SRD-1 in AWA neurons is the receptor for female volatile sex pheromones in C. elegans males.

Pheromones are critical cues for attracting mating partners for successful reproduction. Sexually mature Caenorhabditis remanei virgin females and self-sperm-depleted Caenorhabditis elegans hermaphrodites produce volatile sex pheromones to attract adult males of both species from afar. The chemoresponsive receptor in males has remained unknown. Here, we show that the male chemotactic behavior requires amphid sensory neurons (AWA neurons) and the G-protein-coupled receptor SRD-1. SRD-1 expression in AWA neurons is sexually dimorphic, with the levels being high in males but undetectable in hermaphrodites. Notably, srd-1 mutant males lack the chemotactic response and pheromone-induced excitation of AWA neurons, both of which can be restored in males and hermaphrodites by AWA-specific srd-1 expression, and ectopic expression of srd-1 in AWB neurons in srd-1 mutants results in a repulsive behavioral response in both sexes. Furthermore, we show that the C-terminal region of SRD-1 confers species-specific differences in the ability to perceive sex pheromones between C. elegans and C. remanei These findings offer an excellent model for dissecting how a single G-protein-coupled receptor expressed in a dimorphic neural system contributes to sex-specific behaviors in animals.

Generation and characterization of pathogenic Mab21l2(R51C) mouse model.

MAB21L2(R51C) is one of the five documented MAB21L2 mutations in human patients with bilateral eye malformations identified via whole exome sequencing. In addition to the eye abnormality, patients with MAB21L2 R51C/+ mutation also have skeletal dysplasia and intellectual disability. To evaluate the pathology of this mutant allele systematically in understanding the functional role of MAB21L2 in human development, we introduce the R51C mutation into the mouse genome by CRISPR/Cas9 system to generate a mouse model for detailed characterization. The Mab21l2 R51C/+ mice have eyeless phenotype and skeletal abnormalities. Micro-computed tomography (micro-CT) analysis showed the Mab21l2 R51C/+ mice have no eye balls but with two abnormal tissues underneath the brain. Histological analysis revealed that the early eye development in the mutant embryos is interrupted. In addition, Mab21l2 R51C/+ mice also have joint fusion phenotype; the humerus is fused with radius, whereas femur is fused with tibia. Limbs in the mutant animals are distinctly shorter than the wild type; and deltoid tuberosities in humeri are absent in these Mab21l2 R51C/+ mice. In summary, we showed that our Mab21l2 R51C/+ mutant mice have recapitulated the pathological features in eye and bone of human patients. Further analyses of the mutant phenotype with molecular markers will provide insight on how MAB21L2 guides the optic differentiation and skeletogenesis, revealing specific underlying pathogenic mechanism of the MAB21L2(R51C) mutation.

Three-dimensional self-assembling peptide matrix enhances the formation of embryoid bodies and their neuronal differentiation.

We have tailored the properties of a self-assembling peptide (SAP) matrix to direct embryonic stem cells towards neuronal differentiation by adopting a three-dimensional (3D) culture, matching mechanical strength with that of neural tissue, and incorporating fixed laminin-derived pentapeptide signals (IKVAV). We report here that such a matrix alone can induce mouse embryonic stem (ES) cells to first develop into embryoid body (EB) and increase their propensity for subsequent neuronal differentiation. Embryoid bodies were observed by day 5 of culture in SAP matrix. ßIII-tubulin as an early neuronal commitment marker was more prominently detected in cells cultured in the matrix containing IKVAV signals. Interestingly, ES-derived cells did not display distinct neuron morphology within the 3D culture; however, 55 ± 10% of those cells within IKVAV conjugated matrix and 38 ± 6% of those within base matrix displayed higher potential towards neuronal differentiation after 7 days. When retrieved and recultured on a tissue culture plate, they exhibited extended neurite outgrowths and networks in the absence of any additional neuronal differentiation growth factor. The up-regulated expression of neuronal development markers (MAP2 and MeCP2) and the down-regulation of glial marker (GFAP) support that further neuronal differentiation takes place upon reculture. The results showed that an artificial matrix composed of designer SAPs could prompt the formation of EB and provides the cues favoring neuronal differentiation of ES cells.

Factorial analysis of adaptable properties of self-assembling peptide matrix on cellular proliferation and neuronal differentiation of pluripotent embryonic carcinoma.

An integrative and quantitative approach for systematically studying the effects of changing the matrix environment on pluripotent cell viability and neuronal differentiation was demonstrated. This approach, based on factorial analysis and a self-assembling peptide (SAP) matrix, was exemplified using P19 as a pluripotent cell model. In a two-level, three-factor factorial design of experiments, three niche factors, namely, culture dimensionality, fixed biochemical signal and mechanical stiffness, were simultaneously investigated. We found that cell growth was slowed in matrices containing IKVAV epitopes on the SAP constructs, and neuronal differentiation was promoted synergistically by culturing in a three-dimensional matrix and in the presence of IKVAV. Variation of the storage modulus from around 262 Pa to 672 Pa had no significant effect on either viability or differentiation. This approach should be applicable to studying how niche properties that are tunable using SAPs affect the behavior of pluripotent cells in general, thus generating guidelines for constructing artificial matrices. FROM THE CLINICAL EDITOR: In this basic science study, an integrative and quantitative approach to study the effects of matrix environment on pluripotent cell viability and neuronal differentiation is demonstrated. Approaches, like the one described in this paper, are applicable to studying how self assembling peptides affect the behavior of pluripotent cells in general.

Cell culture medium as an alternative to conventional simulated body fluid.

Simulated body fluid (SBF) has been widely used for bioactivity assessment of biomaterials. Many different recipes have been developed, but there is still room for improvement. We have suggested the use of cell culture medium to overcome the drawbacks of conventional SBF. Compared with conventional SBF, cell culture medium is easy to prepare. The normal practices in cell culturing, such as filtering, can eliminate insoluble precipitates in the medium and incubation at 37 °C in an atmosphere with 5% CO(2) also better simulates the in vivo environment. After 4 days immersion in carbonate buffered Dulbecco's modified Eagle's medium (DMEM), precipitates were found to have formed on the surfaces of hydroxyapatite (HA), α-tricalcium phosphate (α-TCP) and ß-tricalcium phosphate (ß-TCP). In order to further verify the use of cell culture medium for SBF studies, we carried out computational thermodynamic and kinetic analyses of the precipitation reaction to reveal the effect of pH and ion concentrations on the driving force and nucleation rate of precipitation of different calcium phosphates (CaP). In general, a slight increase in pH of the cell culture medium from physiological pH (pH 7.4) would favor CaP precipitation thermodynamically and increase the rate, as in the case of r-SBF reported previously. [Ca] and [P] have more impact on precipitation compared with other ions, but the effect is consistent among different materials, indicating that other cell culture media with slightly different compositions may also be used. This study also shows that matching the buffer with the environment is required and fetal bovine serum (FBS) slows down surface CaP precipitation on HA.

Is macroporosity absolutely required for preliminary in vitro bone biomaterial study? A comparison between porous materials and flat materials.

Porous materials are highly preferred for bone tissue engineering due to space for blood vessel ingrowth, but this may introduce extra experimental variations because of the difficulty in precise control of porosity. In order to decide whether it is absolutely necessary to use porous materials in in vitro comparative osteogenesis study of materials with different chemistries, we carried out osteoinductivity study using C3H/10T1/2 cells, pluripotent mesenchymal stem cells (MSCs), on seven material types: hydroxyapatite (HA), α-tricalcium phosphate (α-TCP) and b-tricalcium phosphate (ß-TCP) in both porous and dense forms and tissue culture plastic. For all materials under test, dense materials give higher alkaline phosphatase gene (Alp) expression compared with porous materials. In addition, the cell density effects on the 10T1/2 cells were assessed through alkaline phosphatase protein (ALP) enzymatic assay. The ALP expression was higher for higher initial cell plating density and this explains the greater osteoinductivity of dense materials compared with porous materials for in vitro study as porous materials would have higher surface area. On the other hand, the same trend of Alp mRNA level (HA > ß-TCP > α-TCP) was observed for both porous and dense materials, validating the use of dense flat materials for comparative study of materials with different chemistries for more reliable comparison when well-defined porous materials are not available. The avoidance of porosity variation would probably facilitate more reproducible results. This study does not suggest porosity is not required for experiments related to bone regeneration application, but emphasizes that there is often a tradeoff between higher clinical relevance, and less variation in a less complex set up, which facilitates a statistically significant conclusion. Technically, we also show that the base of normalization for ALP activity may influence the conclusion and there may be ALP activity from serum, necessitating the inclusion of "no cell" control in ALP activity assay with materials. These explain the opposite conclusions drawn by different groups on the effect of porosity.

Cryopreservation of mammalian embryos: Advancement of putting life on hold.

Rodent transgenesis and human-assisted reproductive programs involve multistep handling of preimplantation embryos. The efficacy of production and quality of results from conventionally scheduled programs are limited by temporal constraints other than the quality and quantities of embryos per se. The emergence of vitrification, a water ice-free cryopreservation technique, as a reliable way to arrest further growth of preimplantation embryos, provides an option to eliminate the time constraint. In this article, current and potential applications of cryopreservation to facilitate laboratory animal experiments, colony management, and human-assisted reproductive programs are reviewed. Carrier devices developed for vitrification in the last two decades are compared with an emphasis on their physical properties that infer cooling rate of samples and sterility assurance. Biological impacts of improved cryopreservation on preimplantation embryos are also discussed.

mab-31 and the TGF-beta pathway act in the ray lineage to pattern C. elegans male sensory rays.

BACKGROUND: C. elegans TGF-beta-like Sma/Mab signaling pathway regulates both body size and sensory ray patterning. Most of the components in this pathway were initially identified by genetic screens based on the small body phenotype, and many of these mutants display sensory ray patterning defect. At the cellular level, little is known about how and where these components work although ray structural cell has been implicated as one of the targets. Based on the specific ray patterning abnormality, we aim to identify by RNAi approach additional components that function specifically in the ray lineage to elucidate the regulatory role of TGF-beta signaling in ray differentiation. RESULT: We report here the characterization of a new member of the Sma/Mab pathway, mab-31, recovered from a genome-wide RNAi screen. mab-31 mutants showed ray cell cluster patterning defect and mis-specification of the ray identity. mab-31 encodes a nuclear protein expressed in descendants of ray precursor cells impacting on the ray cell's clustering properties, orientation of cell division plane, and fusion of structural cells. Genetic experiments also establish its relationship with other Sma/Mab pathway components and transcription factors acting upstream and downstream of the signaling event. CONCLUSION: mab-31 function is indispensable in Sma/Mab signal recipient cells during sensory rays specification. Both mab-31 and sma-6 are required in ray lineage at the late larval stages. They act upstream of C. elegans Pax-6 homolog and repress its function. These findings suggested mab-31 is a key factor that can integrate TFG-beta signals in male sensory ray lineage to define organ identity.

Mouse embryo cryopreservation utilizing a novel high-capacity vitrification spatula.

Embryo cryopreservation is an indispensable technique in reproductive programs and in animal facilities where genetically modified mice are used extensively. Here we report the use of a vitrification spatula (VS) that can be readily homemade and has a large holding capacity to vitrify preimplantation mammalian embryos in a micro-drop employing ultra-rapid cooling in liquid nitrogen (LN2). Vitrified one-cell embryos and morulae have high survival rates after thawing, and the fertility of the derived progeny is comparable to that of the control unvitrified group. The large holding capacity (up to 50 embryos per VS) does not only allow rapid expansion of storage capacity for additional mouse strains but also opens up the possibility to streamline transgenic mice generation procedures in transgenic facilities.

Study of hydroxyapatite osteoinductivity with an osteogenic differentiation of mesenchymal stem cells.

Osteoinductivity of hydroxyapatite (HA) was investigated using uncommitted pluripotent mouse stem cells, C3H10T1/2 in an in vitro differentiation assay. For comparative analysis, the cells were cultured on substrates made of osteoinductive HA, with biocompatible titanium and plastics as the negative control. HA exhibited the ability to induce expression of osteo-specific genes in C3H10T1/2, including alkaline phosphatase (ALP), type I collagen, and osteocalcin; compared with its insignificant up-regulation of the same genes in osteoblast-like cells, Saos-2. HA osteoinductivity exhibited in C3H10T1/2 was comparable to that of a bone morphogenetic protein (BMP) with reference to the up-regulation of osteo-specific genes except the core binding factor 1 (Cbfa1, Runx). This result implies a difference in osteogenic induction pathway initiated by HA and BMP. Using this mesenchymal stem cells (MSC) culture assay, osteoinductivity was also demonstrated to be present in the conditioned medium derived from MSC cultured on HA substrates. This conditioned medium exhibited excellent ability to up-regulate ALP in the absence of HA and BMP. The results suggest that the HA can interact with the cells and generate potent inductive substance released into the medium. Such substance in turn is able to induce uncommitted cells to differentiate into the osteolineage.

Improvement of heavy metal stress and toxicity assays by coupling a transgenic reporter in a mutant nematode strain.

Previous studies have demonstrated that wild type Caenorhabditis elegans displays high sensitivity to heavy metals in a lethality test at a level comparable to that of other bioindicator organisms. Taking advantage of the genetics of this model organism, we have tested a number of mutant strains for enhanced sensitivity in heavy metal induced lethality and stress response. These mutants are defective in genes controlling dauer formation, longevity or response to reactive oxygen species (ROS). Among the tested mutants, a double mutant daf-16 unc-75 strain was identified to have superior sensitivity. It has a 6-, 3- and 2-fold increase in sensitivity to cadmium, copper and zinc, respectively, as compared with that of wild type animals. When a fluorescent reporter transgene was coupled with this double mutant for stress detection, a 10-fold enhancement of sensitivity to cadmium over the wild type strain was observed. These transgenic animals, superior to most of the model organisms currently used in bioassays for environmental pollutants, offer a fast and economic approach to reveal the bioavailability of toxic substance in field samples. This study also demonstrates that combination of genetic mutations and transgenesis is a viable approach to identify sensitive indicator animals for environmental monitoring.

A mutation at the start codon defines the differential requirement of dpy-11 in Caenorhabditis elegans body hypodermis and male tail.

dpy-11 encodes a thioredoxin-like molecule that is important for both body and male sensory ray morphogenesis in Caenorhabditis elegans. A mutant allele, s287, has a point mutation with its start codon, AUG, converted into AUA, presumably leading to null function. Since only a weak loss-of-function phenotype was observed, we tested whether an alternative start codon or the converted AUA could be used for translation initiation with reduced efficiency. Based on a functional assay of mutant phenotype complementation and biochemical analysis examining the in vivo synthesis of wild-type and mutant proteins, we conclude that AUA can be used as a less-efficient start codon for initiating translation of DPY-11. Our results also provide direct evidence that the body hypodermis and male tail of C. elegans have differential requirements of dpy-11 activity for their respective normal morphogenesis.

Synergistic toxicity of multiple heavy metals is revealed by a biological assay using a nematode and its transgenic derivative.

Caenorhabditis elegans, a free-living nematode species, was adopted for a toxicity bioassay of 10 heavy metals. The lethal concentration (LC) of these metals was determined. Based on these data, we conducted pairwise and triple metal combination testing and demonstrated that these heavy metals displayed synergistic killing effects on C. elegans larvae. Drastic increases in mortality rate up to 100% could be observed at low metal concentrations. The results illustrate the complexity of toxicity tests in biological systems and show that physical-chemical monitoring of toxicants may underestimate biohazards in environmental samples. We also demonstrate that a transgenic derivative nematode strain, KC136, carrying a heat shock promoter driven gfp reporter gene could be used to reduce the duration of an assay so that the synergistic effects among toxicants could be revealed. This derivative strain allows rapid and frequent monitoring of environmental hazards, which usually requires the handling of a large number samples.

Expression of zebrafish mab21 genes marks the differentiating eye, midbrain and neural tube.

Mab21 homolog in Caenorhabditis elegans is expressed in the sensory ray cells and determines the ray identity (Dev. Dyn. 221 (2001) 422). Similarly, vertebrate homologs of this gene have been shown to express in the craniofacial tissues, limb bud and neural tissues. We report here the cloning and expression analysis of two zebrafish mab21 genes. Both genes have a conserved open reading frame of 1080 nucleotides. Phylogenetic analysis suggested that the two family subgroups existed prior to the divergence of vertebrates. Their expression profiles revealed that mab21l1 was turned on in embryos as early as 8 h post-fertilization (hpf) while mab21l2 was first detected at around 11 hpf. In situ hybridization results showed that expression of these mab21 genes marked the early differentiating olfactory bulbs, eye primordia, midbrain and subsequently the branchial pouches and neural tube.

A novel thioredoxin-like protein encoded by the C. elegans dpy-11 gene is required for body and sensory organ morphogenesis.

Sensory ray morphogenesis in C. elegans requires active cellular interaction regulated by multiple genetic activities. We report here the cloning of one of these genes, dpy-11, which encodes a membrane-associated thioredoxin-like protein. The DPY-11 protein is made exclusively in the hypodermis and resides in the cytoplasmic compartment. Whereas the TRX domain of DPY-11 displays a catalytic activity in vitro, mapping of lesions in different mutant alleles and functional analysis of deletion transgenes reveal that both this enzymatic activity and transmembrane topology are essential for determining body shape and ray morphology. Based on the abnormal features in both the expressing and non-expressing ray cells, we propose that the DPY-11 is required in the hypodermis for modification of its substrates. In turn, ray cell interaction and the whole morphogenetic process can be modulated by these substrate molecules.

Why are there males in the hermaphroditic species Caenorhabditis elegans?

The free-living nematode worm Caenorhabditis elegans reproduces primarily as a self-fertilizing hermaphrodite, yet males are maintained in wild-type populations at low frequency. To determine the role of males in C. elegans, we develop a mathematical model for the genetic system of hermaphrodites that can either self-fertilize or be fertilized by males and we perform laboratory observations and experiments on both C. elegans and a related dioecious species C. remanei. We show that the mating efficiency of C. elegans is poor compared to a dioecious species and that C. elegans males are more attracted to C. remanei females than they are to their conspecific hermaphrodites. We postulate that a genetic mutation occurred during the evolution of C. elegans hermaphrodites, resulting in the loss of an attracting sex pheromone present in the ancestor of both C. elegans and C. remanei. Our findings suggest that males are maintained in C. elegans because of the particular genetic system inherited from its dioecious ancestor and because of nonadaptive spontaneous nondisjunction of sex chromosomes, which occurs during meiosis in the hermaphrodite. A theoretical argument shows that the low frequency of male mating observed in C. elegans can support male-specific genes against mutational degeneration. This results in the continuing presence of functional males in a 99.9% hermaphroditic species in which outcrossing is disadvantageous to hermaphrodites.