Functions of LKB1 in neural crest development: The story unfolds.

Neural crest cells (NCCs) are highly motile, multipotent, embryonic cells that delaminate from the dorsal edges of the neural tube. NCCs follow stereotypical long-range migratory pathways to reach target organs during development, where they give rise to multiple derivatives. The identification of reservoirs of neural crest stem cells that persist to adulthood has recently aroused renewed interest in the biology of NCCs. In this context, several recent studies have demonstrated the essential role of the metabolic kinase LKB1 in NCC establishment. This review surveys how LKB1 governs the formation and maintenance of several neural crest derivatives, including facial bones, melanocytes, Schwann cells, and the enteric nervous system. We also detail the underlying molecular mechanisms that involve downstream effectors of LKB1, in particular the contribution of the AMPK-mTOR signaling pathway to both polarity and metabolic processes. Collectively, these recent discoveries open promising perspectives for new therapeutic applications for the treatment of neural crest disorders.

MEX3A regulates Lgr5+ stem cell maintenance in the developing intestinal epithelium.

Intestinal stem cells (ISCs) fuel the lifelong self-renewal of the intestinal tract and are paramount for epithelial repair. In this context, the Wnt pathway component LGR5 is the most consensual ISC marker to date. Still, the effort to better understand ISC identity and regulation remains a challenge. We have generated a Mex3a knockout mouse model and show that this RNA-binding protein is crucial for the maintenance of the Lgr5+ ISC pool, as its absence disrupts epithelial turnover during postnatal development and stereotypical organoid maturation ex vivo. Transcriptomic profiling of intestinal crypts reveals that Mex3a deletion induces the peroxisome proliferator-activated receptor (PPAR) pathway, along with a decrease in Wnt signalling and loss of the Lgr5+ stem cell signature. Furthermore, we identify PPARγ activity as a molecular intermediate of MEX3A-mediated regulation. We also show that high PPARγ signalling impairs Lgr5+ ISC function, thus uncovering a new layer of post-transcriptional regulation that critically contributes to intestinal homeostasis.

LKB1 signaling in cephalic neural crest cells is essential for vertebrate head development.

Head development in vertebrates proceeds through a series of elaborate patterning mechanisms and cell-cell interactions involving cephalic neural crest cells (CNCC). These cells undergo extensive migration along stereotypical paths after their separation from the dorsal margins of the neural tube and they give rise to most of the craniofacial skeleton. Here, we report that the silencing of the LKB1 tumor suppressor affects the delamination of pre-migratory CNCC from the neural primordium as well as their polarization and survival, thus resulting in severe facial and brain defects. We further show that LKB1-mediated effects on the development of CNCC involve the sequential activation of the AMP-activated protein kinase (AMPK), the Rho-dependent kinase (ROCK) and the actin-based motor protein myosin II. Collectively, these results establish that the complex morphogenetic processes governing head formation critically depends on the activation of the LKB1 signaling network in CNCC.

The RNA-binding protein Mex3b regulates the spatial organization of the Rap1 pathway.

The four related mammalian MEX-3 RNA-binding proteins are evolutionarily conserved molecules for which the in vivo functions have not yet been fully characterized. Here, we report that male mice deficient for the gene encoding Mex3b are subfertile. Seminiferous tubules of Mex3b-deficient mice are obstructed as a consequence of the disrupted phagocytic capacity of somatic Sertoli cells. In addition, both the formation and the integrity of the blood-testis barrier are compromised owing to mislocalization of N-cadherin and connexin 43 at the surface of Sertoli cells. We further establish that Mex3b acts to regulate the cortical level of activated Rap1, a small G protein controlling phagocytosis and cell-cell interaction, through the activation and transport of Rap1GAP. The active form of Rap1 (Rap1-GTP) is abnormally increased at the membrane cortex and chemically restoring Rap1-GTP to physiological levels rescues the phagocytic and adhesion abilities of Sertoli cells. Overall, these findings implicate Mex3b in the spatial organization of the Rap1 pathway that orchestrates Sertoli cell functions.

Patched dependence receptor triggers apoptosis through ubiquitination of caspase-9.

Patched (Ptc), the main receptor for Sonic Hedgehog, is a tumor suppressor. Ptc has been shown to be a dependence receptor, and as such triggers apoptosis in the absence of its ligand. This apoptosis induction occurs through the recruitment by the Ptc intracellular domain of a caspase-activating complex, which includes the adaptor proteins DRAL and TUCAN, and the apical caspase-9. We show here that this caspase-activating complex also includes the E3 ubiquitin ligase NEDD4. We demonstrate that Ptc-mediated apoptosis and Ptc-induced caspase-9 activation require NEDD4. We show that Ptc, but not Bax, the prototypical inducer of the intrinsic cell-death pathway, triggers polyubiquitination of caspase-9. Moreover, a caspase-9 mutant that could not be ubiquitinated failed to mediate Ptc-induced apoptosis. Taken together, these data support the view that the Ptc dependence receptor specifically allows the activation of caspase-9 via its ubiquitination, which occurs via the recruitment by Ptc of NEDD4.

Alix is involved in caspase 9 activation during calcium-induced apoptosis.

The cytoplasmic protein Alix/AIP1 (ALG-2 interacting protein X) is involved in cell death through mechanisms which remain unclear but require its binding partner ALG-2 (apoptosis-linked gene-2). The latter was defined as a regulator of calcium-induced apoptosis following endoplasmic reticulum (ER) stress. We show here that Alix is also a critical component of caspase 9 activation and apoptosis triggered by calcium. Indeed, expression of Alix dominant-negative mutants or downregulation of Alix afford significant protection against cytosolic calcium elevation following thapsigargin (Tg) treatment. The function of Alix in this paradigm requires its interaction with ALG-2. In addition, we demonstrate that caspase 9 activation is necessary for apoptosis induced by Tg and that this activation is impaired by knocking down Alix. Altogether, our findings identify, for the first time, Alix as a crucial mediator of Ca(2+) induced caspase 9 activation.

LKB1 specifies neural crest cell fates through pyruvate-alanine cycling.

Metabolic processes underlying the development of the neural crest, an embryonic population of multipotent migratory cells, are poorly understood. Here, we report that conditional ablation of the Lkb1 tumor suppressor kinase in mouse neural crest stem cells led to intestinal pseudo-obstruction and hind limb paralysis. This phenotype originated from a postnatal degeneration of the enteric nervous ganglia and from a defective differentiation of Schwann cells. Metabolomic profiling revealed that pyruvate-alanine conversion is enhanced in the absence of Lkb1. Mechanistically, inhibition of alanine transaminases restored glial differentiation in an mTOR-dependent manner, while increased alanine level directly inhibited the glial commitment of neural crest cells. Treatment with the metabolic modulator AICAR suppressed mTOR signaling and prevented Schwann cell and enteric defects of Lkb1 mutant mice. These data uncover a link between pyruvate-alanine cycling and the specification of glial cell fate with potential implications in the understanding of the molecular pathogenesis of neural crest diseases.

[Even the Warburg effect can be oxidized: metabolic cooperation and tumor development]. / Même l'effet Warburg est oxydable - Coopération métabolique et développement tumoral.

During tumor development, malignant cells rewire their metabolism to meet the biosynthetic needs required to increase their biomass and to overcome their microenvironment constraints. The sustained activation of aerobic glycolysis, also called Warburg effect, is one of these adaptative mechanisms. The progresses in this area of research have revealed the flexibility of cancer cells that alternate between glycolytic and oxidative metabolism to cope with their conditions of development while sharing their energetic resources. In this survey, we review these recent breakthroughs and discuss a model that likens tumor to an evolutive metabolic ecosystem. We further emphasize the ensuing therapeutic applications that target metabolic weaknesses of neoplastic cells.

Dependence receptors: mechanisms of an announced death.

Dependence receptors form a family of functionally related receptors which are all able to induce two completely opposite intracellular signals depending on the availability of their ligand. Indeed, in its presence, they mediate a positive, classical signal transduction of survival, differentiation or migration but without it, they trigger a negative signal which leads to cell death. The molecular mechanisms involved in triggering cell death in the absence of ligand are starting to be unravelled: dependence receptors are recruited at well-defined domains at the plasma membrane, they trigger cell death through a monomeric form, they are cleaved by caspases and they recruit a caspase activating complex.

The Patched dependence receptor triggers apoptosis through a DRAL-caspase-9 complex.

Sonic hedgehog (Shh) and its main receptor, Patched (Ptc), are implicated in both neural development and tumorigenesis. Besides its classic morphogenic activity, Shh is also a survival factor. Along this line, Ptc has been shown to function as a dependence receptor; it induces apoptosis in the absence of Shh, whereas its pro-apoptotic activity is blocked in the presence of Shh. Here we show that, in the absence of its ligand, Ptc interacts with the adaptor protein DRAL (downregulated in rhabdomyosarcoma LIM-domain protein; also known as FHL2). DRAL is required for the pro-apoptotic activity of Ptc both in immortalized cells and during neural tube development in chick embryos. We demonstrate that, in the absence of Shh, Ptc recruits a protein complex that includes DRAL, one of the caspase recruitment (CARD)-domain containing proteins TUCAN (family member, 8) or NALP1 (NLR family, pyrin domain containing 1) and apical caspase-9. Ptc triggers caspase-9 activation and enhances cell death through a caspase-9-dependent mechanism. Thus, we propose that in the absence of its ligand Shh the dependence receptor Ptc serves as the anchor for a caspase-activating complex that includes DRAL, and caspase-9.

Morphogens and cell survival during development.

The notion of "morphogens" is an important one in developmental biology. By definition, a morphogen is a molecule that emanates from a specific set of cells that is present in a concentration gradient and that specifies the fate of each cell along this gradient. The strongest candidate morphogens are members of the transforming growth factor-beta (TGF-beta), Hedgehog (Hh), and Wnt families. While these morphogens have been extensively described as differentiation inducers, some reports also suggest their possible involvement in cell death and cell survival. It is frequently speculated that the cell death induction that is found associated with experimental removal of morphogens is the manifestation of abnormal differentiation signals. However, several recent reports have raised controversy about this death by default, suggesting that cell death regulation is an active process for shaping tissues and organs. In this review, we will present morphogens, with a specific emphasis on Sonic Hedgehog, a mammalian member of the Hh family, not as a positive regulators of cell differentiation but as key regulators of cell survival.

Inhibition of neuroepithelial patched-induced apoptosis by sonic hedgehog.

During early development in vertebrates, Sonic hedgehog (Shh) is produced by the notochord and the floor plate. A ventrodorsal gradient of Shh directs ventrodorsal patterning of the neural tube. However, Shh is also required for the survival of neuroepithelial cells. We show that Patched (Ptc) induces apoptotic cell death unless its ligand Shh is present to block the signal. Moreover, the blockade of Ptc-induced cell death partly rescues the chick spinal cord defect provoked by Shh deprivation. Thus, the proapoptotic activity of unbound Ptc and the positive effect of Shh-bound Ptc on cell differentiation probably cooperate to achieve the appropriate spinal cord development.