New directions in craniofacial morphogenesis

The vertebrate head is an extremely complicated structure: development of the head requires tissue-tissue interactions between derivates of all the germ layers and coordinated morphogenetic movements in three dimensions. In this review, we highlight a number of recent embryological studies, using chicken, frog, zebrafish and mouse, which have identified crucial signaling centers in the embryonic face. These studies demonstrate how small variations in growth factor signaling can lead to a diversity of phenotypic outcomes. We also discuss novel genetic studies, in human, mouse and zebrafish, which describe cell biological mechanisms fundamental to the growth and morphogenesis of the craniofacial skeleton. Together, these findings underscore the complex interactions leading to species-specific morphology. These and future studies will improve our understanding of the genetic and environmental influences underlying human craniofacial anomalies.     

The Tumor Suppressor pVHL Down-regulates Never-in-Mitosis A-related Kinase 8 via Hypoxia-inducible Factors to Maintain Cilia in Human Renal Cancer Cells

NEK8 (never in mitosis gene A (NIMA)-related kinase 8) is involved in cytoskeleton, cilia, and DNA damage response/repair. Abnormal expression and/or dysfunction of NEK8 are related to cancer development and progression. However, the mechanisms that regulate NEK8 are not well declared. We demonstrated here that pVHL may be involved in regulating NEK8. We found that CAK-I cells with wild-type vhl expressed a lower level of NEK8 than the cells loss of vhl, such as 786-O, 769-P, and A-498 cells. Moreover, pVHL overexpression down-regulated the NEK8 protein in 786-O cells, whereas pVHL knockdown up-regulated NEK8 in CAK-I cells. In addition, we found that the positive hypoxia response elements (HREs) are located in the promoter of the nek8 sequence and hypoxia could induce nek8 expression in different cell types. Consistent with this, down-regulation of hypoxia-inducible factors α (HIF-1α or HIF-2α) by isoform-specific siRNA reduced the ability of hypoxia inducing nek8 expression. In vivo, NEK8 and HIF-1α expression were increased in kidneys of rats subjected to an experimental hypoxia model of ischemia and reperfusion. Furthermore, NEK8 siRNA transfection significantly blocked pVHL-knockdown-induced cilia disassembling, through impairing the pVHL-knockdown-up-regulated NEK8 expression. These results support that nek8 may be a novel hypoxia-inducible gene. In conclusion, our findings show that nek8 may be a new HIF target gene and pVHL can down-regulate NEK8 via HIFs to maintain the primary cilia structure in human renal cancer cells.     

Cilia on bovine mammary epithelium: ultrastructural observations

Ultrastructural examination of bovine mammary tissues revealed the presence of 9+0 or primary cilia protruding from surfaces of alveolar epithelial and myoepithelial cells. Cilia of epithelial cells protruded approximately 1200 nm into lumina of alveoli and arose from a basal body centriole, the associated centriole of the diplosome, and an accessory rootlet system. Cilia on epithelial cells were more frequently observed than cilia on myoepithelial cells. Occasional cilia made contact with macrophages in the alveolar lumen. The structures were more commonly found in tissues from nonlactating cows, and most were observed in the ventral portion of the mammary gland.     

Neuronal organization of the ascidian (Urochordata) branchial basket revealed by cholinesterase activity

Summary Innervation of the ascidian branchial basket and other structures is demonstrated by staining for cholinesterase. Cholinesterase activity is not restricted to synaptic sites but is present throughout the neurons. Primary and secondary axonal bundles form a bilaterally symmetric innervation pattern around the large dorsal visceral nerve. These bundles continue to split into progressively smaller bundles as they course throughout the basket. Axons are suspended in a fibrous matrix and run within the blood sinuses on the atrial side of the basket. Stigmatal ciliated cells of the branchial basket are innervated by highly branched distal portions of neurons, whose cell bodies are located in the ganglion. Synaptic boutons, containing electron-lucent vesicles, are found at nearly all stigmatal ciliated cells. NiCl₂backfills of the visceral nerve reveal a distinct population of central neurons, some of which presumably control ciliary arrest.     

Gap junctions suggest epithelial conduction within the comb plates of the ctenophore Pleurobrachia bachei

Intercellular gap junctions occur between the ciliated cells that make up the comb plates of the ctenophore Pleurobrachia. Similar junctions are found within the ciliated grooves which run from the apical organ to the first plate of each comb row, as well as throughout the endoderm of the meridional canals. Gap junctions were not found in the ectodermal tissue between the comb rows. The distribution of junctions suggests that excitation conduction within the ciliated grooves, comb plates and meridional canal endoderm may be epithelial.     

Studies on the size,location and turnover of calcium pools accessible to growing Tetrahymena cells

Tetrahymena pyriformis cells in the logarithmic phase of growth accumulate 2.5–3.75 times as much calcium per unit volume as is present in the growth medium. It appears that most of this calcium is stored in a non-ionic form, with approximately 30% existing in the cilia, near its site of action in effecting ciliary reversal. The exchange of extracellular ⁴⁵Ca²⁺ with the major internal pools is extremely rapid, exhibiting a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of less than 0.5 h. Sites located on the cilia are responsible for 35–50% of Ca²⁺ influx, with the remainder entering through other positions on the cell surface.     

Ciliary function of the frog oro-pharyngeal epithelium

Summary The palate epithelium of the frog was examined by scanning electron microscopy, light microscopy and high speed cine micrography. The cilia remain stationary for much of the time in the end-of-effective stroke position. Each beat cycle begins with a forwardly-directed recovery stroke lasting about 60 ms, followed by an effective stroke towards the oesophagus lasting about 12 ms. Activity can often be correlated with the presence of mucus, which is carried as strands on the tips of the ciliary effective strokes whilst the recovery strokes move beneath the mucus. Coordination of ciliary activity was very variable; local antiplectic metachrony of the recovery strokes could almost always be seen, and on very active epithelia effective strokes were associated with approximately diaplectic waves (either to left or right), but any particular pattern of coordinated activity was transient and quickly transformed to another pattern. Beating and coordination of these short cilia were compared with those of cilia propelling water.     

Low concentrations of dimethyl sulphoxide accelerate conjugation and incorporation of glycine and glucosamine intetrahymena

Summary Dimethyl sulphoxide at relatively low comentrations, 0.01 to 1 mM, enhanced the conjugation and cell-to-cell adhesion of complementary strains of matingTetrahymena thermophila. The time required to form stable conjugates was reduced by dimethyl sulphoxide. This chemical stimulated the uptake of glycine and glucosamine from the suspending media. Incorporation of 2-¹⁴C-glycine and 6-³H-D-glucosamine into protein and glycoprotein was enhanced in whole cells, surface membrane and cilia. Incorporation of glucosamine into the microsomal fraction was increased in the dimethyl sulphoxidetreated cells while there was little change in glycine incorporation. There were no detectable changes in glycine and glucosamine incorporation into the nuclear fractions isolated from conjugatingTetrahymena exposed to dimethyl sulphoxide.     

Ultrastructure of the grasshopper proximal femoral chordotonal organ

Summary Calpasoma dactyloptera, a tentacled form of minute, freshwater coelenterate, has been investigated by light and electron microscopy and time-lapse cinematography. Each tentacle consists of a protrusion from a single ectodermal epithelial cell termed a tentaculocyte. hin tentaculocyte vesicles which represent invaginations of the plasma membrane. A cnidocil protrudes into the external medium. The bottom of each nematocyte is elongated as a stalk which extends to the tentacle base, coursing through tubular membrane lined channels within the tentaculocyte. A network of fibers and microtubules, originating in the cnidocil, extends to the base of the nematocyte stalk.Supported by PHS Research Career Development Award 1-K04-GM42595 and NSF Research Grant GB 29284.     

Sequence and Properties of Cagein,a Coiled-Coil Scaffold Protein Linking Basal Bodies in the Polykinetids of the Ciliate Euplotes aediculatus

In the hypotrich ciliate Euplotes, many individual basal bodies are grouped together in tightly packed clusters, forming ventral polykinetids. These groups of basal bodies (which produce compound ciliary organelles such as cirri and oral membranelles) are cross-linked into ordered arrays by scaffold structures known as “basal-body cages.” The major protein comprising Euplotes cages has been previously identified and termed “cagein.” Screening a E. aediculatus cDNA expression library with anti-cagein antisera identified a DNA insert containing most of a putative cagein gene; standard PCR techniques were used to complete the sequence. Probes designed from this gene identified a macronuclear “nanochromosome” of ca. 1.5 kb in Southern blots against whole-cell DNA. The protein derived from this sequence (463 residues) is predicted to be hydrophilic and highly charged; however, the native cage structures are highly resistant to salt/detergent extraction. This insolubility could be explained by the coiled-coil regions predicted to extend over much of the length of the derived cagein polypeptide. One frameshift sequence is found within the gene, as well as a short intron. BLAST searches find many ciliates with evident homologues to cagein within their derived genomic sequences.