- Email: [email protected]
Expression profile of proneural genes in developing hypothalamus
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Abstracts / Int. J. Devl Neuroscience 47 (2015) 1–131
ISDN2014 0379
ISDN2014 0380
Innervation of peripheral and central auditory tissues by human embryonic stem cell-derived neurons in vitro
Expression profile of proneural genes in developing hypothalamus
T. Hyakumura 1,∗ , K. Needham 1 , M. Dottori 2 , B.A. Nayagam 1,3 1 Department of Otolaryngology, University of Melbourne, Australia 2 Centre for Neural Engineering, NICTA, University of Melbourne, Australia 3 Department of Audiology and Speech Pathology, University of Melbourne, Australia E-mail address: [email protected] (T. Hyakumura).
The loss of auditory neurons that occurs with profound hearing loss is irreversible in humans. Stem cell therapy for replacement of the lost auditory neurons thereby offers a potential to restore hearing in patients with profound deafness. We have previously shown that neurons derived from human embryonic stem cells (ESCs) by treatment with Noggin and small molecule Y27632 are electrically active, and capable of innervating rat sensory hair cells in vitro. This project aims to further investigate the formation of synaptic connections between hESC-derived neurons and target auditory tissues in vitro, as stem cells must be capable of synapse formation for functional improvement after deafness. Human ESC-derived neurons were co-cultured with their peripheral target tissue, sensory hair cells, from postnatal day 1–3 rats (n = 60), or with their central target tissue, cochlear nucleus slices, from postnatal day 9-12 rats (n = 12) in vitro for 2 weeks. Synapse formation was examined using immunofluorescence and confocal microscopy. Human ESC-derived neurons innervated the rows of sensory hair cells in all co-cultures. Immunoreactivity to pre-synaptic markers synapsin 1 and vesicular glutamate transporter-1 was found near the site of innervation, similarly to auditory neurons reinnervating the hair cells. Numerous synapsin 1-positive neurites and vesicular glutamate transporter-1-positive neurites of human ESC-derived neurons also innervated cochlear nucleus slices. Human ESC-derived neurons are capable of innervating both peripheral and central targets in vitro, and these newly formed synapses contain pre-synaptic markers, and the correct glutamatergic phenotype. This assay will allow investigation of the timing and further charactrisation of synaptic connections between ESC-derived neurons with their peripheral and central targets in vitro. Synapse formation is an essential component in the development of a stem cell therapy for profound hearing loss.
S. Aslan Pour 1,∗ , N. Klenin 2 , D.M. Kurrasch 1,3 1 Department of Neuroscience, Alberta Children’s Hospital Research Institute, Hotchkiss Brain Institute, University of Calgary, Calgary, Canada 2 Department Biochemistry & Molecular Biology, Alberta Children’s Hospital Research Institute, Hotchkiss Brain Institute, University of Calgary, Calgary, Canada 3 Department of Medical Genetics, Alberta Children’s Hospital Research Institute, Hotchkiss Brain Institute, University of Calgary, Calgary, Canada E-mail address: [email protected] (S. Aslan Pour).
Hypothalamus is the controlling centre for many different essential physiological functions such as hunger, reproduction, body temperature and circadian rhythms, however its developmental aspects remain poorly understood. Since proneural genes are one of the key drivers in neurogenesis throughout the brain, we investigated whether proneural genes play a role in embryonic hypothalamus development. Proneural genes encode class II bHLH transcription factors that are uniquely expressed in different hypothalamic progenitor pools, leading us to reason that each proneural gene might give rise to specific hypothalamic neuronal subpopulation. We chose Ascl1 (Mash1), Neurogenin1 (Ngn1) and Neurogenin2 (Ngn2) as target genes of this study since they play an important role in specifying of cortical neuron and are also expressed in hypothalamic progenitors. To investigate more fully the expression pattern of these genes in embryonic hypothalamus we dissected wild type (WT) mice at different embryonic stages from e10.5 to P0, the period that encompassed hypothalamic neurogenesis and migration. We detected Ngn1 and Ngn2 expression at early (e10.5) and peak (e12.5) developmental stages, with reduced expression at later developmental stages (e14.5) and reaching undetectable levels by late developmental stages (e16.5, P0). In contrast to Ngn1 and Ngn2, Ascl1 was expressed poorly at early developmental stages (e10.5), however transcript levels rapidly increased by e12.5 and reached maximum expression by late developmental stages (e14.5, 16.5). Consistent with Ngn1 and Ngn2, Ascl1 was not expressed in mature nuclei (P0). In conclusion, here we showed that Ascl1, Ngn1 and Ngn2 have distinct temporal expression profiles. This suggests that different proneural genes might play a role in establishing hypothalamic neuronal identities during development.
http://dx.doi.org/10.1016/j.ijdevneu.2015.04.299 http://dx.doi.org/10.1016/j.ijdevneu.2015.04.300
Abstracts / Int. J. Devl Neuroscience 47 (2015) 1–131
ISDN2014 0379
ISDN2014 0380
Innervation of peripheral and central auditory tissues by human embryonic stem cell-derived neurons in vitro
Expression profile of proneural genes in developing hypothalamus
T. Hyakumura 1,∗ , K. Needham 1 , M. Dottori 2 , B.A. Nayagam 1,3 1 Department of Otolaryngology, University of Melbourne, Australia 2 Centre for Neural Engineering, NICTA, University of Melbourne, Australia 3 Department of Audiology and Speech Pathology, University of Melbourne, Australia E-mail address: [email protected] (T. Hyakumura).
The loss of auditory neurons that occurs with profound hearing loss is irreversible in humans. Stem cell therapy for replacement of the lost auditory neurons thereby offers a potential to restore hearing in patients with profound deafness. We have previously shown that neurons derived from human embryonic stem cells (ESCs) by treatment with Noggin and small molecule Y27632 are electrically active, and capable of innervating rat sensory hair cells in vitro. This project aims to further investigate the formation of synaptic connections between hESC-derived neurons and target auditory tissues in vitro, as stem cells must be capable of synapse formation for functional improvement after deafness. Human ESC-derived neurons were co-cultured with their peripheral target tissue, sensory hair cells, from postnatal day 1–3 rats (n = 60), or with their central target tissue, cochlear nucleus slices, from postnatal day 9-12 rats (n = 12) in vitro for 2 weeks. Synapse formation was examined using immunofluorescence and confocal microscopy. Human ESC-derived neurons innervated the rows of sensory hair cells in all co-cultures. Immunoreactivity to pre-synaptic markers synapsin 1 and vesicular glutamate transporter-1 was found near the site of innervation, similarly to auditory neurons reinnervating the hair cells. Numerous synapsin 1-positive neurites and vesicular glutamate transporter-1-positive neurites of human ESC-derived neurons also innervated cochlear nucleus slices. Human ESC-derived neurons are capable of innervating both peripheral and central targets in vitro, and these newly formed synapses contain pre-synaptic markers, and the correct glutamatergic phenotype. This assay will allow investigation of the timing and further charactrisation of synaptic connections between ESC-derived neurons with their peripheral and central targets in vitro. Synapse formation is an essential component in the development of a stem cell therapy for profound hearing loss.
S. Aslan Pour 1,∗ , N. Klenin 2 , D.M. Kurrasch 1,3 1 Department of Neuroscience, Alberta Children’s Hospital Research Institute, Hotchkiss Brain Institute, University of Calgary, Calgary, Canada 2 Department Biochemistry & Molecular Biology, Alberta Children’s Hospital Research Institute, Hotchkiss Brain Institute, University of Calgary, Calgary, Canada 3 Department of Medical Genetics, Alberta Children’s Hospital Research Institute, Hotchkiss Brain Institute, University of Calgary, Calgary, Canada E-mail address: [email protected] (S. Aslan Pour).
Hypothalamus is the controlling centre for many different essential physiological functions such as hunger, reproduction, body temperature and circadian rhythms, however its developmental aspects remain poorly understood. Since proneural genes are one of the key drivers in neurogenesis throughout the brain, we investigated whether proneural genes play a role in embryonic hypothalamus development. Proneural genes encode class II bHLH transcription factors that are uniquely expressed in different hypothalamic progenitor pools, leading us to reason that each proneural gene might give rise to specific hypothalamic neuronal subpopulation. We chose Ascl1 (Mash1), Neurogenin1 (Ngn1) and Neurogenin2 (Ngn2) as target genes of this study since they play an important role in specifying of cortical neuron and are also expressed in hypothalamic progenitors. To investigate more fully the expression pattern of these genes in embryonic hypothalamus we dissected wild type (WT) mice at different embryonic stages from e10.5 to P0, the period that encompassed hypothalamic neurogenesis and migration. We detected Ngn1 and Ngn2 expression at early (e10.5) and peak (e12.5) developmental stages, with reduced expression at later developmental stages (e14.5) and reaching undetectable levels by late developmental stages (e16.5, P0). In contrast to Ngn1 and Ngn2, Ascl1 was expressed poorly at early developmental stages (e10.5), however transcript levels rapidly increased by e12.5 and reached maximum expression by late developmental stages (e14.5, 16.5). Consistent with Ngn1 and Ngn2, Ascl1 was not expressed in mature nuclei (P0). In conclusion, here we showed that Ascl1, Ngn1 and Ngn2 have distinct temporal expression profiles. This suggests that different proneural genes might play a role in establishing hypothalamic neuronal identities during development.
http://dx.doi.org/10.1016/j.ijdevneu.2015.04.299 http://dx.doi.org/10.1016/j.ijdevneu.2015.04.300