Providing Level-of-Match Information to Perfectly Matched Unrelated Stem Cell Donors: Evaluating Acceptability and Potential Changes in Donor Availability.

Patients with blood-related diseases often cannot identify a matched related donor and must seek donors in unrelated donor registries. These registries face the challenge of ensuring that potential donors are available when contacted. Donor attrition is especially problematic when there is only a single perfectly matched potential donor. One way to improve donor availability might be to present perfectly matched donors (high-priority donors [HPDs]) with more precise information about their match status. This project evaluated the impact of providing such information to HPDs at the National Marrow Donor Program (NMDP)/Be The Match. Objectives were to determine the acceptability of the new messaging to both HPDs and the donor contact representatives (DCRs) who delivered the message, consistency of message delivery, and whether the new messaging was associated with improved donor availability. Mixed methods were used to collect telephone interview data from HPDs, matched samples of non-HPDs, and DCRs. Donor availability data came from NMDP records. Key findings were as follows: (1) the HPD message was acceptable to potential donors and did not seem to produce undue pressure, (2) the message was acceptable to DCRs who became more comfortable and consistent in delivering the message over time, but (3) the new messaging did not significantly increase availability. Despite the lack of evidence for increased availability, there may be ethical benefits and little harm to providing well-matched donors with more information about their degree of matching. Research should examine stronger match status messages and delivery of new messaging to additional highly-matched donor groups.

Recent insights into spindle function in mammalian oocytes and early embryos.

Errors in chromosome segregation in oocytes and early embryos lead to embryo aneuploidy, which contributes to early pregnancy loss. At the heart of chromosome segregation is the spindle, a dynamic biomechanical machine fashioned from microtubules, which is tasked with gathering and sorting chromosomes and dispatching them to the daughter cells at the time of cell division. Understanding the causes of segregation error in the oocyte and early embryo will undoubtedly hinge on a thorough understanding of the mechanism of spindle assembly and function in these highly specialized cellular environments. The recent advent of live imaging approaches to observe chromosome segregation in real-time in oocytes and embryos, paired with gene-silencing techniques and specific inhibition for assessing the function of a protein of interest, has led to a substantial advance in our understanding of chromosome segregation in early mammalian development. These studies have uncovered numerous mechanistic differences between oocytes, embryos, and traditional model systems. In addition, a flurry of recent studies using naturally aged mice as the model for human aging have begun to shed light on the increased levels of aneuploidy seen in embryos from older mothers. Here we review these recent developments and consider what has been learned about the causes of chromosome missegregation in early development.

A non-canonical mode of microtubule organization operates throughout pre-implantation development in mouse.

In dividing animal cells, the centrosome, comprising centrioles and surrounding pericentriolar-material (PCM), is the major interphase microtubule-organizing center (MTOC), arranging a polarized array of microtubules (MTs) that controls cellular architecture. The mouse embryo is a unique setting for investigating the role of centrosomes in MT organization, since the early embryo is acentrosomal, and centrosomes emerge de novo during early cleavages. Here we use embryos from a GFP::CETN2 transgenic mouse to observe the emergence of centrosomes and centrioles in embryos, and show that unfocused acentriolar centrosomes first form in morulae (~16-32-cell stage) and become focused at the blastocyst stage (~64-128 cells) concomitant with the emergence of centrioles. We then used high-resolution microscopy and dynamic tracking of MT growth events in live embryos to examine the impact of centrosome emergence upon interphase MT dynamics. We report that pre-implantation mouse embryos of all stages employ a non-canonical mode of MT organization that generates a complex array of randomly oriented MTs that are preferentially nucleated adjacent to nuclear and plasmalemmal membranes and cell-cell interfaces. Surprisingly, however, cells of the early embryo continue to employ this mode of interphase MT organization even after the emergence of centrosomes. Centrosomes are found at MT-sparse sites and have no detectable impact upon interphase MT dynamics. To our knowledge, the early embryo is unique among proliferating cells in adopting an acentrosomal mode of MT organization despite the presence of centrosomes, revealing that the transition to a canonical mode of interphase MT organization remains incomplete prior to implantation.

Diet and energy-sensing inputs affect TorC1-mediated axon misrouting but not TorC2-directed synapse growth in a Drosophila model of tuberous sclerosis.

The Target of Rapamycin (TOR) growth regulatory system is influenced by a number of different inputs, including growth factor signaling, nutrient availability, and cellular energy levels. While the effects of TOR on cell and organismal growth have been well characterized, this pathway also has profound effects on neural development and behavior. Hyperactivation of the TOR pathway by mutations in the upstream TOR inhibitors TSC1 (tuberous sclerosis complex 1) or TSC2 promotes benign tumors and neurological and behavioral deficits, a syndrome known as tuberous sclerosis (TS). In Drosophila, neuron-specific overexpression of Rheb, the direct downstream target inhibited by Tsc1/Tsc2, produced significant synapse overgrowth, axon misrouting, and phototaxis deficits. To understand how misregulation of Tor signaling affects neural and behavioral development, we examined the influence of growth factor, nutrient, and energy sensing inputs on these neurodevelopmental phenotypes. Neural expression of Pi3K, a principal mediator of growth factor inputs to Tor, caused synapse overgrowth similar to Rheb, but did not disrupt axon guidance or phototaxis. Dietary restriction rescued Rheb-mediated behavioral and axon guidance deficits, as did overexpression of AMPK, a component of the cellular energy sensing pathway, but neither was able to rescue synapse overgrowth. While axon guidance and behavioral phenotypes were affected by altering the function of a Tor complex 1 (TorC1) component, Raptor, or a TORC1 downstream element (S6k), synapse overgrowth was only suppressed by reducing the function of Tor complex 2 (TorC2) components (Rictor, Sin1). These findings demonstrate that different inputs to Tor signaling have distinct activities in nervous system development, and that Tor provides an important connection between nutrient-energy sensing systems and patterning of the nervous system.

MCAK regulates chromosome alignment but is not necessary for preventing aneuploidy in mouse oocyte meiosis I.

Errors in chromosome segregation in mammalian oocytes lead to aneuploid eggs that are developmentally compromised. In mitotic cells, mitotic centromere associated kinesin (MCAK; KIF2C) prevents chromosome segregation errors by detaching incorrect microtubule-kinetochore interactions. Here, we examine whether MCAK is involved in spindle function in mouse oocyte meiosis I, and whether MCAK is necessary to prevent chromosome segregation errors. We find that MCAK is recruited to centromeres, kinetochores and chromosome arms in mid-meiosis I, and that MCAK depletion, or inhibition using a dominant-negative construct, causes chromosome misalignment. However, the majority of oocytes complete meiosis I and the resulting eggs retain the correct number of chromosomes. Moreover, MCAK-depleted oocytes can recover from mono-orientation of homologous kinetochores in mid-meiosis I to segregate chromosomes correctly. Thus, MCAK contributes to chromosome alignment in meiosis I, but is not necessary for preventing chromosome segregation errors. Although other correction mechanisms may function in mammalian meiosis I, we speculate that late establishment of kinetochore microtubules in oocytes reduces the likelihood of incorrect microtubule-kinetochore interactions, bypassing the requirement for error correction.

Mechanisms of TSC-mediated control of synapse assembly and axon guidance.

Tuberous sclerosis complex is a dominant genetic disorder produced by mutations in either of two tumor suppressor genes, TSC1 and TSC2; it is characterized by hamartomatous tumors, and is associated with severe neurological and behavioral disturbances. Mutations in TSC1 or TSC2 deregulate a conserved growth control pathway that includes Ras homolog enriched in brain (Rheb) and Target of Rapamycin (TOR). To understand the function of this pathway in neural development, we have examined the contributions of multiple components of this pathway in both neuromuscular junction assembly and photoreceptor axon guidance in Drosophila. Expression of Rheb in the motoneuron, but not the muscle of the larval neuromuscular junction produced synaptic overgrowth and enhanced synaptic function, while reductions in Rheb function compromised synapse development. Synapse growth produced by Rheb is insensitive to rapamycin, an inhibitor of Tor complex 1, and requires wishful thinking, a bone morphogenetic protein receptor critical for functional synapse expansion. In the visual system, loss of Tsc1 in the developing retina disrupted axon guidance independently of cellular growth. Inhibiting Tor complex 1 with rapamycin or eliminating the Tor complex 1 effector, S6 kinase (S6k), did not rescue axon guidance abnormalities of Tsc1 mosaics, while reductions in Tor function suppressed those phenotypes. These findings show that Tsc-mediated control of axon guidance and synapse assembly occurs via growth-independent signaling mechanisms, and suggest that Tor complex 2, a regulator of actin organization, is critical in these aspects of neuronal development.