Molecular analyses of zebrafish V0v spinal interneurons and identification of transcriptional regulators downstream of Evx1 and Evx2 in these cells.

BACKGROUND: V0v spinal interneurons are highly conserved, glutamatergic, commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However, we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. METHODS: To identify candidate members of V0v gene regulatory networks, we FAC-sorted wild-type and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression-profiled them using microarrays and single cell RNA-seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 double mutants and wild-type siblings. RESULTS: Our data reveal two molecularly distinct subtypes of zebrafish V0v spinal interneurons at 48 h and suggest that, by this stage of development, evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons, or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons, plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly, we show that Hmx2/3a, repress dI2 interneuron expression of skor1a and nefma, two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. CONCLUSIONS: This study identifies two molecularly distinct subsets of zebrafish V0v spinal interneurons, as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of these cells. Our data further suggest that in the absence of both Evx1 and Evx2, V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then, later, start to express markers of distinct types of inhibitory spinal interneurons, or motoneurons. Taken together, our findings significantly increase our knowledge of V0v and spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type.

Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in these Cells.

Background: V0v spinal interneurons are highly conserved, glutamatergic, commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However, we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks, we FAC-sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression-profiled them using microarrays and single cell RNA-seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 double mutants and wild-type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that, by this stage of development, evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons, or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons, plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly, we show that Hmx2/3a, repress dI2 interneuronal expression of skor1a and nefma, two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons, as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of these cells. Our data further suggest that in the absence of both Evx1 and Evx2, V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then, later, start to express markers of distinct types of inhibitory spinal interneurons, or motoneurons. Taken together, our findings significantly increase our knowledge of V0v and spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type.

Pericentriolar matrix (PCM) integrity relies on cenexin and polo-like kinase (PLK)1.

Polo-like-kinase (PLK) 1 activity is associated with maintaining the functional and physical properties of the centrosome's pericentriolar matrix (PCM). In this study, we use a multimodal approach of human cells (HeLa), zebrafish embryos, and phylogenic analysis to test the role of a PLK1 binding protein, cenexin, in regulating the PCM. Our studies identify that cenexin is required for tempering microtubule nucleation by maintaining PCM cohesion in a PLK1-dependent manner. PCM architecture in cenexin-depleted zebrafish embryos was rescued with wild-type human cenexin, but not with a C-terminal cenexin mutant (S796A) deficient in PLK1 binding. We propose a model where cenexin's C terminus acts in a conserved manner in eukaryotes, excluding nematodes and arthropods, to sequester PLK1 that limits PCM substrate phosphorylation events required for PCM cohesion.

Imaging the early zebrafish embryo centrosomes following injection of small-molecule inhibitors to understand spindle formation.

During the earliest division stages, zebrafish embryos have large cells that divide rapidly and synchronously to create a cellular layer on top of the yolk. Here, we describe a protocol for monitoring spindle dynamics during these early embryonic divisions. We outline techniques for injecting zebrafish embryos with small-molecule inhibitors toward polo-like kinases, preparing and mounting embryos for three-dimensional imaging using confocal microscopy. These techniques are used to understand how the early zebrafish embryo's centrosome constructs the mitotic spindle. For complete details on the use and execution of this protocol, please refer to Rathbun et al. (2020).

Factors associated with overweight and obesity in preschool children.

Aim To determine factors associated with overweight/obesity in preschool children. Methods This cross-sectional study was carried out in paediatric outpatient departments of the Public Institution Primary Health Care Centre of Canton Sarajevo, Bosnia and Herzegovina. The study included 300 preschool children who were divided into two groups: normal weight (n=150) and overweight/obese (n=150). Children were weighed and measured and body mass index was calculated. Data concerning potential childhood overweight/obesity risk factors (sociodemographic characteristics of child and mother, physical activity and sedentary behaviour of the child) were collected using a designed questionnaire filled in by the mothers. Logistic regression analysis was used to estimate the association between potential risk factors and overweight/obesity in preschool children. Results Male gender of the children (p=0.043) and maternal overweight (p=0.000) were positively associated with child overweight/obesity. Male children were 1.6 times more likely to be overweight/obese than female (95% CI: 1.01-2.53). Children of overweight mothers were 3.34 times more likely to be overweight/ obese than children with mothers of normal weight (95% CI: 1.77- 6.28). Physical activity <60 minutes/day ) (p=0.014), screen time >180 minutes/day (p=0.020), regular snacking while watching television, using computer/tablet/ mobile phones (p=0.000) were associated with overweight/obesity status of the preschool children. Conclusion Public health programs that aim to reduce overweight/ obesity in preschool children should mainly help mothers understand the serious risk of childhood obesity and the importance of them creating a healthy lifestyle in childhood.

Preventive health services utilization in patients treated by family physicians.

Aim To explore preventive health service utilization in patients treated by family physicians and the factors associated with their use. Methods This cross-sectional study was carried out in family medicine outpatient departments of the Primary Health Care Canter of Canton Sarajevo, Bosnia and Herzegovina. The study included 300 patients (150 males and 150 females). A questionnaire for the evaluation of patients' socio-demographic characteristics, health profile and use of preventive health services was used. Results Females visited family physicians significantly more often than males (p=0.001). About 51.2% of males reported undergoing a prostate examination within the past 2 years, 77% of females had an examination of the breast within the past 2 years, 9.8% of males and 10.5% of females had received influenza immunization in past 12 months. A number of visits to family physicians in the last twelve months was significantly associated with having had a prostate examination (digital rectal examination and/or prostate-specific antigen testing) (B=1.413, SE=0.171; p=0.043) and an examination of the breast (clinical breast examination and/or mammography) (B=1.817, SE=0.307; p=0.041). Advancement in age was positively associated with influenza immunization (B=2.901, SE=0.026; p=0.000). Conclusion A visit to family physicians was an important step along the causal pathway to receiving preventive services (a prostate examination, an examination of breast). Adults aged 18-64 years were not well protected against influenza and comprehensive strategies are needed.

The relationship between education and self-reported mental and physical health.

Aim To investigate the relationship between educational level and self-reported physical and mental health in the population of Sarajevo Canton. Methods This cross-sectional study was carriedout in family medicine outpatient departments of the Primary Health Care Centre of Sarajevo Canton, Bosnia and Herzegovina. The study included 300 respondents who were divided into lower- and higher-education groups (≤12 years and >12 years of education, respectively). The SF-36 questionnaire for self-assessment of mental and physical health and a questionnaire for the evaluation of socio-demographic characteristics were used. Results The mean values for the mental component summary (MCS) were significantly lower in the lower education (56.86±23.02) than in the higher education group (65.08±20.91) (p=0.001). The mean values for the physical component summary (PCS) were significantly lower in the lower education (61.77 ±21.60) than in the higher education group (74.26 ±17.89) (p=0.000). On average, females had significantly lower scores than males on both the PCS (p=0.00) and the MCS (p=0.00). There was significant relationship of low education with self-reported poor mental (B=6.547, SE=2.481; p=0.009) and physical health (B=10.870, SE=2.248; p=0.024). Increased age was associated with poorer PCS and better MCS. Conclusion Educational level is a strong determinant of perceived health. The importance of education should be emphasized to children as vitally important for their future health.