A simple slice culture system for the imaging of nerve development in embryonic mouse

Brachmann, Isabel; Jakubick, Vera Catherine; Shakèd, Maya; Unsicker, Klaus; Tucker, Kerry L.

doi:10.1002/dvdy.21386

Cited by 13 publications

(13 citation statements)

References 35 publications

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“…Embryos, sections, and stainings were performed as described (Brachmann et al, 2007). Primary antibodies (Ab): rabbit anti-␤-galactosidase (clone 55976, ICN/Cappel) 1:2000, mouse anti-nestin (Rat 401 clone; BD PharMingen) 1:500, RC2 (DSHB) 1:10, mouse anti-␤ tubulin III (TuJ1 clone; Covance) 1:1500, rabbit anti-phospho-histone H3 (Ser10, rabbit 06 -570; Upstate) 1:200, and rabbit anti-Pax6 (AB5409, Millipore) 1:3000.…”

Section: Immunohistochemical Analysismentioning

confidence: 99%

A Crucial Role for Primary Cilia in Cortical Morphogenesis

Willaredt¹,

Hasenpusch‐Theil²,

Gardner³

et al. 2008

J. Neurosci.

128

152

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Primary cilia are important sites of signal transduction involved in a wide range of developmental and postnatal functions. Proteolytic processing of the transcription factor Gli3, for example, occurs in primary cilia, and defects in intraflagellar transport (IFT), which is crucial for the maintenance of primary cilia, can lead to severe developmental defects and diseases. Here we report an essential role of primary cilia in forebrain development. Uncovered by N-ethyl-N-nitrosourea-mutagenesis, cobblestone is a hypomorphic allele of the IFT gene Ift88, in which Ift88 mRNA and protein levels are reduced by 70 -80%. cobblestone mutants are distinguished by subpial heterotopias in the forebrain. Mutants show both severe defects in the formation of dorsomedial telencephalic structures, such as the choroid plexus, cortical hem and hippocampus, and also a relaxation of both dorsal-ventral and rostral-caudal compartmental boundaries. These defects phenocopy many of the abnormalities seen in the Gli3 mutant forebrain, and we show that Gli3 proteolytic processing is reduced, leading to an accumulation of the full-length activator isoform. In addition, we observe an upregulation of canonical Wnt signaling in the neocortex and in the caudal forebrain. Interestingly, the ultrastructure and morphology of ventricular cilia in the cobblestone mutants remains intact. Together, these results indicate a critical role for ciliary function in the developing forebrain.

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Section: Immunohistochemical Analysismentioning

confidence: 99%

A Crucial Role for Primary Cilia in Cortical Morphogenesis

Willaredt¹,

Hasenpusch‐Theil²,

Gardner³

et al. 2008

J. Neurosci.

128

152

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“…Culture conditions that facilitate neuron survival and cell body migration in embryo slice cultures of higher vertebrates are thus required. Currently, spinal cord slice cultures of higher vertebrates have been used to seed dissociated neurons on top of the slices [12][13][14] , investigate fluorescently labelled axons in the periphery of the slice 18 , or of progenitor cell behavior in early spinal cord development 19 . Slice culture conditions for later spinal cords, particularly those that maintain motor neuron development are currently lacking.…”

Section: Representative Resultsmentioning

confidence: 99%

Chicken Embryo Spinal Cord Slice Culture Protocol

Tubby

Norval

Price

2013

JoVE

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Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

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“…The following primary Abs were used, with clone name, source, and dilution indicated: mouse anti-NeuN (MAB377, Millipore, 1:1000), mouse anti-parvalbumin (clone PARV-19, Sigma, 1:2000), rabbit anti-calretinin (7699/4, Swant, 1:3000), mouse anti-calbindin (300, Swant, 1:3000), rabbit anti-cleaved-caspase-3 (clone 5A1, Cell Signaling Technologies, 1:200), rabbit anti-phospho-histone H3 (Ser10, rabbit 06-570; Upstate) 1:200, and rabbit anti-ER81 (kind gift of Silvia Arber, 1:200). For caspase-3, phospho-histone H3, and ER81 stains, secondary Abs were used as described (Brachmann et al, 2007). …”

Section: Methodsmentioning

confidence: 99%

“…The following primary antibodies were used: FITC-coupled goat anti-GFP (G8965-12A, US Biologicals, 1:2000), mouse anti-HA-tag (clone 6E2, Cell Signaling Technologies, 1:200), mouse anti-c-Myc-tag (clone 9E10, sc-40, Santa Cruz Biotechnology, 1:100), rabbit anti-cleaved-caspase-3 (Asp175, clone 5A1, Cell Signaling Technologies, 1:200. Secondary Abs were used as described (Brachmann et al, 2007). DAPI (2 μg/ml) was used to stain nuclei.…”

Section: Methodsmentioning

confidence: 99%

Control of Postnatal Apoptosis in the Neocortex byRhoA-Subfamily GTPases Determines Neuronal Density

Sanno¹,

Shen²,

Kuru³

et al. 2010

J. Neurosci.

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Apoptosis of neurons in the maturing neocortex has been recorded in a wide variety of mammals, but very little is known about its effects on cortical differentiation. Recent research has implicated the RhoA GTPase subfamily in the control of apoptosis in the developing nervous system and in other tissue types. Rho GTPases are important components of the signaling pathways linking extracellular signals to the cytoskeleton. To investigate the role of the RhoA GTPase subfamily in neocortical apoptosis and differentiation, we have engineered a mouse line in which a dominant-negative RhoA mutant (N19 -RhoA) is expressed from the Mapt locus, such that all neurons of the developing nervous system are expressing the N19 -RhoA inhibitor. Postnatal expression of N19 -RhoA led to no major changes in neocortical anatomy. Six layers of the neocortex developed and barrels (whisker-related neural modules) formed in layer IV. However, the density and absolute number of neurons in the somatosensory cortex increased by 12-26% compared with wild-type littermates. This was not explained by a change in the migration of neurons during the formation of cortical layers but rather by a large decrease in the amount of neuronal apoptosis at postnatal day 5, the developmental maximum of cortical apoptosis. In addition, overexpression of RhoA in cortical neurons was seen to cause high levels of apoptosis. These results demonstrate that RhoA-subfamily members play a major role in developmental apoptosis in postnatal neocortex of the mouse but that decreased apoptosis does not alter cortical cytoarchitecture and patterning.

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A simple slice culture system for the imaging of nerve development in embryonic mouse

Cited by 13 publications

References 35 publications

A Crucial Role for Primary Cilia in Cortical Morphogenesis

A Crucial Role for Primary Cilia in Cortical Morphogenesis

Chicken Embryo Spinal Cord Slice Culture Protocol

Control of Postnatal Apoptosis in the Neocortex byRhoA-Subfamily GTPases Determines Neuronal Density

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