Moody SA et al. (2013), On becoming neural: what the embryo can tell us...

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Am J Stem Cells 2013 Jun 30;22:74-94.

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On becoming neural: what the embryo can tell us about differentiating neural stem cells.

Moody SA , Klein SL , Karpinski BA , Maynard TM , Lamantia AS .

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THE EARLIEST STEPS OF EMBRYONIC NEURAL DEVELOPMENT ARE ORCHESTRATED BY SETS OF TRANSCRIPTION FACTORS THAT CONTROL AT LEAST THREE PROCESSES: the maintenance of proliferative, pluripotent precursors that expand the neural ectoderm; their transition to neurally committed stem cells comprising the neural plate; and the onset of differentiation of neural progenitors. The transition from one step to the next requires the sequential activation of each gene set and then its down-regulation at the correct developmental times. Herein, we review how these gene sets interact in a transcriptional network to regulate these early steps in neural development. A key gene in this regulatory network is FoxD4L1, a member of the forkhead box (Fox) family of transcription factors. Knock-down experiments in Xenopus embryos show that FoxD4L1 is required for the expression of the other neural transcription factors, whereas increased FoxD4L1 levels have three different effects on these genes: up-regulation of neural ectoderm precursor genes; transient down-regulation of neural plate stem cell genes; and down-regulation of neural progenitor differentiation genes. These different effects indicate that FoxD4L1 maintains neural ectodermal precursors in an immature, proliferative state, and counteracts premature neural stem cell and neural progenitor differentiation. Because it both up-regulates and down-regulates genes, we characterized the regions of the FoxD4L1 protein that are specifically involved in these transcriptional functions. We identified a transcriptional activation domain in the N-terminus and at least two domains in the C-terminus that are required for transcriptional repression. These functional domains are highly conserved in the mouse and human homologues. Preliminary studies of the related FoxD4 gene in cultured mouse embryonic stem cells indicate that it has a similar role in promoting immature neural ectodermal precursors and delaying neural progenitor differentiation. These studies in Xenopus embryos and mouse embryonic stem cells indicate that FoxD4L1/FoxD4 has the important function of regulating the balance between the genes that expand neural ectodermal precursors and those that promote neural stem/progenitor differentiation. Thus, regulating the level of expression of FoxD4 may be important in stem cell protocols designed to create immature neural cells for therapeutic uses.

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Species referenced: Xenopus
Genes referenced: foxd4l1.1 gmnn h3-3a krt12.4 lif mapre2 nsd1 sox11 sox2 sox3 tle5 zic2

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	Fugure 1. Several transcription factor expression domains overlap in the newly induced neural ectoderm. Upper left cartoon diagrams the overlapping expression domains of 12 neural transcription factors from a dorsal view in an early gastrulating Xenopus embryo. Similarly oriented embryos show the broad expression domains of Gmnn and Sox3, and the slightly smaller domains of FoxD4L1, Zic2 and Sox2. At this stage of development, the Irx genes are weakly expressed in the same domain as FoxD4L1/Zic/Sox2, with higher expression in lateral patches.
	Figure 2. After the neural ectoderm is induced, its fate is stabilized by transcription factors that oppose BMP and Wnt signals. Top: Side view of a late gastrula Xenopus embryo after neural induction has occurred. The ectoderm is divided into two domains: neural (blue), which will give rise to the neural plate, and non-neural (white), which will give rise to the epidermis. Ventrally, BMP and Wnt signals activate epidermis specific transcription factors (e.g., Msx, Dlx, FoxI, AP2) and Vent transcription factors that repress neural ectoderm. Dorsally, neural induction activates transcription factors (e.g., FoxD4, Gmnn, Sox, Irx) that stabilize neural fate by opposing BMP and Wnt activities. Bottom: The two transcriptionally different ectodermal domains are illustrated by expression of epidermis-specific Cytokeratin in the epidermis (Epi) and Sox2 expression in the neural plate (NP).
	Figure 3. FoxD4L1 increases the number of proliferative cells when expressed in either neural ectoderm or epidermis. Clones of cells, identified by blue cytoplasmic expression of the beta-galactosidase lineage marker, were immunostained for the mitosis marker, phosphorylated histone H3 (PH3). The mean number of labeled cells in the clone (arrows in top panel) was counted. Expression of FoxD4L1 in the clone significantly increased the number of PH3-positive cells over controls (p<0.05; t-test).
	Figure 4. Neural transcription factors can be divided into three groups that act sequentially during the transition from a newly induced neural ectoderm (left side) to the onset of neuronal differentiation (right side). At early stages, neural ectodermal precursors (green) express high levels of FoxD4L1, which directly up-regulates Gmnn and Zic2, transiently down-regulates the Sox neural plate stem cell genes (yellow) directly and/or indirectly, and down-regulates the neural progenitor genes (red). FoxD4L1 also opposes epidermal fate by down-regulating the BMP pathway. As FoxD4L1, Gmnn and Zic2 levels decrease, the neural ectoderm transitions to the neural plate and high levels of Sox gene expression. Finally, as the neural plate stem cells begin to differentiate, neural precursor gene expression predominates.
	Figure 5. The neural ectoderm gene regulatory network that is active as cells transition from neural ectoderm precursors to neural plate stem cells and then to differentiating neural progenitors. Knock-down experiments demonstrate that FoxD4L1 is required for the expression of the other genes in this network. FoxD4L1 directly up-regulates Geminin, Zic2 and Sox11 (blue arrows). These three genes also regulate each other. Together, the NE precursor genes delay the expression of neural plate stem cell genes (Sox2, Sox3) and down-regulate neural progenitor genes (Zic, Irx), which are required for the expression of the bHLH neuronal differentiation genes. Zic and Irx also feedback to down-regulate FoxD4L1. Arrows depict up-regulation; bars depict down-regulation; diamonds depict delayed expression.
	Figure 6. Functional domains of the FoxD4L1 protein are conserved across human, mouse, fish and frog. A: Within the first 40 amino acids in the N-terminus of FoxD4/FoxD4L1 proteins there is an acidic blob (AB) region (red line) that contains a short Î²-strand (IDIL/IDVL) flanked by negatively-charged (-) acidic amino acids (e.g., D, E). Structure-function studies carried out in Xenopus embryos show that this region is responsible for the transcriptional activation activity of FoxD4L1, and that a glycine residue (G) downstream of the Î²-strand is required for flexibility in the protein to bring the two acidic domains in proximity. B: Within the C-terminal region of the FoxD4/FoxD4L1 proteins is a conserved Eh-1 motif (green bar), which binds the Groucho/Grg transcriptional repressor co-factor, and a conserved region that is predicted to form an Î±-helical structure (blue bar). Deletion of the entire region (ÎRII-Cterm; red bar) eliminates the transcriptional repressive activity of FoxD4L1. Mutation of the Eh-1 motif (called A6) that changes the sequence from FSIENIM to AAAAAAM prevents Grg binding and reduces repression. In addition, mutation of a single amino acid predicted to break the Î±-helix (P to Q) reduces repression. These experiments indicate that both Grg binding and the Î±-helical structure are required for full repressive activity of the protein. Arrows at conserved L and Q indicate control mutations near the Î±-helix that had no effect on repressive function of the protein.
	Figure 7. The timing of gene expression in a neuronal differentiation protocol of mouse embryonic stem cells is similar to that in a frog embryo. A: Mouse embryonic stem cells (ESC) are grown as colonies on a STO fibroblast feeder layer in a medium that contains 15% serum, LIF and Î²-mercaptoethanol. They form embryoid bodies (EB) when removed from the feeder layer, cultured in a medium that contains 10% serum and no LIF or Î²-mercaptoethanol, and shaken to keep them non-adherent. If EBs are treated for two days with retinoic acid (RA), and then cultured for an additional 3-5 days without RA they differentiate into neurons that express the neuron-specific Class III Î²-tubulin identified by the TuJ1 antibody, indicated by red immunofluorescent staining. Nuclei are stained blue with DAPI. B: The neuronal differentiation protocol used, as described above, indicating the number of days in culture (EB1, EB2, etc.) and demarcating the 2-day period of exposure to RA. Below the protocol is a summary of gene expression results from real-time quantitative PCR assays (qPCR) that indicate that mouse FoxD4 is activated immediately upon RA treatment, and it subsides shortly after RA removal from the culture. The up-regulation of FoxD4 coincides with the loss of expression of pluripotency genes (FoxD3, Oct4). The down-regulation of FoxD4 coincides with the expression of neural stem cell (N-Cadherin) and neural progenitor (Zic1) genes. Above the protocol is a summary of gene expression results using immunofluorescence assays that counted the percentage of cells in the EB that expressed various proteins. In agreement with the qPCR assays, the up-regulation of FoxD4 coincides with loss of expression of a pluripotency protein (Nanog); the down-regulation of FoxD4 coincides with expression of a neural stem cell protein (Nestin), ultimately resulting in the production of mature neurons, as indicated by antibodies against neuron-specific Î²-tubulin (TuJ1) and neurofilament (NF) proteins.

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Aruga, Zic1 promotes the expansion of dorsal neural progenitors in spinal cord by inhibiting neuronal differentiation. 2002, Pubmed