Iain Shepherd - Academia.edu (original) (raw)

Papers by Iain Shepherd

Research paper thumbnail of The zebrafish mutantlessen: an experimental model for congenital enteric neuropathies

Neurogastroenterology and Motility, Dec 20, 2015

The aim of the study was to characterize the enteric phenotype of the zebrafish mutant lessen. • ... more The aim of the study was to characterize the enteric phenotype of the zebrafish mutant lessen. • Wild-type and lessen zebrafish were used in vivo at 3-6 days post fertilization to investigate the development of intestinal motility patterns, while the intrinsic innervation and the ICC network were investigated in the intestinal segments using immunohistochemistry. • Mutant zebrafish show a disturbed and delayed onset of intestinal motility, accompanied by a disturbed formation of the ENS and ICC network. • The distal parts of the intestine were most affected in the lessen mutant, while the proximal and mid parts were less affected. • The zebrafish mutant lessen shows a defect in the development of the ENS, ICC network and intestinal motility, and may serve as an appropriate animal model to investigate the pathogenesis of CEN.

Research paper thumbnail of Unbiased characterization of the larval zebrafish enteric nervous system at a single cell transcriptomic level

Research paper thumbnail of Additional file 5: Table S4. of Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

Comparison of de novo mutation rates. (XLSX 9 kb)

Research paper thumbnail of Additional file 2: of Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

Supplementary methods and Figures S1â S11. (PDF 4258 kb)

Research paper thumbnail of Additional file 11: Table S10. of Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

gRNA and primers for zebrafish knockout and T7E1 assay. (XLSX 9 kb)

Research paper thumbnail of Unbiased intestinal single cell transcriptomics reveals previously uncharacterized enteric nervous system populations in larval zebrafish

The enteric nervous system (ENS) regulates many gastrointestinal functions including peristalsis,... more The enteric nervous system (ENS) regulates many gastrointestinal functions including peristalsis, immune regulation and uptake of nutrients. Defects in the ENS can lead to severe enteric neuropathies such as Hirschsprung disease (HSCR), which is caused by defective ENS development. Zebrafish have proven to be fruitful in the identification of novel genes involved in ENS development and HSCR pathology. However, the composition and specification of enteric neurons and glial subtypes of the larval zebrafish at a single cell level, remains mainly unexplored. Here, we performed single cell RNA sequencing of zebrafish ENS at 5 days post-fertilization. We identified both vagal neural crest progenitors and Schwann cell precursors, as well as four clusters of early differentiated neurons. Interestingly, since we took an unbiased approach where we sequenced total intestines, an elavl3+/phox2bb- population of neurons and the presence of cx43+/phox2bb- enteric glia were identified in larval zeb...

Research paper thumbnail of Additional file 10: Table S9. of Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

Sequences and dosages of antisense morpholinos. (XLSX 10 kb)

Research paper thumbnail of The Netherlands

syndrome to microtubule dynamics and neuronal differentiation

Research paper thumbnail of The long Filamin-A isoform is required for intestinal development and motility: implications for chronic intestinal pseudo-obstruction

Human Molecular Genetics

Filamin A (FLNA) is a cytoplasmic actin binding protein, recently shown to be expressed as a long... more Filamin A (FLNA) is a cytoplasmic actin binding protein, recently shown to be expressed as a long and short isoform. Mutations in FLNA are associated with a wide spectrum of disorders, including an X-linked form of chronic intestinal pseudo-obstruction (CIPO). However, the role of FLNA in intestinal development and function is largely unknown. In this study, we show that FLNA is expressed in the muscle layer of the small intestine from early human fetal stages. Expression of FLNA variants associated with CIPO, blocked expression of the long flna isoform and led to an overall reduction of RNA and protein levels. As a consequence, contractility of human intestinal smooth muscle cells was affected. Lastly, our transgenic zebrafish line showed that the flna long isoform is required for intestinal elongation and peristalsis. Histological analysis revealed structural and architectural changes in the intestinal smooth muscle of homozygous fish, likely triggered by the abnormal expression o...

Research paper thumbnail of Additional file 6: Table S5. of Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

Joint distribution of common and rare variants for each trio proband. (XLSX 13 kb)

Research paper thumbnail of Additional file 4: Table S3. of Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

Statistical models for mutation prediction. (XLSX 9 kb)

Research paper thumbnail of Additional file 3: Table S2. of Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

Quality metrics for sequencing reads and variants from different cohorts. (XLSX 9 kb)

Research paper thumbnail of Additional file 12: Table S11. of Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

Primers for expression analysis in zebrafish. (XLSX 9 kb)

Research paper thumbnail of Additional file 9: Table S8. of Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

Characteristics of 116 ENS-related HSCR candidate genes. (XLSX 32 kb)

Research paper thumbnail of Additional file 8: Table S7. of Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

Bioinformatics prediction of the functional impact of DNMs. (XLSX 11 kb)

Research paper thumbnail of Additional file 7: Table S6. of Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

Gene recurrence and burden test. (XLSX 14 kb)

Research paper thumbnail of Genetic model system studies of the development of the enteric nervous system, gut motility and Hirschsprung’s disease

Neurogastroenterology & Motility, 2009

The enteric nervous system (ENS) is the largest and most complicated subdivision of the periphera... more The enteric nervous system (ENS) is the largest and most complicated subdivision of the peripheral nervous system. Its action is necessary to regulate many of the functions of the gastrointestinal tract including its motility. Whilst the ENS has been studied extensively by developmental biologists, neuroscientists and physiologists for several decades it has only been since the early 1990s that the molecular and genetic basis of ENS development has begun to emerge. Central to this understanding has been the use of genetic model organisms. In this article, we will discuss recent advances that have been achieved using both mouse and zebrafish model genetic systems that have led to new insights into ENS development and the genetic basis of HirschsprungÕs disease.

Research paper thumbnail of KBP interacts with SCG10, linking Goldberg-Shprintzen syndrome to microtubule dynamics and neuronal differentiation

Human Molecular Genetics, 2010

Goldberg–Shprintzen syndrome (GOSHS) is a rare clinical disorder characterized by central and ent... more Goldberg–Shprintzen syndrome (GOSHS) is a rare clinical disorder characterized by central and enteric nervous system defects. This syndrome is caused by inactivating mutations in the Kinesin Binding Protein (KBP) gene, which encodes a protein of which the precise function is largely unclear. We show that KBP expression is up-regulated during neuronal development in mouse cortical neurons. Moreover, KBP-depleted PC12 cells were defective in nerve growth factor-induced differentiation and neurite outgrowth, suggesting that KBP is required for cell differentiation and neurite development. To identify KBP interacting proteins, we performed a yeast two-hybrid screen and found that KBP binds almost exclusively to microtubule associated or related proteins, specifically SCG10 and several kinesins. We confirmed these results by validating KBP interaction with one of these proteins: SCG10, a microtubule destabilizing protein. Zebrafish studies further demonstrated an epistatic interaction be...

Research paper thumbnail of Additional file 1: Table S1. of Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

Information on samples included in the study. (XLSX 10 kb)

Research paper thumbnail of Assessment of left ventricular function in chronic alcoholics by real-time three-dimensional echocardiography

Medicine

Chronic alcohol consumption may lead to progressive cardiac dysfunction. The aim of this study wa... more Chronic alcohol consumption may lead to progressive cardiac dysfunction. The aim of this study was to evaluate the feasibility of using real-time 3-dimensional echocardiography (3DE) on assessing left ventricular (LV) function in chronic alcoholics. We classified 92 male alcoholics into mild, moderate, and severe groups; 30 age-matched controls were also recruited. LV enddiastolic volume (LVEDV), LV end-systolic volume (LVESV), LV ejection fraction (LVEF), LV mass (LVM), LV mass index (LVMI), and systolic dyssynchrony index (SDI) were measured by 3DE and 2-dimensional echocardiography (2DE). Compared to the control group, LV volume and mass were higher in the moderate and severe alcoholic groups (P < 0.05). The severe alcoholic (symptomatic) group demonstrated decreased LVEF and increased SDI (detected by 3DE) (P < 0.05). Real-time 3DE can detect the increases of LV volumes and mass in asymptomatic alcoholics, and the changes of LVEF and systolic synchrony index in symptomatic alcoholics. Abbreviations: 2DE = 2-dimensional echocardiography, 3DE = 3-dimensional echocardiography, ACM = alcoholic cardiomyopathy, BSA = body surface area, CMR = cardiac magnetic resonance, ICC = intraclass correlation coefficient, LVEDV = left ventricular end-diastolic volume, LVEF = left ventricular ejection fraction, LVESV = left ventricular end-systolic volume, LVM = left ventricular mass, LVMI = left ventricular mass index, SD = standard deviation, SDI = systolic dyssynchrony index.

Research paper thumbnail of The zebrafish mutantlessen: an experimental model for congenital enteric neuropathies

Neurogastroenterology and Motility, Dec 20, 2015

The aim of the study was to characterize the enteric phenotype of the zebrafish mutant lessen. • ... more The aim of the study was to characterize the enteric phenotype of the zebrafish mutant lessen. • Wild-type and lessen zebrafish were used in vivo at 3-6 days post fertilization to investigate the development of intestinal motility patterns, while the intrinsic innervation and the ICC network were investigated in the intestinal segments using immunohistochemistry. • Mutant zebrafish show a disturbed and delayed onset of intestinal motility, accompanied by a disturbed formation of the ENS and ICC network. • The distal parts of the intestine were most affected in the lessen mutant, while the proximal and mid parts were less affected. • The zebrafish mutant lessen shows a defect in the development of the ENS, ICC network and intestinal motility, and may serve as an appropriate animal model to investigate the pathogenesis of CEN.

Research paper thumbnail of Unbiased characterization of the larval zebrafish enteric nervous system at a single cell transcriptomic level

Research paper thumbnail of Additional file 5: Table S4. of Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

Comparison of de novo mutation rates. (XLSX 9 kb)

Research paper thumbnail of Additional file 2: of Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

Supplementary methods and Figures S1â S11. (PDF 4258 kb)

Research paper thumbnail of Additional file 11: Table S10. of Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

gRNA and primers for zebrafish knockout and T7E1 assay. (XLSX 9 kb)

Research paper thumbnail of Unbiased intestinal single cell transcriptomics reveals previously uncharacterized enteric nervous system populations in larval zebrafish

The enteric nervous system (ENS) regulates many gastrointestinal functions including peristalsis,... more The enteric nervous system (ENS) regulates many gastrointestinal functions including peristalsis, immune regulation and uptake of nutrients. Defects in the ENS can lead to severe enteric neuropathies such as Hirschsprung disease (HSCR), which is caused by defective ENS development. Zebrafish have proven to be fruitful in the identification of novel genes involved in ENS development and HSCR pathology. However, the composition and specification of enteric neurons and glial subtypes of the larval zebrafish at a single cell level, remains mainly unexplored. Here, we performed single cell RNA sequencing of zebrafish ENS at 5 days post-fertilization. We identified both vagal neural crest progenitors and Schwann cell precursors, as well as four clusters of early differentiated neurons. Interestingly, since we took an unbiased approach where we sequenced total intestines, an elavl3+/phox2bb- population of neurons and the presence of cx43+/phox2bb- enteric glia were identified in larval zeb...

Research paper thumbnail of Additional file 10: Table S9. of Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

Sequences and dosages of antisense morpholinos. (XLSX 10 kb)

Research paper thumbnail of The Netherlands

syndrome to microtubule dynamics and neuronal differentiation

Research paper thumbnail of The long Filamin-A isoform is required for intestinal development and motility: implications for chronic intestinal pseudo-obstruction

Human Molecular Genetics

Filamin A (FLNA) is a cytoplasmic actin binding protein, recently shown to be expressed as a long... more Filamin A (FLNA) is a cytoplasmic actin binding protein, recently shown to be expressed as a long and short isoform. Mutations in FLNA are associated with a wide spectrum of disorders, including an X-linked form of chronic intestinal pseudo-obstruction (CIPO). However, the role of FLNA in intestinal development and function is largely unknown. In this study, we show that FLNA is expressed in the muscle layer of the small intestine from early human fetal stages. Expression of FLNA variants associated with CIPO, blocked expression of the long flna isoform and led to an overall reduction of RNA and protein levels. As a consequence, contractility of human intestinal smooth muscle cells was affected. Lastly, our transgenic zebrafish line showed that the flna long isoform is required for intestinal elongation and peristalsis. Histological analysis revealed structural and architectural changes in the intestinal smooth muscle of homozygous fish, likely triggered by the abnormal expression o...

Research paper thumbnail of Additional file 6: Table S5. of Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

Joint distribution of common and rare variants for each trio proband. (XLSX 13 kb)

Research paper thumbnail of Additional file 4: Table S3. of Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

Statistical models for mutation prediction. (XLSX 9 kb)

Research paper thumbnail of Additional file 3: Table S2. of Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

Quality metrics for sequencing reads and variants from different cohorts. (XLSX 9 kb)

Research paper thumbnail of Additional file 12: Table S11. of Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

Primers for expression analysis in zebrafish. (XLSX 9 kb)

Research paper thumbnail of Additional file 9: Table S8. of Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

Characteristics of 116 ENS-related HSCR candidate genes. (XLSX 32 kb)

Research paper thumbnail of Additional file 8: Table S7. of Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

Bioinformatics prediction of the functional impact of DNMs. (XLSX 11 kb)

Research paper thumbnail of Additional file 7: Table S6. of Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

Gene recurrence and burden test. (XLSX 14 kb)

Research paper thumbnail of Genetic model system studies of the development of the enteric nervous system, gut motility and Hirschsprung’s disease

Neurogastroenterology & Motility, 2009

The enteric nervous system (ENS) is the largest and most complicated subdivision of the periphera... more The enteric nervous system (ENS) is the largest and most complicated subdivision of the peripheral nervous system. Its action is necessary to regulate many of the functions of the gastrointestinal tract including its motility. Whilst the ENS has been studied extensively by developmental biologists, neuroscientists and physiologists for several decades it has only been since the early 1990s that the molecular and genetic basis of ENS development has begun to emerge. Central to this understanding has been the use of genetic model organisms. In this article, we will discuss recent advances that have been achieved using both mouse and zebrafish model genetic systems that have led to new insights into ENS development and the genetic basis of HirschsprungÕs disease.

Research paper thumbnail of KBP interacts with SCG10, linking Goldberg-Shprintzen syndrome to microtubule dynamics and neuronal differentiation

Human Molecular Genetics, 2010

Goldberg–Shprintzen syndrome (GOSHS) is a rare clinical disorder characterized by central and ent... more Goldberg–Shprintzen syndrome (GOSHS) is a rare clinical disorder characterized by central and enteric nervous system defects. This syndrome is caused by inactivating mutations in the Kinesin Binding Protein (KBP) gene, which encodes a protein of which the precise function is largely unclear. We show that KBP expression is up-regulated during neuronal development in mouse cortical neurons. Moreover, KBP-depleted PC12 cells were defective in nerve growth factor-induced differentiation and neurite outgrowth, suggesting that KBP is required for cell differentiation and neurite development. To identify KBP interacting proteins, we performed a yeast two-hybrid screen and found that KBP binds almost exclusively to microtubule associated or related proteins, specifically SCG10 and several kinesins. We confirmed these results by validating KBP interaction with one of these proteins: SCG10, a microtubule destabilizing protein. Zebrafish studies further demonstrated an epistatic interaction be...

Research paper thumbnail of Additional file 1: Table S1. of Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

Information on samples included in the study. (XLSX 10 kb)

Research paper thumbnail of Assessment of left ventricular function in chronic alcoholics by real-time three-dimensional echocardiography

Medicine

Chronic alcohol consumption may lead to progressive cardiac dysfunction. The aim of this study wa... more Chronic alcohol consumption may lead to progressive cardiac dysfunction. The aim of this study was to evaluate the feasibility of using real-time 3-dimensional echocardiography (3DE) on assessing left ventricular (LV) function in chronic alcoholics. We classified 92 male alcoholics into mild, moderate, and severe groups; 30 age-matched controls were also recruited. LV enddiastolic volume (LVEDV), LV end-systolic volume (LVESV), LV ejection fraction (LVEF), LV mass (LVM), LV mass index (LVMI), and systolic dyssynchrony index (SDI) were measured by 3DE and 2-dimensional echocardiography (2DE). Compared to the control group, LV volume and mass were higher in the moderate and severe alcoholic groups (P < 0.05). The severe alcoholic (symptomatic) group demonstrated decreased LVEF and increased SDI (detected by 3DE) (P < 0.05). Real-time 3DE can detect the increases of LV volumes and mass in asymptomatic alcoholics, and the changes of LVEF and systolic synchrony index in symptomatic alcoholics. Abbreviations: 2DE = 2-dimensional echocardiography, 3DE = 3-dimensional echocardiography, ACM = alcoholic cardiomyopathy, BSA = body surface area, CMR = cardiac magnetic resonance, ICC = intraclass correlation coefficient, LVEDV = left ventricular end-diastolic volume, LVEF = left ventricular ejection fraction, LVESV = left ventricular end-systolic volume, LVM = left ventricular mass, LVMI = left ventricular mass index, SD = standard deviation, SDI = systolic dyssynchrony index.