An essential role for Fgfs in endodermal pouch formation influences later craniofacial skeletal patterning

Fibroblast growth factor (Fgf) proteins are important regulators of pharyngeal arch development. Analyses of Fgf8 function in chick and mouse and Fgf3 function in zebrafish have demonstrated a role for Fgfs in the differentiation and survival of postmigratory neural crest cells (NCC) that give rise to the pharyngeal skeleton. Here we describe, in zebrafish, an earlier, essential function for Fgf8 and Fgf3 in regulating the segmentation of the pharyngeal endoderm into pouches. Using time-lapse microscopy, we show that pharyngeal pouches form by the directed lateral migration of discrete clusters of endodermal cells. In animals doubly reduced for Fgf8 and Fgf3, the migration of pharyngeal endodermal cells is disorganized and pouches fail to form. Transplantation and pharmacological experiments show that Fgf8 and Fgf3 are required in the neural keel and cranial mesoderm during early somite stages to promote first pouch formation. In addition, we show that animals doubly reduced for Fgf8 and Fgf3 have severe reductions in hyoid cartilages and the more posterior branchial cartilages. By examining early pouch and later cartilage phenotypes in individual animals hypomorphic for Fgf function, we find that alterations in pouch structure correlate with later cartilage defects. We present a model in which Fgf signaling in the mesoderm and segmented hindbrain organizes the segmentation of the pharyngeal endoderm into pouches. Moreover, we argue that the Fgf-dependent morphogenesis of the pharyngeal endoderm into pouches is critical for the later patterning of pharyngeal cartilages.     

Role of endothelin in the human craniofacial morphogenesis

Human craniofacial morphogenesis is a complex biological event: it is mediated by several factors and different types tissue interaction. Recent studies on animal models have led to an improved understanding of human craniofacial malformations. In particular, the endothelins, peptides that are involved in various biological functions in many tissues and organs, have been shown to play a crucial role in the development of the first branchial-arch-derived structures in mice [Kurihara et al., Nature 368:703-710, 1994]. We previously reported the identification and localization of endothelin-1 (ET-1) and its receptors in human fetal jaw [Barni et al., Dev Biol 168:373-377, 1995]. In the present study, the gene expression of ET-1 and its receptors were demonstrated in human jaw from 11-12-week-old fetuses. By using in situ hybridization, mRNA for ET-1 was localized in the epithelial cells of the oral mucosa: mRNA for ET receptors (ETA and ETB subtypes) was expressed in the mesenchyme. In situ binding experiments confirmed the presence of ETA and ETB receptors in the cells involved in the osteogenesis of the mandible. Furthermore, ET-1 was able to stimulate thymidine uptake and the expression of the oncoprotein c-fos in the same cell types. Our results indicate that ET-1 may play a putative role in epithelium-mesenchyme interaction during human craniofacial morphogenesis. Our findings are in complete accord with those of the most recent works by Yanagisawa [Yanagisawa H et al., 1998] and Clouthier [Clouthier et al., Development 125:813-824, 1998]. They most probably confirm the primary role of ET-1 in the development of the pharyngeal arches.     

Role of essential genes in mitochondrial morphogenesis in Saccharomyces cerevisiae

Mitochondria are essential organelles of eukaryotic cells. Inheritance and maintenance of mitochondrial structure depend on cytoskeleton-mediated organelle transport and continuous membrane fusion and fission events. However, in Saccharomyces cerevisiae most of the known components involved in these processes are encoded by genes that are not essential for viability. Here we asked which essential genes are required for mitochondrial distribution and morphology. To address this question, we performed a systematic screen of a yeast strain collection harboring essential genes under control of a regulatable promoter. This library contains 768 yeast mutants and covers approximately two thirds of all essential yeast genes. A total of 119 essential genes were found to be required for maintenance of mitochondrial morphology. Among these, genes were highly enriched that encode proteins involved in ergosterol biosynthesis, mitochondrial protein import, actin-dependent transport processes, vesicular trafficking, and ubiquitin/26S proteasome-dependent protein degradation. We conclude that these cellular pathways play an important role in mitochondrial morphogenesis and inheritance.     

Misexpression of genes in brain vesicles by in ovo electroporation

Transfection to living chick embryos in ovo by electroporation has been recently developed. In this mini-review, misexpression in brain vesicles is introduced. To transfect, expression plasmid is inserted in the brain vesicle, and the square pulse of 25 V, 50 ms was charged five times. The translation product of the transfected gene is detected 2 h after electroporation, and reaches the peak at 24 h after electroporation. Transfection is so effective that this method is contributing greatly to the study of the molecular mechanisms of morphogenesis.     

NK-2 class homeobox genes and pharyngeal/oral patterning: Nkx2-3 is required for salivary gland and tooth morphogenesis

Head development in vertebrates requires reciprocal patterning interactions between cranial neural crest and the ectodermal, mesodermal and endodermal components of the branchial arches. Patterning elements within the pharyngeal endoderm and oral ectoderm appear to play defining roles in this process. Several homeobox genes of the NK-2 class (Nkx2-1, Nkx2-3, Nkx2-5 and Nkx2-6) are expressed regionally in the developing pharynx, and Nkx2-1 mutants and Nkx2-5/Nkx2-6 double mutants show loss of thyroid and distal lung progenitors, and pharyngeal cell viability, respectively. Here we examined the expression and genetic role of Nkx2-3 in pharyngeal development. Nkx2-3 was expressed in the pharyngeal floor and pouches, as well as in oral and branchial arch ectoderm. Expression persisted in the developing thyroid until birth, in mucous-forming cells of the lingual and sublingual salivary glands, and in odontogenic epithelium of the mandible. Examination of Nkx2-3 null mice revealed defects in maturation and cellular organisation of the sublingual glands. Furthermore, cusps were absent from mandibular molars and the third molar was occasionally missing. These data suggest roles for Nkx2-3 during pharyngeal organogenesis, although the considerable potential for genetic redundancy within and outside of this gene family may mask earlier functions in organ specification.     

Twist controls skeletal development and dorsoventral patterning by regulating runx2 in zebrafish

Background

Twist1a and twist1b are the principal components of twists that negatively regulate a number of cellular signaling events. Expression of runx2 and downstream targets is essential for skeletal development and ventral organizer formation and specification in early vertebrate embryos, but what controls ventral activity of maternal runx2 and how twists function in zebrafish embryogenesis still remain unclear.

Methodology/Principal Findings

By studying the loss of twist induced by injection of morpholino-oligonucleotide in zebrafish, we found that twist1a and twist1b, but not twist2 or twist3, were required for proper skeletal development and dorsoventral patterning in early embryos. Overexpression of twist1a or twist1b following mRNA injection resulted in deteriorated skeletal development and formation of typical dorsalized embryos, whereas knockdown of twist1a and twist1b led to the formation of abnormal embryos with enhanced skeletal formation and typical ventralized patterning. Overexpression of twist1a or twist1b decreased the expression of runx2b, whereas twist1a and twist1b knockdown increased runx2b expression. We have further demonstrated that phenotypes induced by twist1a and twist1b knockdown were rescued by runx2b knockdown.

Conclusions/Significance

Together, these results suggest that twist1a and twist1b control skeletal development and dorsoventral patterning by regulating runx2b in zebrafish and provide potential targets for the treatment of diseases or syndromes associated with decreased skeletal development.     

Oscillations of the snail genes in the presomitic mesoderm coordinate segmental patterning and morphogenesis in vertebrate somitogenesis

The segmented body plan of vertebrate embryos arises through segmentation of the paraxial mesoderm to form somites. The tight temporal and spatial control underlying this process of somitogenesis is regulated by the segmentation clock and the FGF signaling wavefront. Here, we report the cyclic mRNA expression of Snail 1 and Snail 2 in the mouse and chick presomitic mesoderm (PSM), respectively. Whereas Snail genes' oscillations are independent of NOTCH signaling, we show that they require WNT and FGF signaling. Overexpressing Snail 2 in the chick embryo prevents cyclic Lfng and Meso 1 expression in the PSM and disrupts somite formation. Moreover, cells mis-expressing Snail 2 fail to express Paraxis, remain mesenchymal, and are thereby inhibited from undergoing the epithelialization event that culminates in the formation of the epithelial somite. Thus, Snail genes define a class of cyclic genes that coordinate segmentation and PSM morphogenesis.     

Differential expression of orthologous Dlx genes in zebrafish and mice: implications for the evolution of the Dlx homeobox gene family

Dlx homeobox genes of vertebrates are often organised as physically linked pairs in which the two genes are transcribed convergently (tail-to-tail arrangement). Three such Dlx pairs have been found in mouse, human, and zebrafish and are thought to have originated from the duplication of an ancestral gene pair. These pairs include Dlx1/Dlx2, Dlx7/Dlx3, and Dlx6/Dlx5 (the zebrafish orthologue of Dlx5 is named dlx4). Expression patterns of physically linked Dlx genes overlap extensively. Furthermore, orthologous Dlx genes often show highly similar expression patterns. We analysed Dlx expression during the gastrula and early somitogenesis of the mouse and zebrafish. It was found that expression of the mouse Dlx6 gene takes place in the rostral ectoderm and presumptive olfactory and otic placodes with patterns similar to the previously reported expression of the physically linked Dlx5 gene. However, we observed only very weak expression of the mouse Dlx3 gene at the same stage. This contrasts with the expression of dlx genes in zebrafish where dlx3 and dlx7, but not dlx4 and dlx6 are expressed during gastrulation in the rostral ectoderm and presumptive placodes. Thus, Dlx expression patterns at early stages are better conserved between paralogous pairs of physically linked genes than between orthologous pairs. This suggests that early expression of Dlx genes existed prior to the duplications that led to the multiple pairs of physically linked genes but was differentially conserved in different paralogs in zebrafish and mice.     

Temporal requirement of Hoxa2 in cranial neural crest skeletal morphogenesis

Little is known about the spatiotemporal requirement of Hox gene patterning activity in vertebrates. In Hoxa2 mouse mutants, the hyoid skeleton is replaced by a duplicated set of mandibular and middle ear structures. Here, we show that Hoxa2 is selectively required in cranial neural crest cells (NCCs). Moreover, we used a Cre-ERT2 recombinase system to induce a temporally controlled Hoxa2 deletion in the mouse. Hoxa2 inactivation after cranial NCC migration into branchial arches resulted in homeotic transformation of hyoid into mandibular arch skeletal derivatives, reproducing the conventional Hoxa2 knockout phenotype, and induced rapid changes in Alx4, Bapx1, Six2 and Msx1 expression patterns. Thus, hyoid NCCs retain a remarkable degree of plasticity even after their migration in the arch, and require Hoxa2 as an integral component of their morphogenetic program. Moreover, subpopulations of postmigratory NCCs required Hoxa2 at discrete time points to pattern distinct derivatives. This study provides the first temporal inactivation of a vertebrate Hox gene and illustrates Hox requirement during late morphogenetic processes.     

Genetic dissection of Pitx2 in craniofacial development uncovers new functions in branchial arch morphogenesis, late aspects of tooth morphogenesis and cell migration

Pitx2, a paired-related homeobox gene that encodes multiple isoforms, is the gene mutated in the haploinsufficient Rieger Syndrome type 1 that includes dental, ocular and abdominal wall anomalies as cardinal features. Previous analysis of the craniofacial phenotype of Pitx2-null mice revealed that Pitx2 was both a positive regulator of Fgf8 and a repressor of Bmp4-signaling, suggesting that Pitx2 may function as a coordinator of craniofacial signaling pathways. We show that Pitx2 isoforms have interchangeable functions in branchial arches and that Pitx2 target pathways respond to small changes in total Pitx2 dose. Analysis of Pitx2 allelic combinations that encode varying levels of Pitx2 showed that repression of Bmp signaling requires high Pitx2 while maintenance of Fgf8 signaling requires only low Pitx2. Fate-mapping studies with a Pitx2 cre recombinase knock in allele revealed that Pitx2 daughter cells are migratory and move aberrantly in the craniofacial region of Pitx2 mutant embryos. Our data reveal that Pitx2 function depends on total Pitx2 dose and rule out the possibility that the differential sensitivity of target pathways was a consequence of isoform target specificity. Moreover, our results uncover a new function of Pitx2 in regulation of cell motility in craniofacial development.     

Multiple joint and skeletal patterning defects caused by single and double mutations in the mouse Gdf6 and Gdf5 genes

Growth/differentiation factors 5, 6, and 7 (GDF5/6/7) represent a distinct subgroup within the bone morphogenetic protein (BMP) family of secreted signaling molecules. Previous studies have shown that the Gdf5 gene is expressed in transverse stripes across developing skeletal elements and is one of the earliest known markers of joint formation during embryonic development. Although null mutations in this gene disrupt formation of some bones and joints in the skeleton, many sites are unaffected. Here, we show that the closely related family members Gdf6 and Gdf7 are expressed in different subsets of developing joints. Inactivation of the Gdf6 gene causes defects in joint, ligament, and cartilage formation at sites distinct from those seen in Gdf5 mutants, including the wrist and ankle, the middle ear, and the coronal suture between bones in the skull. Mice lacking both Gdf5 and Gdf6 show additional defects, including severe reduction or loss of some skeletal elements in the limb, additional fusions between skeletal structures, scoliosis, and altered cartilage in the intervertebral joints of the spinal column. These results show that members of the GDF5/6/7 subgroup are required for normal formation of bones and joints in the limbs, skull, and axial skeleton. The diverse effects on joint development and the different types of joints affected in the mutants suggest that members of the GDF family play a key role in establishing boundaries between many different skeletal elements during normal development. Some of the skeletal defects seen in single or double mutant mice resemble defects seen in human skeletal diseases, which suggests that these genes may be candidates that underlie some forms of carpal/tarsal coalition, conductive deafness, scoliosis, and craniosynostosis.     

GPS: Identification of disease genes by rank aggregation of multi-genomic scoring schemes

In solving the gene prioritization problem, ranking candidate genes from most to least promising is attempted before further experimental validation. Integrating the results of various data sources and methods tends to result in a better performance when solving the gene prioritization problem. Therefore, a wide range of datasets and algorithms was investigated; these included topological features of protein networks, physicochemical characteristics and blast similarity scores of protein sequences, gene ontology, biological pathways, and tissue-based data sources. The novelty of this study lies in how the best-performing methods and reliable multi-genomic data sources were applied in an efficient two-step approach. In the first step, various multi-genomic data sources and algorithms were evaluated and seven best-performing rankers were then applied to prioritize candidate genes in different ways. In the second step, global prioritization was obtained by aggregating several scoring schemes.The results showed that protein networks, functional linkage networks, gene ontology, and biological pathway data sources have a significant impact on the quality of the gene prioritization approach. The findings also demonstrated a direct relationship between the degree of genes and the ranking quality of the evaluated tools. This approach outperformed previously published algorithms (e.g., DIR, GPEC, GeneDistiller, and Endeavour) in all evaluation metrices and led to the development of GPS software. Its user-friendly interface and accuracy makes GPS a powerful tool for the identification of human disease genes. GPS is available at http://gpsranker.com and http://LBB.ut.ac.ir.     

Elevated CO2 influences the expression of floral-initiation genes in Arabidopsis thaliana

Atmospheric CO(2) concentration ([CO(2)]) is rising on a global scale and is known to affect flowering time. Elevated [CO(2)] may be as influential as temperature in determining future changes in plant developmental timing, but little is known about the molecular mechanisms that control altered flowering times at elevated [CO(2)]. Using Arabidopsis thaliana, the expression patterns were compared of floral-initiation genes between a genotype that was selected for high fitness at elevated [CO(2)] and a nonselected control genotype. The selected genotype exhibits pronounced delays in flowering time when grown at elevated [CO(2)], whereas the control genotype is unaffected by elevated [CO(2)]. Thus, this comparison provides an evolutionarily relevant system for gaining insight into the responses of plants to future increases in [CO(2)]. Evidence is provided that elevated [CO(2)] influences the expression of floral-initiation genes. In addition, it is shown that delayed flowering at elevated [CO(2)] is associated with sustained expression of the floral repressor gene, FLOWERING LOCUS C (FLC), in an elevated CO(2)-adapted genotype. Understanding the mechanisms that account for changes in plant developmental timing at elevated [CO(2)] is critical for predicting the responses of plants to a high-CO(2) world of the near future.