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In JoVE (1)

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Articles by Sally A. Moody in JoVE

 JoVE Biology

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

1Department of Biological Sciences, The George Washington University, 2Department of Anatomy and Regenerative Biology, The George Washington University


JoVE 4458

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

Other articles by Sally A. Moody on PubMed

Cloning and Characterization of the 5'-flanking Region of the Rat Neuron-specific Class III Beta-tubulin Gene

The promoter regions of several neuron-specific structural proteins (e.g. neurofilaments, peripherin, Talpha1-tubulin) have revealed potential regulatory elements that could contribute to the choice of a neuronal phenotype during development. We initiated study of the 5'-flanking region of the rat Class III neuron-specific beta-tubulin gene (betaIII-tubulin) because this gene is expressed at the time of terminal mitosis only in neurons and thus its promoter should be an excellent tool for studying neuron-specific gene expression during the transition from proliferative progenitor cell to early neuronal differentiation. We identified the minimal promoter region needed to drive expression of the betaIII-tubulin gene. This minimal region contains multiple putative binding sites for the transcription factors SP1 and AP2, as well as a central nervous system enhancer regulatory element and an E-box. A primer extension analysis identifies a single transcription start site. We highlight several putative regulatory elements that may modulate the expression of the betaIII-tubulin gene in a stage- and tissue-specific manner. In addition, we show that the first 490 bp of the promoter are sufficient to regulate betaIII-tubulin gene expression during neuronal differentiation of PCC7 cells.

Multiple Maternal Influences on Dorsal-ventral Fate of Xenopus Animal Blastomeres

Molecular asymmetries in the animal-vegetal axis of the Xenopus oocyte are well known to regulate the formation of gametes and germ layers. Likewise, many transplantation and explant studies demonstrate that maternal dorsalizing activities are localized to the future dorsal side of the embryo after fertilization, but to date only a few of the molecules involved in this process have been shown to be asymmetrically distributed. In this report, we identify two new aspects of the maternal regulation of dorsal-ventral fate asymmetry in Xenopus blastomeres: cytoplasmic polyadenylation of dorsal maternal mRNAs and localized Wnt8b signaling. Previous studies demonstrated that there are maternal, dorsal axis-inducing RNAs localized to dorsal animal blastomeres that become activated between the 8- and 16-cell stage (Hainski and Moody [1992] Development 116:347-355; Hainski and Moody [1996] Dev. Genet. 19:210-221). We report herein that the activation of these axis-inducing dorsal mRNAs is regulated by cytoplasmic polyadenylation. We also show that maternal wnt8b mRNA is concentrated in ventral animal blastomeres. These ventral cells and exogenous Wnt8b both inhibit the dorsal fate of neighboring blastomeres in culture, indicating that a maternal Wnt signal also contributes to segregating dorsal and ventral fates.

Neural Induction, Neural Fate Stabilization, and Neural Stem Cells

The promise of stem cell therapy is expected to greatly benefit the treatment of neurodegenerative diseases. An underlying biological reason for the progressive functional losses associated with these diseases is the extremely low natural rate of self-repair in the nervous system. Although the mature CNS harbors a limited number of self-renewing stem cells, these make a significant contribution to only a few areas of brain. Therefore, it is particularly important to understand how to manipulate embryonic stem cells and adult neural stem cells so their descendants can repopulate and functionally repair damaged brain regions. A large knowledge base has been gathered about the normal processes of neural development. The time has come for this information to be applied to the problems of obtaining sufficient, neurally committed stem cells for clinical use. In this article we review the process of neural induction, by which the embryonic ectodermal cells are directed to form the neural plate, and the process of neural-fate stabilization, by which neural plate cells expand in number and consolidate their neural fate. We will present the current knowledge of the transcription factors and signaling molecules that are known to be involved in these processes. We will discuss how these factors may be relevant to manipulating embryonic stem cells to express a neural fate and to produce large numbers of neurally committed, yet undifferentiated, stem cells for transplantation therapies.

Dual Phosphorylation Controls Cdc25 Phosphatases and Mitotic Entry

Negative regulation of the Cdc25C protein phosphatase by phosphorylation on Ser 216, the 14-3-3-binding site, is an important regulatory mechanism used by cells to block mitotic entry under normal conditions and after DNA damage. During mitosis, Cdc25C is not phosphorylated on Ser 216 and ionizing radiation (IR) does not induce either phosphorylation of Ser 216, or binding to 14-3-3. Here, we show that Cdc25C is phosphorylated on Ser 214 during mitosis, which in turn prevents phosphorylation of Ser 216. Mutation of Ser 214 to Ala reconstitutes Ser 216 phosphorylation and 14-3-3 binding during mitosis. Introduction of exogenous Cdc25C(S214A) into HeLa cells depleted of endogenous Cdc25C results in a substantial delay to mitotic entry. This effect was fully reversed in a S214A/S216A double-mutant, implying that the inhibitory effect of S214A mutant was entirely dependent on Ser 216 phosphorylation. A similar regulatory mechanism may also apply to another mitotic phosphatase, Cdc25B, as well as mitotic phosphatases of other species, including Xenopus laevis. We propose that this pathway ensures that Cdc2 remains active once mitosis is initiated and is a key control mechanism for maintaining the proper order of cell-cycle transitions.

Phosphorylation of Xenopus Cdc25C at Ser285 Interferes with Ability to Activate a DNA Damage Replication Checkpoint in Pre-midblastula Embryos

We have recently demonstrated that negative regulation of human Cdc25 protein phosphatases by phosphorylation at their 14-3-3 site can be antagonized through phosphorylation at an adjacent site in the -2 position.1 Based on structural homology for different Cdc25 phosphatases, a similar regulatory pathway also could be conserved in Xenopus embryos, where cell cycle checkpoints are not operational prior to the Midblastula Transition (MBT). Here, we demonstrate that before MBT, XeCdc25C is phosphorylated on Ser285, an analogous site to Ser214 in human Cdc25C or Ser307 Cdc25B.(1) Phosphorylation of Ser285 prevents subsequent inhibitory phosphorylation of XeCdc25C on Ser287, thus maintaining XeCdc25C in an active form. Mutation of Ser285 to alanine allows the reconstitution of a DNA damage replication checkpoint. This effect is completely dependent on Ser287 phosphorylation as additional mutation of Ser287 to alanine fully reversed the cell cycle inhibitory effect of Ser285A XeCdc25C. We propose that phosphorylation of XeCdc25C Ser285 may account for the lack of a DNA replication checkpoint in cleaving Xenopus embryos prior to the MBT.

Morphogenesis During Xenopus Gastrulation Requires Wee1-mediated Inhibition of Cell Proliferation

Major developmental events in early Xenopus embryogenesis coincide with changes in the length and composition of the cell cycle. These changes are mediated in part through the regulation of CyclinB/Cdc2 and they occur at the first mitotic cell cycle, the mid-blastula transition (MBT) and at gastrulation. In this report, we investigate the contribution of maternal Wee1, a kinase inhibitor of CyclinB/Cdc2, to these crucial developmental transitions. By depleting Wee1 protein levels using antisense morpholino oligonucleotides, we show that Wee1 regulates M-phase entry and Cdc2 tyrosine phosphorylation in early gastrula embryos. Moreover, we find that Wee1 is required for key morphogenetic movements involved in gastrulation, but is not needed for the induction of zygotic transcription. In addition, Wee1 is positively regulated by tyrosine autophosphorylation in early gastrula embryos and this upregulation of Wee1 activity is required for normal gastrulation. We also show that overexpression of Cdc25C, a phosphatase that activates the CyclinB/Cdc2 complex, induces gastrulation defects that can be rescued by Wee1, providing additional evidence that cell cycle inhibition is crucial for the gastrulation process. Together, these findings further elucidate the developmental function of Wee1 and demonstrate the importance of cell cycle regulation in vertebrate morphogenesis.

Morphogenetic Movements Underlying Eye Field Formation Require Interactions Between the FGF and EphrinB1 Signaling Pathways

The definitive retinal progenitors of the eye field are specified by transcription factors that both promote a retinal fate and control cell movements that are critical for eye field formation. However, the molecular signaling pathways that regulate these movements are largely undefined. We demonstrate that both the FGF and ephrin pathways impact eye field formation. Activating the FGF pathway before gastrulation represses cellular movements in the presumptive anterior neural plate and prevents cells from expressing a retinal fate, independent of mesoderm induction or anterior-posterior patterning. Inhibiting the FGF pathway promotes cell dispersal and significantly increases eye field contribution. ephrinB1 reverse signaling is required to promote cellular movements into the eye field, and can rescue the FGF receptor-induced repression of retinal fate. These results indicate that FGF modulation of ephrin signaling regulates the positioning of retinal progenitor cells within the definitive eye field.

To Differentiate or Not to Differentiate: Regulation of Cell Fate Decisions by Being in the Right Place at the Right Time

Cell fate decisions are influenced by intrinsic factors, expression of specific transcription factor genes, and cell-to-cell signaling. In addition, a recent paper by Moore et al. demonstrates that cell movements during development influence whether a cell becomes competent to express a particular fate. Interactions between the FGF and EphrinB1 signaling pathways regulate whether embryonic cells move into the eye field and ultimately contribute to the retina. This study illustrates the importance of being located in the right place at the right time during development.

Regulation of Primary Spinal Neuron Lineages After Deletion of a Major Progenitor

Vertebrate embryos are able to reconstitute the body plan when early blastomeres are deleted, but it is not known whether this is accomplished by cells local to the lesion or by a readjustment of the entire pattern of the embryo. We distinguished between these two possibilities by studying which embryonic cells change primary spinal neuronal fates after deletion of a major spinal cord progenitor. After ablation of the V1.2 blastomere of the 16-cell Xenopus embryo, the spinal cord contained normal numbers of Rohon-Beard neurons and primary motoneurons, indicating that the remaining blastomeres numerically reconstituted these populations. Using lineage-tracing techniques we revealed a global response: 10 out of the 15 remaining blastomeres significantly changed the number of one or both neuronal types they produced. This widespread response indicates that position in the early embryo plays an important role in regulating the production of primary spinal neurons. However, not all cells are influenced solely by position; a vegetal cell transplanted into the position of the deleted V1.2 did not take on the neuronal fate of its new position. Thus, restitution of pattern relies on a combination of positional cues and intrinsic fate restrictions.

Xenopus Flotillin1, a Novel Gene Highly Expressed in the Dorsal Nervous System

The two paralogues of the Xenopus flotillin1 gene (flotillin1A and flotillin1B), which encodes a putative membrane-associated protein, were cloned from egg, cleavage, and tadpole cDNA libraries. Both mRNAs are present during oogenesis and cleavage stages. After the onset of zygotic transcription, flotillin1 transcripts are first expressed throughout the embryonic ectoderm and become enhanced in the presumptive neural ectoderm as the neural plate forms. As the neural tube forms and differentiates, flotillin1 transcripts become enriched in the dorsal half, with particularly high expression in dorsal primary neurons. At early tail bud stages, there is additional expression in the paraxial mesoderm. At late tail bud stages, flotillin1A is expressed in branchial arch mesenchyme, the overlying branchial ectoderm and in dorsal somitic mesoderm, whereas flotillin1B expression is more restricted in the dorsal neural tube and head sensory structures. This report is the first comprehensive developmental description in any animal of the expression pattern of this gene, whose protein product in several systems plays important roles in signal transduction events.

Six1 Promotes a Placodal Fate Within the Lateral Neurogenic Ectoderm by Functioning As Both a Transcriptional Activator and Repressor

Cranial placodes, which give rise to sensory organs in the vertebrate head, are important embryonic structures whose development has not been well studied because of their transient nature and paucity of molecular markers. We have used markers of pre-placodal ectoderm (PPE) (six1, eya1) to determine that gradients of both neural inducers and anteroposterior signals are necessary to induce and appropriately position the PPE. Overexpression of six1 expands the PPE at the expense of neural crest and epidermis, whereas knock-down of Six1 results in reduction of the PPE domain and expansion of the neural plate, neural crest and epidermis. Using expression of activator and repressor constructs of six1 or co-expression of wild-type six1 with activating or repressing co-factors (eya1 and groucho, respectively), we demonstrate that Six1 inhibits neural crest and epidermal genes via transcriptional repression and enhances PPE genes via transcriptional activation. Ectopic expression of neural plate, neural crest and epidermal genes in the PPE demonstrates that these factors mutually influence each other to establish the appropriate boundaries between these ectodermal domains.

Stem Cells: Cell and Developmental Biology in Regenerative Medicine

Induction and Specification of the Vertebrate Ectodermal Placodes: Precursors of the Cranial Sensory Organs

The sensory organs of the vertebrate head derive from two embryological structures, the neural crest and the ectodermal placodes. Although quite a lot is known about the secreted and transcription factors that regulate neural crest development, until recently little was known about the molecular pathways that regulate placode development. Herein we review recent findings on the induction and specification of the pre-placodal ectoderm, and the transcription factors that are involved in regulating placode fate and initial differentiation.

Step-wise Specification of Retinal Stem Cells During Normal Embryogenesis

The specification of embryonic cells to produce the retina begins at early embryonic stages as a multi-step process that gradually restricts fate potentials. First, a subset of embryonic cells becomes competent to form retina by their lack of expression of endo-mesoderm-specifying genes. From these cells, a more restricted subset is biased to form retina by virtue of their close proximity to sources of bone morphogenetic protein antagonists during neural induction. During gastrulation, the definitive RSCs (retinal stem cells) are specified as the eye field by interactions with underlying mesoderm and the expression of a network of retina-specifying genes. As the eye field is transformed into the optic vesicle and optic cup, a heterogeneous population of RPCs (retinal progenitor cells) forms to give rise to the different domains of the retina: the optic stalk, retinal pigmented epithelium and neural retina. Further diversity of RPCs appears to occur under the influences of cell-cell interactions, cytokines and combinations of regulatory genes, leading to the differentiation of a multitude of different retinal cell types. This review examines what is known about each sequential step in retinal specification during normal vertebrate development, and how that knowledge will be important to understand how RSCs might be manipulated for regenerative therapies to treat retinal diseases.

Dishevelled Mediates EphrinB1 Signalling in the Eye Field Through the Planar Cell Polarity Pathway

An important step in retinal development is the positioning of progenitors within the eye field where they receive the local environmental signals that will direct their ultimate fate. Recent evidence indicates that ephrinB1 functions in retinal progenitor movement, but the signalling pathway is unclear. We present evidence that ephrinB1 signals through its intracellular domain to control retinal progenitor movement into the eye field by interacting with Xenopus Dishevelled (Xdsh), and by using the planar cell polarity (PCP) pathway. Blocking Xdsh translation prevents retinal progeny from entering the eye field, similarly to the morpholino-mediated loss of ephrinB1 (ref. 2). Overexpression of Xdsh can rescue the phenotype induced by loss of ephrinB1, and this rescue (as well as a physical association between Xdsh and ephrinB1) is completely dependent on the DEP (Dishevelled, Egl-10, Pleckstrin) domain of Xdsh. Similar gain- and loss-of-function experiments suggest that Xdsh associates with ephrinB1 and mediates ephrinB1 signalling through downstream members of the PCP pathway during eye field formation.

Noggin Signaling from Xenopus Animal Blastomere Lineages Promotes a Neural Fate in Neighboring Vegetal Blastomere Lineages

In Xenopus, localized factors begin to regionalize embryonic fates prior to the inductive interactions that occur during gastrulation. We previously reported that an animal-to-vegetal signal that occurs prior to gastrulation promotes primary spinal neuron fate in vegetal equatorial (C-tier) blastomere lineages. Herein we demonstrate that maternal mRNA encoding noggin is enriched in animal tiers and at low concentrations in the C-tier, suggesting that the neural fates of C-tier blastomeres may be responsive to early signaling from their neighboring cells. In support of this hypothesis, experimental alteration of the levels of Noggin from animal equatorial (B-tier) or BMP4 from vegetal (D-tier) blastomeres significantly affects the numbers of primary spinal neurons derived from their neighboring C-tier blastomeres. These effects are duplicated in blastomere explants isolated at cleavage stages and cultured in the absence of gastrulation interactions. Co-culture with animal blastomeres enhanced the expression of zygotic neural markers in C-tier blastomere explants, whereas co-culture with vegetal blastomeres repressed them. The expression of these markers in C-tier explants was promoted when Noggin was transiently added to the culture during cleavage/morula stages, and repressed with the transient addition of BMP4. Reduction of Noggin translation in B-tier blastomeres by antisense morpholino oligonucleotides significantly reduced the efficacy of neural marker induction in C-tier explants. These experiments indicate that early anti-BMP signaling from the animal hemisphere recruits vegetal equatorial cells into the neural precursor pool prior to interactions that occur during gastrulation.

Changes in Rx1 and Pax6 Activity at Eye Field Stages Differentially Alter the Production of Amacrine Neurotransmitter Subtypes in Xenopus

Both rx1 and pax6 are expressed during the initial formation of the vertebrate eye field, and they are thought to be crucial for maintenance of the retinal stem cells in the ciliary marginal zone. However, both genes continue to be expressed in different layers of the differentiating retina, suggesting that they have additional roles in cell type specification. Because previous work suggested that amacrine cell subtypes are derived from biased progenitors in the eye field, we tested whether altering Rx1 or Pax6 activity during eye field stages affects the production of three neurotransmitter subtypes of amacrine cells.

The Competence of Xenopus Blastomeres to Produce Neural and Retinal Progeny is Repressed by Two Endo-mesoderm Promoting Pathways

Only a subset of cleavage stage blastomeres in the Xenopus embryo is competent to contribute cells to the retina; ventral vegetal blastomeres do not form retina even when provided with neuralizing factors or transplanted to the most retinogenic position of the embryo. These results suggest that endogenous maternal factors in the vegetal region repress the ability of blastomeres to form retina. Herein we provide three lines of evidence that two vegetal-enriched maternal factors (VegT, Vg1), which are known to promote endo-mesodermal fates, negatively regulate which cells are competent to express anterior neural and retinal fates. First, both molecules can repress the ability of dorsal-animal retinogenic blastomeres to form retina, converting the lineage from neural/retinal to non-neural ectodermal and endo-mesodermal fates. Second, reducing the endogenous levels of either factor in dorsal-animal retinogenic blastomeres expands expression of neural/retinal genes and enlarges the retina. The dorsal-animal repression of neural/retinal fates by VegT and Vg1 is likely mediated by Sox17alpha and Derriere but not by XNr1. VegT and Vg1 likely exert their effects on neural/retinal fates through at least partially independent pathways because Notch1 can reverse the effects of VegT and Derriere but not those of Vg1 or XNr1. Third, reduction of endogenous VegT and/or Vg1 in ventral vegetal blastomeres can induce a neural fate, but only allows expression of a retinal fate when both BMP and Wnt signaling pathways are concomitantly repressed.

Alterations of Rx1 and Pax6 Expression Levels at Neural Plate Stages Differentially Affect the Production of Retinal Cell Types and Maintenance of Retinal Stem Cell Qualities

rx1 and pax6 are necessary for the establishment of the vertebrate eye field and for the maintenance of the retinal stem cells that give rise to multiple retinal cell types. They also are differentially expressed in cellular layers in the retina when cell fates are being specified, and their expression levels differentially affect the production of amacrine cell subtypes. To determine whether rx1 and pax6 expression after the eye field is established simply maintains stem cell-like qualities or affects cell type differentiation, we used hormone-inducible constructs to increase or decrease levels/activity of each protein at two different neural plate stages. Our results indicate that rx1 regulates the size of the retinal stem cell pool because it broadly affected all cell types, whereas pax6 regulates more restricted retinal progenitor cells because it selectively affected different cell types in a time-dependent manner. Analysis of rx1 and pax6 effects on proliferation, and expression of stem cell or differentiation markers demonstrates that rx1 maintains cells in a stem cell state by promoting proliferation and delaying expression of neural identity and differentiation markers. Although pax6 also promotes proliferation, it differentially regulates neural identity and differentiation genes. Thus, these two genes work in parallel to regulate different, but overlapping aspects of retinal cell fate determination.

Eya1 and Six1 Promote Neurogenesis in the Cranial Placodes in a SoxB1-dependent Fashion

Genes of the Eya family and of the Six1/2 subfamily are expressed throughout development of vertebrate cranial placodes and are required for their differentiation into ganglia and sense organs. How they regulate placodal neurogenesis, however, remains unclear. Through loss of function studies in Xenopus we show that Eya1 and Six1 are required for neuronal differentiation in all neurogenic placodes. The effects of overexpression of Eya1 or Six1 are dose dependent. At higher levels, Eya1 and Six1 expand the expression of SoxB1 genes (Sox2, Sox3), maintain cells in a proliferative state and block expression of neuronal determination and differentiation genes. At lower levels, Eya1 and Six1 promote neuronal differentiation, acting downstream of and/or parallel to Ngnr1. Our findings suggest that Eya1 and Six1 are required for both the regulation of placodal neuronal progenitor proliferation, through their effects on SoxB1 expression, and subsequent neuronal differentiation.

FoxD5 Plays a Critical Upstream Role in Regulating Neural Ectodermal Fate and the Onset of Neural Differentiation

foxD5 is expressed in the nascent neural ectoderm concomitant with several other neural-fate specifying transcription factors. We used loss-of-function and gain-of-function approaches to analyze the functional position of foxD5 amongst these other factors. Loss of FoxD5 reduces the expression of sox2, sox11, soxD, zic1, zic3 and Xiro1-3 at the onset of gastrulation, and of geminin, sox3 and zic2, which are maternally expressed, by late gastrulation. At neural plate stages most of these genes remain reduced, but the domains of zic1 and zic3 are expanded. Increased FoxD5 induces geminin and zic2, weakly represses sox11 at early gastrula but later (st12) induces it; weakly represses sox2 and sox3 transiently and strongly represses soxD, zic1, zic3 and Xiro1-3. The foxD5 effects on zic1, zic3 and Xiro1-3 involve transcriptional repression, whereas those on geminin and zic2 involve transcriptional activation. foxD5's effects on geminin, sox11 and zic2 occur at the onset of gastrulation, whereas the other genes require earlier foxD5 activity. geminin, sox11 and zic2, each of which is up-regulated directly by foxD5, are all required to account for foxD5 phenotypes, indicating that this triad constitutes a transcriptional network rather than linear path that coordinately up-regulates genes that promote an immature neural fate and inhibits genes that promote the onset of neural differentiation. We also show that foxD5 promotes an ectopic neural fate in the epidermis by reducing BMP signaling. Several of the genes that are repressed by foxD5 in turn reduce foxD5 expression, contributing to the medial-lateral patterning of the neural plate.

Notch Signaling Downstream of FoxD5 Promotes Neural Ectodermal Transcription Factors That Inhibit Neural Differentiation

We investigated the role of the Notch signaling pathway in regulating several transcription factors that stabilize a neural fate and expand the neural plate. Increased Notch signaling in a neural lineage via a constitutively activated form (NICD) up-regulated geminin and zic2 in a cell-autonomous manner, and expanded the neural plate domains of sox11, sox2, and sox3. Loss- and gain-of-function assays show that foxD5 acts upstream of notch1 gene expression. Decreasing Notch signaling with an anti-morphic form of a Notch ligand (X-Delta-1(STU)) showed that the foxD5-mediated expansion of the sox gene neural plate domains requires Notch signaling. However, geminin and zic2 appear to be dually regulated by foxD5 and Notch1 signaling. These studies demonstrate that: (1) Notch signaling acts downstream of foxD5 to promote the expression of a subset of neural ectodermal transcription factors; and (2) Notch signaling and the foxD5 transcriptional pathway together maintain the neural plate in an undifferentiated state. Developmental Dynamics 238:1358-1365, 2009. (c) 2009 Wiley-Liss, Inc.

Neural Induction and Factors That Stabilize a Neural Fate

The neural ectoderm of vertebrates forms when the bone morphogenetic protein (BMP) signaling pathway is suppressed. Herein, we review the molecules that directly antagonize extracellular BMP and the signaling pathways that further contribute to reduce BMP activity in the neural ectoderm. Downstream of neural induction, a large number of "neural fate stabilizing" (NFS) transcription factors are expressed in the presumptive neural ectoderm, developing neural tube and ultimately in neural stem cells. Herein, we review what is known about their activities during normal development to maintain a neural fate and regulate neural differentiation. Further elucidation of how the NFS genes interact to regulate neural specification and differentiation should ultimately prove useful for regulating the expansion and differentiation of neural stem and progenitor cells.

The Genesis of New and Exciting Developmental Genetics Research

Highlighted Article: "E(nos)/CG4699 is Required for Nanos Function in the Female Germ Line of Drosophila" by Yu, Song and Wharton

Microarray Identification of Novel Downstream Targets of FoxD4L1/D5, a Critical Component of the Neural Ectodermal Transcriptional Network

FoxD4L1/D5 is a forkhead transcription factor that functions as both a transcriptional activator and repressor. FoxD4L1/D5 acts upstream of several other neural transcription factors to maintain neural fate, regulate neural plate patterning, and delay the expression of neural differentiation factors. To identify a more complete list of downstream genes that participate in these earliest steps of neural ectodermal development, we carried out a microarray analysis comparing gene expression in control animal cap ectodermal explants (ACs), which will form epidermis, to that in FoxD4L1/D5-expressing ACs. Forty-four genes were tested for validation by RT-PCR of ACs and/or in situ hybridization assays in embryos; 86% of those genes up-regulated and 100% of those genes down-regulated in the microarray were altered accordingly in one of these independent assays. Eleven of these 44 genes are of unknown function, and we provide herein their developmental expression patterns to begin to reveal their roles in ectodermal development.

Developmental Expression Patterns of Candidate Cofactors for Vertebrate Six Family Transcription Factors

Six family transcription factors play important roles in craniofacial development. Their transcriptional activity can be modified by cofactor proteins. Two Six genes and one cofactor gene (Eya1) are involved in the human Branchio-otic (BO) and Branchio-otic-renal (BOR) syndromes. However, mutations in Six and Eya genes only account for approximately half of these patients. To discover potential new causative genes, we searched the Xenopus genome for orthologues of Drosophila cofactor proteins that interact with the fly Six-related factor, SO. We identified 33 Xenopus genes with high sequence identity to 20 of the 25 fly SO-interacting proteins. We provide the developmental expression patterns of the Xenopus orthologues for 11 of the fly genes, and demonstrate that all are expressed in developing craniofacial tissues with at least partial overlap with Six1/Six2. We speculate that these genes may function as Six-interacting partners with important roles in vertebrate craniofacial development and perhaps congenital syndromes.

Special Issue on Craniofacial Development. Editorial

Yes-associated Protein 65 (YAP) Expands Neural Progenitors and Regulates Pax3 Expression in the Neural Plate Border Zone

Yes-associated protein 65 (YAP) contains multiple protein-protein interaction domains and functions as both a transcriptional co-activator and as a scaffolding protein. Mouse embryos lacking YAP did not survive past embryonic day 8.5 and showed signs of defective yolk sac vasculogenesis, chorioallantoic fusion, and anterior-posterior (A-P) axis elongation. Given that the YAP knockout mouse defects might be due in part to nutritional deficiencies, we sought to better characterize a role for YAP during early development using embryos that develop externally. YAP morpholino (MO)-mediated loss-of-function in both frog and fish resulted in incomplete epiboly at gastrulation and impaired axis formation, similar to the mouse phenotype. In frog, germ layer specific genes were expressed, but they were temporally delayed. YAP MO-mediated partial knockdown in frog allowed a shortened axis to form. YAP gain-of-function in Xenopus expanded the progenitor populations in the neural plate (sox2(+)) and neural plate border zone (pax3(+)), while inhibiting the expression of later markers of tissues derived from the neural plate border zone (neural crest, pre-placodal ectoderm, hatching gland), as well as epidermis and somitic muscle. YAP directly regulates pax3 expression via association with TEAD1 (N-TEF) at a highly conserved, previously undescribed, TEAD-binding site within the 5' regulatory region of pax3. Structure/function analyses revealed that the PDZ-binding motif of YAP contributes to the inhibition of epidermal and somitic muscle differentiation, but a complete, intact YAP protein is required for expansion of the neural plate and neural plate border zone progenitor pools. These results provide a thorough analysis of YAP mediated gene expression changes in loss- and gain-of-function experiments. Furthermore, this is the first report to use YAP structure-function analyzes to determine which portion of YAP is involved in specific gene expression changes and the first to show direct in vivo evidence of YAP's role in regulating pax3 neural crest expression.

Using 32-cell Stage Xenopus Embryos to Probe PCP Signaling

Use of loss-of function (via antisense Morpholino oligonucleotides (MOs)) or over-expression of proteins in epithelial cells during early embryogenesis of Xenopus embryos, can be a powerful tool to understand how signaling molecules can affect developmental events. The techniques described here are useful for examining the roles of proteins in cell-cell adhesion, and planar cell polarity (PCP) signaling in cell movement. We describe how to target specific regions within the embryos by injecting an RNA encoding a tracer molecule along with RNA encoding your protein of interest or an antisense MO to knock-down a particular protein within a specific blastomere of the embryo. Effects on cell-cell adhesion, cell movement, and endogenous or exogenous protein localization can be assessed at later stages in specific targeted tissues using fluorescent microscopy and immunolocalization.

Specific Domains of FoxD4/5 Activate and Repress Neural Transcription Factor Genes to Control the Progression of Immature Neural Ectoderm to Differentiating Neural Plate

FoxD4/5, a forkhead transcription factor, plays a critical role in establishing and maintaining the embryonic neural ectoderm. It both up-regulates genes that maintain a proliferative, immature neural ectoderm and down-regulates genes that promote the transition to a differentiating neural plate. We constructed deletion and mutant versions of FoxD4/5 to determine which domains are functionally responsible for these opposite activities, which regulate the critical developmental transition of neural precursors to neural progenitors to differentiating neural plate cells. Our results show that up-regulation of genes that maintain immature neural precursors (gem, zic2) requires the Acidic blob (AB) region in the N-terminal portion of the protein, indicating that the AB is the transactivating domain. Additionally, down-regulation of those genes that promote the transition to neural progenitors (sox) and those that lead to neural differentiation (zic, irx) involves: 1) an interaction with the Groucho co-repressor at the Eh-1 motif in the C-terminus; and 2) sequence downstream of this motif. Finally, the ability of FoxD4/5 to induce the ectopic expression of neural precursor genes in the ventral ectoderm also involves both the AB region and the Eh-1 motif; FoxD4/5 accomplishes ectopic neural induction by both activating neural precursor genes and repressing BMP signaling and epidermal genes. This study identifies the specific, conserved domains of the FoxD4/5 protein that allow this single transcription factor to regulate a network of genes that controls the transition of a proliferative neural ectodermal population to a committed neural plate population poised to begin differentiation.

Targeted Microinjection of Synthetic MRNAs to Alter Retina Gene Expression in Xenopus Embryos

The individual cells of the Xenopus cleavage-stage embryo have been fate mapped, revealing which of these cells contribute to the retina. Using this retina fate map, one can specifically modulate levels of gene expression in retina lineages to determine the function of proteins in various aspects of early retinal development, such as formation of the eye fields and determination of specific cell fates. This chapter presents the techniques for identifying specific retina blastomere precursor cells, and injecting them with lineage tracers, mRNAs encoding wild-type and mutant constructs or morpholino antisense oligonucleotides to alter gene expression.

Testing Retina Fate Commitment in Xenopus by Blastomere Deletion, Transplantation, and Explant Culture

The lineages of individual cells of the Xenopus cleavage-stage embryo have been fate-mapped to reveal the subset of blastomeres that are the major and minor precursors of the retina. Using this retina fate map, one can test the commitment of each of these cells to various retinal cell fates by manipulating the environment in which they develop. This chapter presents the techniques for identifying specific retina blastomere precursor cells, deleting them to test whether they are required for producing specific kinds of retinal cells, transplanting them to novel embryonic locations in host embryos to test whether they are committed to produce specific kinds of retinal cells, and growing them in explant culture to determine if their ability to produce specific kinds of retinal cells is autonomous.

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