The ALS-associated KIF5A P986L variant is not pathogenic for Drosophila motoneurons.

Amyotrophic lateral sclerosis (ALS) is a devastating paralytic disorder caused by the death of motoneurons. Several mutations in the KIF5A gene have been identified in patients with ALS. Some mutations affect the splicing sites of exon 27 leading to its deletion (Δ27 mutation). KIF5A Δ27 is aggregation-prone and pathogenic for motoneurons due to a toxic gain of function. Another mutation found to be enriched in ALS patients is a proline/leucine substitution at position 986 (P986L mutation). Bioinformatic analyses strongly suggest that this variant is benign. Our study aims to conduct functional studies in Drosophila to classify the KIF5A P986L variant. When expressed in motoneurons, KIF5A P986L does not modify the morphology of larval NMJ or the synaptic transmission. In addition, KIF5A P986L is uniformly distributed in axons and does not disturb mitochondria distribution. Locomotion at larval and adult stages is not affected by KIF5A P986L. Finally, both KIF5A WT and P986L expression in adult motoneurons extend median lifespan compared to control flies. Altogether, our data show that the KIF5A P986L variant is not pathogenic for motoneurons and may represent a hypomorphic allele, although it is not causative for ALS.

ALS-Associated KIF5A Mutation Causes Locomotor Deficits Associated with Cytoplasmic Inclusions, Alterations of Neuromuscular Junctions, and Motor Neuron Loss.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons. Recently, genome-wide association studies identified KIF5A as a new ALS-causing gene. KIF5A encodes a protein of the kinesin-1 family, allowing the anterograde transport of cargos along the microtubule rails in neurons. In ALS patients, mutations in the KIF5A gene induce exon 27 skipping, resulting in a mutated protein with a new C-terminal region (KIF5A Δ27). To understand how KIF5A Δ27 underpins the disease, we developed an ALS-associated KIF5A Drosophila model. When selectively expressed in motor neurons, KIF5A Δ27 alters larval locomotion as well as morphology and synaptic transmission at neuromuscular junctions in both males and females. We show that the distribution of mitochondria and synaptic vesicles is profoundly disturbed by KIF5A Δ27 expression. That is consistent with the numerous KIF5A Δ27-containing inclusions observed in motor neuron soma and axons. Moreover, KIF5A Δ27 expression leads to motor neuron death and reduces life expectancy. Our in vivo model reveals that a toxic gain of function underlies the pathogenicity of ALS-linked KIF5A mutant.SIGNIFICANCE STATEMENT Understanding how a mutation identified in patients with amyotrophic lateral sclerosis (ALS) causes the disease and the loss of motor neurons is crucial to fight against this disease. To this end, we have created a Drosophila model based on the motor neuron expression of the KIF5A mutant gene, recently identified in ALS patients. KIF5A encodes a kinesin that allows the anterograde transport of cargos. This model recapitulates the main features of ALS, including alterations of locomotion, synaptic neurotransmission, and morphology at neuromuscular junctions, as well as motor neuron death. KIF5A mutant is found in cytoplasmic inclusions, and its pathogenicity is because of a toxic gain of function.

Can childbearing spinal cord injury women with continent cutaneous urinary diversion have child?

Over the last half century, significant improvements in health outcomes for individuals with spinal cord injury (SCI) and growing recognition those women with SCI can become pregnant. However, pregnancy must be rated as high risk and requires multidisciplinary medical care as higher rate of complication compare to general population. Most of published literature grouped all patients with lower urinary tract reconstruction (LUTR) like exstrophy-epispadias complex, spina bifida, interstitial cystitis urogenital sinus or fistula, but our article is focusing in the childbearing SCI women who undergone cutaneous continent urinary diversion (CCUD) with mitrofanoff procedure. We report two cases of three successful pregnancies in this population.

Amyotrophic Lateral Sclerosis Genes in Drosophila melanogaster.

Amyotrophic lateral sclerosis (ALS) is a devastating adult-onset neurodegenerative disease characterized by the progressive degeneration of upper and lower motoneurons. Most ALS cases are sporadic but approximately 10% of ALS cases are due to inherited mutations in identified genes. ALS-causing mutations were identified in over 30 genes with superoxide dismutase-1 (SOD1), chromosome 9 open reading frame 72 (C9orf72), fused in sarcoma (FUS), and TAR DNA-binding protein (TARDBP, encoding TDP-43) being the most frequent. In the last few decades, Drosophila melanogaster emerged as a versatile model for studying neurodegenerative diseases, including ALS. In this review, we describe the different Drosophila ALS models that have been successfully used to decipher the cellular and molecular pathways associated with SOD1, C9orf72, FUS, and TDP-43. The study of the known fruit fly orthologs of these ALS-related genes yielded significant insights into cellular mechanisms and physiological functions. Moreover, genetic screening in tissue-specific gain-of-function mutants that mimic ALS-associated phenotypes identified disease-modifying genes. Here, we propose a comprehensive review on the Drosophila research focused on four ALS-linked genes that has revealed novel pathogenic mechanisms and identified potential therapeutic targets for future therapy.

Impact on quality of life and sexual satisfaction of continent cystostomy with enterocystoplasty in an adult neurologic population.

AIMS: To evaluate long-term general and urinary quality of life (QOL) and sexual satisfaction in adult neurologic patients undergoing continent cystostomy surgery associated with a bladder enlargement to treat neurogenic lower urinary tract dysfunction. METHODS: Monocentre, retrospective series of adult neurologic patients who underwent continent cystostomy with bladder enlargement and followed-up in the long-term. We assessed during follow-up, urinary and renal function and patients filled QOL questionnaires on general QOL, sexuality and urinary (short form Qualiveen) disability. RESULTS: Fifty-three patients were included and followed-up 77 months on average. Rates of patients' satisfaction, stomal and urethral continences were respectively of 98.7% (n = 51), 94.1% (n = 48), and 80.4% (n = 41). Impact of surgery on general QOL and autonomy were strong and positive (respective mean scores of 4.8 and 4.7 on a scale ranging from 1 to 5). Mean overall urinary Qualiveen QOL score was 0.8 (0.09-2.67) indicating a low negative impact of urinary disability on QOL. In patients <45 years, 52.6% (n = 10) reported a moderate to important improvement of their sexuality after surgery. Renal function remained stable during follow-up. CONCLUSION: In the long-term, continent cystostomy with bladder enlargement provides great satisfaction to almost most patients. It has a strong positive impact on general and specific urinary QOL, patients' autonomy and urinary continence. In young patients a positive impact on sexuality was also noticed. These encouraging data, that need to be confirmed, constitute interesting information to provide to neurologic patients to help them deciding whether they are willing to undergo continent cystostomy surgery.

A New Behavioral Test and Associated Genetic Tools Highlight the Function of Ventral Abdominal Muscles in Adult Drosophila.

The function of the nervous system in complex animals is reflected by the achievement of specific behaviors. For years in Drosophila, both simple and complex behaviors have been studied and their genetic bases have emerged. The neuromuscular junction is maybe one of the prototypal simplest examples. A motor neuron establishes synaptic connections on its muscle cell target and elicits behavior: the muscle contraction. Different muscles in adult fly are related to specific behaviors. For example, the thoracic muscles are associated with flight and the leg muscles are associated with locomotion. However, specific tools are still lacking for the study of cellular physiology in distinct motor neuron subpopulations. Here we decided to use the abdominal muscles and in particular the ventral abdominal muscles (VAMs) in adult Drosophila as new model to link a precise behavior to specific motor neurons. Hence, we developed a new behavioral test based on the folding movement of the adult abdomen. Further, we performed a genetic screen and identify two specific Gal4 lines with restricted expression patterns to the adult motor neurons innervating the VAMs or their precursor cells. Using these genetic tools, we showed that the lack of the VAMs or the loss of the synaptic transmission in their innervating motor neurons lead to a significant impairment of the abdomen folding behavior. Altogether, our results allow establishing a direct link between specific motor neurons and muscles for the realization of particular behavior: the folding behavior of the abdomen in Drosophila.

Identification of DmTTLL5 as a Major Tubulin Glutamylase in the Drosophila Nervous System.

Microtubules (MTs) play crucial roles during neuronal life. They are formed by heterodimers of alpha and beta-tubulins, which are subjected to several post-translational modifications (PTMs). Amongst them, glutamylation consists in the reversible addition of a variable number of glutamate residues to the C-terminal tails of tubulins. Glutamylation is the most abundant MT PTM in the mammalian adult brain, suggesting that it plays an important role in the nervous system (NS). Here, we show that the previously uncharacterized CG31108 gene encodes an alpha-tubulin glutamylase acting in the Drosophila NS. We show that this glutamylase, which we named DmTTLL5, initiates MT glutamylation specifically on alpha-tubulin, which are the only glutamylated tubulin in the Drosophila brain. In DmTTLL5 mutants, MT glutamylation was not detected in the NS, allowing for determining its potential function. DmTTLL5 mutants are viable and we did not find any defect in vesicular axonal transport, synapse morphology and larval locomotion. Moreover, DmTTLL5 mutant flies display normal negative geotaxis behavior and their lifespan is not altered. Thus, our work identifies DmTTLL5 as the major enzyme responsible for initiating neuronal MT glutamylation specifically on alpha-tubulin and we show that the absence of MT glutamylation is not detrimental for Drosophila NS function.

Long-term efficacy and safety of tension free vaginal tape in a historic cohort of 463 women with stress urinary incontinence.

INTRODUCTION AND HYPOTHESIS: We report retrospective data on the long-term safety and efficacy of the retropubic midurethral sling (MUS) in a large series of women with stress urinary incontinence. METHODS: In all, 517 patients were treated during the period January 2005 to June 2012 at a single centre in France. The Urinary Symptoms Profile score was used to identify women who were subjectively cured or improved or in whom treatment had failed. The rates of peroperative, and early (<30 days) and late postoperative complications were recorded. RESULTS: A total of 463 patients were evaluable at a mean (±SD) follow-up of 71 ± 23 months. At the last follow-up, 344 patients (74.3 %) demonstrated subjective cure, 55 (11.9 %) were improved and 64 (13.8 %) had treatment failure. Bladder perforations occurred in 33 patients (7.1 %); however, this had no effect on cure rate. In the early postoperative period, temporary intermittent self-catheterization was required in 10 patients (2.2 %) due to voiding difficulties. The most frequent long-term postoperative complication was de novo urge incontinence that was reported by 59 patients (12.7 %); seven patients (1.5 %) needed tape excision due to voiding difficulties and six (1.3 %) needed tape removal due to erosion or chronic pain. CONCLUSIONS: The retropubic MUS was shown to be durable at a mean follow-up of 71 ± 23 months, with a high success/improvement rate and no serious long-term tape-induced adverse effects.

Honey Bee Allatostatins Target Galanin/Somatostatin-Like Receptors and Modulate Learning: A Conserved Function?

Sequencing of the honeybee genome revealed many neuropeptides and putative neuropeptide receptors, yet functional characterization of these peptidic systems is scarce. In this study, we focus on allatostatins, which were first identified as inhibitors of juvenile hormone synthesis, but whose role in the adult honey bee (Apis mellifera) brain remains to be determined. We characterize the bee allatostatin system, represented by two families: allatostatin A (Apime-ASTA) and its receptor (Apime-ASTA-R); and C-type allatostatins (Apime-ASTC and Apime-ASTCC) and their common receptor (Apime-ASTC-R). Apime-ASTA-R and Apime-ASTC-R are the receptors in bees most closely related to vertebrate galanin and somatostatin receptors, respectively. We examine the functional properties of the two honeybee receptors and show that they are transcriptionally expressed in the adult brain, including in brain centers known to be important for learning and memory processes. Thus we investigated the effects of exogenously applied allatostatins on appetitive olfactory learning in the bee. Our results show that allatostatins modulate learning in this insect, and provide important insights into the evolution of somatostatin/allatostatin signaling.

Dual mechanism for bitter avoidance in Drosophila.

In flies and humans, bitter chemicals are known to inhibit sugar detection, but the adaptive role of this inhibition is often overlooked. At best, this inhibition is described as contributing to the rejection of potentially toxic food, but no studies have addressed the relative importance of the direct pathway that involves activating bitter-sensitive cells versus the indirect pathway represented by the inhibition of sugar detection. Using toxins to selectively ablate or inactivate populations of bitter-sensitive cells, we assessed the behavioral responses of flies to sucrose mixed with strychnine (which activates bitter-sensitive cells and inhibits sugar detection) or with L-canavanine (which only activates bitter-sensitive cells). As expected, flies with ablated bitter-sensitive cells failed to detect L-canavanine mixed with sucrose in three different feeding assays (proboscis extension responses, capillary feeding, and two-choice assays). However, such flies were still able to avoid strychnine mixed with sucrose. By means of electrophysiological recordings, we established that bitter molecules differ in their potency to inhibit sucrose detection and that sugar-sensing inhibition affects taste cells on the proboscis and the legs. The optogenetic response of sugar-sensitive cells was not reduced by strychnine, thus suggesting that this inhibition is linked directly to sugar transduction. We postulate that sugar-sensing inhibition represents a mechanism in insects to prevent ingesting harmful substances occurring within mixtures.

Gαo is required for L-canavanine detection in Drosophila.

Taste is an essential sense for the survival of most organisms. In insects, taste is particularly important as it allows to detect and avoid ingesting many plant toxins, such as L-canavanine. We previously showed that L-canavanine is toxic for Drosophila melanogaster and that flies are able to detect this toxin in the food. L-canavanine is a ligand of DmXR, a variant G-protein coupled receptor (GPCR) belonging to the metabotropic glutamate receptor subfamily that is expressed in bitter-sensitive taste neurons of Drosophila. To transduce the signal intracellularly, GPCR activate heterotrimeric G proteins constituted of α, ß and γ subunits. The aim of this study was to identify which Gα protein was required for L-canavanine detection in Drosophila. By using a pharmacological approach, we first demonstrated that DmXR has the best coupling with Gαo protein subtype. Then, by using genetic, behavioral assays and electrophysiology, we found that Gαo47A is required in bitter-sensitive taste neurons for L-canavanine sensitivity. In conclusion, our study revealed that Gαo47A plays a crucial role in L-canavanine detection.

A prospective comparison of surgical and pathological outcomes obtained after robot-assisted or pure laparoscopic partial nephrectomy in moderate to complex renal tumours: results from a French multicentre collaborative study.

OBJECTIVE: To prospectively compare the surgical and pathological outcomes obtained with robot-assisted laparoscopic partial nephrectomy (RAPN) or laparoscopic PN (LPN) for renal cell carcinoma in a multicentre cohort. PATIENTS AND METHODS: Between 2007 and 2011, 265 nephron-sparing surgeries were performed at six French urology departments. The patients underwent either RAPN (n = 220) or LPN (n = 45) procedures. The operative data included operative duration, warm ischaemia time (WIT) and estimated blood loss (EBL). The postoperative outcomes included length of stay (LOS), creatinine variation (Modification of Diet in Renal Disease group), Clavien complications and pathological results. The complexity of the renal tumour was classified using the R.E.N.A.L. nephrometry scoring system. Student's t-test and chi-squared tests were used to compare variables. RESULTS: The median follow-ups for the RAPN and LPN groups were 7 and 18 months, respectively (P < 0.001). Age and American Society of Anesthesiology score were significantly higher in the LPN group (P = 0.02 and P = 0.004, respectively). These variables were lower in the RAPN group: WIT [mean (sd) 20.4 (9.7) vs 24.3 (15.2) min; P = 0.03], operative duration [mean (sd) 168.1 (55.5) vs 199.7 (51.2) min; P < 0.001], operating room occupation time [mean (sd) 248.3 (66.7) vs 278.2 (71.3) min; P = 0.008], EBL [mean (sd) 244.8 (365.4) vs 268.3 (244.9) mL; P = 0.01], use of haemostatic agents [used in 78% of RAPNs and 100% of LPNs; P < 0.001] and LOS [mean (sd) 5.5 (4.3) vs 6.8 (3.2) days; P = 0.05). There were no significant differences between pre- and postoperative creatinine levels, pathology report or complication rates between the groups. The main limitation was due to the study's non-randomised design. CONCLUSION: RAPN is not inferior to pure LPN for perioperative outcomes (i.e. EBL, operative duration, WIT, LOS). Only a randomised study with a longer follow-up can now provide further insight into oncological outcomes.

Long-term results of artificial urinary sphincter for women with type III stress urinary incontinence.

BACKGROUND: The use of the artificial urinary sphincter (AUS) in women is limited. OBJECTIVE: To analyse long-term results and mechanical survival of the AUS (AMS 800; American Medical Systems, Minnetonka, MN, USA) in women with stress urinary incontinence (SUI) due to intrinsic sphincter deficiency (ISD). DESIGN, SETTING, AND PARTICIPANTS: Women with SUI who were treated between January 1987 and March 2007 were included in this prospective study. Only women with low closure pressure at urethral profile and negative continence tests, indicators of severe ISD, were included. INTERVENTIONS: An AUS was implanted. The surgical technique was modified in 1999, involving opening the endopelvic fascia on both sides and dissection in contact with the vaginal wall. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Assessment of complications was made pre- and postoperatively and continence status was based on pad usage. Kaplan-Meier survival curves were used to calculate mechanical survival of the device. Student t test and the chi-square test were used to compare continence and complication rates. RESULTS AND LIMITATIONS: A total of 376 AUS were implanted in 344 patients, whose mean age was 57 yr. The mean follow-up, plus or minus standard deviation, was 9.6±4.0 yr. At last follow-up, postoperative continence rates, assessed as fully continent (no leakage), socially incontinent (some drops but no pad), or incontinent (one pad or more), were 85.6%, 8.8%, and 5.6%, respectively. The 3-, 5-, and 10-yr device survival rates were 92.0%, 88.6%, and 69.2%, respectively. The mean mechanical survival was 176 mo (14.7 yr). Three risk factors for AUS survival were the number of previous incontinence surgeries, the presence of neurogenic bladder, and simultaneous augmentation enterocystoplasty. Principal limitation of the study is the absence of validated incontinence questionnaire. CONCLUSIONS: The AUS provides excellent outcome in women with ISD, with low explantation rate and very good device survival.

A novel role of the glial fate determinant glial cells missing in hematopoiesis.

Glial cell deficient/Glial cells missing (Glide/Gcm) transcription factor is expressed in all glial precursors of the Drosophila embryo. Gcm is necessary and sufficient to induce glial differentiation but also plays a role in other cell types, by interacting with specific factors. To find potential partners of Gcm which trigger these other pathways, we performed a yeast two-hybrid screen and identified dpias, a gene involved in post-embryonic hematopoiesis. dpias larvae show melanotic tumors due to excess of lamellocytes, a hemocyte lineage that is involved in non-self recognition. We here show that blocking Gcm activity also triggers melanotic tumors and that gcm interacts genetically with dpias. Moreover, the members of the Janus Kinase (JAK)/ Signal Transducer and Activator of Transcription (STAT) pathway, which are known for their role in the vertebrate and invertebrate immune system and are required for dpias-dependent tumor formation, act downstream of Gcm. Altogether, this study identifies an unpredicted role of Gcm, dictated by its cofactor dpias, allowing Gcm to act in a specific pathway. Together with the recent finding that glia act as scavengers during development and in pathological conditions, our data open new perspectives onto the cellular and molecular pathways involved in non-self recognition within and outside the nervous system.

Plant insecticide L-canavanine repels Drosophila via the insect orphan GPCR DmX.

For all animals, the taste sense is crucial to detect and avoid ingesting toxic molecules. Many toxins are synthesized by plants as a defense mechanism against insect predation. One example of such a natural toxic molecule is L-canavanine, a nonprotein amino acid found in the seeds of many legumes. Whether and how insects are informed that some plants contain L-canavanine remains to be elucidated. In insects, the taste sense relies on gustatory receptors forming the gustatory receptor (Gr) family. Gr proteins display highly divergent sequences, suggesting that they could cover the entire range of tastants. However, one cannot exclude the possibility of evolutionarily independent taste receptors. Here, we show that L-canavanine is not only toxic, but is also a repellent for Drosophila. Using a pharmacogenetic approach, we find that flies sense food containing this poison by the DmX receptor. DmXR is an insect orphan G-protein-coupled receptor that has partially diverged in its ligand binding pocket from the metabotropic glutamate receptor family. Blockade of DmXR function with an antagonist lowers the repulsive effect of L-canavanine. In addition, disruption of the DmXR encoding gene, called mangetout (mtt), suppresses the L-canavanine repellent effect. To avoid the ingestion of L-canavanine, DmXR expression is required in bitter-sensitive gustatory receptor neurons, where it triggers the premature retraction of the proboscis, thus leading to the end of food searching. These findings show that the DmX receptor, which does not belong to the Gr family, fulfills a gustatory function necessary to avoid eating a natural toxin.

UV laser mediated cell selective destruction by confocal microscopy.

Analysis of cell-cell interactions, cell function and cell lineages greatly benefits selective destruction techniques, which, at present, rely on dedicated, high energy, pulsed lasers and are limited to cells that are detectable by conventional microscopy. We present here a high resolution/sensitivity technique based on confocal microscopy and relying on commonly used UV lasers. Coupling this technique with time-lapse enables the destruction and following of any cell(s) in any pattern(s) in living animals as well as in cell culture systems.

Control of gcm RNA stability is necessary for proper glial cell fate acquisition.

The control of RNA stability is an important post-transcriptional event. While neural development is known to require proteins that bind AU-rich elements (ARE) and affect RNA half-life, the role of specific RNA stability in this process remains elusive. In the Drosophila embryo, glial fate acquisition is triggered by glial cells missing (gcm) master gene, which is transiently expressed in all gliogenic stem cells and submitted to tight transcriptional regulation. By using in vitro and in vivo site directed mutagenesis, we have discovered that gcm RNA is unstable and that its 3'UTR confers labile properties to RNA due to the presence of an ARE motif. Moreover, we show that the gliogenic potential of Gcm transcription factor increases when ARE is abolished and demonstrate the importance of gcm RNA stability in the acquisition of the glial fate. Thus, control of a single RNA half-life is crucial for nervous system differentiation.

Novel gcm-dependent lineages in the postembryonic nervous system of Drosophila melanogaster.

glial cells missing genes (gcm and gcm2) act as the glial fate determinants in the Drosophila embryo. However, their requirement in the adult central nervous system (CNS) is at present not known, except for their role in lamina glia. This is particularly important with respect to two recent sets of data. Adult glial subpopulations differentiate through embryonic glia proliferation. Also, gcm-gcm2 are required for the differentiation of specific adult neurons. We here show that gcm is expressed in precursors and postmitotic, migrating, cells of the medulla neuropile glia (mng) lineage. It is also expressed in a thoracic glial lineage and in neurons of the ventral nerve cord (VNC). Finally, while gcm is required for gliogenesis in medulla and VNC, it does not seem to be required for the generation of VNC neurons.

Neurogenic role of Gcm transcription factors is conserved in chicken spinal cord.

Although glial cells missing (gcm) genes are known as glial determinants in the fly embryo, the role of vertebrate orthologs in the central nervous system is still under debate. Here we show for the first time that the chicken ortholog of fly gcm (herein referred to as c-Gcm1), is expressed in early neuronal lineages of the developing spinal cord and is required for neural progenitors to differentiate as neurons. Moreover, c-Gcm1 overexpression is sufficient to trigger cell cycle exit and neuronal differentiation in neural progenitors. Thus, c-Gcm1 expression constitutes a crucial step in the developmental cascade that prompts progenitors to generate neurons: c-Gcm1 acts downstream of proneural (neurogenin) and progenitor (Sox1-3) factors and upstream of NeuroM neuronal differentiation factor. Strikingly, this neurogenic role is not specific to the vertebrate gene, as fly gcm and gcm2 are also sufficient to induce the expression of neuronal markers. Interestingly, the neurogenic role is restricted to post-embryonic stages and we identify two novel brain neuronal lineages expressing and requiring gcm genes. Finally, we show that fly gcm and the chick and mouse orthologs induce expression of neural markers in HeLa cells. These data, which demonstrate a conserved neurogenic role for Gcm transcription factors, call for a re-evaluation of the mode of action of these genes during evolution.

Glial differentiation and the Gcm pathway.

One of the most challenging issues in developmental biology is to understand how cell diversity is generated. The Drosophila nervous system provides a model of choice for unraveling this process. First, many neural stem cells and lineages have been identified. Second, major molecular pathways involved in neural development and associated mutations have been characterized extensively in recent years. In this review, we focus on the cellular and molecular mechanisms underlying the generation of glia. This cell population relies on the expression of gcm fate determinant, which is necessary and sufficient to induce glial differentiation. We also discuss the recently identified role of gcm genes in Drosophila melanogaster and vertebrate neurogenesis. Finally, we will consider the Gcm pathway in the context of neural stem cell differentiation.