Denise Al Alam

Fgf10 -positive cells represent a progenitor cell population during lung development and postnatally

The lung mesenchyme consists of a widely heterogeneous population of cells that play crucial roles during development and homeostasis after birth. These cells belong to myogenic, adipogenic, chondrogenic, neuronal and other lineages. Yet, no clear hierarchy for these lineages has been established. We have previously generated a novel Fgf10iCre knock-in mouse line that allows lineage tracing of Fgf10-positive cells during development and postnatally. Using these mice, we hereby demonstrate the presence of two waves of Fgf10 expression during embryonic lung development: the first wave, comprising Fgf10-positive cells residing in the submesothelial mesenchyme at early pseudoglandular stage (as well as their descendants); and the second wave, comprising Fgf10-positive cells from late pseudoglandular stage (as well as their descendants). Our lineage-tracing data reveal that the first wave contributes to the formation of parabronchial and vascular smooth muscle cells as well as lipofibroblasts at later developmental stages, whereas the second wave does not give rise to smooth muscle cells but to lipofibroblasts as well as an Nkx2.1- E-Cad- Epcam+ Pro-Spc+ lineage that requires further in-depth analysis. During alveologenesis, Fgf10-positive cells give rise to lipofibroblasts rather than alveolar myofibroblasts, and during adult life, a subpopulation of Fgf10-expressing cells represents a pool of resident mesenchymal stromal (stem) cells (MSCs) (Cd45- Cd31- Sca-1+). Taken together, we show for the first time that Fgf10-expressing cells represent a pool of mesenchymal progenitors in the embryonic and postnatal lung. Our findings suggest that Fgf10-positive cells could be useful for developing stem cell-based therapies for treating interstitial lung diseases.

Contrasting Expression of Canonical Wnt Signaling Reporters TOPGAL, BATGAL and Axin2LacZ during Murine Lung Development and Repair

Canonical Wnt signaling plays multiple roles in lung organogenesis and repair by regulating early progenitor cell fates: investigation has been enhanced by canonical Wnt reporter mice, TOPGAL, BATGAL and Axin2LacZ. Although widely used, it remains unclear whether these reporters convey the same information about canonical Wnt signaling. We therefore compared beta-galactosidase expression patterns in canonical Wnt signaling of these reporter mice in whole embryo versus isolated prenatal lungs. To determine if expression varied further during repair, we analyzed comparative pulmonary expression of beta-galactosidase after naphthalene injury. Our data show important differences between reporter mice. While TOPGAL and BATGAL lines demonstrate Wnt signaling well in early lung epithelium, BATGAL expression is markedly reduced in late embryonic and adult lungs. By contrast, Axin2LacZ expression is sustained in embryonic lung mesenchyme as well as epithelium. Three days into repair after naphthalene, BATGAL expression is induced in bronchial epithelium as well as TOPGAL expression (already strongly expressed without injury). Axin2LacZ expression is increased in bronchial epithelium of injured lungs. Interestingly, both TOPGAL and Axin2LacZ are up regulated in parabronchial smooth muscle cells during repair. Therefore the optimal choice of Wnt reporter line depends on whether up- or down-regulation of canonical Wnt signal reporting in either lung epithelium or mesenchyme is being compared.

Signaling by FGFR2b controls the regenerative capacity of adult mouse incisors

Rodent incisors regenerate throughout the lifetime of the animal owing to the presence of epithelial and mesenchymal stem cells in the proximal region of the tooth. Enamel, the hardest component of the tooth, is continuously deposited by stem cell-derived ameloblasts exclusively on the labial, or outer, surface of the tooth. The epithelial stem cells that are the ameloblast progenitors reside in structures called cervical loops at the base of the incisors. Previous studies have suggested that FGF10, acting mainly through fibroblast growth factor receptor 2b (FGFR2b), is crucial for development of the epithelial stem cell population in mouse incisors. To explore the role of FGFR2b signaling during development and adult life, we used an rtTA transactivator/tetracycline promoter approach that allows inducible and reversible attenuation of FGFR2b signaling. Downregulation of FGFR2b signaling during embryonic stages led to abnormal development of the labial cervical loop and of the inner enamel epithelial layer. In addition, postnatal attenuation of signaling resulted in impaired incisor growth, characterized by failure of enamel formation and degradation of the incisors. At a cellular level, these changes were accompanied by decreased proliferation of the transit-amplifying cells that are progenitors of the ameloblasts. Upon release of the signaling blockade, the incisors resumed growth and reformed an enamel layer, demonstrating that survival of the stem cells was not compromised by transient postnatal attenuation of FGFR2b signaling. Taken together, our results demonstrate that FGFR2b signaling regulates both the establishment of the incisor stem cell niches in the embryo and the regenerative capacity of incisors in the adult.

FGF10 controls the patterning of the tracheal cartilage rings via Shh

During embryonic development, appropriate dorsoventral patterning of the trachea leads to the formation of periodic cartilage rings from the ventral mesenchyme and continuous smooth muscle from the dorsal mesenchyme. In this work, we have investigated the role of two crucial morphogens, fibroblast growth factor 10 and sonic hedgehog, in the formation of periodically alternating cartilaginous and non-cartilaginous domains in the ventral mesenchyme. Using a combination of gain- and loss-of-function approaches for FGF10 and SHH, we demonstrate that precise spatio-temporal patterns and appropriate levels of expression of these two signaling molecules in the ventral area are crucial between embryonic day 11.5 and 13.5 for the proper patterning of the cartilage rings. We conclude that the expression level of FGF10 in the mesenchyme has to be within a critical range to allow for periodic expression of Shh in the ventral epithelium, and consequently for the correct patterning of the cartilage rings. We propose that disturbed balances of Fgf10 and Shh may explain a subset of human tracheomalacia without tracheo-esophageal fistula or tracheal atresia.

Eya1 controls cell polarity, spindle orientation, cell fate and Notch signaling in distal embryonic lung epithelium

Cell polarity, mitotic spindle orientation and asymmetric division play a crucial role in the self-renewal/differentiation of epithelial cells, yet little is known about these processes and the molecular programs that control them in embryonic lung distal epithelium. Herein, we provide the first evidence that embryonic lung distal epithelium is polarized with characteristic perpendicular cell divisions. Consistent with these findings, spindle orientation-regulatory proteins Insc, LGN (Gpsm2) and NuMA, and the cell fate determinant Numb are asymmetrically localized in embryonic lung distal epithelium. Interfering with the function of these proteins in vitro randomizes spindle orientation and changes cell fate. We further show that Eya1 protein regulates cell polarity, spindle orientation and the localization of Numb, which inhibits Notch signaling. Hence, Eya1 promotes both perpendicular division as well as Numb asymmetric segregation to one daughter in mitotic distal lung epithelium, probably by controlling aPKCζ phosphorylation. Thus, epithelial cell polarity and mitotic spindle orientation are defective after interfering with Eya1 function in vivo or in vitro. In addition, in Eya1−/− lungs, perpendicular division is not maintained and Numb is segregated to both daughter cells in mitotic epithelial cells, leading to inactivation of Notch signaling. As Notch signaling promotes progenitor cell identity at the expense of differentiated cell phenotypes, we test whether genetic activation of Notch could rescue the Eya1−/− lung phenotype, which is characterized by loss of epithelial progenitors, increased epithelial differentiation but reduced branching. Indeed, genetic activation of Notch partially rescues Eya1−/− lung epithelial defects. These findings uncover novel functions for Eya1 as a crucial regulator of the complex behavior of distal embryonic lung epithelium.

Nanostructured Assemblies for Dental Application

Millions of teeth are saved each year by root canal therapy. Although current treatment modalities offer high levels of success for many conditions, an ideal form of therapy might consist of regenerative approaches in which diseased or necrotic pulp tissues are removed and replaced with healthy pulp tissue to revitalize teeth. Melanocortin peptides (alpha-MSH) possess anti-inflammatory properties in many acute and chronic inflammatory models. Our recent studies have shown that alpha-MSH covalently coupled to poly-l-glutamic acid (PGA-alpha-MSH) retains anti-inflammatory properties on rat monocytes. This study aimed to define the effects of PGA-alpha-MSH on dental pulp fibroblasts. Lipopolysaccharide (LPS)-stimulated fibroblasts incubated with PGA-alpha-MSH showed an early time-dependent inhibition of TNF-alpha, a late induction of IL-10, and no effect on IL-8 secretion. However, in the absence of LPS, PGA-alpha-MSH induced IL-8 secretion and proliferation of pulp fibroblasts, whereas free alpha-MSH inhibited this proliferation. Thus, PGA-alpha-MSH has potential effects in promoting human pulp fibroblast adhesion and cell proliferation. It can also reduce the inflammatory state of LPS-stimulated pulp fibroblasts observed in gram-negative bacterial infections. These effects suggest a novel use of PGA-alpha-MSH as an anti-inflammatory agent in the treatment of endodontic lesions. To better understand these results, we have also used the multilayered polyelectrolyte films as a reservoir for PGA-alpha-MSH by using not only PLL (poly-l-lysine) but also the Dendri Graft poly-l-lysines (DGL(G4)) to be able to adsorb more PGA-alpha-MSH. Our results indicated clearly that, by using PGA-alpha-MSH, we increase not only the viability of cells but also the proliferation. We have also analyzed at the nanoscale by atomic force microscopy these nanostructured architectures and shown an increase of thickness and roughness in the presence of PGA-alpha-MSH incorporated into the multilayered film (PLL-PGA-alpha-MSH)(10) or (DGL(G4)-PGA-alpha-MSH)(10) in accordance with the increase of the proliferation of the cells growing on the surface of these architectures. We report here the first use of nanostructured and functionalized multilayered films containing alpha-MSH as a new active biomaterial for endodontic regeneration.

Fibroblast growth factor 10 plays a causative role in the tracheal cartilage defects in a mouse model of Apert syndrome.

Patients with Apert syndrome (AS) display a wide range of congenital malformations including tracheal stenosis, which is a disease characterized by a uniform cartilaginous sleeve in place of a normally ribbed cartilagenous trachea. We have studied the cellular and molecular basis of this phenotype in a mouse model of AS (Fgfr2c+/Δ mice), which shows ectopic expression of Fgfr2b in mesenchymal tissues. Here we report that tracheal stenosis is associated with increased proliferation of mesenchymal cells, where the expression of Fgf10 and its upstream regulators Tbx4 and Tbx5 are abnormally elevated. We show that Fgf10 has a critical inductive role in tracheal stenosis, as genetic knockdown of Fgf10 in Fgfr2c+/Δ mice rescues this phenotype. These novel findings demonstrate a regulatory role for Fgf10 in tracheal development and shed more light on the underlying cause of tracheal defects in AS.

Eyes absent 1 (Eya1) is a critical coordinator of epithelial, mesenchymal and vascular morphogenesis in the mammalian lung

The proper level of proliferation and differentiation along the proximodistal axis is crucial for lung organogenesis. Elucidation of the factors that control these processes will therefore provide important insights into embryonic lung development and regeneration. Eya1 is a transcription factor/protein phosphatase that regulates cell lineage specification and proliferation. Yet its functions during lung development are unknown. In this paper we show that Eya1(-/-) lungs are severely hypoplastic with reduced epithelial branching and increased mesenchymal cellularity. Eya1 is expressed at the distal epithelial tips of branching tubules as well as in the surrounding distal mesenchyme. Eya1(-/-) lung epithelial cells show loss of progenitor cell markers with increased expression of differentiation markers and cell cycle exit. In addition, Eya1(-/-) embryos and newborn mice exhibit severe defects in the smooth muscle component of the bronchi and major pulmonary vessels with decreased Fgf10 expression. These defects lead to rupture of the major vessels and hemorrhage into the lungs after birth. Treatment of Eya1(-/-) epithelial explants in culture with recombinant Fgf10 stimulates epithelial branching. Since Shh expression and activity are abnormally increased in Eya1(-/-) lungs, we tested whether genetically lowering Shh activity could rescue the Eya1(-/-) lung phenotype. Indeed, genetic reduction of Shh partially rescues Eya1(-/-) lung defects while restoring Fgf10 expression. This study provides the first evidence that Eya1 regulates Shh signaling in embryonic lung, thus ensuring the proper level of proliferation and differentiation along the proximodistal axis of epithelial, mesenchymal and endothelial cells. These findings uncover novel functions for Eya1 as a critical upstream coordinator of Shh-Fgf10 signaling during embryonic lung development. We conclude, therefore, that Eya1 function is critical for proper coordination of lung epithelial, mesenchymal and vascular development.

Characterization of a Novel Fibroblast Growth Factor 10 (Fgf10) Knock-In Mouse Line to Target Mesenchymal Progenitors during Embryonic Development

Fibroblast growth factor 10 (Fgf10) is a key regulator of diverse organogenetic programs during mouse development, particularly branching morphogenesis. Fgf10-null mice suffer from lung and limb agenesis as well as cecal and colonic atresia and are thus not viable. To date, the Mlcv1v-nLacZ-24 transgenic mouse strain (referred to as Fgf10LacZ), which carries a LacZ insertion 114 kb upstream of exon 1 of Fgf10 gene, has been the only strain to allow transient lineage tracing of Fgf10-positive cells. Here, we describe a novel Fgf10Cre-ERT2 knock-in line (Fgf10iCre) in which a Cre-ERT2-IRES-YFP cassette has been introduced in frame with the ATG of exon 1 of Fgf10 gene. Our studies show that Cre-ERT2 insertion disrupts Fgf10 function. However, administration of tamoxifen to Fgf10iCre; Tomatoflox double transgenic embryos or adult mice results in specific labeling of Fgf10-positive cells, which can be lineage-traced temporally and spatially. Moreover, we show that the Fgf10iCre line can be used for conditional gene inactivation in an inducible fashion during early developmental stages. We also provide evidence that transcription factors located in the first intron of Fgf10 gene are critical for maintaining Fgf10 expression over time. Thus, the Fgf10iCre line should serve as a powerful tool to explore the functions of Fgf10 in a controlled and stage-specific manner.

Six1 transcription factor is critical for coordination of epithelial, mesenchymal and vascular morphogenesis in the mammalian lung

Six1 is a member of the six-homeodomain family of transcription factors. Six1 is expressed in multiple embryonic cell types and plays important roles in proliferation, differentiation and survival of precursor cells of different organs, yet its function during lung development was hitherto unknown. Herein we show that Six1(-/-) lungs are severely hypoplastic with greatly reduced epithelial branching and increased mesenchymal cellularity. Six1 is expressed at the distal epithelial tips of branching tubules as well as in the surrounding distal mesenchyme. Six1(-/-) lung epithelial cells show increased expression of differentiation markers, but loss of progenitor cell markers. Six1 overexpression in MLE15 lung epithelial cells in vitro inhibited cell differentiation, but increases the expression of progenitor cell markers. In addition, Six1(-/-) embryos and newborn mice exhibit mesenchymal overproliferation, decreased Fgf10 expression and severe defects in the smooth muscle component of the bronchi and major pulmonary vessels. These defects lead to rupture of major vessels in mutant lungs after birth. Treatment of Six1(-/-) epithelial explants in culture with recombinant Fgf10 protein restores epithelial branching. As Shh expression is abnormally increased in Six1(-/-) lungs, we also treated mutant mesenchymal explants with recombinant Shh protein and found that these explants were competent to respond to Shh and continued to grow in culture. Furthermore, inhibition of Shh signaling with cyclopamine stimulated Six1(-/-) lungs to grow and branch in culture. This study provides the first evidence for the requirement of Six1 in coordinating Shh-Fgf10 signaling in embryonic lung to ensure proper levels of proliferation and differentiation along the proximodistal axis of epithelial, mesenchymal and endothelial cells. These findings uncover novel and essential functions for Six1 as a critical coordinator of Shh-Fgf10 signaling during embryonic lung development. We propose that Six1 is hence critical for coordination of proper lung epithelial, mesenchymal and vascular development.