[Show abstract][Hide abstract] ABSTRACT: Guiding axon growth cones towards their targets is a fundamental process that occurs in a developing nervous system. Several major signaling systems are involved in axon-guidance, and disruption of these systems causes axon-guidance defects. However, the specific role of the environment in which axons navigate in regulating axon-guidance has not been examined in detail. In Drosophila, the ventral nerve cord is divided into segments, and half-segments and the precursor neuroblasts are formed in rows and columns in individual half-segments. The row-wise expression of segment-polarity genes within the neuroectoderm provides the initial row-wise identity to neuroblasts. Here, we show that in embryos mutant for the gene midline, which encodes a T-box DNA binding protein, row-2 neuroblasts and their neuroectoderm adopt a row-5 identity. This reiteration of row-5 ultimately creates a non-permissive zone or a barrier, which prevents the extension of interneuronal longitudinal tracts along their normal anterior-posterior path. While we do not know the nature of the barrier, the axon tracts either stall when they reach this region or project across the midline or towards the periphery along this zone. Previously, we had shown that midline ensures ancestry-dependent fate specification in a neuronal lineage. These results provide the molecular basis for the axon guidance defects in midline mutants and the significance of proper specification of the environment to axon-guidance. These results also reveal the importance of segmental polarity in guiding axons from one segment to the next, and a link between establishment of broad segmental identity and axon guidance.
[Show abstract][Hide abstract] ABSTRACT: Parkinson's disease (PD) is one of the most common neurodegenerative disease characterized by the clinical triad: tremor, akinesia and rigidity. Several studies have suggested that PD patients show disturbances in olfaction at the earliest onset of the disease. The fruit fly Drosophila melanogaster is becoming a powerful model organism to study neurodegenerative diseases. We sought to use this system to explore olfactory dysfunction, if any, in PINK1 mutants, which is a model for PD. PINK1 mutants display many important diagnostic symptoms of the disease such as akinetic motor behavior. In the present study, we describe for the first time, to the best of our knowledge, neurophysiological and neuroanatomical results concerning the olfactory function in PINK1 mutant flies. Electroantennograms were recorded in response to synthetic and natural volatiles (essential oils) from groups of PINK1 mutant adults at three different time points in their life cycle: one from 3-5 day-old flies, from 15-20 and from 27-30 days. The results obtained were compared with the same age-groups of wild type flies. We found that mutant adults showed a decrease in the olfactory response to 1-hexanol, α-pinene and essential oil volatiles. This olfactory response in mutant adults decreased even more as the flies aged. Immunohistological analysis of the antennal lobes in these mutants revealed structural abnormalities, especially in the expression of Bruchpilot protein, a marker for synaptic active zones. The combination of electrophysiological and morphological results suggests that the altered synaptic organization may be due to a neurodegenerative process. Our results indicate that this model can be used as a tool for understanding PD pathogensis and pathophysiology. These results help to explore the potential of using olfaction as a means of monitoring PD progression and developing new treatments.
PLoS ONE 01/2013; 8(8):e73156. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Neurosteroids are synthesized in the brain and modulate brain excitability. There is increasing evidence of their sedative, anesthetic and antiseizure properties, as well as their influence on mood. Currently neurosteroids are classified as pregnane neurosteroids (allopregnanolone and allotetrahydrodeoxycorticosterone), androstane neurosteroids (androstanediol and etiocholanone) or sulfated neurosteroids (pregnenolone sulfate and dehydroepiandrosterone sulfate). Both preclinical and clinical findings indicate that progesterone derivative neurosteroids such as allopregnanolone and allotetrahydrodeoxycorticosterone play a role in mood disorders. Clozapine and olanzapine, which were shown to be effective in stabilizing bipolar disorder, elevate pregnenolone levels in rat hippocampus, cerebral cortex, and serum. In lithium-treated mice, the blood levels of allopregnanolone and pregnenolone were elevated compared to control levels. Women diagnosed with bipolar disorder typically show symptomatic exacerbation in relation to the menstrual cycle, and show vulnerability to the onset or recurrence of mood disorders immediately after giving birth, when the levels of neurosteroid derivatives of progesterone drop. Whereas in women who had recovered from bipolar disorder, the plasma concentration of allopregnanolone was elevated compared to either healthy controls or women with major depressive disorder during the premenstrual period. During depressive episodes, blood level of allopregnanolone is low. Treatment with fluoxetine tends to stabilize the levels of neurosteroids in depression. These findings converge to suggest that these steroids have significant mood-stabilizing effect. This hypothesis is consistent with the observation that a number of anticonvulsants are effective therapies for bipolar disorder, a finding also consistent with the antiseizure properties of neurosteroids. Further exploration of action of neuroactive steroids is likely to open new frontiers in the investigation of the etiology and treatment of mood disorders, particularly bipolar disorders.
Behavioral and Brain Functions 12/2012; 8(1):61. · 2.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to determine the risk for Bipolar Disorder (BD) in Wilson's disease (WD) and to measure the impaired Quality of Life (QL) in BD with WD using standardized psychiatric diagnostic tools and a case control design.
This was a case control study. The cases were 23 consecutive patients with WD treated at the University Hospital in Cagliari, Italy, and the controls were 92 sex- and age-matched subjects with no diagnosis of WD who were randomly selected from a database used previously for an epidemiological study. Psychiatric diagnoses according to DSM-IV criteria were determined by physicians using structured interview tools (ANTAS-SCID). QL was measured by means of SF-12.
Compared to controls, WD patients had lower scores on the SF-12 and higher lifetime prevalence of DSM-IV major depressive disorders (OR = 5.7, 95% CI 2.4-17.3) and bipolar disorders (OR = 12.9, 95% CI 3.6-46.3). BD was associated with lower SF-12 in WD patients.
This study was the first to show an association between BD and WD using standardized diagnostic tools and a case control design. Reports in the literature about increased schizophrenia-like psychosis in WD and a lack of association with bipolar disorders may thus have been based on a more inclusive diagnosis of schizophrenia in the past. Our findings may explain the frequent reports of loss of emotional control, hyperactivity, loss of sexual inhibition, and irritability in WD patients. This study was limited by a small sample size.
[Show abstract][Hide abstract] ABSTRACT: The objective of the study was to determine if community surveys, conducted 3 times over a period of 20years in a small district of Sardinia (Italy), confirm the increase in depressive disorders reported in the recent literature.
Three community surveys were carried out on randomized samples of the same Sardinian mining area in 1988, 1998 and 2008. The surveys were conducted using the interview "Present State Examination" in 1988 (depression diagnosed with ICD-IX) and the CIDI-S in 1998 and 2008 (major depression diagnosed with ICD-X). The three surveys produced estimates of one-month prevalence and of lifetime prevalence in 1998 and 2008.
Our work found a substantial decrease in depressive disorders from the survey conducted in 1998 to the survey in 2008 using a similar methodology, except in the youngest age group, which showed an increase in the rate. A decrease in the frequency of depressive disorders compared to what was found 20years ago was also observed. However, in this case the comparison is more problematic because of use of different diagnostic systems.
The research seems to show a decrease in depressive disorders over the past two decades. While the small population examined makes it difficult to generalize the overall findings, this study suggests that the hypothesis of an increase in the incidence of depressive disorders since the 1980s in western countries, should have exceptions. A complex interaction between socio-economic (mining closure and large migration) and biological factors (possible selective migration) is likely to influence changes in the prevalence of mood disorders. However, due to certain limitations of this study, this hypothesis may be considered from a heuristic perspective.
Journal of affective disorders 05/2012; 141(2-3):255-60. · 3.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A recent survey put forward the hypothesis that the emigration that occurred from Sardinia from the 1960's to the 1980's, selected people with a hypomanic temperament. The paper aims to verify if the people who migrated from Sardinia in that period have shown a high risk of mood disorders in the surveys carried out in their host countries, and if the results are consistent with this hypothesis.
This is systematic review.
In the 1970's when examining the attitudes towards migration in Sardinian couples waiting to emigrate, Rudas found that the decision to emigrate was principally taken by males. Female showed lower self-esteem than male emigrants. A study on Sardinian immigrants in Argentina carried out in 2001-02, at the peak of the economic crisis, found a high risk of depressive disorders in women only. These results were opposite to the findings recorded ten years earlier in a survey on Sardinian immigrants in Paris, where the risk of Depressive Episode was higher in young men only.
Data point to a bipolar disorder risk for young (probably hypomanic) male migrants in competitive, challenging conditions; and a different kind of depressive episodes for women in trying economic conditions. The results of the survey on Sardinian migrants are partially in agreement with the hypothesis of a selective migration of people with a hypomanic temperament. Early motivations and self-esteem seem related to the ways mood disorders are expressed, and to the vulnerability to specific triggering situations in the host country.
Clinical Practice and Epidemiology in Mental Health 01/2012; 8:175-9.
[Show abstract][Hide abstract] ABSTRACT: We discuss recent evidences about schizophrenia (frequency, onset, course, risk factors and genetics) and their influences to some epidemiological myths about schizophrenia diffuse between psychiatric and psychopathology clinicians. The scope is to evaluate if the new acquisitions may change the rehabilitation approaches to schizophrenia modifying the balance about the neurodevelopmental hypothesis of schizophrenia accepting that the cognitive deficits are produced by errors during the normal development of the brain (neurodevelopmental hypothesis) that remains stable in the course of illness and the neurodegenerative hypothesis according of which they derived from a degenerative process that goes on inexorably. RESEARCH METHOD/DESIGN: A review of the literature about epidemiology of schizophrenia has been performed and the contributions of some of these evidence to neurodevelopmental hypothesis and to rehabilitation has been described.
It cannot be definitively concluded for or against the neurodevelopmental or degenerative hypothesis, but efforts in understanding basis of schizophrenia must go on. Until now, rehabilitation programs are based on the vulnerability-stress model: supposing an early deficit that go on stable during the life under favorable circumstances. So, rehabilitation approaches (as neuro-cognitive approaches, social skill training, cognitive-emotional training) are focused on the individual and micro-group coping skills, aiming to help people with schizophrenia to cope with environmental stress factors.
Coping of cognitive deficits in schizophrenia may represents the starting-point for further research on schizophrenia, cohort studies and randomized trials are necessary to defined the range of effectiveness and the outcome of the treatments.
Clinical Practice and Epidemiology in Mental Health 01/2012; 8:52-66.
[Show abstract][Hide abstract] ABSTRACT: Systemic sclerosis (SSc) is a rare conjunctive tissue disorder characterized by fibrosis of the skin and internal organs, and vascular obliteration phenomena. Patients with SSc often experience elevated symptoms of psychological distress, determined by the disfiguration, the pain, the fatigue sensation, and the difficult in daily life occupations. The characteristics of the disease may influence the perceived quality of life (QoL) in people with SSc.
This is a narrative review aiming to define the amount of impairment of Quality of Life in patients with Systemic Sclerosis and the component of this impairment due to depressive or other psychiatric symptoms. The search of the significant articles was carried out in PubMed for the key words "Psychiatric symptoms and Systemic Sclerosis"; "Quality of life and Systemic Sclerosis"; "Depressive Disorders and Systemic Sclerosis".
Psychiatric symptoms are frequents in patients with SSc, but pain, fatigue, disability, body changes don't appear to explain the high prevalence of psychiatric comorbidity in SSc. Many studies founded a significant impairment in SSc patients' QoL, and despite the undeniable correlation between physical symptoms and SSc patients' QoL, mental health was found significantly impaired.
The high rate of depression seems to strictly correlate with poor quality of life, and this finding needs more research to establish the cause of such a correlation. Patients' point of view regarding their health-related QoL could help physicians to enlarge the knowledge about physical and mental correlates of the disease, and to fit therapies as patient required. Particular attention must be given to provide the patient with correct information, in order to mitigate the anxious state on disease course, and to enhance coping skills of the patients.
Clinical Practice and Epidemiology in Mental Health 01/2012; 8:30-5.
[Show abstract][Hide abstract] ABSTRACT: This study aims to evaluate relationship between three different clinical conditions: Major Depressive Disorders (MDD), Hashimoto Thyroiditis (HT) and reduction in regional Cerebral Blood Flow (rCBF) in order to explore the possibility that patients with HT and MDD have specific pattern(s) of cerebral perfusion.
Design: Analysis of data derived from two separate data banks.Sample: 54 subjects, 32 with HT (29 women, mean age 38.8 ± 13.9); 22 without HT (19 women, mean age 36.5 ± 12.25). Assessment: Psychiatric diagnosis was carried out by Simplified Composite International Diagnostic Interview (CIDIS) using DSM-IV categories; cerebral perfusion was measured by (99 m)Tc-ECD SPECT. Statistical analysis was done through logistic regression.
MDD appears to be associated with left frontal hypoperfusion, left temporal hypoperfusion, diffuse hypoperfusion and parietal perfusion asymmetry. A statistically significant association between parietal perfusion asymmetry and MDD was found only in the HT group.
In HT, MDD is characterized by a parietal flow asymmetry. However, the specificity of rCBF in MDD with HT should be confirmed in a control sample with consideration for other health conditions. Moreover, this should be investigated with a longitudinally designed study in order to determine a possible pathogenic cause. Future studies with a much larger sample size should clarify whether a particular perfusion pattern is associated with a specific course or symptom cluster of MDD.
[Show abstract][Hide abstract] ABSTRACT: Cytoplasmic polyadenylation element binding (CPEB) proteins bind mRNAs to regulate their localization and translation. While the first CPEBs discovered were germline specific, subsequent studies indicate that CPEBs also function in many somatic tissues including the nervous system. Drosophila has two CPEB family members. One of these, orb, plays a key role in the establishment of polarity axes in the developing egg and early embryo, but has no known somatic functions or expression outside of the germline. Here we characterize the other Drosophila CPEB, orb2. Unlike orb, orb2 mRNA and protein are found throughout development in many different somatic tissues. While orb2 mRNA and protein of maternal origin are distributed uniformly in early embryos, this pattern changes as development proceeds and by midembryogenesis the highest levels are found in the CNS and PNS. In the embryonic CNS, Orb2 appears to be concentrated in cell bodies and mostly absent from the longitudinal and commissural axon tracts. In contrast, in the adult brain, the protein is seen in axonal and dendritic terminals. Lethal effects are observed for both RNAi knockdowns and orb2 mutant alleles while surviving adults display locomotion and behavioral defects. We also show that orb2 funtions in asymmetric division of stem cells and precursor cells during the development of the embryonic nervous system and mesoderm.
[Show abstract][Hide abstract] ABSTRACT: The Hem/Kette/Nap1 protein is involved in many biological processes. We have recently reported that Hem is required for the normal migration of neurons in the Drosophila embryo. In this paper, we report that Hem regulates the asymmetric division of neural precursor cells. We find that a well-studied Hem/Kette mutant allele produces at least two main, but possibly more, phenotypic classes of mutant embryos, and these phenotypes correlate with variable levels of maternal wild type Hem protein in the developing embryo. While the weaker class exhibits weak axon guidance defect and the mis-migration of neurons, the stronger class causes severe axon guidance defects, mis-migration of neurons and symmetric division of ganglion mother cells (GMC) of the RP2/sib lineage. We also show that the basis for the loss of asymmetric division is due to non-localization of Inscuteable and Numb in GMC-1. A non-asymmetric Numb segregates to both daughter cells of GMC-1, which then prevents Notch signaling from specifying a sib fate. This causes both cells to adopt an RP2 fate. Furthermore, loss of function for Abelson tyrosine kinase also causes loss of asymmetric localization of Inscuteable and Numb and symmetric division of GMC-1, the loss of function for WAVE has a very weakly penetrant loss of asymmetry defect. These results define another role for Hem/Kette/Nap1 in a neural precursor cell during neurogenesis.
Mechanisms of development 09/2011; 128(7-10):483-95. · 2.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the nervous system, neurons form in different regions, then they migrate and occupy specific positions. We have previously shown that RP2/sib, a well-studied neuronal pair in the Drosophila ventral nerve cord (VNC), has a complex migration route. Here, we show that the Hem protein, via the WAVE complex, regulates migration of GMC-1 and its progeny RP2 neuron. In Hem or WAVE mutants, RP2 neuron either abnormally migrates, crossing the midline from one hemisegment to the contralateral hemisegment, or does not migrate at al and fail to send out its axon projection. We report that Hem regulates neuronal migration through stabilizing WAVE. Since Hem and WAVE normally form a complex, our data argues that in the absence of Hem, WAVE, which is presumably no longer in a complex, becomes susceptible to degradation. We also find that Abelson tyrosine kinase affects RP2 migration in a similar manner as Hem and WAVE, and appears to operate via WAVE. However, while Abl negatively regulates the levels of WAVE, it regulates migration via regulating the activity of WAVE. Our results also show that during the degradation of WAVE, Hem function is opposite to that of and downstream of Abl.
[Show abstract][Hide abstract] ABSTRACT: In the CNS, the evolutionarily conserved Notch pathway regulates asymmetric cell fate specification to daughters of ganglion mother cells (GMCs). The E3 Ubiquitin ligase protein Neuralized (Neur) is thought to activate Notch-signaling by the endocytosis of Delta and the Delta-bound extracellular domain of Notch. The intracellular Notch then initiates Notch-signaling. Numb blocks N-signaling in one of the two daughters of a GMC, allowing that cell to adopt a different identity. Numb is asymmetrically localized in a GMC and is segregated to only one of the two daughter cells. In the typical GMC-1→RP2/sib lineage, we found that loss of Neur activity causes symmetric division of GMC-1 into two RP2s. We further found that Neur asymmetrically localizes in a late GMC-1 to the Numb domain and Neur mediates asymmetric division via two distinct, sequential mechanisms: by promoting the asymmetric localization of Numb in a GMC-1 via down-regulation of the transcription factor Pdm1, followed by enhancing the Notch-signaling via trans-potentiation of Notch in a cell committed to become a sib. In neur mutants the GMC-1 identity is not altered but Numb is non-asymmetrically localized due to an up-regulation of Pdm1. Thus, both its daughters inherit Numb, which prevents Notch from specifying a sib identity. Neur also enhances Notch since in neur; numb double mutants, both sibling cells often adopt a mixed fate as opposed to an RP2 fate observed in Notch; numb double mutants. Furthermore, over-expression of Neur can induce both cells to adopt a sib fate similar to gain of function Notch. Our results tie Numb and Notch-signaling through a single player, Neur, thus giving us a more complete picture of the events surrounding asymmetric division of precursor cells. We also show that Neur and Numb are interdependent for their asymmetric-localizations.
[Show abstract][Hide abstract] ABSTRACT: To determine the use of antidepressants (ADs) in people with sub-threshold depression (SD); the lifetime prevalence of mania and hypomania in SD and the link between ADs use, bipolarity and anxiety disorders in SD.
Study design: community survey. Study population: samples randomly drawn, after stratification from the adult population of municipal records. Sample size: 4999 people from seven areas within six Italian regions. Tools: Questionnaire on psychotropic drug consumption, prescription; Structured Clinical Interview NP for DSM-IV modified (ANTAS); Hamilton Depression Rating Scale (HAM-D); Mood Disorder Questionnaire (MDQ); Short Form Health Survey (SF-12). SD definition: HAM-D > 10 without lifetime diagnosis of Depressive Episode (DE).
SD point prevalence is 5.0%. The lifetime prevalence of mania and hypomania episodes in SD is 7.3%. Benzodiazepines (BDZ) consumption in SD is 24.1%, followed by ADs (19.7%). In SD, positive for MDQ and comorbidity with Panic Disorder (PD) or Generalized Anxiety Disorders (GAD) are associated with ADs use, whereas the association between a positive MDQ and ADs use, without a diagnosis of PD or GAD, is not significant. Only in people with DE the well-being (SF-12) is higher among those using first-line antidepressants compared to those not using any medication. In people with SD no significant differences were found in terms of SF-12 score according to drug use.
This study suggests caution in prescribing ADs to people with SD. In people with concomitant anxiety disorders and SD, it should be mandatory to perform a well-designed assessment and evaluate the presence of previous manic or hypomanic symptoms prior to prescribing ADs.
[Show abstract][Hide abstract] ABSTRACT: The tumor suppressor morphogen, Patched (Ptc), has an extensive homology to the Niemann-Pick-C 1 (NPC1) protein. The NPC disease is a paediatric, progressive and fatal neurodegenerative disorder thought to be due to an abnormal accumulation of cholesterol in neurons. Here, we report that patched mutant adults develop a progressive neurodegenerative disease and their brain contains membranous and lamellar inclusions. There is also a significant reduction in the number of synaptic terminals in the brain of the mutant adults. Interestingly, feeding cholesterol to wild type flies generates inclusions in the brain, but does not cause the disease. However, feeding cholesterol to mutant flies increases synaptic connections and suppresses the disease. Our results suggest that sequestration of cholesterol in the mutant brain in the form of membranous material and inclusions affects available pool of cholesterol for cellular functions. This, in turn, negatively affects the synaptic number and contributes to the disease-state. Consistent with this, in ptc mutants there is a reduction in the pool of cholesterol esters, and cholesterol-mediated suppression of the disease accompanies an increase in cholesterol esters. We further show that Ptc does not function directly in this process since gain of function for Hedgehog also induces the same disease with a reduction in the level of cholesterol esters. We believe that loss of function for ptc causes neurodegeneration via two distinct ways: de-repression of genes that interfere with lipid trafficking, and de-repression of genes outside of the lipid trafficking; the functions of both classes of genes ultimately converge on synaptic connections.
[Show abstract][Hide abstract] ABSTRACT: In the Drosophila CNS, combinatorial, interdependent, sequential genetic programs in neuroectodermal (NE) cells, prior to the formation of neuroblasts (NBs), determine the initial identity of NBs. Temporal factors are then sequentially expressed to change the temporal identity. It is unclear at what levels this positional and temporal information integrates to determine progeny cell identity. One idea is that this is a top-down linear process: the identity of a NB determines the identity of its daughter, the ganglion mother cell (GMC), the asymmetric division of the GMC and the fate specification of daughter cells of the GMC. Our results with midline (mid), which encodes a T-box protein, in a typical lineage, NB4-2-->GMC-1-->RP2/sib, suggest that at least part of the process operates in GMCs. That is, a GMC or a neuronal identity need not be determined at the NB or NE level. This is demonstrated by showing that Mid is expressed in a row 5 GMC (M-GMC), but not in its parent NB or NE cell. In mid mutants, M-GMC changes into GMC-1 and generates an RP2 and a sib without affecting the expression of key genes at the NE/NB levels. Expression of Mid in the M-GMC in mid mutants rescues the fate change, indicating that Mid specifies neurons at the GMC level. Moreover, we found a significant plasticity in the temporal window in which a neuronal lineage can develop. Although the extra GMC-1 in mid mutants is born approximately 2 hours later than the bona fide GMC-1, it follows the same developmental pattern as the bona fide GMC-1. Thus, a GMC identity can be independent of parental identity and GMC formation and elaboration need not be strictly time-bound.
Development 12/2008; 136(2):263-74. · 6.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Neurons and their precursor cells are formed in different regions within the developing CNS, but they migrate and occupy very specific sites in the mature CNS. The ultimate position of neurons is crucial for establishing proper synaptic connectivity in the brain. In Drosophila, despite its extensive use as a model system to study neurogenesis, we know almost nothing about neuronal migration or its regulation. In this paper, I show that one of the most studied neuronal pairs in the Drosophila nerve cord, RP2/sib, has a complicated migratory route. Based on my studies on Wingless (Wg) signaling, I report that the neuronal migratory pattern is determined at the precursor cell stage level. The results show that Wg activity in the precursor neuroectodermal and neuroblast levels specify neuronal migratory pattern two divisions later, thus, well ahead of the actual migratory event. Moreover, at least two downstream genes, Cut and Zfh1, are involved in this process but their role is at the downstream neuronal level. The functional importance of normal neuronal migration and the requirement of Wg signaling for the process are indicated by the finding that mislocated RP2 neurons in embryos mutant for Wg-signaling fail to properly send out their axon projection.
[Show abstract][Hide abstract] ABSTRACT: Netrin and Slit signaling systems play opposing roles during the positioning of longitudinal tracts along the midline in the ventral nerve cord of Drosophila embryo. It has been hypothesized that a gradient of Slit from the midline interacts with three different Robo receptors to specify the axon tract positioning. However, no such gradient has been detected. Moreover, overexpression of Slit at the midline has no effect on the positioning of these lateral tracts. In this article, we show that Slit is present outside of the midline along the longitudinal and commissural tracts. Sli from the midline, in a Robo-independent manner, is initially taken up by the commissural axon tracts when they cross the midline and is transported along the commissural tracts into the longitudinal connectives. These results are not consistent with a Sli gradient model. We also find that sli mRNA is maternally deposited and embryos that are genetically null for sli can have weaker guidance defects. Moreover, in robo or robo3 mutants, embryos with normal axon tracts are found and such robo embryos reach pupal stages and die, while robo3 mutant embryos develop into normal individuals and produce eggs. Interestingly, embryos from robo3 homozygous individuals fail to develop but have axon tracts ranging from normal to various defects: robo3 phenotype, robo phenotype, and slit-like phenotype, suggesting a more complex functional role for these genes than what has been proposed. Finally, our previous results indicated that netrin phenotype is epistatic to sli or robo phenotypes. However, it seems likely that this previously reported epistatic relationship might be due to the partial penetrance of the sli, robo, robo3 (or robo2) phenotypes. Our results argue that double mutant epistasis is most definitive only if the penetrance of the phenotypes of the mutants involved is complete.