bud muscle precursor cells. In the present study we have used a combination of comparative genomics, mouse transgenic analysis, in situ hybridisation and immunohistochemistry to identify a highly conserved and tissue-specific regulatory sub-domain within the previously characterised Msx1 gene proximal enhancer element that supports the expression of the Msx1 gene in Pax3-expressing mouse limb pre-muscle masses. Furthermore, using a combination of in situ hybridisation, in vivo ChIP assay and transgenic explant culture analysis we provide evidence that Msx1 expression in limb bud muscle precursor cells is dependent on the canonical Wnt/TCF signalling pathway that is important in muscle shape formation. The results of these studies provide evidence of a mechanistic link between the Wnt/TCF and the Msx1/Pax3/MyoD pathways within limb bud muscle precursor cells.