Expression of five frizzleds during zebrafish craniofacial development

Wnt/Planar Cell Polarity (PCP) signaling is critical for proper animal development. While initially identified in Drosophila, this pathway is also essential for the proper development of vertebrates. Zebrafish mutants, defective in the Wnt/PCP pathway, frequently display defects in convergence and extension gastrulation movements and additional later abnormalities including problems with craniofacial cartilage morphogenesis. Although multiple Frizzled (Fzd) homologues, Wnt receptors, were identified in zebrafish, it is unknown which Fzd plays a role in shaping the early larvae head skeleton. In an effort to determine which Frizzleds are involved in this process, we analyzed the expression of five zebrafish frizzled homologues fzd2, 6, 7a, 7b, and 8a from 2–4 days post-fertilization (dpf). During the analyzed developmental time points fzd2 and fzd6 are broadly expressed throughout the head, while the expression of fzd7a, 7b and 8a is much more restricted. Closer examination revealed that fzd7b is expressed in the neural crest and the mesodermal core of the pharyngeal arches and in the chondrocytes of newly stacked craniofacial cartilage elements. However, fzd7a is only expressed in the neural crest of the pharyngeal arches and fzd8a is expressed in the pharyngeal endoderm.     

Dynamic assembly of tight junction-associated proteins ZO-1, ZO-2, ZO-3 and occludin during mouse tooth development

Tight junctions might play a role during tissue morphogenesis and cell differentiation. In order to address these questions, we have studied the distribution pattern of the tight junction-associated proteins ZO-1, ZO-2, ZO-3 and occludin in the developing mouse tooth as a model. A specific temporal and spatial distribution of tight junction-associated proteins during tooth development was observed. ZO-1 appeared discontinuously in the cell membrane of enamel organ and dental mesenchyme cells. However, endothelial cells of the dental mesenchyme capillaries displayed a continuous fluorescence at the cell membrane. Inner dental epithelium first showed an evident signal for ZO-1 at the basal pole of the cells at bud/cap stage, but ZO-1 was accumulated at the basal and apical pole of preameloblast/ameloblasts at late bell stage. Surprisingly, in the incisor ZO-1 decreased as the inner dental epithelium differentiated, and was re-expressed in secretory and mature ameloblasts. On the contrary, ZO-2 was confined to continuous cell-cell contacts of the enamel organ in both molars and incisors. The lateral cell membrane of inner dental epithelial cells was specifically ZO-2 labeled. However, ZO-3 was expressed in oral epithelium whereas dental embryo tissues were negative. In addition, occludin was hardly detected in dental tissues at the early stage of tooth development, but was distributed continuously at the cell membrane of endothelial cells of ED19.5 dental mesenchyme. In incisors, occludin was detected at the cell membrane of the secretory pole of ameloblasts. The occurrence and relation during tooth development of tight junction proteins ZO-1, ZO-2 and occludin, but not ZO-3, suggests a combinatory assembly in tooth morphogenesis and cell differentiation.     

Expression patterns of group-I aristaless-related genes during craniofacial and limb development

Aristaless-related proteins are structurally defined by the presence of a paired-type homeodomain and an additional conserved domain, known as aristaless domain or OAR-domain. These proteins can be further categorized in three groups (Int. J. Dev. Biol., 43 (1999) 651). Group-I aristaless-related genes are linked to functions in the development of the craniofacial and appendicular skeleton and are expressed predominantly in the mesenchyme in stages from gastrulation through at least mid-gestation (Mech. Dev., 48 (1994) 245; Mech. Dev., 52 (1995) 51; Development, 124 (1997) 3999; Dev. Biol., 199 (1998) 11; Development, 126 (1999) 495). In view of the highly redundant character of the functions of these genes in patterning craniofacial and limb structures, we found it important to directly compare their expression patterns at critical stages of craniofacial and limb development.     

Differential regulation of TIMP-1, -2, and -3 mRNA and protein expressions during mouse incisor development

Tissue inhibitors of metalloproteinases (TIMPs) possess multiple functions, in addition to their matrix metalloproteinase (MMP) inhibitory activity. The continuously growing incisor of mouse possesses a stem cell compartment at the apical end of the epithelium (the apical loop) and thus provides an excellent tool to analyze the mechanisms of organogenesis and cytodifferentiation. To understand the functions of TIMPs in tooth development, we have analyzed the gene expression and protein localization of TIMP-1, -2, and -3 during mouse incisor development, from embryonic day 13 (E13) to postnatal day 3 (P3). TIMP-1 was present on the basement membrane during early developmental stages. At P2, TIMP-1 was strongly detected along the apical loop, transiently disappeared from the basement membrane in the cytodifferentiation zone, and later reappeared at the distal end of functional ameloblasts. Expression of TIMP-2 protein was restricted to the outer part of the apical loop throughout the examined stages. At P2, TIMP-2 was present on the basement membrane at the outer part of the apical loop. The dental follicle also expressed Timp-2, and the corresponding protein was abundant within the extracellular matrix. Timp-3 mRNA was highly expressed in the mesenchyme surrounding the apical loop. During matrix formation, Timp-3 was expressed by subodontoblasts, and the protein was detected in this layer and between odontoblasts. Distinct temporal and spatial expression patterns of TIMPs suggest divergent functions of these factors in incisor organogenesis. This work was supported by INSERM, CNRS, ARC, French Ministry of Research (ACI), Japanese Ministry of Education, Culture, Sports, Science, and Technology, and Niigata University Research Projects.     

Differential expression of transforming growth factor-beta1, connective tissue growth factor,phosphorylated-SMAD2/3 and phosphorylated-ERK1/2 during mouse tooth development

Connective tissue growth factor (CTGF) is a downstream mediator of transforming growth factor-beta 1 (TGF-β₁) and TGF-β₁-induced CTGF expression is regulated through SMAD and mitogen-activated protein kinase (MAPK) signaling pathways. The fine modulation of TGF-β₁ signaling is very important to the process of tooth development. However, little is known about the localization of CTGF, MAPK and SMAD in the context of TGF-β₁ signaling during odontogenesis. Hence, we aimed to investigate the expression of TGF-β₁, CTGF, phosphorylated-SMAD2/3 (p-SMAD2/3) and phosphorylated-ERK1/2 (p-ERK1/2). ICR mice heads of embryonic (E) day 13.5, E14.5, E16.5, postnatal (PN) day 0.5 and PN3.5 were processed for immunohistochemistry. Results revealed that at E13.5, TGF-β₁ and CTGF were strongly expressed in dental epithelium (DE) and dental mesenchyme (DM), while p-SMAD2/3 was intensely expressed in the internal side of DE. p-ERK1/2 was not present in DE or DM. At E14.5 and E16.5, strong staining for TGF-β₁ and CTGF was detected in enamel knot (EK) and dental papilla (DPL). DPL was intensely stained for p-ERK1/2 but negatively stained for p-SMAD2/3. There was no staining for p-SMAD2/3 and p-ERK1/2 in EK. At PN0.5 and PN3.5, moderate to intense staining for TGF-β₁ and CTGF was evident in preameloblasts (PA), secretary ameloblasts (SA) and dental pulp (DP). p-SMAD2/3 was strongly expressed in SA and DP but sparsely localized in PA. p-ERK1/2 was intensely expressed in DP, although negative staining was observed in PA and SA. These data demonstrate that TGF-β₁ and CTGF show an identical expression pattern, while p-SMAD2/3 and p-ERK1/2 exhibit differential expression, and indicate that p-SMAD2/3 and p-ERK1/2 might play a regulatory role in TGF-β₁ induced CTGF expression during tooth development.     

Cbf beta regulates Runx2 function isoform-dependently in postnatal bone development

Runx2 and Cbfbeta are essential for skeletal development during the embryonic stage. Runx2 has two isoforms with different N-termini. We examined the functions of the Runx2 isoforms and Cbfbeta in postnatal bone development. On luciferase and electrophoretic mobility shift assays, Runx2-I was less active than Runx2-II in the absence of Cbfb, but the two Runx2 isoforms had similar activity levels in the presence of Cbfb. We generated Runx2-I transgenic mice under the control of Col1a1 promoter and Runx2-I/Cbfb and Runx2-II/Cbfb double transgenic mice. Runx2-I transgenic mice showed less severe osteopenia and fragility than Runx2-II transgenic mice due to milder inhibition of both osteoblast maturation and transition to osteocytes, even though the former mice showed higher transgene expression. However, Runx2-I/Cbfb and Runx2-II/Cbfb double transgenic mice had enhanced inhibition of osteoblast maturation, resulting in similar severity of osteopenia and fragility, although the latter mice had less osteocytes. These findings indicate that (1) Runx2-II more strongly inhibits osteoblast maturation and transition to osteocytes than Runx2-I; (2) Cbfbeta regulates Runx2 function isoform-dependently; and (3) Runx2-I activity is highly dependent on Cbfbeta. These findings demonstrate that Runx2 isoforms exert their functions through at least partly different mechanisms and Cbfbeta regulates bone development by regulating Runx2 function isoform-dependently.     

Expression of rat, renal NHE2 and NHE3 during postnatal development

In apical membrane vesicles from beef tracheal epithelia expressing up to 30% of the proteins as functional cystic fibrosis transmembrane conductance regulator (CFTR)-- i.e. a voltage-independent and PKA-sensitive 36Cl- flux--an ATPase activity, different from P, F0F1 and V types, was reproducibly detected. Its specific activity averaged 20 micromol Pi h(-1) mg(-1) with an apparent affinity for ATP of 530 +/- 30 microM. Its possible involvement in CFTR functions was supported by (1) the linear relationship between the ATPase activity and the magnitude of 36Cl- fluxes (turnover rate: 3 ATP hydrolyzed per CFTR per second), (2) the same rank of potency of ATP, ITP, GTP, UTP and CTP to be hydrolyzed and to open CFTR chloride channels, (3) the similar and parallel inhibition of the ATPase and CFTR Cl- fluxes by NS004 (IC50: 60 microM) and (4) the potency of anti-R domain antibodies to increase by 18% the ATPase activity.     

Expression of Wnt signalling pathway genes during tooth development.

We have carried out comparative in situ hybridisation analysis of six Wnt genes Wnts-3, -4, -5a, -6, -7b, and 10b together with Wnt receptor MFz6 and receptor agonist/antagonists MFrzb1 and Mfrp2 during murine odontogenesis from the earliest formation of the epithelial thickening to the early bell stage. Expression of Wnt-4, Wnt-6, and one Wnt receptor MFz6 was observed in the facial, oral and dental epithelium. Wnt10b was localised specifically to the presumptive dental epithelium. Wnts-3 and -7b were expressed in oral epithelium but showed no expression in the presumptive dental epithelium. Wnt-3 also showed no expression in the epithelial cells of the molar bud stage tooth germs, but showed restricted expression in the enamel knots which are signalling centres believed to be involved in regulating tooth shape. Wnts -6, -10b and MFz6 were also detected in the primary and secondary enamel knots. Wnt-5a and agonist/antagonists MFrzb1 and Mfrp2 were expressed in a graded proximo-distal (P-D) manner in mesenchymal cells during the early stages of tooth development with no overlying expression in the oral or dental epithelium. Wnt-5a and MFrzb1 show strong expression in the dental papilla mesenchyme.