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In JoVE (4)
- Mouse Adrenal Chromaffin Cell Isolation
- Growth Factor-Coated Bead Placement on Dorsal Forebrain Explants
- Culture of Mouse Neural Stem Cell Precursors
- Flash Freezing and Cryosectioning E12.5 Mouse Brain
Other Publications (8)
- Development (Cambridge, England)
- Development (Cambridge, England)
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Proceedings of the National Academy of Sciences of the United States of America
- Cell Stem Cell
- Proceedings of the National Academy of Sciences of the United States of America
Articles by D. Spencer Currle in JoVE
Mouse Adrenal Chromaffin Cell Isolation
Aaron Kolski-Andreaco1, Haijiang Cai2,3, D. Spencer Currle4, K. George Chandy1, Robert H. Chow2,3
1Department of Physiology and Biophysics, University of California, Irvine (UCI), 2Department of Physiology and Biophysics, University of Southern California, Keck School of Medicine, 3Zilkha Neurogenetic Institute, University of Southern California, Keck School of Medicine, 4Department of Developmental and Cell Biology, University of California, Irvine (UCI)
Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting. This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex. The digestion of the medulla into single chromaffin cells is then demonstrated.
Growth Factor-Coated Bead Placement on Dorsal Forebrain Explants
D. Spencer Currle1, Aaron Kolski-Andreaco2, Edwin S. Monuki3
1Department of Developmental and Cell Biology, University of California, Irvine (UCI), 2Department of Physiology and Biophysics, University of California, Irvine (UCI), 3Department of Pathology, University of California, Irvine (UCI)
This video demonstrates two methods for preparing and placing beads, which have been coated with growth factor, on explants of the developing cerebral cortex. These beads can be used to induce spatially restricted gene expression on developing neural tissue such as forebrain explants. Methods are given for using both Affi-Gel beads and heparin acryllic beads.
Culture of Mouse Neural Stem Cell Precursors
D. Spencer Currle1, Jia Sheng Hu2, Aaron Kolski-Andreaco3, Edwin S. Monuki2
1Department of Developmental and Cell Biology, University of California, Irvine (UCI), 2Department of Pathology, University of California, Irvine (UCI), 3Department of Physiology and Biophysics, University of California, Irvine (UCI)
This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.
Flash Freezing and Cryosectioning E12.5 Mouse Brain
D. Spencer Currle, Edwin S. Monuki
Department of Developmental and Cell Biology, University of California, Irvine (UCI)
Demonstrated in this video are the techniques for flash freezing and sectioning embryonic brain tissue from mouse. Useful tips for using the cryostat are given, including troubleshooting methods that can be used while cutting to ensure that the resultant tissues sections are free of cracks and other distortions.
Other articles by D. Spencer Currle on PubMed
Development (Cambridge, England). Aug, 2005 | Pubmed ID: 15975937
Choroid plexus (CP) produces the cerebrospinal fluid (CSF) of the central nervous system (CNS), but little is known about the mechanisms underlying development of this important tissue. CP forms in the hindbrain (4th ventricle), diencephalon (3rd ventricle) and dorsomedial telencephalon bilaterally (lateral ventricles). All of these sites lie at or near the embryonic dorsal midline (DM), which acts as a CNS patterning center. We therefore examined DM-CP relationships using normal and Gdf7 (Bmp12) transgenic embryos to fate map or ablate DM cells. These studies revealed a Gdf7 fate map that includes most CP epithelial (CPe) cells of the hindbrain and diencephalon. In the telencephalon, Gdf7 cell lineages were found in the small anterior domain of telencephalic CPe (tCPe), but its large posterior domain was devoid of these lineages. Anterior and posterior tCPe domains, which arise within a contiguous field separate from diencephalic CPe, also exhibited different patterns of apoptosis. Despite lacking Gdf7 cell lineages, the posterior tCPe domain failed to form after ablating Gdf7-expressing DM cells at neural tube stages. The tCPe loss was associated with abrogation of high-level bone morphogenetic protein (Bmp) signaling, which is known to be required for tCPe induction. Taken together, these studies demonstrate intimate DM-CPe relationships throughout the CNS and highlight two distinct tCPe domains, including a posterior domain whose genesis depends on DM cells in a non-cell-autonomous fashion.
Development (Cambridge, England). Aug, 2006 | Pubmed ID: 16790481
During embryogenesis, the isthmic organizer, a well-described signaling center at the junction of the mid-hindbrain, establishes the cerebellar territory along the anterior/posterior axis of the neural tube. Mechanisms specifying distinct populations within the early cerebellar anlage are less defined. Using a newly developed gene expression map of the early cerebellar anlage, we demonstrate that secreted signals from the rhombomere 1 roof plate are both necessary and sufficient for specification of the adjacent cerebellar rhombic lip and its derivative fates. Surprisingly, we show that the roof plate is not absolutely required for initial specification of more distal cerebellar cell fates, but rather regulates progenitor proliferation and cell position within the cerebellar anlage. Thus, in addition to the isthmus, the roof plate represents an important signaling center controlling multiple aspects of cerebellar patterning.
The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jul, 2006 | Pubmed ID: 16855091
The roof plate is a well known signaling center in CNS development, but its roles in the developing telencephalon and the common holoprosencephaly (HPE) malformation have been uncertain. Using cellular ablations in mice, we show that roof plate cell loss causes failed midline induction and HPE in the dorsal telencephalon. This morphologic phenotype is accompanied by selective deficits in midline gene expression and a reduced activity gradient for bone morphogenetic proteins (Bmps), the major signals produced by the roof plate. In dissociated cells and mutant explants, exogenous Bmp4 is sufficient to mimic roof plate selectivity in midline gene regulation and to rescue roof plate-dependent midline patterning. Previously unrecognized neuroanatomical defects predicted by the mouse model are then confirmed in human HPE patients. These findings establish selective roles for roof plate-dependent Bmp signaling in dorsal telencephalic patterning and HPE and define novel candidate genes for the human disorder.
Proceedings of the National Academy of Sciences of the United States of America. Mar, 2008 | Pubmed ID: 18292231
Conversions of signaling gradients into sharp "all-or-none" borders are fundamental to tissue and organismal development. However, whether such conversions can be meaningfully reduced to dissociated cells in culture has been uncertain. Here we describe ultrasensitivity, the phenomenon equivalent to an all-or-none response, in dissociated neural precursor cells (NPCs) exposed to bone morphogenetic protein 4 (Bmp4). NPC ultrasensitivity is evident at the population and single-cell levels based on Msx1 induction, a well known Bmp target response, and occurs in the context of gene expression changes consistent with Bmp4 activity as a morphogen. Dissociated NPCs also display immediate early kinetics and irreversibility for Msx1 induction after brief Bmp4 exposure, which are attractive features for initial border formation. Relevance to border formation in vivo is provided by Bmp4 gain-of-function studies in explants and evidence for single-cell ultrasensitivity in normal and mutant Bmp gradient contexts in the developing forebrain. Together, these studies demonstrate relatively simple, robust, and reducible cell-intrinsic properties that contribute to developmental border formation within a signaling gradient.
Nature. Jan, 2009 | Pubmed ID: 19092805
Cancer stem cells are remarkably similar to normal stem cells: both self-renew, are multipotent and express common surface markers, for example, prominin 1 (PROM1, also called CD133). What remains unclear is whether cancer stem cells are the direct progeny of mutated stem cells or more mature cells that reacquire stem cell properties during tumour formation. Answering this question will require knowledge of whether normal stem cells are susceptible to cancer-causing mutations; however, this has proved difficult to test because the identity of most adult tissue stem cells is not known. Here, using an inducible Cre, nuclear LacZ reporter allele knocked into the Prom1 locus (Prom1(C-L)), we show that Prom1 is expressed in a variety of developing and adult tissues. Lineage-tracing studies of adult Prom1(+/C-L) mice containing the Rosa26-YFP reporter allele showed that Prom1(+) cells are located at the base of crypts in the small intestine, co-express Lgr5 (ref. 2), generate the entire intestinal epithelium, and are therefore the small intestinal stem cell. Prom1 was reported recently to mark cancer stem cells of human intestinal tumours that arise frequently as a consequence of aberrant wingless (Wnt) signalling. Activation of endogenous Wnt signalling in Prom1(+/C-L) mice containing a Cre-dependent mutant allele of beta-catenin (Ctnnb1(lox(ex3))) resulted in a gross disruption of crypt architecture and a disproportionate expansion of Prom1(+) cells at the crypt base. Lineage tracing demonstrated that the progeny of these cells replaced the mucosa of the entire small intestine with neoplastic tissue that was characterized by focal high-grade intraepithelial neoplasia and crypt adenoma formation. Although all neoplastic cells arose from Prom1(+) cells in these mice, only 7% of tumour cells retained Prom1 expression. Our data indicate that Prom1 marks stem cells in the adult small intestine that are susceptible to transformation into tumours retaining a fraction of mutant Prom1(+) tumour cells.
Lmx1a Regulates Fates and Location of Cells Originating from the Cerebellar Rhombic Lip and Telencephalic Cortical Hem
Proceedings of the National Academy of Sciences of the United States of America. Jun, 2010 | Pubmed ID: 20498066
The cerebellar rhombic lip and telencephalic cortical hem are dorsally located germinal zones which contribute substantially to neuronal diversity in the CNS, but the mechanisms that drive neurogenesis within these zones are ill defined. Using genetic fate mapping in wild-type and Lmx1a(-/-) mice, we demonstrate that Lmx1a is a critical regulator of cell-fate decisions within both these germinal zones. In the developing cerebellum, Lmx1a is expressed in the roof plate, where it is required to segregate the roof plate lineage from neuronal rhombic lip derivatives. In addition, Lmx1a is expressed in a subset of rhombic lip progenitors which produce granule cells that are predominantly restricted to the cerebellar posterior vermis. In the absence of Lmx1a, these cells precociously exit the rhombic lip and overmigrate into the anterior vermis. This overmigration is associated with premature regression of the rhombic lip and posterior vermis hypoplasia in Lmx1a(-/-) mice. These data reveal molecular organization of the cerebellar rhombic lip and introduce Lmx1a as an important regulator of rhombic lip cell-fate decisions, which are critical for maintenance of the entire rhombic lip and normal cerebellar morphogenesis. In the developing telencephalon Lmx1a is expressed in the cortical hem, and in its absence cortical hem progenitors contribute excessively to the adjacent hippocampus instead of producing Cajal-Retzius neurons. Thus, Lmx1a activity is critical for proper production of cells originating from both the cerebellar rhombic lip and the telencephalic cortical hem.
Nature. Dec, 2010 | Pubmed ID: 21150899
Medulloblastoma encompasses a collection of clinically and molecularly diverse tumour subtypes that together comprise the most common malignant childhood brain tumour. These tumours are thought to arise within the cerebellum, with approximately 25% originating from granule neuron precursor cells (GNPCs) after aberrant activation of the Sonic Hedgehog pathway (hereafter, SHH subtype). The pathological processes that drive heterogeneity among the other medulloblastoma subtypes are not known, hindering the development of much needed new therapies. Here we provide evidence that a discrete subtype of medulloblastoma that contains activating mutations in the WNT pathway effector CTNNB1 (hereafter, WNT subtype) arises outside the cerebellum from cells of the dorsal brainstem. We found that genes marking human WNT-subtype medulloblastomas are more frequently expressed in the lower rhombic lip (LRL) and embryonic dorsal brainstem than in the upper rhombic lip (URL) and developing cerebellum. Magnetic resonance imaging (MRI) and intra-operative reports showed that human WNT-subtype tumours infiltrate the dorsal brainstem, whereas SHH-subtype tumours are located within the cerebellar hemispheres. Activating mutations in Ctnnb1 had little impact on progenitor cell populations in the cerebellum, but caused the abnormal accumulation of cells on the embryonic dorsal brainstem which included aberrantly proliferating Zic1(+) precursor cells. These lesions persisted in all mutant adult mice; moreover, in 15% of cases in which Tp53 was concurrently deleted, they progressed to form medulloblastomas that recapitulated the anatomy and gene expression profiles of human WNT-subtype medulloblastoma. We provide the first evidence, to our knowledge, that subtypes of medulloblastoma have distinct cellular origins. Our data provide an explanation for the marked molecular and clinical differences between SHH- and WNT-subtype medulloblastomas and have profound implications for future research and treatment of this important childhood cancer.