Fezf2 overexpression in murine cortical progenitors in vivo
Ontology highlight
ABSTRACT: Corticospinal motor neurons (CSMN) are one specialized class of cortical excitatory neurons, which connect layer Vb of the cortex to the spinal cord. a master transcription factor –Forebrain expressed zinc finger 2 (Fezf2) – has been identified that is necessary for the fate specification of CSMN. Fezf2 alone can cell-autonomously instruct the acquisition of CSMN-specific features when expressed in diverse, permissive cellular contexts, in vivo. In order to understand the molecular logic underlying the acquisition of CSMN traits upon Fezf2 expression, we compared the in vivo gene expression of FACS-purified cortical progenitors that ectopically expressed Fezf2 to control progenitors. We used in utero electroporation to deliver Fezf2GFP or CtrlGFP expression vectors to neocortical progenitors at E14.5, when they primarily generate CPN of the upper layers. Overexpression of Fezf2 in these progenitors is sufficient to instruct a fate-switch resulting in the generation of CSMN and other subtypes of corticofugal projection neurons. Fezf2GFP- and CtrlGFP -electroporated progenitors were FACS-purified at 24 and 48 hours after surgery and acutely profiled by microarrays.
Project description:Corticospinal motor neurons (CSMN) are one specialized class of cortical excitatory neurons, which connect layer Vb of the cortex to the spinal cord. a master transcription factor –Forebrain expressed zinc finger 2 (Fezf2) – has been identified that is necessary for the fate specification of CSMN. Fezf2 alone can cell-autonomously instruct the acquisition of CSMN-specific features when expressed in diverse, permissive cellular contexts, in vivo. In order to understand the molecular logic underlying the acquisition of CSMN traits upon Fezf2 expression, we compared the in vivo gene expression of FACS-purified cortical progenitors that ectopically expressed Fezf2 to control progenitors.
Project description:RNA Sequencing of E14.5 mouse cortical neurospheres in response to Fezf2 over-expression 2 replicates each of GFP-transfected or Fezf2/GFP-transfected E14.5 mouse cortical neurospheres. Paired-end sequencing 101bp.
Project description:Cortical projection neurons comprise classes of neurons connecting the cerebral cortex to distant targets in the nervous system. Each projection neuron class acquires a distinct molecular identity and develops characteristic patterns of axonal projection and dendritic arborization that determine their inputs, outputs, and distinct functions. How different aspects of neuron identity, including axonal and dendritic morphology, are coordinated appropriately in each specific neuron type is not known. A network of transcription factors, including the selector gene Fezf2, is central to specifying cortical projection neuron fates. However, regulation down-stream of these fate-determinant transcription factors to control different aspects of neuron identity is not understood, particularly as it relates to the development of distinct dendritic arbors that determine the inputs to each projection neuron class. Here we show that the miR-193b~365 microRNA cluster downstream of Fezf2 cooperatively represses the signaling molecule Mapk8 to regulate dendritic development in a neuron subtype-specific manner.
Project description:Amyotrophic Lateral Sclerosis is clinically defined as the combined degeneration of the corticospinal and corticobulbar neurons (CSN) along with the bulbar and spinal motor neurons (SMN). While a growing body of evidence points to the motor cortex, where CSN are located, as the potential initiation site of ALS, little is known about how CSN degenerate. To gain insights into the molecular mechanisms behind CSN selective degeneration, we first developed an approach to purify this neuronal population from the cerebral cortex of adult wild-type and Sod1G86R mice, combining retrograde labelling and Fluorescence Activated Cell Sorting. In parallel, a second population of cortical neurons, the callosal projection neurons (CPN) located in the layers II/III of the cerebral cortex were also purified. CPN and CSN are both cortical excitatory projection neurons but as opposed to CSN, CPN do not degenerate in Sod1G86R mice, and served as control population. CSN and CPN were purified from same animals, at two presymptomatic ages (3 and 60 days) and two symptomatic ages, 90 and 105 days. A total of 57 samples were further processed and analysed (CSN: 30d: n=4 WT and 3 Sod1G86R; 60d: n=4 WT and 4 Sod1G86R; 90d: n=4 WT and 4 Sod1G86R; 105d: n=4 WT and 2 Sod1G86R; CPN: 30d: n=4 WT and 3 Sod1G86R; 60d: n=2 WT and 3 Sod1G86R; 90d: n=4 WT and 4 Sod1G86R; 105d: n=4 WT and 4 Sod1G86R).
Project description:The goal of the study was to compare gene expression of P0 wild-type, P0 Fezf2-/- cortices, and Fezf2-/-; Fezf2-EnR cortices. Total RNAs were isolated from P0 cortices dissected from wild-type (n=3), Fezf2-/- mice (n=4), and Fezf2-/-; Fezf2-EnR cortices (n=2), following Qiagen RNAeasy kit instruction.Sequence libraries were made following Illumina RNA PolyA library preparation guide.The libaries were pair-end sequenced (150nt per end). Differentially expressed genes were identified by DESEQ.
Project description:3 subtypes of cortical projection neurons were purified by fluorescence-activated cell sorting (FACS) at 4 different stages of development from mouse cortex. A detailed description of the data set is described in Arlotta, P et al (2005) and Molyneaux, BJ et al (2009). The hybridization cocktails used here were originally applied to the Affymetrix mouse 430A arrays and submitted as GEO accession number GSE2039. The same hybridization cocktails were then applied to the Affymetrix mouse 430 2.0 arrays, and those data are contained in this series. Experiment Overall Design: Three subtypes of cortical neurons were purified by FACS at multiple stages of mouse brain development. The neuron subtypes are: corticospinal motor neurons (CSMN), callosal projection neurons (CPN), and corticotectal projection neurons (CTPN). The stages of development included embryonic day 18 (E18), postnatal day 3 (P3), postnatal day 6 (P6), and postnatal day 14 (P14). CSMN and CPN were analyzed at all four stages, while CTPN were only analyzed at P14. The replicates included in the data set are all true biological replicates with independent sample collection for each.
Project description:The cerebral cortex contains layers of neurons sequentially generated by distinct lineage-related progenitors. At the onset of corticogenesis, the first-born progenitors are apical progenitors (APs) whose asymmetric division give birth directly to neurons. Later, they switch to indirect neurogenesis by generating intermediate progenitors (IPs), which give rise to projection neurons of all cortical layers. While a direct lineage relationship between APs and IPs has been established, the molecular mechanism that controls their transition remains elusive. Our data suggest that interfering with codon translation speed triggers endoplasmic reticulum stress and the unfolded protein response (UPR), further impairing the generation of IPs and leading to microcephaly. Moreover, we demonstrate that a progressive downregulation of UPR in cortical progenitors acts as physiological signal to amplify IPs and promotes indirect neurogenesis. Thus, our findings reveal a hitherto unrecognized contribution of UPR to cell fate acquisition during mammalian brain development. Ribosome profiling and RNA-Seq of forebrains from E14.5 mouse embryos from wild type animals and mutants carrying a conditional knockout of ELP3 in cortical progenitors
Project description:Fezf2 is highly and specifically expressed in mTECs in mouse thymus and Fezf2 deficiency (Fezf2 KO) in the thymus leads to autoimmunity. However, it is unclear how Fezf2 contributes to thymic gene expression. We collected WT and Fezf2 KO mTECs by FACS, and performed microarrays to determine genes regulated by Fezf2. mTECs were subjected to RNA extraction (from WT or Fezf2 KO mTECs) and hybridization on Affymetrix microarrays.
Project description:Fezf2 is highly and specifically expressed in mTECs in mouse thymus and Fezf2 deficiency (Fezf2 KO) in the thymus leads to autoimmunity. However, it is unclear how Fezf2 contributes to thymic gene expression. We collected WT and Fezf2 KO mTECs by FACS, and performed microarrays to determine genes regulated by Fezf2.