The Metalloproteinase adam19b Is Required for Sensory Axon Guidance in the Hindbrain.

Little is known about the molecular and cellular mechanisms involved in the formation of the cranial peripheral sensory system in vertebrates. To identify genes involved in the formation of these circuits, we performed a forward genetic screen utilizing a transgenic zebrafish line (p2rx3.2:gfpsl1) that expresses green fluorescent protein (gfp) in sensory neurons of the Vth, VIIth, IXth and Xth cranial ganglia. Here, we describe a novel zebrafish mutant in which a missense mutation in the adam19b gene selectively affects the epibranchial sensory circuits.

Transcriptome Analysis of Chemically-Induced Sensory Neuron Ablation in Zebrafish.

Peripheral glia are known to have a critical role in the initial response to axon damage and degeneration. However, little is known about the cellular responses of non-myelinating glia to nerve injury. In this study, we analyzed the transcriptomes of wild-type and mutant (lacking peripheral glia) zebrafish larvae that were treated with metronidazole. This treatment allowed us to conditionally and selectively ablate cranial sensory neurons whose axons are ensheathed only by non-myelinating glia. While transcripts representing over 27,000 genes were detected by RNAseq, only a small fraction (~1% of genes) were found to be differentially expressed in response to neuronal degeneration in either line at either 2 hrs or 5 hrs of metronidazole treatment. Analysis revealed that most expression changes (332 out of the total of 458 differentially expressed genes) occurred over a continuous period (from 2 to 5 hrs of metronidazole exposure), with a small number of genes showing changes limited to only the 2 hr (55 genes) or 5 hr (71 genes) time points. For genes with continuous alterations in expression, some of the most meaningful sets of enriched categories in the wild-type line were those involving the inflammatory TNF-alpha and IL6 signaling pathways, oxidoreductase activities and response to stress. Intriguingly, these changes were not observed in the mutant line. Indeed, cluster analysis indicated that the effects of metronidazole treatment on gene expression was heavily influenced by the presence or absence of glia, indicating that the peripheral non-myelinating glia play a significant role in the transcriptional response to sensory neuron degeneration. This is the first transcriptome study of metronidazole-induced neuronal death in zebrafish and the response of non-myelinating glia to sensory neuron degeneration. We believe this study provides important insight into the mechanisms by which non-myelinating glia react to neuronal death and degeneration in sensory circuits.

The relationship between children's sensory processing patterns and their leisure preferences and participation patterns.

BACKGROUND: Sensory processing patterns may be associated with children's preferences for different activities; however, knowledge about how different sensory processing patterns may relate to children's participation in leisure activities is scarce. PURPOSE: This study investigated in what leisure activities children with extreme sensory processing patterns participate and if relationships exist between children's sensory processing patterns and their leisure preferences and participation patterns. METHOD: This correlational study analyzed data from children's Sensory Profiles and reported play and leisure preferences. All 91 children in the sample completed the Children's Assessment for Participation and Enjoyment (CAPE) and the Preferences for Activities of Children (PAC). Parents of children ages 6 to 10 years completed the Sensory Profile, and children ages 11 to 14 years completed the Adolescent/Adult Sensory Profile. FINDINGS: Children with different sensory processing patterns preferred both similar and distinct leisure activities. Low-registration quadrant summary z scores negatively correlated with CAPE overall diversity scores (rs=-.23, p=.03), sensitivity quadrant summary z scores negatively correlated with preferences for social activities (rs=-.23, p=.03) and preferences for skill-based activities (rs=-.22, p=.04), and avoiding quadrant summary z scores negatively correlated with preferences for social activities (rs=-.26, p=.01). IMPLICATIONS: Children's sensory preferences are related to leisure preferences and participation.

Novel role for carbamoyl phosphate synthetase 2 in cranial sensory circuit formation.

In zebrafish, cranial sensory circuits form by 4 days post-fertilization. We used a forward genetic screen to identify genes involved in the formation of these circuits. In one mutant allele, sl23, axons arising from the epibranchial sensory ganglia do not form their stereotypical terminal fields in the hindbrain. These embryos also had small eyes and deformed jaws, suggesting a pleiotropic effect. Using positional cloning, a 20-nucleotide deletion in the carbamoyl-phosphate-synthetase2-aspartate-transcarbamylase-dihydroorotase (cad) gene was found. Injection of a CAD morpholino phenocopied the mutant and mutants were rescued by injection of cad RNA. Cad activity is required for pyrimidine biosynthesis, and thus is a prerequisite for nucleic acid production and UDP-dependent protein glycosylation. Perturbation of nucleic acid biosynthesis can result in cell death. sl23 mutants did not exhibit elevated cell death, or gross morphological changes, in their hindbrains. To determine if defective protein glycosylation was involved in the aberrant targeting of sensory axons, we treated wild type embryos with tunicamycin, which blocks N-linked protein glycosylation. Interference with glycosylation via tunicamycin treatment mimicked the sl23 phenotype. Loss of cad reveals a critical role for protein glycosylation in cranial sensory circuit formation.

Offenders who are mothers with and without experience in prostitution: differences in historical trauma, current stressors, and physical and mental health differences.

BACKGROUND: Women offenders are a growing population in the criminal justice system; most are mothers. A subset of these women have a history of prostitution. Despite more recent research identifying the needs of women offenders who are mothers, those with and without experience in prostitution are still generally represented in the literature as a homogenous group. METHODS: This study examined the differences between mothers who indicated that they had engaged in prostitution with those who had not. The data were from a survey of offending mothers in a Midwestern city and was based on 889 respondents. Approximately 20% of the women indicated that they had engaged in prostitution at some point in their lives. FINDINGS: Mothers with histories of prostitution reported more exposure to violence, witnessing crime, living in areas with high drug activity, and having a higher rate of physical and mental health problems. CONCLUSIONS: Health care professionals who interact with mothers in the criminal justice system who have histories of prostitution should be careful to assess for a history of trauma and its psychological consequences. Along with increased health care needs, interventions are needed to help these women obtain basic needs such as stable housing outside of high crime and high drug-use areas and to receive targeted psychological services that respond to the unique trauma suffered by this subpopulation of offenders.

A zebrafish SKIV2L2-enhancer trap line provides a useful tool for the study of peripheral sensory circuit development.

The zebrafish is an ideal model for elucidating the cellular and molecular mechanisms that underlie development of the peripheral nervous system. A transgenic line that selectively labels all the sensory circuits would be a valuable tool for such investigations. In this study, we describe such a line: the enhancer trap zebrafish line Tg(SKIV2L2:gfp)(j1775) which expresses green fluorescent protein (gfp) in the peripheral sensory ganglia. We show that this transgene marks all peripheral ganglia and sensory nerves, beginning at the time when the neurons are first extending their processes, but does not label the efferent nerves. The trapped reporter is inserted just upstream of a previously poorly described gene: lhfpl4 on LG6. The expression pattern of this gene by in situ hybridization reveals a different, but overlapping, pattern of expression compared to that of the transgene. This pattern also does not mimic that of the gene (skiv2l2), which provided the promoter element in the construct. These findings indicate that reporter expression is not dictated by an endogenous enhancer element, but instead arises through an unknown mechanism. Regardless, this reporter line should prove to be a valuable tool in the investigation of peripheral nervous system formation in the zebrafish.

Diverse mechanisms for assembly of branchiomeric nerves.

The formation of branchiomeric nerves (cranial nerves V, VII, IX and X) from their sensory, motor and glial components is poorly understood. The current model for cranial nerve formation is based on the Vth nerve, in which sensory afferents are formed first and must enter the hindbrain in order for the motor efferents to exit. Using transgenic zebrafish lines to discriminate between motor neurons, sensory neurons and peripheral glia, we show that this model does not apply to the remaining three branchiomeric nerves. For these nerves, the motor efferents form prior to the sensory afferents, and their pathfinding show no dependence on sensory axons, as ablation of cranial sensory neurons by ngn1 knockdown had no effect. In contrast, the sensory limbs of the IXth and Xth nerves (but not the Vth or VIIth) were misrouted in gli1 mutants, which lack hindbrain bmn, suggesting that the motor efferents are crucial for appropriate sensory axon projection in some branchiomeric nerves. For all four nerves, peripheral glia were the intermediate component added and had a critical role in nerve integrity but not in axon guidance, as foxd3 null mutants lacking peripheral glia exhibited defasciculation of gVII, gIX, and gX axons. The bmn efferents were unaffected in these mutants. These data demonstrate that multiple mechanisms underlie formation of the four branchiomeric nerves. For the Vth, sensory axons initiate nerve formation, for the VIIth the sensory and motor limbs are independent, and for the IXth/Xth the motor axons initiate formation. In all cases the glia are patterned by the initiating set of axons and are needed to maintain axon fasciculation. These results reveal that coordinated interactions between the three neural cell types in branchiomeric nerves differ according to their axial position.

Sirolimus therapy for angiomyolipoma in tuberous sclerosis and sporadic lymphangioleiomyomatosis: a phase 2 trial.

PURPOSE: Renal angiomyolipomas are a frequent manifestation of tuberous sclerosis and sporadic lymphangioleiomyomatosis (LAM). These disorders are associated with mutations of TSC1 or TSC2 that lead to overactivation of mTOR complex 1 (mTORC1), suggesting an opportunity for targeted therapy by using mTORC1 inhibitors. This study investigated the efficacy and safety of the mTORC1 inhibitor sirolimus for treatment of renal angiomyolipomas in patients with these disorders. EXPERIMENTAL DESIGN: In this multicenter phase 2 nonrandomized open label trial, 16 patients with tuberous sclerosis or sporadic LAM and renal angiomyolipoma(s) were treated with oral sirolimus for up to 2 years. Steady-state blood levels were 3 to 10 ng/mL. The primary outcome was change in size of renal angiomyolipomas measured by MRI and assessed by Response Evaluation Criteria in Solid Tumors (RECIST) criteria. Secondary outcomes included safety, neurocognitive function, and pulmonary function. RESULTS: The response rate, by RECIST criteria, was 50%. Summated angiomyolipoma diameters were reduced in all 16 patients and by 30% or more in eight (all from the per protocol group of 10). Forty-one of 48 angiomyolipomas were smaller at the last measurement than at baseline. Most shrinkage occurred during the first year of treatment. There was little change in pulmonary function. Recall memory improved in seven of eight patients with tuberous sclerosis. Adverse events were consistent with the known toxicities of sirolimus. CONCLUSIONS: This study showed sustained regression of renal angiomyolipomas in patients with tuberous sclerosis or sporadic LAM receiving 2 years of sirolimus treatment. Possible effects on pulmonary function and neurocognition require further investigation.

Ectodermal P2X receptor function plays a pivotal role in craniofacial development of the zebrafish.

P2X receptors are non-selective cation channels operated by extracellular ATP. Currently, little is known concerning the functions of these receptors during development. Previous work from our lab has shown that zebrafish have two paralogs of the mammalian P2X3 receptor subunit. One paralog, p2rx3.1, is expressed in subpopulations of neural and ectodermal cells in the embryonic head. To investigate the role of this subunit in early cranial development, we utilized morpholino oligonucleotides to disrupt its translation. Loss of this subunit resulted in craniofacial defects that included malformation of the pharyngeal skeleton. During formation of these structures, there was a marked increase in cell death within the branchial arches. In addition, the epibranchial (facial, glossopharyngeal, and vagal) cranial sensory ganglia and their circuits were perturbed. These data suggest that p2rx3.1 function in ectodermal cells is involved in purinergic signaling essential for proper craniofacial development and sensory circuit formation in the embryonic and larval zebrafish.

Molecular characterization and embryonic expression of the family of N-methyl-D-aspartate receptor subunit genes in the zebrafish.

We present the cloning of 10 N-methyl-D-aspartate (NMDA) receptor subunits from the zebrafish. These subunits fall into five subtypes, each containing two paralogous genes. Thus, we report two NMDAR1 genes (NR1.1 and NR1.2), and eight NMDAR2 genes, designated NR2A.1 and NR2A.2, NR2B.1 and NR2B.2, NR2C.1 and NR2C.2, and NR2D.1 and NR2D.2. The predicted sequences of the NR1 paralogs display 90% identity to the human protein. The NR2 subunits show less identity, differing most at the N- and C-termini. The NR1 genes are both expressed embryonically, although in a nonidentical manner. NR1.1 is found in brain, retina, and spinal cord at 24 hours postfertilization (hpf). NR1.2 is expressed in the brain at 48 hpf but not in the spinal cord. NR2 developmental gene expression varies: both paralogs of the NR2A are expressed at 48 hpf in the retina, only one paralog of the NR2B is expressed at low levels in the heart at 48 hpf. Neither of the NR2C is expressed embryonically. Both paralogs of the NR2D are expressed: 2D.1 is in the forebrain, retina, and spinal cord at 24 hpf, whereas the 2D.2 is only found in the retina. Our findings demonstrate that the zebrafish can serve as a useful model system for investigating the role of NMDA receptors in the development of the nervous system.

Molecular structure of P2X receptors.

P2X receptors are ligand-gated ion channels that transduce many of the physiological effects of extracellular ATP. There has been a dramatic increase in awareness of these receptors over the past 5 or so years, in great part due to their molecular cloning and characterization. The availability of cDNA clones for the various subunits has led to rapid progress in identifying their tissue-specific expression, resulting in new ideas concerning the functional roles these receptors might play in physiological and pathophysiological processes. In addition, molecular approaches have yielded much information regarding the structure and function of the receptor proteins themselves. In this review we seek to review recent findings concerning the molecular determinants of receptor-channel function, with particular focus on ligand binding and gating, ion selectivity, and subunit assembly.

Cloning and characterization of two novel zebrafish P2X receptor subunits.

In this report we describe the cloning and characterization of two P2X receptor subunits cloned from the zebrafish (Danio rerio). Primary sequence analysis suggests that one cDNA encodes an ortholog of the mammalian P2X(4) subunit and the second cDNA encodes the ortholog of the mammalian P2X(5) subunit. The zP2X(4) subunit forms a homo-oligomeric receptor that displays a low affinity for ATP (EC(50)=274+/-48 microM) and very low affinity (EC(50)>500 microM) for other purinergic ligands such as alphabetameATP, suramin, and PPADS. As seen with the mammalian orthologs, the zP2X(5) subunit forms a homo-oligomeric receptor that yields very small whole-cell currents (<20pA), making determination of an EC(50) problematic. Both subunit genes were physically mapped onto the zebrafish genome using radiation hybrid analysis of the T51 panel, with the zp2x4 localized to LG21 and zp2x5 to LG5.