An efficient method for immortalizing mouse embryonic fibroblasts.

Mouse embryonic fibroblasts (MEFs) derived from genetically modified mice are a valuable resource for studying gene function and regulation. The MEF system can also be combined with rescue studies to characterize the function of mutant genes/proteins, such as disease-causing variants. However, primary MEFs undergo senescence soon after isolation and passaging, making long-term genetic manipulations difficult.

The tale of capturing Norrin.

Detailed binding experiments reveal new insights into the Norrin/Wnt signaling pathway that helps to control vascularization in the retina.

Structure and function of the ROR2 cysteine-rich domain in vertebrate noncanonical WNT5A signaling.

The receptor tyrosine kinase ROR2 mediates noncanonical WNT5A signaling to orchestrate tissue morphogenetic processes, and dysfunction of the pathway causes Robinow syndrome, brachydactyly B, and metastatic diseases. The domain(s) and mechanisms required for ROR2 function, however, remain unclear. We solved the crystal structure of the extracellular cysteine-rich (CRD) and Kringle (Kr) domains of ROR2 and found that, unlike other CRDs, the ROR2 CRD lacks the signature hydrophobic pocket that binds lipids/lipid-modified proteins, such as WNTs, suggesting a novel mechanism of ligand reception.

Non-canonical WNT5A-ROR signaling: New perspectives on an ancient developmental pathway.

Deciphering non-canonical WNT signaling has proven to be both fascinating and challenging. Discovered almost 30 years ago, non-canonical WNT ligands signal independently of the transcriptional co-activator β-catenin to regulate a wide range of morphogenetic processes during development. The molecular and cellular mechanisms that underlie non-canonical WNT function, however, remain nebulous.

WNT5a-ROR Signaling Is Essential for Alveologenesis.

WNT5a is a mainly "non-canonical" WNT ligand whose dysregulation is observed in lung diseases such as idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD) and asthma. Germline deletion of Wnt5a disrupts embryonic lung development. However, the temporal-specific function of WNT5a remains unknown.

Agrin-Lrp4-Ror2 signaling regulates adult hippocampal neurogenesis in mice.

Adult neurogenesis in the hippocampus may represent a form of plasticity in brain functions including mood, learning and memory. However, mechanisms underlying neural stem/progenitor cells (NSPCs) proliferation are not well understood. We found that Agrin, a factor critical for neuromuscular junction formation, is elevated in the hippocampus of mice that are stimulated by enriched environment (EE).

Oscillatory cortical forces promote three dimensional cell intercalations that shape the murine mandibular arch.

Multiple vertebrate embryonic structures such as organ primordia are composed of confluent cells. Although mechanisms that shape tissue sheets are increasingly understood, those which shape a volume of cells remain obscure. Here we show that 3D mesenchymal cell intercalations are essential to shape the mandibular arch of the mouse embryo.

Wnt/PCP Signaling Contribution to Carcinoma Collective Cell Migration and Metastasis.

Our understanding of the cellular mechanisms governing carcinoma invasiveness and metastasis has evolved dramatically over the last several years. The previous emphasis on the epithelial-mesenchymal transition as a driver of the migratory properties of single cells has expanded with the observation that carcinoma cells often invade and migrate collectively as adherent groups. Moreover, recent analyses suggest that circulating tumor cells within the vasculature often exist as multicellular clusters and that clusters more efficiently seed metastatic lesions than single circulating tumor cells.

Genetic interactions between Ror2 and Wnt9a, Ror1 and Wnt9a and Ror2 and Ror1: Phenotypic analysis of the limb skeleton and palate in compound mutants.

Mutations in the human receptor tyrosine kinase ROR2 are associated with Robinow syndrome (RRS) and brachydactyly type B1. Amongst others, the shortened limb phenotype associated with RRS is recapitulated in Ror2 mutant mice. In contrast, Ror1 mutant mice are viable and show no limb phenotype.

Whole genome variant association across 100 dogs identifies a frame shift mutation in DISHEVELLED 2 which contributes to Robinow-like syndrome in Bulldogs and related screw tail dog breeds.

Domestic dog breeds exhibit remarkable morphological variations that result from centuries of artificial selection and breeding. Identifying the genetic changes that contribute to these variations could provide critical insights into the molecular basis of tissue and organismal morphogenesis. Bulldogs, French Bulldogs and Boston Terriers share many morphological and disease-predisposition traits, including brachycephalic skull morphology, widely set eyes and short stature.

Quantitative Live-cell Reporter Assay for Noncanonical Wnt Activity.

Noncanonical Wnt signaling functions independently of the β-catenin pathway to control diverse developmental processes, and dysfunction of the pathway contributes to a number of human pathological conditions, including birth defects and metastatic cancer. Progress in the field, however, has been hampered by the scarcity of functional assays for measuring noncanonical Wnt signaling activity. We recently described the Wnt5a-Ror-Kif26b (WRK) reporter assay, which directly monitors a post-transcriptional regulatory event in noncanonical Wnt signaling.

Identification of a WNT5A-Responsive Degradation Domain in the Kinesin Superfamily Protein KIF26B.

Noncanonical WNT pathways function independently of the β-catenin transcriptional co-activator to regulate diverse morphogenetic and pathogenic processes. Recent studies showed that noncanonical WNTs, such as WNT5A, can signal the degradation of several downstream effectors, thereby modulating these effectors' cellular activities. The protein domain(s) that mediates the WNT5A-dependent degradation response, however, has not been identified.

Kinesin superfamily protein Kif26b links Wnt5a-Ror signaling to the control of cell and tissue behaviors in vertebrates.

Wnt5a-Ror signaling constitutes a developmental pathway crucial for embryonic tissue morphogenesis, reproduction and adult tissue regeneration, yet the molecular mechanisms by which the Wnt5a-Ror pathway mediates these processes are largely unknown. Using a proteomic screen, we identify the kinesin superfamily protein Kif26b as a downstream target of the Wnt5a-Ror pathway. Wnt5a-Ror, through a process independent of the canonical Wnt/β-catenin-dependent pathway, regulates the cellular stability of Kif26b by inducing its degradation via the ubiquitin-proteasome system.

The Ror1 receptor tyrosine kinase plays a critical role in regulating satellite cell proliferation during regeneration of injured muscle.

The Ror family receptor tyrosine kinases, Ror1 and Ror2, play important roles in regulating developmental morphogenesis and tissue- and organogenesis, but their roles in tissue regeneration in adult animals remain largely unknown. In this study, we examined the expression and function of Ror1 and Ror2 during skeletal muscle regeneration. Using an skeletal muscle injury model, we show that expression of Ror1 and Ror2 in skeletal muscles is induced transiently by the inflammatory cytokines, TNF-α and IL-1β, after injury and that inhibition of TNF-α and IL-1β by neutralizing antibodies suppresses expression of and in injured muscles.

Planar cell polarity signaling in the uterus directs appropriate positioning of the crypt for embryo implantation.

Blastocyst implantation is a complex process requiring coordination of a dynamic sequence of embryo-uterine interactions. Blood vessels enter the uterus from the mesometrium, demarcating the uterus into mesometrial (M) and antimesometrial (AM) domains. Implantation occurs along the uterine longitudinal axis within specialized implantation chambers (crypts) that originate within the evaginations directed from the primary lumen toward the AM domain.

Unidirectional Eph/ephrin signaling creates a cortical actomyosin differential to drive cell segregation.

Cell segregation is the process by which cells self-organize to establish developmental boundaries, an essential step in tissue formation. Cell segregation is a common outcome of Eph/ephrin signaling, but the mechanisms remain unclear. In craniofrontonasal syndrome, X-linked mosaicism for ephrin-B1 expression has been hypothesized to lead to aberrant Eph/ephrin-mediated cell segregation.

Meiotic onset is reliant on spatial distribution but independent of germ cell number in the mouse ovary.

Mouse ovarian germ cells enter meiosis in a wave that propagates from anterior to posterior, but little is known about contribution of germ cells to initiation or propagation of meiosis. In a Ror2 mutant with diminished germ cell number and migration, we find that overall timing of meiotic initiation is delayed at the population level. We use chemotherapeutic depletion to exclude a profoundly reduced number of germ cells as a cause for meiotic delay.

EphB1 and EphB2 intracellular domains regulate the formation of the corpus callosum and anterior commissure.

The two cortical hemispheres of the mammalian forebrain are interconnected by major white matter tracts, including the corpus callosum (CC) and the posterior branch of the anterior commissure (ACp), that bridge the telencephalic midline. We show here that the intracellular signaling domains of the EphB1 and EphB2 receptors are critical for formation of both the ACp and CC. We observe partial and complete agenesis of the corpus callosum, as well as highly penetrant ACp misprojection phenotypes in truncated EphB1/2 mice that lack intracellular signaling domains.

Appropriate crypt formation in the uterus for embryo homing and implantation requires Wnt5a-ROR signaling.

Embryo homing and implantation occur within a crypt (implantation chamber) at the antimesometrial (AM) pole along the uterus. The mechanism by which this is achieved is not known. Here, we show that villi-like epithelial projections from the main uterine lumen toward the AM pole at regularly spaced intervals that form crypts for embryo implantation were disrupted in mice with uterine loss or gain of function of Wnt5a, or loss of function of both Ror1 and Ror2.

Vangl1 and Vangl2: planar cell polarity components with a developing role in cancer.

Cancers commonly reactivate embryonic developmental pathways to promote the aggressive behavior of their cells, resulting in metastasis and poor patient outcome. While developmental pathways such as canonical Wnt signaling and epithelial-to-mesenchymal transition have received much attention, our understanding of the role of the planar cell polarity (PCP) pathway in tumor progression remains rudimentary. Protein components of PCP, including a subset that overlaps with the canonical Wnt pathway, partition in polarized epithelial cells along the planar axis and are required for the establishment and maintenance of lateral epithelial polarity.

EphB receptor forward signaling regulates area-specific reciprocal thalamic and cortical axon pathfinding.

In early brain development, ascending thalamocortical axons (TCAs) navigate through the ventral telencephalon (VTel) to reach their target regions in the young cerebral cortex. Descending, deep-layer cortical axons subsequently target appropriate thalamic and subcortical target regions. However, precisely how and when corticothalamic axons (CTAs) identify their appropriate, reciprocal thalamic targets remains unclear.

An autocrine Wnt5a-Ror signaling loop mediates sympathetic target innervation.

During nervous system development, axon branching at nerve terminals is an essential step in the formation of functional connections between neurons and target cells. It is known that target tissues exert control of terminal arborization through secretion of trophic factors. However, whether the in-growing axons themselves produce diffusible cues to instruct target innervation remains unclear.

A chemical genetic approach reveals distinct EphB signaling mechanisms during brain development.

EphB receptor tyrosine kinases control multiple steps in nervous system development. However, it remains unclear whether EphBs regulate these different developmental processes directly or indirectly. In addition, given that EphBs signal through multiple mechanisms, it has been challenging to define which signaling functions of EphBs regulate particular developmental events.