C. Michael Jones

The DVR gene family in embryonic development

The DVR gene family consists of at least 15 members, including decapentaplegic from Drosophila, Xenopus Vg1 and the mammalian bone morphogenetic protein genes, encoding secreted proteins closely related to transforming growth factor beta Genetic and biochemical evidence supports the idea that DVR proteins form part of a cascade of extracellular signalling molecules mediating inductive tissue interactions during development.

Functional coupling between the extracellular matrix and nuclear lamina by Wnt signaling in Progeria

The segmental premature aging disease Hutchinson-Gilford Progeria (HGPS) is caused by a truncated and farnesylated form of Lamin A. In a mouse model for HGPS, a similar Lamin A variant causes the proliferative arrest and death of postnatal, but not embryonic, fibroblasts. Arrest is due to an inability to produce a functional extracellular matrix (ECM), because growth on normal ECM rescues proliferation. The defects are associated with inhibition of canonical Wnt signaling, due to reduced nuclear localization and transcriptional activity of Lef1, but not Tcf4, in both mouse and human progeric cells. Defective Wnt signaling, affecting ECM synthesis, may be critical to the etiology of HGPS because mice exhibit skeletal defects and apoptosis in major blood vessels proximal to the heart. These results establish a functional link between the nuclear envelope/lamina and the cell surface/ECM and may provide insights into the role of Wnt signaling and the ECM in aging.

WLS-dependent secretion of WNT3A requires Ser209 acylation and vacuolar acidification

Wnt proteins are secreted post-translationally modified proteins that signal locally to regulate development and proliferation. The production of bioactive Wnts requires a number of dedicated factors in the secreting cell whose coordinated functions are not fully understood. A screen for small molecules identified inhibitors of vacuolar acidification as potent inhibitors of Wnt secretion. Inhibition of the V-ATPase or disruption of vacuolar pH gradients by diverse drugs potently inhibited Wnt/β-catenin signaling both in cultured human cells and in vivo, and impaired Wnt-regulated convergent extension movements in Xenopus embryos. WNT secretion requires its binding to the carrier protein wntless (WLS); we find that WLS is ER-resident in human cells and WNT3A binding to WLS requires PORCN-dependent lipid modification of WNT3A at serine 209. Inhibition of vacuolar acidification results in accumulation of the WNT3A–WLS complex both in cells and at the plasma membrane. Modeling predictions suggest that WLS has a lipid-binding β-barrel that is similar to the lipocalin-family fold. We propose that WLS binds Wnts in part through a lipid-binding domain, and that vacuolar acidification is required to release palmitoylated WNT3A from WLS in secretory vesicles, possibly to facilitate transfer of WNT3A to a soluble carrier protein.

Expression and modification of Hox 2.1 protein in mouse embryos

A polyclonal antibody, alpha Hox 2.1a, has been generated and used to immunolocalize Hox 2.1 protein in mouse embryos. Protein is present in nuclei of all tissues previously shown to express Hox 2.1 RNA. In addition, protein is seen in somites and proximal regions of the limb buds, tissues in which Hox 2.1 RNA expression was not clearly detected previously by in situ hybridization. At the 7 somite stage, protein is detectable in the neural tube up to the level of somite 1, but later retracts to a more posterior position. Immunoblot, in vitro translation, and immunoprecipitation experiments were carried out to characterize the Hox 2.1 protein. The results show that the Hox 2.1 gene produces at least two related phosphorylated proteins present in different proportions in different tissues.

WLS Retrograde Transport to the Endoplasmic Reticulum during Wnt Secretion

Wnts are transported to the cell surface by the integral membrane protein WLS (also known as Wntless, Evi, and GPR177). Previous studies of WLS trafficking have emphasized WLS movement from the Golgi to the plasma membrane (PM) and then back to the Golgi via retromer-mediated endocytic recycling. We find that endogenous WLS binds Wnts in the endoplasmic reticulum (ER), cycles to the PM, and then returns to the ER through the Golgi. We identify an ER-targeting sequence at the carboxyl terminus of native WLS that is critical for ER retrograde recycling and contributes to Wnt secretory function. Golgi-to-ER recycling of WLS requires the COPI regulator ARF as well as ERGIC2, an ER-Golgi intermediate compartment protein that is also required for the retrograde trafficking of the KDEL receptor and certain toxins. ERGIC2 is required for efficient Wnt secretion. ER retrieval is an integral part of the WLS transport cycle.

Neural tube derived Wnt signals cooperate with FGF signaling in the formation and differentiation of the trigeminal placodes

BackgroundNeurogenic placodes are focal thickenings of the embryonic ectoderm that form in the vertebrate head. It is within these structures that the precursors of the majority of the sensory neurons of the cranial ganglia are specified. The trigeminal placodes, the ophthalmic and maxillomandibular, form close to the midbrain-hindbrain boundary and many lines of evidence have shown that signals emanating from this level of the neuraxis are important for the development of the ophthalmic placode.ResultsHere, we provide the first evidence that both the ophthalmic and maxillomandibular placodes form under the influence of isthmic Wnt and FGF signals. Activated Wnt signals direct development of the Pax3 expressing ophthalmic placodal field and induce premature differentiation of both the ophthalmic and the maxillomandibular placodes. Similarly, overexpression of Fgf8 directs premature differentiation of the trigeminal placodes. Wnt signals require FGF receptor activity to initiate Pax3 expression and, subsequently, the expression of neural markers, such as Brn3a, within the cranial ectoderm. Furthermore, fibroblast growth factor signaling via the mitogen activated protein kinase pathway is required to maintain early neuronal differentiation within the trigeminal placodes.ConclusionWe demonstrate the identity of inductive signals that are necessary for trigeminal ganglion formation. This is the first report that describes how isthmic derived Wnt signals act in concert with fibroblast growth factor signaling. Together, both are necessary and sufficient for the establishment and differentiation of the ophthalmic and maxillomandibular placodes and, consequently, the trigeminal ganglion.

Xhex-expressing endodermal tissues are essential for anterior patterning in Xenopus.

Two regions expressing Hex in the early gastrula contribute to organizing the anterior of the vertebrate embryo. In Xenopus, these include the anterior yolky endoderm and the suprablastoporal endoderm (SBE), which is fated to form the epithelial lining of the gut. These tissues may correspond to the anterior visceral endoderm and anterior definitive endoderm of amniotes. Genetic studies in mice have demonstrated the important roles of these tissues in producing anterior identity in the adjacent neural ectoderm. In Xenopus, both the anterior endoderm and the SBE have anterior inducing properties; furthermore, the SBE can organize a full anterior-posterior axis. Inhibition of Xhex function shows that both these Xhex-expressing endodermal tissues are required for anterior development in Xenopus.

Measurement of photon statistics with live photoreceptor cells.

We study responses of live retinal photoreceptors to light sources with different photon statistics. We show the ability of the cells to discriminate between the sources, down to the level of single photons.

FGFR1 Signaling Stimulates Proliferation of Human Mesenchymal Stem Cells by Inhibiting the Cyclin‐Dependent Kinase Inhibitors p21Waf1 and p27Kip1

Signaling through fibroblast growth factor receptor one (FGFR1) is a known inducer of proliferation in both embryonic and human adult mesenchymal stem cells (hMSCs) and positively regulates maintenance of stem cell viability. Leveraging the mitogenic potential of FGF2/FGFR1 signaling in stem cells for therapeutic applications necessitates a mechanistic understanding of how this receptor stimulates cell cycle progression. Using small interfering RNA (siRNA) depletion, antibody‐inhibition, and small molecule inhibition, we establish that FGFR1 activity is rate limiting for self‐renewal of hMSCs. We show that FGFR1 promotes stem cell proliferation through multiple mechanisms that unite to antagonize cyclin‐dependent kinase (CDK) inhibitors. FGFR1 not only stimulates c‐Myc to suppress transcription of the CDK inhibitors p21Waf1 and p27Kip1, thus promoting cell cycle progression but also increases the activity of protein kinase B (AKT) and the level of S‐phase kinase‐associated protein 2 (Skp2), resulting in the nuclear exclusion and reduction of p21Waf1. The in vivo importance of FGFR1 signaling for the control of proliferation in mesenchymal progenitor populations is underscored by defects in ventral mesoderm formation during development upon inhibition of its signaling. Collectively, these studies demonstrate that FGFR1 signaling mediates the continuation of MSC growth and establishes a receptor target for enhancing the expansion of mesenchymal progenitors while maintaining their multilineage potential. Stem Cells 2013;31:2724–2736

Method of targeted delivery of laser beam to isolated retinal rods by fiber optics.

A method of controllable light delivery to retinal rod cells using an optical fiber is described. Photo-induced current of the living rod cells was measured with the suction electrode technique. The approach was tested with measurements relating the spatial distribution of the light intensity to photo-induced current. In addition, the ion current responses of rod cells to polarized light at two different orientation geometries of the cells were studied.