Neural crest cell‐specific inactivation of Nipbl or Mau2 during mouse development results in a late onset of craniofacial defects

Nipbl (Scc2) and Mau2 (Scc4) encode evolutionary conserved proteins that play a vital role for loading the cohesin complex onto chromosomes, thereby ensuring accurate chromosome segregation during cell division. While mutations in human NIPBL are known to cause the developmental disorder Cornelia de Lange syndrome, the functions of Nipbl and Mau2 in mammalian development are poorly defined. Here we generated conditional alleles for both genes in mice and show that neural crest cell‐specific inactivation of Nipbl or Mau2 strongly affects craniofacial development. Surprisingly, the early phase of neural crest cell proliferation and migration is only moderately affected in these mutants. Moreover, we found that Mau2 single homozygous mutants exhibited a more severe craniofacial phenotype when compared to that of Nipbl;Mau2 double homozygous mutants. This raises the possibility that the Mau2/Nipbl protein interaction is not only required for cohesin loading, but may also be required to restrict the level of Nipbl involved in regulating gene expression. Together, the data suggest that proliferating neural crest cells tolerate a substantial reduction of cohesin loading proteins and we propose that the successive decrease of cohesin loading proteins in neural crest cells may alter developmental gene regulation in a highly dynamic manner. genesis 52:687–694, 2014. © 2014 Wiley Periodicals, Inc.     

Stable individual differences in separation calls during early development in cats and mice

Background

The development of ethologically meaningful test paradigms in young animals is an essential step in the study of the ontogeny of animal personality. Here we explore the possibility to integrate offspring separation (distress) calls into the study of consistent individual differences in behaviour in two species of mammals, the domestic cat (Felis silvestris catus) and the mound-building mouse (Mus spicilegus). Such vocal responses in young mammals are a potentially useful test option as they represent an important element of mother-offspring communication with strong implications for offspring survival. In addition, the neural control of vocalisation is closely associated with emotional state.

Results

We found marked similarities in the pattern of individual responses of the young of both species to separation from their mother and littermates. In the domestic cat as well as in the mound-building mouse, individual differences in the frequency of calls and to a lesser extent in locomotor activity were repeatable across age, indicating the existence of personality types. Such consistencies across age were also apparent when only considering relative individual differences among litter siblings. In both species, however, individual patterns of vocalisation and locomotor activity were unrelated. This suggests that these two forms of behavioural responses to isolation represent different domains of personality, presumably based on different underlying neurophysiological mechanisms.

Conclusions

Brief separation experiments in young mammals, and particularly the measurement of separation calls, provide a promising approach to study the ontogeny of personality traits. Future long-term studies are needed to investigate the association of these traits with biologically meaningful and potentially repeatable elements of behaviour during later life.

     

Modeling the Ecological and Phenological Predictors of Fruit Consumption by Gibbons (Hylobates albibarbis)

Understanding the ecological interactions between plant reproductive strategies and frugivore feeding behavior can offer insight into the maintenance of tropical forest biodiversity. We examined the role of plant ecological and phenological characteristics in influencing fruit consumption by the White‐bearded gibbon (Hylobates albibarbis) in Gunung Palung National Park, Indonesian Borneo. Gibbons are widespread across Borneo, highly frugivorous and perform important seed dispersal services. We compare multiple models using information criteria to identify the ecological and phenological predictors that most strongly influence gibbon fruit use of 154 plant genera. The most important predictors of resource use were the overall abundance of a genus and the consistency of fruit availability. Plant genera can maintain constant fruit availability as a result of (1) individual stems fruiting often or (2) stems fruiting out of synchrony with each other (asynchrony). Our results demonstrate that gibbons prefer to feed on plant genera that provide consistent fruit availability due to fruiting asynchrony. Because gibbons feed more often on genera that fruit asynchronously, gibbons are more likely to disperse seeds of plant genera with this reproductive strategy. Research on other frugivorous species is needed to determine whether the results for gibbons are generalizable more broadly. Finally, these results suggest that asynchronously fruiting plant genera may be particularly important for habitat restoration in tropical forests designed for frugivore conservation.     

Involvement of connexin43 in the EGF/EGFR signalling during self-renewal and differentiation of neural progenitor cells

Neural progenitor cells (NPCs) are sensitive to epidermal growth factor (EGF), which is essential for their self-renewal. Recently we showed that high level of connexin43 (Cx43) expression and gap junctional intercellular communication (GJIC) are also required to maintain NPCs in a proliferative state. In this study the connection between EGF/EGFR signalling and Cx43 expression was investigated during proliferation and differentiation of cultured ReNcell VM197 human NPCs. We found that EGF, but not basic fibroblast growth factor (bFGF), strongly stimulated both Cx43 expression and GJIC in proliferating cells. This stimulatory effect was blocked by AG1478, a specific inhibitor for EGFR kinase. Notably, knockdown of Cx43 strongly inhibited the cell proliferation promoted by EGF/EGFR signalling. High sensitivity to EGF was still maintained in differentiated NPCs. Administration of EGF to differentiating cells led to a pronounced increase (9-fold) of Cx43 expression and a re-induction of proliferation. This strong impact of EGF was found to correlate with a surprisingly massive 60-fold up-regulation of EGFR expression in differentiated cells. Our data argue for a mutual regulation between Cx43 expression and EGF/EGFR signalling during self-renewal and differentiation of NPCs.     

Exclusion mapping of the X-linked dominant chondrodysplasia punctata/ichthyosis/cataract/short stature (Happle) syndrome: possible involvement of an unstable pre-mutation

Summary Homology with the mouse bare patches mutant suggests that the gene for the X-linked dominant chondrodysplasia punctata / ichthyosis / cataract / short stature syndrome (Happle syndrome) is located in the human Xq28 region. To test this hypothesis, we performed a linkage study in three families comprising a total of 12 informative meioses. Multiple recombinations appear to exclude the Xq28 region as the site of the gene. Surprisingly, multiple crossovers were also found with 26 other markers spread along the rest of the X chromosome. Two-point linkage analysis and analysis of recombination chromosomes seem to exclude the gene from the entire X chromosome. Three different mechanisms are discussed that could explain the apparent exclusion of an X-linked gene from the X chromosome by linkage analysis: (a) different mutations on the X chromosome disturbing X inactivation, (b) metabolic interference, i.e. allele incompatibility of an X-linked gene, and (c) an unstable pre-mutation that can become silent in males. We favour the last explanation, as it would account for the unexpected sex ratio (MF) of 1.21 among surviving siblings, and for the striking clinical variability of the phenotype, including stepwise increases in disease expression in successive generations.     

Effect of milk- and TRIS-based extenders on the fertility of sheep inseminated vaginally once or twice with liquid semen

We studied the influence of two different extenders, a milk-based versus a TRIS-based extender, using a split-sample technique, on fertility after single and double vaginal inseminations in natural estrous in Norwegian Crossbred ewes. Semen from 21 Norwegian Crossbred rams, all aged approximately 0.5 years, was used for AI of totally 561 Norwegian Crossbred ewes housed at 37 different farms. The farmers performed the inseminations themselves. The ewes were allocated to four parallel groups based on the two extenders and single or double inseminations (2 x 2). The farmers were recommended to inseminate the ewes between 12 and 24 h after detection of natural standing estrous. Vaginal insemination with cooled liquid semen diluted in the milk-based extender resulted in a statistically significant (P<0.01) better fertility of about 10% units both as 25-day NR (non return rate)-and lambing rates, compared with semen diluted in the TRIS-based extender. Double inseminations gave significantly higher (P=0.03) fertility results for both extenders expressed as 25-day NR results, but was not quite statistically significant when expressed as lambing rates (P=0.06) compared with single insemination. The overall 25-day NR results for the milk-based extender (66.4%) after single inseminations is in accordance with both the national results (67.1%) based on vaginal inseminations of 11,377 ewes, as well as with the results from a previous study in the same region achieving a 25-day NR results of 63.3%. In conclusion, liquid ram semen diluted in a milk-based extender and vaginally inseminated once in natural heat, with a semen dose of 150 x 10(6) spermatozoa, gave acceptable fertility results and is to be recommended as the method of choice in Norway.     

Real-time single cell analysis of Smac/DIABLO release during apoptosis

We examined the temporal and causal relationship between Smac/DIABLO release, cytochrome c (cyt-c) release, and caspase activation at the single cell level during apoptosis. Cells treated with the broad-spectrum caspase inhibitor z-VAD-fmk, caspase-3 (Casp-3)-deficient MCF-7 cells, as well as Bax-deficient DU-145 cells released Smac/DIABLO and cyt-c in response to proapoptotic agents. Real-time confocal imaging of MCF-7 cells stably expressing Smac/DIABLO-yellow fluorescent protein (YFP) revealed that the average duration of Smac/DIABLO-YFP release was greater than that of cyt-c-green fluorescent protein (GFP). However, there was no significant difference in the time to the onset of release, and both cyt-c-GFP and Smac/DIABLO-YFP release coincided with mitochondrial membrane potential depolarization. We also observed no significant differences in the Smac/DIABLO-YFP release kinetics when z-VAD-fmk-sensitive caspases were inhibited or Casp-3 was reintroduced. Simultaneous measurement of DEVDase activation and Smac/DIABLO-YFP release demonstrated that DEVDase activation occurred within 10 min of release, even in the absence of Casp-3.     

Side-by-side comparability of batch and continuous downstream for the production of monoclonal antibodies

Continuous processing is the future production method for monoclonal antibodies (mAbs). A fully continuous, fully automated downstream process based on disposable equipment was developed and implemented inside the MoBiDiK pilot plant. However, a study evaluating the comparability between batch and continuous processing based on product quality attributes was not conducted before. The work presented fills this gap comparing both process modes experimentally by purifying the same harvest material (side-by-side comparability). Samples were drawn at different time points and positions in the process for batch and continuous mode. Product quality attributes, product-related impurities, as well as process-related impurities were determined. The resulting polished material was processed to drug substance and further evaluated regarding storage stability and degradation behavior. The in-process control data from the continuous process showed the high degree of accuracy in providing relevant process parameters such as pH, conductivity, and protein concentration during the entire process duration. Minor differences between batch and continuous samples are expected as different processing conditions are unavoidable due to the different nature of batch and continuous processing. All tests revealed no significant differences in the intermediates and comparability in the drug substance between the samples of both process modes. The stability study of the final product also showed no differences in the stability profile during storage and forced degradation. Finally, online data analysis is presented as a powerful tool for online-monitoring of chromatography columns during continuous processing.