Ethan David Cohen

Wnt2/2b and β-Catenin Signaling Are Necessary and Sufficient to Specify Lung Progenitors in the Foregut

Patterning of the primitive foregut promotes appropriate organ specification along its anterior-posterior axis. However, the molecular pathways specifying foregut endoderm progenitors are poorly understood. We show here that Wnt2/2b signaling is required to specify lung endoderm progenitors within the anterior foregut. Embryos lacking Wnt2/2b expression exhibit complete lung agenesis and do not express Nkx2.1, the earliest marker of the lung endoderm. In contrast, other foregut endoderm-derived organs, including the thyroid, liver, and pancreas, are correctly specified. The phenotype observed is recapitulated by an endoderm-restricted deletion of beta-catenin, demonstrating that Wnt2/2b signaling through the canonical Wnt pathway is required to specify lung endoderm progenitors within the foregut. Moreover, activation of canonical Wnt/beta-catenin signaling results in the reprogramming of esophagus and stomach endoderm to a lung endoderm progenitor fate. Together, these data reveal that canonical Wnt2/2b signaling is required for the specification of lung endoderm progenitors in the developing foregut.

A Gata6-Wnt pathway required for epithelial stem cell development and airway regeneration

Epithelial organs, including the lung, are known to possess regenerative abilities through activation of endogenous stem cell populations, but the molecular pathways regulating stem cell expansion and regeneration are not well understood. Here we show that Gata6 regulates the temporal appearance and number of bronchioalveolar stem cells (BASCs) in the lung, its absence in Gata6-null lung epithelium leading to the precocious appearance of BASCs and concurrent loss in epithelial differentiation. This expansion of BASCs was the result of a pronounced increase in canonical Wnt signaling in lung epithelium upon loss of Gata6. Expression of the noncanonical Wnt receptor Fzd2 was downregulated in Gata6 mutants and increased Fzd2 or decreased β-catenin expression rescued, in part, the lung epithelial defects in Gata6 mutants. During lung epithelial regeneration, canonical Wnt signaling was activated in the niche containing BASCs and forced activation of Wnt signaling led to a large increase in BASC numbers. Moreover, Gata6 was required for proper lung epithelial regeneration, and postnatal loss of Gata6 led to increased BASC expansion and decreased differentiation. Together, these data demonstrate that Gata6-regulated Wnt signaling controls the balance between progenitor expansion and epithelial differentiation required for both lung development and regeneration.

Wnt signaling: an essential regulator of cardiovascular differentiation, morphogenesis and progenitor self-renewal.

Emerging evidence indicates that Wnt signaling regulates crucial aspects of cardiovascular biology (including cardiac morphogenesis, and the self-renewal and differentiation of cardiac progenitor cells). The ability of Wnt signaling to regulate such diverse aspects of cardiovascular development rests on the multifarious downstream and tangential targets affected by this pathway. Here, we discuss the roles for Wnt signaling in cardiac and vascular development, and focus on the emerging role of Wnt signaling in cardiovascular morphogenesis and progenitor cell self-renewal.

Wnt signaling regulates smooth muscle precursor development in the mouse lung via a tenascin C/PDGFR pathway

Paracrine signaling from lung epithelium to the surrounding mesenchyme is important for lung SMC development and function and is a contributing factor in an array of pulmonary diseases such as bronchopulmonary dysplasia, pulmonary hypertension, and asthma. Wnt7b, which is exclusively expressed in the lung epithelium, is important for lung vascular smooth muscle integrity, but the underlying mechanism by which Wnt signaling regulates lung SMC development is unclear. In this report, we have demonstrated that Wnt7b regulates a program of mesenchymal differentiation in the mouse lung that is essential for SMC development. Genetic loss-of-function studies showed that Wnt7b and beta-catenin were required for expression of Pdgfralpha and Pdgfrbeta and proliferation in pulmonary SMC precursors. In contrast, gain-of-function studies showed that activation of Wnt signaling increased the expression of both Pdgfralpha and Pdgfrbeta as well as the proliferation of SMC precursors. We further showed that the effect on Pdgfr expression was, in part, mediated by direct transcriptional regulation of the ECM protein tenascin C (Tnc), which was necessary and sufficient for Pdgfralpha/beta expression in lung explants. Moreover, this pathway was highly upregulated in a mouse model of asthma and in lung tissue from patients with pulmonary hypertension. Together, these data define a Wnt/Tnc/Pdgfr signaling axis that is critical for smooth muscle development and disease progression in the lung.

Characterization and In Vivo Pharmacological Rescue of a Wnt2-Gata6 Pathway Required for Cardiac Inflow Tract Development

Little is understood about the molecular mechanisms underlying the morphogenesis of the posterior pole of the heart. Here we show that Wnt2 is expressed specifically in the developing inflow tract mesoderm, which generates portions of the atria and atrio-ventricular canal. Loss of Wnt2 results in defective development of the posterior pole of the heart, resulting in a phenotype resembling the human congenital heart syndrome complete common atrio-ventricular canal. The number and proliferation of posterior second heart field progenitors is reduced in Wnt2(-/-) mutants. Moreover, these defects can be rescued in a temporally restricted manner through pharmacological inhibition of Gsk-3beta. We also show that Wnt2 works in a feedforward transcriptional loop with Gata6 to regulate posterior cardiac development. These data reveal a molecular pathway regulating the posterior cardiac mesoderm and demonstrate that cardiovascular defects caused by loss of Wnt signaling can be rescued pharmacologically in vivo.

Wnt5a and Wnt11 are essential for second heart field progenitor development

Wnt/β-catenin has a biphasic effect on cardiogenesis, promoting the induction of cardiac progenitors but later inhibiting their differentiation. Second heart field progenitors and expression of the second heart field transcription factor Islet1 are inhibited by the loss of β-catenin, indicating that Wnt/β-catenin signaling is necessary for second heart field development. However, expressing a constitutively active β-catenin with Islet1-Cre also inhibits endogenous Islet1 expression, reflecting the inhibitory effect of prolonged Wnt/β-catenin signaling on second heart field development. We show that two non-canonical Wnt ligands, Wnt5a and Wnt11, are co-required to regulate second heart field development in mice. Loss of Wnt5a and Wnt11 leads to a dramatic loss of second heart field progenitors in the developing heart. Importantly, this loss of Wnt5a and Wnt11 is accompanied by an increase in Wnt/β-catenin signaling, and ectopic Wnt5a/Wnt11 inhibits β-catenin signaling and promotes cardiac progenitor development in differentiating embryonic stem cells. These data show that Wnt5a and Wnt11 are essential regulators of the response of second heart field progenitors to Wnt/β-catenin signaling and that they act by restraining Wnt/β-catenin signaling during cardiac development.

Wnt2 signaling is necessary and sufficient to activate the airway smooth muscle program in the lung by regulating myocardin/Mrtf-B and Fgf10 expression

Smooth muscle in the lung is thought to derive from the developing lung mesenchyme. Smooth muscle formation relies upon coordination of both autocrine and paracrine signaling between the budding epithelium and adjacent mesenchyme to govern its proliferation and differentiation. However, the pathways initiating the earliest aspects of smooth muscle specification and differentiation in the lung are poorly understood. Here, we identify the Wnt2 ligand as a critical regulator of the earliest aspects of lung airway smooth muscle development. Using Wnt2 loss and gain of function models, we show that Wnt2 signaling is necessary and sufficient for activation of a transcriptional and signaling network critical for smooth muscle specification and differentiation including myocardin/Mrtf-B and the signaling factor Fgf10. These studies place Wnt2 high in a hierarchy of signaling molecules that promote the earliest aspects of lung airway smooth muscle development.

Parallel Circuits from Cones to the On‐Beta Ganglion Cell

Neural integration depends critically upon circuit architecture; yet the architecture has never been established quantitatively (numbers of cells and synapses) for any vertebrate local circuit. Here we describe circuits in the cat retina that connect cones to the on‐beta ganglion cell. This cell type is important because on‐ and off‐beta cells contribute about 50% of the optic nerve fibres and the major retinal input to the striate cortex. Three adjacent on‐beta cells in the area centralis and their bipolar connections to cones were reconstructed from electron micrographs of 279 serial sections. The beta dendritic field is 34±2 μm in diameter and encompasses 35 cones. All of these cones connect to the beta cell via 14–17 bipolar cells. These bipolar cells were shown previously by cluster analysis to be of four types (b1‐b4); three of these types (b1′ b2 and b3) provided 97% of the bipolar contacts to the beta cell, in the ratio 4:2:1. On average, bipolar cells nearest the centre of the beta dendritic field contribute more synapses than those towards the edge, but the peaked distribution of bipolar synapses across the dendritic field is only slightly broader than the optical pointspread function of the cats eye, and is narrower by half than the centre of the ganglion cell receptive field. This implies that the distribution of bipolar synapses across the beta cell dendritic field contributes little to the extent or shape of the receptive field. Since all three bipolar circuits connect to the same set of cones, they must carry the same spatial and chromatic information; they might convey different temporal frequencies. The numbers of bipolar synapses (mean ± SD = 154±8) and amacrine synapses (59±5) converging on three adjacent beta cells are remarkably constant (SD=±5% of the mean). Thus, as the circuits repeat locally, the fundamental design is accurately reproduced.

Wnt ligands signal in a cooperative manner to promote foregut organogenesis

Endoderm-mesenchyme cross-talk is a central process in the development of foregut-derived organs. How signaling pathways integrate the activity of multiple ligands to guide organ development is poorly understood. We show that two Wnt ligands, Wnt2 and Wnt7b, cooperatively induce Wnt signaling without affecting the stabilization of the Wnt canonical effector β-catenin despite it being necessary for Wnt2–Wnt7b cooperativity. Wnt2–Wnt7b cooperation is specific for mesenchymal cell lineages and the combined loss of Wnt2 and Wnt7b leads to more severe developmental defects in the lung than loss of Wnt2 or Wnt7b alone. High-throughput small-molecule screens and biochemical assays reveal that the Pdgf pathway is required for cooperative Wnt2-Wnt7b signaling. Inhibition of Pdgf signaling in cell culture reduces Wnt2–Wnt7b cooperative signaling. Moreover, inhibition of Pdgf signaling in lung explant cultures results in decreased Wnt signaling and lung smooth-muscle development. These data suggest a model in which Pdgf signaling potentiates Wnt2–Wnt7b signaling to promote high levels of Wnt activity in mesenchymal progenitors that is required for proper development of endoderm-derived organs, such as the lung.

The Importance of Wnt Signaling in Cardiovascular Development

Cardiac development is comprised of a series of morphological events tightly controlled both spatially and temporally. The molecular pathways controlling early cardiac differentiation are poorly understood, but Wnt signaling is emerging as a critical pathway for multiple aspects of early cardiovascular development. The Wnt pathway plays multiple roles in regulating cellular behavior including proliferation, differentiation, cell migration, and cell polarity. Recent data have demonstrated that Wnt activity is important for early precardiac mesoderm differentiation but must be inhibited in subsequent steps for cardiomyocyte differentiation to proceed. Given the important role that Wnt signaling plays in both the differentiation of cardiomyocytes from pluripotential stem cells and tissue regeneration in general, an increased understanding of this pathway is likely to enhance our knowledge about both cardiovascular development and reparative mechanisms.