Proximal-distal Polarity Research Articles

Loss of dlx5a/dlx6a Locus Alters Non-Canonical Wnt Signaling and Meckel's Cartilage Morphology.

The dlx genes encode transcription factors that establish a proximal-distal polarity within neural crest cells to bestow a regional identity during craniofacial development. The expression regions of dlx paralogs are overlapping yet distinct within the zebrafish pharyngeal arches and may also be involved in progressive morphologic changes and organization of chondrocytes of the face. However, how each dlx paralog of dlx1a, dlx2a, dlx5a and dlx6a affects craniofacial development is still largely unknown. We report here that the average lengths of the Meckel's, palatoquadrate and ceratohyal cartilages in different dlx mutants were altered. Mutants for dlx5a-/- and dlx5i6-/-, where the entire dlx5a/dlx6a locus was deleted, have the shortest lengths for all three structures at 5 days post fertilization (dpf). This phenotype was also observed in 14 dpf larvae. Loss of dlx5i6 also resulted in increased proliferation of neural crest cells and expression of chondrogenic markers. Additionally, altered expression and function of non-canonical Wnt signaling were observed in these mutants suggesting a novel interaction between dlx5i6 locus and non-canonical Wnt pathway regulating ventral cartilage morphogenesis.

Structures of SAS-6 coiled coil hold implications for the polarity of the centriolar cartwheel

SummaryCentrioles are eukaryotic organelles that template the formation of cilia and flagella, as well as organize the microtubule network and the mitotic spindle in animal cells. Centrioles have proximal-distal polarity and a 9-fold radial symmetry imparted by a likewise symmetrical central scaffold, the cartwheel. The spindle assembly abnormal protein 6 (SAS-6) self-assembles into 9-fold radially symmetric ring-shaped oligomers that stack via an unknown mechanism to form the cartwheel. Here, we uncover a homo-oligomerization interaction mediated by the coiled-coil domain of SAS-6. Crystallographic structures of Chlamydomonas reinhardtii SAS-6 coiled-coil complexes suggest this interaction is asymmetric, thereby imparting polarity to the cartwheel. Using a cryoelectron microscopy (cryo-EM) reconstitution assay, we demonstrate that amino acid substitutions disrupting this asymmetric association also impair SAS-6 ring stacking. Our work raises the possibility that the asymmetric interaction inherent to SAS-6 coiled-coil provides a polar element for cartwheel assembly, which may assist the establishment of the centriolar proximal-distal axis.

Architecture and sedimentary evolution of the southwestern Gissar carbonate platform (Uzbekistan) during the Middle‒Late Jurassic

Abstract Sedimentological investigations have been carried out in the southwestern part of the Gissar Range (Uzbekistan) in order to document the stratigraphic evolution of a vast Middle–Upper Jurassic carbonate platform developed on the northern margin of the Amu Darya Basin. Two major sequences are distinguished, based on their contrasting facies pattern, platform configuration and carbon isotope signature: (1) a Callovian Sequence and (2) an Upper Jurassic Sequence, potentially Oxfordian to Kimmeridgian in age. A carbonate ramp with a double proximal-distal polarity developed during the Callovian Sequence. Oo-bioclastic grainstones were produced in two permanently agitated shallow water areas developing to the north and to the south. Patch and pinnacle reefs mainly built up close to the storm-wave base, passing distally to pellet- and foraminiferal-oncoid-rich mid-to outer-ramp deposits. The top of the Callovian Sequence is a regional discontinuity that could have formed by subaerial exposure of the platform. This probable hiatus could record a major climatic change associated to the Middle‒Late Jurassic transition. The Upper Jurassic Sequence consists of metre-thick alternations of pellet/algal-pack- to grainstones, microbial oncoid floatstones, ooid grainstones, and mudstones, which have likely accumulated in a vast lagoon protected by a large-scale barrier reef observed south of the study area. Gypsum and anhydrite content gradually increased towards the top of the Upper Jurassic Sequence, ending up with a thick chicken-wire gypsum accumulation recording the progradation of a large-scale sabkha over the lagoon. This phenomenon is related to a progressive aridification and restriction of the Amu Darya Basin that could have begun during the Middle/Late Oxfordian and reached its climax during the Tithonian with massive halite deposition. Comparable events occurred in Jurassic platforms located along the Tethyan margins, pointing to regional and global controls on sedimentation.

Basal body structure in Trichonympha.

Trichonympha is a symbiotic flagellate of many species of termites and of the wood-feeding cockroach. Remarkably, this unicellular organism harbors up to over ten thousand flagella on its surface, which serve to propel it through the viscous environment of the host hindgut. In the 1960s, analysis of resin-embedded Trichonympha samples by electron microscopy revealed that the basal bodies that give rise to these flagella are exceptionally long, with a proximal, cartwheel-bearing, region some 50 times longer than that of regular centrioles. In recent years, this salient feature has prompted the analysis of the 3D architecture of Trichonympha basal bodies in the native state using cryo-electron tomography. The resulting ~40 Å resolution map of the basal body proximal region revealed a number of novel features that may be conserved in centrioles of other systems. These include proximal–distal polarity of the pinhead structure that links the cartwheel to centriolar microtubules, as well as of the linker between the A and the C microtubules. Moreover, this work demonstrated that the cartwheel is made of stacked ring-like structures that likely each comprise 18 molecules of SAS-6 proteins.

Nephrons require Rho-kinase for proximal-distal polarity development.

Epithelial tubules must have the right length and pattern for proper function. In the nephron, planar cell polarity controls elongation along the proximal-distal axis. As the tubule lengthens, specialized segments (proximal, distal etc.) begin to differentiate along it. Other epithelia need Rho-kinase for planar cell polarity but it is not known whether Rho-kinase is involved in this way in the nephron. We show that Rho-kinase is essential for the morphogenesis of nephrons, specifically for correct cell orientation and volume. We use fluorescent reporter-models and progenitor-specific markers to demonstrate that inhibition of Rho-kinase prevents proper proximal-distal axis formation, causes segments to develop abnormally, and progenitor-cell segregation to fail. Our data demonstrate the importance of Rho-kinase in normal nephron tubulogenesis and patterning.

DSH-2 regulates asymmetric cell division in the early C. elegans somatic gonad

Like other organs, the C. elegans gonad develops from a simple primordium that must undergo axial patterning to generate correct adult morphology. Proximal/distal (PD) polarity in the C. elegans gonad is established early during gonadogenesis by the somatic gonad precursor cells, Z1 and Z4. Z1 and Z4 each divide asymmetrically to generate one daughter with a proximal fate and one with a distal fate. PD polarity of the Z1/Z4 lineages requires the activity of a Wnt pathway that activates the TCF/LEF homolog pop-1. How the gonadal pathway controlling pop-1 is regulated by upstream factors has been unclear, as neither Wnt nor Dishevelled (Dsh) proteins have been shown to be required. Here we show that the C. elegans dsh homolog dsh-2 controls gonadal polarity. As in pop-1 mutants, dsh-2 hermaphrodites have Z1 and Z4 lineage defects indicative of defective PD polarity and are missing gonadal arms. Males have an elongated but disorganized gonad, also with lineage defects. DSH-2 protein is expressed in the Z1/Z4 gonadal precursor cells. Asymmetric distribution of nuclear GFP::POP-1 in Z1 and Z4 daughter cells is reversed in dsh-2 mutants, with higher levels in distal than proximal daughters. dsh-2 and the frizzled receptor homolog lin-17 have a strong genetic interaction, suggesting that they act in a common pathway. We suggest that DSH-2 functions as an upstream regulator of POP-1 in the somatic gonad to control asymmetric cell division, thereby establishing proximal-distal polarity of the developing organ.

POP-1 controls axis formation during early gonadogenesis in C. elegans.

The shape and polarity of the C. elegans gonad is defined during early gonadogenesis by two somatic gonadal precursor cells, Z1 and Z4, and their descendants. Z1 and Z4 divide asymmetrically to establish the proximal-distal axes of the gonad and to generate regulatory leader cells that control organ shape. In this paper, we report that pop-1, the C. elegans TCF/LEF-1 transcription factor, controls the first Z1/Z4 asymmetric division and hence controls proximal-distal axis formation. We have identified two pop-1(Sys) alleles (for symmetrical sisters) that render the Z1/Z4 divisions symmetrical. The pop-1(q645) allele is fully penetrant for the Sys gonadogenesis defect in hermaphrodites, but affects male gonads weakly; pop-1(q645) alters a conserved amino acid in the beta-catenin binding domain. The pop-1(q624) allele is weakly penetrant for multiple defects and appears to be a partial loss-of-function mutation; pop-1(q624) alters a conserved amino acid in the HMG-box DNA binding domain. Zygotic pop-1(RNAi) confirms the role of pop-1 in Z1/Z4 asymmetry and reveals additional roles of pop-1, including one in leader cell migration. Two other Wnt pathway regulators, wrm-1 and lit-1, have the same effect as pop-1 on Z1/Z4 asymmetry. Therefore, wrm-1 and lit-1 are required for pop-1 function, rather than opposing it as observed in the early embryo. We conclude that POP-1 controls the Z1/Z4 asymmetric division and thereby establishes the proximal-distal axes of the gonad. This control over proximal-distal polarity extends our view of Wnt signaling in C. elegans, which had previously been known to control anterior-posterior polarities.

Nodal signalling in the epiblast patterns the early mouse embryo.

Shortly after implantation the mouse embryo comprises three tissue layers. The founder tissue of the embryo proper, the epiblast, forms a radially symmetric cup of epithelial cells that grows in close apposition to the extra-embryonic ectoderm and the visceral endoderm. This simple cylindrical structure exhibits a distinct molecular pattern along its proximal-distal axis. The anterior-posterior axis of the embryo is positioned later by coordinated cell movements that rotate the pre-existing proximal-distal axis. The transforming growth factor-beta family member Nodal is known to be required for formation of the anterior-posterior axis. Here we show that signals from the epiblast are responsible for the initiation of proximal-distal polarity. Nodal acts to promote posterior cell fates in the epiblast and to maintain molecular pattern in the adjacent extra-embryonic ectoderm. Both of these functions are independent of Smad2. Moreover, Nodal signals from the epiblast also pattern the visceral endoderm by activating the Smad2-dependent pathway required for specification of anterior identity in overlying epiblast cells. Our experiments show that proximal-distal and subsequent anterior-posterior polarity of the pregastrulation embryo result from reciprocal cell-cell interactions between the epiblast and the two extra-embryonic tissues.

Quantitative freeze-fracture analysis of the frog neuromuscular junction synapse--II. Proximal-distal measurements.

Neurotransmitter release from different parts of frog motor nerve terminals is often non-uniform. There is a decrease in release efficacy from the distal regions of frog motor nerve terminal branches. Since release is thought to occur near the double arrays of large intramembranous particles that constitute the pre-synaptic active zones (AZs), we have examined quantitatively the proximal-distal distribution of AZ structure, using a novel freeze-fracture technique that produces replicas of large fractions of terminals, including the region of nerve entry. This enables us to know the proximal distal orientation of each branch. From 23 end-plates we have obtained fractures of 72 branches. For 27 of these branches we have obtained continuous fractures both greater than 25 microm in length and with sufficient information to determine their proximal distal polarity. Only a few of these branches showed a marked distal decrease in AZ length/unit length of terminal, while several junctions had short regions (5-10 microm), either proximally or distally, that exhibited amounts of AZ that were substantially greater or smaller than the mean value for that terminal branch. The terminal area, post-synaptic gutter width and nerve terminal width all exhibit some distal decline concomitant with the distal tapering of nerve terminal branches. AZ length tends to have the least decline compared to the other parameters. Thus, the vast majority of frog motor nerve terminal branches do not display a significant proximal-distal gradient in the amount of AZ structure / microm terminal length. The present data do not provide an obvious ultrastructural correlate for the distal decline in transmitter release that some authors have observed.

Tissue polarity genes of Drosophila regulate the subcellular location for prehair initiation in pupal wing cells.

The Drosophila wing is decorated with a regular array of distally pointing hairs. In the pupal wing, the hairs are formed from micro-villus like prehairs that contain large bundles of actin filaments. The distal orientation of the actin bundles reveals the proximal-distal polarity within the pupal wing epithelium. We have used F-actin staining to examine early stages of prehair development in both wild-type and mutant pupal wings. We have found a striking correlation between hair polarity and the subcellular location for assembly of the prehair. In a wild-type wing, all of the distally pointing hairs are derived from prehairs that are formed at the distal vertex of the hexagonally shaped pupal wing cells. Mutations in six tissue polarity genes result in abnormal hair polarity on the adult wing, and all also alter the subcellular location for prehair initiation. Based on their cellular phenotypes, we can place these six genes into three phenotypic groups. Double mutant analysis indicates that these phenotypic groups correspond to epistasis groups. This suggests that the tissue polarity genes function in or on a pathway that controls hair polarity by regulating the subcellular location for prehair formation.

Fine structure of the protonephridial system in Planaria. I. Flame cells.

Distal to the flame cells, the protonephridial tubules of fresh-water planarians are circumferentially composed of at least two cells attached by septate junctions; and may be divided, on the basis of morphological criteria, into three longitudinal regions. The cells of nonciliated ductules and ciliated collecting ducts appear to be absorbing material from the lumen; and a distinct proximal-distal polarity is present in each individual cell. At the proximal pole are concentrated micropinocytotic caveolae with amorphous and structural coats on the luminal and cytoplasmic surfaces, respectively. Proceeding distally, a Golgi complex is situated between the micropinocytotic vesicles and large, basophilic, paracrystaline granules. This unusual polarity is interpreted to be important to the resorptive function of the cells.

Fine structure of the protonephridial system in Planaria. II. Ductules, collecting ducts, and osmoregulatory cells.

Distal to the flame cells, the protonephridial tubules of fresh-water planarians are circumferentially composed of at least two cells attached by septate junctions; and may be divided, on the basis of morphological criteria, into three longitudinal regions. The cells of nonciliated ductules and ciliated collecting ducts appear to be absorbing material from the lumen; and a distinct proximal-distal polarity is present in each individual cell. At the proximal pole are concentrated micropinocytotic caveolae with amorphous and structural coats on the luminal and cytoplasmic surfaces, respectively. Proceeding distally, a Golgi complex is situated between the micropinocytotic vesicles and large, basophilic, paracrystaline granules. This unusual polarity is interpreted to be important to the resorptive function of the cells.

Induction of polyspermy by treatment of sea urchin eggs with mercurials

This study is intended to be a contribution to our knowledge of the mechanism protecting the sea-urchin egg against polyspermy. Various degrees of polyspermy were observed upon insemination of eggs that had been pretreated with the mercurials para-chloro mercuriphenylsulfonic acid (PCMPS) or salicyl-γ-hydroxymercuri-β-methoxypropylamide-0-acetic acid, mersalyl (salyrgan). The exposure of the unfertilized eggs to the reagents lasted usually for 15 min. Thereafter the eggs were washed and inseminated in sea water. The concentration range of the mercurials was approximately 10 −7−10 −4 M. The effect varied from batch to batch of eggs, indicating a variation of availability of sulfhydryl (SH-) groups in different egg materials. An attempt was made to define a 50 per cent value (−log Km) for the change in the surface that allows one supernumerary spermatozoon to enter. This value would serve the purpose of making experiments with different egg materials more directly comparable (see pp. 162–163). The degree of polyspermy was also dependent on sperm concentration, as exemplified on p. 163. The progress of polyspermy was followed by immersing the eggs for different periods after insemination in 0.001 per cent lauryl sulfate, which stops any further fertilization. Despite the mercurial pretreatment the eggs that remained monospermic developed normally. Pathological development of mercurial-pretreated eggs depended on a polyspermic state (p. 160). Merthiolate had a weaker polyspermy-promoting action than PCMPS and mersalyl. Whereas these latter compounds seemed to act only on the surface layers of the egg, merthiolate (as also p-chloromercuribenzoate and N-ethylmaleimide) also injured the internal processes (p. 167). The effect of mercurials could be removed by a subsequent exposure to cysteine. The concentrations of cysteine necessary were much higher (e.g. 1000 times) than that of mercurial. The mercurials are thus strongly linked, probably to proteins (pp. 167–168). On leaving the eggs in pure sea water after pretreatment with mercurials, their effect is lowered (pp. 168–169). Exposure of the eggs to ~10 −4 Mo-iodosobenzoate (IBA) decreased the effect of a following mercurial treatment. This confirms that SH groups are involved in the action of mercurials on the egg surface (pp. 164–165). Exposure to and insemination in the presence of ATP (1.8 × 10 −5 M) moderated the effect of pretreatment with mercurial (p. 166). Combination of treatment with IBA prior to the exposure to mercurial and after-treatment with ATP gave a higher moderating effect than that brought about by either IBA or ATP (p. 166). Treatment of the eggs with lower and higher doses of trypsin (see [36]) prior to exposure to mercurials did not modify the extent of polyspermy. The combination of low doses of trypsin and mercurials enhanced the suppression of cleavage beyond the effect of the trypsin doses alone (pp. 169–170). The rate of primary fertilization was not modified by the treatment with mercurials (p. 171). Neither does this treatment modify any microscopic changes accompanying fertilization in such a way that these modifications could be considered as the cause of polyspermy (p. 181). The effect of the mercurials must be at a submicroscopic, biochemical level. On the basis of available knowledge the mechanism of protection against polyspermy and changes in S H compounds upon fertilization are reviewed and discussed (pp. 186–190). The results of mercurial treatment applied soon after fertilization are in keeping with an activation of SH enzymes (pp. 185, 189). The site of attachment of supernumerary spermatozoa and their primary effect give further evidence (see [37]) that, prior to fertilization, a proximaldistal polarity is present in the egg (pp. 177, 189). This polarity also means that the eggs are more receptive to spermatozoa in one region, the proximal one. The cortical changes with ensuing protection evolve also more rapidly in the proximal region [37]. These circumstances are considered to be an important factor in the protection of the egg against polyspermy.

Effect of exposure to ribonuclease on the cortical changes occurring upon fertilization: Experiments on sea urchin eggs

Eggs of Paracentrotus lividus—to a smaller extent also eggs of Psammechinus microtuberculatus—were exposed to solutions of ribonuclease (0.02-0.2-0.36 mg/ml). The eggs were inseminated in the presence of ribonuclease and kept in the enzyme solution for a variable time (in general 30–60 min). The effect of the exposure was examined on living material and and on material that had been fixed in 4 per cent formalin at different lengths of time after fertilization (cf. Table I, for a typical experiment). The RNase treatment brings about a considerable delay in the cortical changes that follow upon fertilization. For more than 2 min the eggs remain in a gelated state with numerous connections between egg surface and elevating fertilization membrane; at the inside of the latter, numerous released (cf. Figs. 6–13) but not incorporated cortical granules were present. In the time interval of 2–10 min gradually one or two concavities developed. Only after concavity formation, did the final incorporation of the released cortical granules in the fertilization membrane take place. Numerous delayed cortical granules were present in the perivitelline space and were here converted into rods or plates. The eggs fertilized in the presence of ribonuclease show a pronounced proximal-distal polarity (Prox.-Dist.). The proximal-distal axis is perpendicular to the animal-vegetal axis (An—Veg.). The Prox.-Dist. axis can be recognized by a concavity or a flattening on the egg surface (cf. Fig. 14 and 15). Moreover, the fertilization membrane is more elevated at the proximal than at the distal pole (this constitutes the main marking of the proximal and the distal region). Also during progress of the first mitosis, the proximal and distal regions can be recognized by a main indentation in the egg cortex (cf. Fig. 22). Later, the polarity in the elevation of the fertilization membrane may be used for this purpose. The site of sperm entry can be recognized in ribonuclease-treated eggs (cf. Fig. 20). There may be a preference for sperm entry at the distal region of the egg (Table II) or the sites of sperm entry may be about equally distributed; in some cases the proximal sperm entry dominated. In control eggs the proximal sperm entry dominated (Table III). The tendency for distal sperm entry is characteristic of eggs exposed to ribonuclease. Variations are due to concentration of enzyme and threshold of suceptibility of the eggs. The sperm entry does not induce the proximal-distal polarity but this is preformed in the egg, which thus has two polar axes that exist prior to fertilization (An.-Veg. and Prox.-Dist.). The rate of fertilization is in general lowered when occurring in the presence of ribonuclease (cf. Figs. 27–29). The birefringent fertilization membrane shows an increase in retardation after exposure to ribonuclease (from about 12.0 to 18.3 mμ). Comparisons were made between the effect of ribonuclease and that of some proteins with a still more basic character (lysozyme and clupein). The difference is so great as to exclude the conclusion that the effect of RNase is mainly due to its basic character. Comparison was made between fully active and only partly active ribonuclease and these experiments point also to the conclusion that the action of RNase is mainly enzymatic. Addition of glutathione shortly before fertilization of ribonuclease-treated eggs counteracts to a large extent the effects of the enzyme (cf. Fig. 34). Of particular interest was the almost complete unfolding of the lamellae of delayed cortical granules after combined treatment with ribonuclease and glutathione (cf. Fig. 35). The experimental results indicate that the exposure to ribonuclease favors release of a factor or factors from ribosomes present in the egg surface; this factor causes an increased gelation of cortical cytoplasm and components of the membranes (fertilization membrane and hyaline layer). The gelation is considered to be due to a polymerization, which in the normal cortical changes may alternate with a depolymerization in a way tentatively indicated in Fig. 41. In this figure the effect of ribonuclease and that of ribonuclease combined with glutathione are also represented. Particularly the changes in the cortical granules, the formation of fertilization membrane and of hyaline layer are discussed in the light of the concept of alternating processes of polymerization and depolymerization. Abnormal conditions induced in different ways are caused by a delay or a break in the transition from one state to the other.