Molecular Control of Organogenesis, Cell Fate Specification and Cell Differentiation: Genetic and Experimental Studies in the Mouse

Deciphering the mechanisms controlling normal development sheds light onto the etiopathogenesis of congenital malformations and diseases, and knowledge of the expression patterns of proteins and/or their encoding genes is necessary to understand developmental pocesses. Good models to study developmental processes, such as morphogenesis, tissue patterning, cell fate specification and cell differentiation include developing teeth and tongue. Carbonic anhydrases (CAs) are involved in several physiological processes and diseases, yet which of these enzymes are produced, and which cells express them during odontogenesis is unknown. To fill in this knowledge gap, we used biochemical and molecular analyses in developing mouse teeth. We revealed dynamic expression patterns of eight CAs during tooth formation, and showed that CAs are not produced solely by cells involved in enamel and dentine secretion and biomineralization. Furthermore, we showed that CAXIII protein was enriched in LAMP1/2-expressing vesicles, suggesting lysosomal localization, and that CAIII expression was confined to root odontoblasts. Our data suggest developmental regulation of CA expression, and that CAs participate in several biological events inherent to tooth-forming cells (study I). The Hedgehog (Hh) and retinoic acid (RA) pathways play key roles during embryogenesis and tissue homeostasis. Both pathways are active in same or adjacent tissues. However, whether these pathways interact is largely unexplored. Furthermore, whether Sonic Hedgehog (SHH) signaling triggered by SHH, a Hh ligand, controls tongue development in vivo is unknown. To address these issues, we generated and studied mice genetically lacking SHH signaling (studies II and III). We revealed that in the developing tongue SHH abates RA activity through the RA-degrading enzymes CYP26s, and that epithelial cell fate specification is regulated by antagonistic SHH and RA activities, wherein SHH inhibits, whereas RA promotes taste placode and minor salivary gland formation. Furthermore, we showed that SHH signaling is required to prevent ectopic Merkel cell specification in the lingual epithelium (study II). We also revealed interactions between the Hedgehog and RA pathways in other embryonic structures (study III). Our findings (studies II and III) show that properly calibrated SHH and RA activities are crucial for adequate development, and are expected to be of interest, as deregulation of Hh/SHH signaling leads to congenital malformations and neoplasia

Case Report: Root resorption caused after pulp death of adjacent primary molar [version 1; referees: 2 approved]

Necrotic decayed primary molars with necrotic pulp tissues may show periapical involvement and root resorption. In this case report, a pediatric patient with a very common chief complain and clinical picture of necrotic badly decayed molar, introduced a very interesting case when radiographic investigation was performed, which showed that root resorption of the adjacent healthy molar occurred. The current report is, to the best of our knowledge, the first to report such finding in primary dentition

Relationship between asymmetric dimethylarginine plasma level and left ventricular mass in hemodialysis patients

Left ventricular hypertrophy (LVH) and left ventricular dysfunction are highly prevalent in patients with end-stage renal disease (ESRD). Several studies suggest that left ventricular mass and function is strongly modulated by the nitric oxide (NO) system. Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of endothelial-based NO synthase, is emerging as an important cardiovascular risk factor in ESRD patients. Our objective is to evaluate the relationship between plasma ADMA level and LVH among hemodialysis (HD) patients. Plasma ADMA measurements by enzyme-linked immunesorbent assay and echocardiographic evaluation were performed for 40 patients on regular HD, 20 patients with pre-dialysis chronic kidney disease, 20 hypertensive patients with left ventricular hypertrophy and normal kidney function and 20 healthy age and sex-matched subjects as a control group. Residual renal function (RRF) was measured in HD patients by urea clearance from a urine collection. Mean values of plasma ADMA level were significantly high in all patient groups when compared with the control group (P 0.05) and between ADMA and RRF in HD patients (r = -0.20, P = 0.60). It was also seen that plasma ADMA was not correlated with left ventricular mass index; however, there could be an association between ADMA level and diastolic dysfunction. The plasma ADMA level was found to be high in the three studied patient groups in comparison with the control group. HD is not an effective procedure for adequate removal of ADMA

Loss of BMP2 and BMP4 Signaling in the Dental Epithelium Causes Defective Enamel Maturation and Aberrant Development of Ameloblasts

BMP signaling is crucial for differentiation of secretory ameloblasts, the cells that secrete enamel matrix. However, whether BMP signaling is required for differentiation of maturation-stage ameloblasts (MA), which are instrumental for enamel maturation into hard tissue, is hitherto unknown. To address this, we used an in vivo genetic approach which revealed that combined deactivation of the Bmp2 and Bmp4 genes in the murine dental epithelium causes development of dysmorphic and dysfunctional MA. These fail to exhibit a ruffled apical plasma membrane and to reabsorb enamel matrix proteins, leading to enamel defects mimicking hypomaturation amelogenesis imperfecta. Furthermore, subsets of mutant MA underwent pathological single or collective cell migration away from the ameloblast layer, forming cysts and/or exuberant tumor-like and gland-like structures. Massive apoptosis in the adjacent stratum intermedium and the abnormal cell-cell contacts and cell-matrix adhesion of MA may contribute to this aberrant behavior. The mutant MA also exhibited severely diminished tissue non-specific alkaline phosphatase activity, revealing that this enzyme’s activity in MA crucially depends on BMP2 and BMP4 inputs. Our findings show that combined BMP2 and BMP4 signaling is crucial for survival of the stratum intermedium and for proper development and function of MA to ensure normal enamel maturation

Sonic Hedgehog Signaling Is Required for Cyp26 Expression during Embryonic Development.

Deciphering how signaling pathways interact during development is necessary for understanding the etiopathogenesis of congenital malformations and disease. In several embryonic structures, components of the Hedgehog and retinoic acid pathways, two potent players in development and disease are expressed and operate in the same or adjacent tissues and cells. Yet whether and, if so, how these pathways interact during organogenesis is, to a large extent, unclear. Using genetic and experimental approaches in the mouse, we show that during development of ontogenetically different organs, including the tail, genital tubercle, and secondary palate, Sonic hedgehog (SHH) loss-of-function causes anomalies phenocopying those induced by enhanced retinoic acid signaling and that SHH is required to prevent supraphysiological activation of retinoic signaling through maintenance and reinforcement of expression of the Cyp26 genes. Furthermore, in other tissues and organs, disruptions of the Hedgehog or the retinoic acid pathways during development generate similar phenotypes. These findings reveal that rigidly calibrated Hedgehog and retinoic acid activities are required for normal organogenesis and tissue patterning

Sonic Hedgehog Signaling Is Required for Cyp26 Expression during Embryonic Development

Deciphering how signaling pathways interact during development is necessary for understanding the etiopathogenesis of congenital malformations and disease. In several embryonic structures, components of the Hedgehog and retinoic acid pathways, two potent players in development and disease are expressed and operate in the same or adjacent tissues and cells. Yet whether and, if so, how these pathways interact during organogenesis is, to a large extent, unclear. Using genetic and experimental approaches in the mouse, we show that during development of ontogenetically different organs, including the tail, genital tubercle, and secondary palate, Sonic hedgehog (SHH) loss-of-function causes anomalies phenocopying those induced by enhanced retinoic acid signaling and that SHH is required to prevent supraphysiological activation of retinoic signaling through maintenance and reinforcement of expression of the Cyp26 genes. Furthermore, in other tissues and organs, disruptions of the Hedgehog or the retinoic acid pathways during development generate similar phenotypes. These findings reveal that rigidly calibrated Hedgehog and retinoic acid activities are required for normal organogenesis and tissue patterning

Cell fate specification in the lingual epithelium is controlled by antagonistic activities of Sonic hedgehog and retinoic acid

<div><p>The interaction between signaling pathways is a central question in the study of organogenesis. Using the developing murine tongue as a model, we uncovered unknown relationships between Sonic hedgehog (SHH) and retinoic acid (RA) signaling. Genetic loss of SHH signaling leads to enhanced RA activity subsequent to loss of SHH-dependent expression of <i>Cyp26a1</i> and <i>Cyp26c1</i>. This causes a cell identity switch, prompting the epithelium of the tongue to form heterotopic minor salivary glands and to overproduce oversized taste buds. At developmental stages during which <i>Wnt10b</i> expression normally ceases and <i>Shh</i> becomes confined to taste bud cells, loss of SHH inputs causes the lingual epithelium to undergo an ectopic and anachronic expression of <i>Shh</i> and <i>Wnt10b</i> in the basal layer, specifying <i>de novo</i> taste placode induction. Surprisingly, in the absence of SHH signaling, lingual epithelial cells adopted a Merkel cell fate, but this was not caused by enhanced RA signaling. We show that RA promotes, whereas SHH, acting strictly within the lingual epithelium, inhibits taste placode and lingual gland formation by thwarting RA activity. These findings reveal key functions for SHH and RA in cell fate specification in the lingual epithelium and aid in deciphering the molecular mechanisms that assign cell identity.</p></div

Early loss of SHH signaling in the tongue impinges upon growth and morphogenesis but is conducive to taste bud differentiation.

<p>(<b>A-N</b>) Tongues and parasagittal tongue sections from control and <i>ShhCreER</i>^<i>T2</i><i>/Shh</i>^<i>f</i> mutant embryos first exposed to tamoxifen at E10.5. (<b>A</b>,<b>B</b>) Anti-Sonic hedgehog-stained (SHH; dark purple) sections of E15.5 control (A) and mutant (B) tongues showing focal epithelial hyperplasia (arrow in B) and severely decreased SHH immunostaining in the mutant. (<b>C</b>,<b>D</b>) <i>Gli1 in situ</i> hybridization (brown) in sections from E13.5 control (C) and mutant (D) tongues showing severe downregulation of <i>Gli1</i> expression in the mutant. (<b>E,F</b>) E15 control (E) and mutant (F) tongues after <i>in situ</i> hybridization with a riboprobe targeting both deleted (exon2) and non-deleted (exon1) <i>Shh</i>-coding sequences (dark purple). Abnormally small mutant tongue with a bifid tip, and exhibiting oversized <i>Shh</i>+ spots (arrowheads in F). (<b>G-L</b>) Sections of E15.5 (G-I) and E17.5 (J-L) control (G,K) and mutant (H-J,L) tongues immunostained (dark purple) for keratin 8 (K8; G-I, K,L) and Rab3c (J). The insets in (K) and (L) are enlarged images of the boxed areas in (K) and (L), respectively. The control and mutant tongues show K8+ taste buds (TB) in fungiform papillae (FuP), and the mutant tongues exhibit K8+ and Rab3c+ ectopic Merkel cells (MC). (<b>M,N</b>) Tongue sections from E18 control (M) and mutant (N) embryos after K8 (green) and P2X2 (red) double staining showing innervated TBs. (<b>O</b>) RT-qPCR analysis for <i>Ptch1</i> relative to <i>Actb</i> (β-actin) in tongues from E13.5 controls (n = 6) and mutants (n = 6) first exposed to tamoxifen (TAM) at E10.5. Severely decreased <i>Ptch1</i> levels in the mutant tongues as compared to the controls (<i>P</i> = 0.0000; mean values ± SD). PD, periderm; CvP, circumvallate papilla; Ic, incisor; RP, rugae palatinae; T, tongue. Scale bars: 500 μm (E,F,K,L), 200 μm (A-D), 100 μm (G,H), 50 μm (I,J), and 25 μm (M,N).</p

Tamoxifen induction at E11.5 causes overproduction of oversized, innervated taste buds in <i>ShhCreER</i>^<i>T2</i><i>/Shh</i>^<i>f</i> mutant tongues.

<p>(<b>A-N</b>) Analyses of tongues from control and <i>ShhCreER</i>^<i>T2</i><i>/Shh</i>^<i>f</i> mutant embryos first exposed to tamoxifen at E11.5. (<b>A,B</b>) <i>In situ</i> hybridization with a riboprobe targeting <i>Shh</i> exon2 (the deleted allele) in E13.5 control (A) and mutant (B) tongues showing severely decreased <i>Shh</i> expression (purple) in the mutant tongue. (<b>C,D</b>) <i>Gli1 in situ</i> hybridization (brown) in parasagittal sections of E14.5 control (C) and mutant (D) tongues showing severe downregulation of <i>Gli1</i> expression in the mutant tongue, except in a small domain (arrows in D). (<b>E</b>) RT-qPCR analysis for <i>Ptch1</i> relative to <i>Actb</i> (β-actin) in tongues from E13.5 controls (n = 9) and mutants (n = 9). Severely decreased <i>Ptch1</i> levels in the mutant tongues as compared to the controls (<i>P</i> = 0.0000; mean values ± SD). (<b>F,G</b>) E15.5 control (F) and mutant (G) tongues after <i>in situ</i> hybridization with a riboprobe targeting both deleted (exon2) and non-deleted (exon1) <i>Shh</i>-coding sequences (brown) showing overproduction of oversized <i>Shh</i>-expressing taste buds in the mutant tongue. (<b>H-L</b>) Immunostaining (dark purple) for Keratin 8 (K8; H-J) and SOX2 (K,L) in parasagittal sections of E18.5 (H-J) and E16 (K,L) control (H,K) and mutant (I,J,L) tongues. The insets in (H) and (I) are enlarged images of the boxed areas in (H) and (I), respectively. The mutant tongues exhibit crowded and oversized taste buds (TB; I,L) and a fungiform papilla (FuP) abnormally harboring three TBs (J). Artefact (asterisk in I). (<b>M,N</b>) Parasagittal sections of E18 control (M) and mutant (N) tongues after K8 (green) and P2X2 (red) double staining showing innervated TBs. CvP, circumvallate papilla; T, tongue. Scale bars: 500 μm (A,B,F,G,H,I), 200 μm (C,D), 50 μm (J-L), and 25μm (M,N).</p