Sp8 controls the anteroposterior patterning at the midbrain-hindbrain border

The specification of neuronal cell types in the developing neural tube is orchestrated by signaling centers. However, how patterned territories of the central nervous system (CNS) are organized into structures with appropriate size and shape is still unclear. We report that in the absence of the mouse transcription factor mBtd/Sp8, a posterior shift of the isthmic organizer (IsO) occurs, suggesting a crucial role for Sp8 in this process. In addition, large patches of cells ectopically expressing Fgf8, Otx2 and/or Wnt1 in the rostral hindbrain are detected in Sp8 mutant embryos. In this context, midbrain dopaminergic neurons are found posterior to the IsO. Furthermore, we provide evidence that cell proliferation in the mid- and hindbrain is tightly controlled by Sp8 activity. Our observations are consistent with a role for Sp8 in restricting Fgf8 expression at the IsO.

Related papers:

title: The Otx family.
abstract: Otx1 and Otx2, the murine homologs of the Drosophila orthodenticle gene, play a remarkable role in specification and regionalization of forebrain and midbrain. Recently, genetic approaches have indicated that OTD, OTX1 and OTX2 have retained reciprocal functional equivalence in evolution, whereas their regulatory control has been remarkably modified. This suggests that during the evolution of the vertebrate brain, regulatory changes modulating the transcriptional and translational control of pre-existing gene functions might have favored the establishment of new morphogenetic pathways.

title: Neural plate patterning: Upstream and downstream of the isthmic organizer
abstract: Two organizing centres operate at long-range distances within the anterior neural plate to pattern the forebrain, midbrain and hindbrain. Important progress has been made in understanding the formation and function of one of these organizing centres, the isthmic organizer, which controls the development of the midbrain and anterior hindbrain. Here we review our current knowledge on the identity, localization and maintenance of the isthmic organizer, as well as on the molecular cascades that underlie the activity of this organizing centre.

title: 
 Divide et Impera – the midbrain–hindbrain boundary and its organizer
abstract: The midbrain-hindbrain organizer (MHO) is a signalling centre that orchestrates development of the mesencephalic and anterior metencephalic primordia. In recent years, details have been revealed about the molecular nature of these signals, their transmission and reception, and the regulatory processes associated with MHO function. This article reviews recent progress in understanding the genetic and molecular components of the MHO, and how they synergize to control brain development.

title: Midbrain development induced by FGF8 in the chick embryo
abstract: VERTEBRATE midbrain development depends on an organizing centre located at the isthmus, a constriction in the embryonic mid/hindbrain region1–3,28. Isthmic tissue grafts transform chick caudal forebrain into an ectopic midbrain that is the mirror image of the normal midbrain4. Here we report that FGF8 protein has the same midbrain-inducing and polarizing effect as isthmic tissue. Moreover, FGF8 induces ectopic expression in the forebrain of genes normally expressed in the isthmus, suggesting that the ectopic midbrain forms under the influence of signals from a new 'isthmus-like' organizing centre induced in the forebrain. Because Fgf8 itself is expressed in the isthmus, our results identify FGF8 as an important signalling molecule in normal midbrain development.

title: Multiple developmental defects in Engrailed-1 mutant mice: an early mid-hindbrain deletion and patterning defects in forelimbs and sternum.
abstract: During mouse development, the homeobox-containing gene En-1 is specifically expressed across the mid-hindbrain junction, the ventral ectoderm of the limb buds, and in regions of the hindbrain, spinal cord, somites and somite-derived tissues. To address the function of En-1 during embryogenesis, we have generated mice homozygous for a targeted deletion of the En-1 homeobox. En-1 mutant mice died shortly after birth and exhibited multiple developmental defects. In the brains of newborn mutants, most of the colliculi and cerebellum were missing and the third and fourth cranial nerves were absent. A deletion of midhindbrain tissue was observed as early as 9.5 days of embryonic development and the phenotype resembles that previously reported for Wnt-1 mutant mice. In addition, patterning of the forelimb paws and sternum was disrupted, and the 13th ribs were truncated. The results of these studies suggest a cell autonomous role for En-1 in generation and/or survival of mid-hindbrain precursor cells and also a non-cell autonomous role in signalling normal development of the limbs and possibly sternum.

title: The Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain
abstract: The Wnt-1 (int-1) proto-oncogene, which encodes a putative signaling molecule, is expressed exclusively in the developing central nervous system and adult testes. To examine the role of Wnt-1, we generated six independent embryonic stem cell lines in which insertion of a neoR gene by homologous recombination inactivated a Wnt-1 allele. Germline chimeras were generated from two lines, and progeny from matings between heterozygous parents were examined. In all day 9.5 fetuses homozygous for mutated Wnt-1 alleles, most of the midbrain and some rostral metencephalon were absent. The remainder of the neural tube and all other tissues were normal. In late-gestation homozygotes, there was virtually no midbrain and no cerebellum, while the rest of the fetus was normal. Homozygotes are born, but die within 24 hr. Thus the normal role of Wnt-1 is in determination or subsequent development of a specific region of the central nervous system.

title: Specification of the anterior hindbrain and establishment of a normal mid/hindbrain organizer is dependent on Gbx2 gene function.
abstract: Analysis of mouse embryos homozygous for a loss-of-function allele of Gbx2 demonstrates that this homeobox gene is required for normal development of the mid/hindbrain region. Gbx2 function appears to be necessary at the neural plate stage for the correct specification and normal proliferation or survival of anterior hindbrain precursors. It is also required to maintain normal patterns of expression at the mid/hindbrain boundary of Fgf8 and Wnt1, genes that encode signaling molecules thought to be key components of the mid/hindbrain (isthmic) organizer. In the absence of Gbx2 function, isthmic nuclei, the cerebellum, motor nerve V, and other derivatives of rhombomeres 1-3 fail to form. Additionally, the posterior midbrain in the mutant embryos appears to be extended caudally and displays abnormalities in anterior/posterior patterning. The failure of anterior hindbrain development is presumably due to the loss of Gbx2 function in the precursors of the anterior hindbrain. However, since Gbx2 expression is not detected in the midbrain it seems likely that the defects in midbrain anterior/posterior patterning result from an abnormal isthmic signaling center. These data provide genetic evidence for a link between patterning of the anterior hindbrain and the establishment of the mid/hindbrain organizer, and identify Gbx2 as a gene required for these processes to occur normally.

title: Cooperation of Pax2 and Pax5 in midbrain and cerebellum development.
abstract: Midbrain and cerebellum development depends on an organizing center that is located at the midbrain-hindbrain junction of the vertebrate embryo. Expression of the two closely related transcription factors Pax2 and Pax5 overlaps spatially and temporally in this region of the developing central nervous system. To study a possible interaction of these transcription factors in midbrain and cerebellum patterning, we have generated Pax5, Krd double mutant mice. The transgene-induced Krd mutation corresponds to an approximately 7-centimorgan chromosome 19 deletion that eliminates the entire Pax2 locus. The heterozygous Krd mutation deleting one Pax2 allele had no effect on midbrain and cerebellum development. Moreover, only minor developmental defects were previously observed at the midline of the inferior colliculus and anterior cerebellum in mice that were homozygous for a targeted Pax5 mutation. Similar morphological alterations were observed in 80% of all compound heterozygous Pax5 (+/-) Krd (+/-) mice. However, in the remaining 20% of compound heterozygotes, the inferior colliculi were missing, and the vermis of the cerebellum was severely disrupted due to the failure of the cerebellar primordia to fuse at the midline. Inactivation of the second Pax5 allele in Pax5 (-/-) Krd (+/-) mice resulted in complete loss of the posterior midbrain and cerebellum, as the tissue originating from the midbrain-hindbrain boundary region was deleted in the embryo as early as day 9.5. On the basis of these data, we propose that the cooperation of Pax2 and Pax5 is essential for normal functioning of the organizing center at the midbrain-hindbrain junction.

title: Fgf8 is mutated in zebrafish acerebellar (ace) mutants and is required for maintenance of midbrain-hindbrain boundary development and somitogenesis.
abstract: We describe the isolation of zebrafish Fgf8 and its expression during gastrulation, somitogenesis, fin bud and early brain development. By demonstrating genetic linkage and by analysing the structure of the Fgf8 gene, we show that acerebellar is a zebrafish Fgf8 mutation that may inactivate Fgf8 function. Homozygous acerebellar embryos lack a cerebellum and the midbrain-hindbrain boundary organizer. Fgf8 function is required to maintain, but not initiate, expression of Pax2.1 and other marker genes in this area. We show that Fgf8 and Pax2.1 are activated in adjacent domains that only later become overlapping, and activation of Fgf8 occurs normally in no isthmus embryos that are mutant for Pax2.1. These findings suggest that multiple signaling pathways are independently activated in the midbrain-hindbrain boundary primordium during gastrulation, and that Fgf8 functions later during somitogenesis to polarize the midbrain. Fgf8 is also expressed in a dorsoventral gradient during gastrulation and ectopically expressed Fgf8 can dorsalize embryos. Nevertheless, acerebellar mutants show only mild dorsoventral patterning defects. Also, in spite of the prominent role suggested for Fgf8 in limb development, the pectoral fins are largely unaffected in the mutants. Fgf8 is therefore required in development of several important signaling centers in the zebrafish embryo, but may be redundant or dispensable for others.

title: Pax2/5 and Pax6 subdivide the early neural tube into three domains
abstract: The nested expression patterns of the paired-box containing transcription factors Pax2/5 and Pax6 demarcate the midbrain and forebrain primordium at the neural plate stage. We demonstrate that, in Pax2/5 deficient mice, the mesencephalon/metencephalon primordium is completely missing, resulting in a fusion of the forebrain to the hindbrain. Morphologically, in the alar plate the deletion is characterized by the substitution of the tectum (dorsal midbrain) and cerebellum (dorsal metencephalon) by the caudal diencephalon and in the basal plate by the replacement of the midbrain tegmentum by the ventral metencephalon (pons). Molecularly, the loss of the tectum is demonstrated by an expanded expression of Pax6, (the molecular determinant of posterior commissure), and a rostral shift of the territory of expression of Gbx2 and Otp (markers for the pons), towards the caudal diencephalon. Our results suggest that an intact territory of expression of Pax2/5 in the neural plate, nested between the rostral and caudal territories of expression of Pax6, is necessary for defining the midbrain vesicle.

title: Hes1 and Hes3 regulate maintenance of the isthmic organizer and development of the mid/hindbrain
abstract: The isthmic organizer, which is located at the midbrain–hindbrain boundary, plays an essential role in development of the midbrain and anterior hindbrain. It has been shown that homeobox genes regulate establishment of the isthmic organizer, but the mechanism by which the organizer is maintained is not well understood. Here, we found that, in mice doubly mutant for the basic helix–loop–helix genes Hes1 and Hes3, the midbrain and anterior hindbrain structures are missing without any significant cell death. In these mutants, the isthmic organizer cells prematurely differentiate into neurons and terminate expression of secreting molecules such as Fgf8 and Wnt1 and the paired box genes Pax2/5, all of which are essential for the isthmic organizer function. These results indicate that Hes1 and Hes3 prevent premature differentiation and maintain the organizer activity of the isthmic cells, thereby regulating the development of the midbrain and anterior hindbrain.

title: Genetic control of brain morphogenesis through Otx gene dosage requirement.
abstract: Understanding the genetic mechanisms that control patterning of the vertebrate brain represents a major challenge for developmental neurobiology. Previous data suggest that Otx1 and Otx2, two murine homologs of the Drosophila orthodenticle (otd) gene, might both contribute to brain morphogenesis. To gain insight into this possibility, the level of OTX proteins was modified by altering in vivo the Otx gene dosage. Here we report that Otx genes may cooperate in brain morphogenesis and that a minimal level of OTX proteins, corresponding either to one copy each of Otx1 and Otx2, or to only two copies of Otx2, is required for proper regionalization and subsequent patterning of the developing brain. Thus, as revealed by anatomical and molecular analyses, only Otx1-/-; Otx2+/- embryos lacked mesencephalon, pretectal area, dorsal thalamus and showed an heavy reduction of the Ammon's horn, while the metencephalon was dramatically enlarged occupying the mesencencephalic area. In 8.5 days post coitum (d.p.c.) Otx1-/-; Otx2+/- embryos, the expression patterns of mesencephalic-metencephalic (mes-met) markers such as En-1 and Wnt-1 confirmed the early presence of the area fated to give rise to mesencephalon and metencephalon while Fgf-8 transcripts were improperly localized in a broader domain. Thus, in Otx1-/-; Otx2+/- embryos, Fgf-8 misexpression is likely to be the consequence of a reduced level of specification between mes-met primitive neuroepithelia that triggers the following repatterning involving the transformation of mesencephalon into metencephalon, the establishment of an isthmic-like structure in the caudal diencephalon and, by 12.5 d.p.c., the telencephalic expression of Wnt-1 and En-2. Taken together these findings support the existence of a molecular mechanism depending on a precise threshold of OTX proteins that is required to specify early regional diversity between adjacent mes-met territories and, in turn, to allow the correct positioning of the isthmic organizer.

title: Cell autonomous and non-cell autonomous functions of Otx2 in patterning the rostral brain.
abstract: Previous studies have shown that the homeobox gene Otx2 is required first in the visceral endoderm for induction of forebrain and midbrain, and subsequently in the neurectoderm for its regional specification. Here, we demonstrate that Otx2 functions both cell autonomously and non-cell autonomously in neurectoderm cells of the forebrain and midbrain to regulate expression of region-specific homeobox and cell adhesion genes. Using chimeras containing both Otx2 mutant and wild-type cells in the brain, we observe a reduction or loss of expression of Rpx/Hesx1, Wnt1, R-cadherin and ephrin-A2 in mutant cells, whereas expression of En2 and Six3 is rescued by surrounding wild-type cells. Forebrain Otx2 mutant cells subsequently undergo apoptosis. Altogether, this study demonstrates that Otx2 is an important regulator of brain patterning and morphogenesis, through its regulation of candidate target genes such as Rpx/Hesx1, Wnt1, R-cadherin and ephrin-A2.

title: A role for Gbx2 in repression of Otx2 and positioning the mid/hindbrain organizer
abstract: The mid/hindbrain (MHB) junction can act as an organizer to direct the development of the midbrain and anterior hindbrain. In mice, Otx2 is expressed in the forebrain and midbrain and Gbx2 is expressed in the anterior hindbrain, with a shared border at the level of the MHB organizer. Here we show that, in Gbx2-/- mutants, the earliest phenotype is a posterior expansion of the Otx2 domain during early somite stages. Furthermore, organizer genes are expressed at the shifted Otx2 border, but not in a normal spatial relationship. To test whether Gbx2 is sufficient to position the MHB organizer, we transiently expressed Gbx2 in the caudal Otx2 domain and found that the Otx2 caudal border was indeed shifted rostrally and a normal appearing organizer formed at this new Otx2 border. Transgenic embryos then showed an expanded hindbrain and a reduced midbrain at embryonic day 9.5–10. We propose that formation of a normal MHB organizer depends on a sharp Otx2 caudal border and that Gbx2 is required to position and sharpen this border.

title: The caudal limit of Otx2 expression positions the isthmic organizer
abstract: The homeobox gene Otx2 is expressed in the anterior neural tube with a sharp limit at the midbrain/hindbrain junction (the isthmic organizer). Otx2 inactivation experiments have shown that this gene is essential for the development of its expression domain. Here we investigate whether the caudal limit of Otx2 expression is instrumental in positioning the isthmic organizer and in specifying midbrain versus hindbrain fate, by ectopically expressing Otx2 in the presumptive anterior hindbrain using a knock-in strategy into the En1 locus. Transgenic offspring display a cerebellar ataxia. Morphological and histological studies of adult transgenic brains reveal that most of the anterior cerebellar vermis is missing, whereas the inferior colliculus is complementarily enlarged. During early neural pattern formation expression of the midbrain markers Wnt1 and Ephrin-A5, the isthmic organizer markers Pax2 and Fgf-8 and the hindbrain marker Gbx2 are shifted caudally in the presumptive hindbrain territory. These findings show that the caudal limit of Otx2 expression is sufficient for positioning the isthmic organizer and encoding caudal midbrain fate within the mid/hindbrain domain.

title: Otx2 and Gbx2 are required for refinement and not induction of mid-hindbrain gene expression.
abstract: Otx2 and Gbx2 are among the earliest genes expressed in the neuroectoderm, dividing it into anterior and posterior domains with a common border that marks the mid-hindbrain junction. Otx2 is required for development of the forebrain and midbrain, and Gbx2 for the anterior hindbrain. Furthermore, opposing interactions between Otx2 and Gbx2 play an important role in positioning the mid-hindbrain boundary, where an organizer forms that regulates midbrain and cerebellum development. We show that the expression domains of Otx2 and Gbx2 are initially established independently of each other at the early headfold stage, and then their expression rapidly becomes interdependent by the late headfold stage. As we demonstrate that the repression of Otx2 by retinoic acid is dependent on an induction of Gbx2 in the anterior brain, molecules other than retinoic acid must regulate the initial expression of Otx2 in vivo. In contrast to previous suggestions that an interaction between Otx2- and Gbx2-expressing cells may be essential for induction of mid-hindbrain organizer factors such as Fgf8, we find that Fgf8 and other essential mid-hindbrain genes are induced in a correct temporal manner in mouse embryos deficient for both Otx2 and Gbx2. However, expression of these genes is abnormally co-localized in a broad anterior region of the neuroectoderm. Finally, we find that by removing Otx2 function, development of rhombomere 3 is rescued in Gbx2(-/-) embryos, showing that Gbx2 plays a permissive, not instructive, role in rhombomere 3 development. Our results provide new insights into induction and maintenance of the mid-hindbrain genetic cascade by showing that a mid-hindbrain competence region is initially established independent of the division of the neuroectoderm into an anterior Otx2-positive domain and posterior Gbx2-positive domain. Furthermore, Otx2 and Gbx2 are required to suppress hindbrain and midbrain development, respectively, and thus allow establishment of the normal spatial domains of Fgf8 and other genes.

title: Distinct regulators control the expression of the mid-hindbrain organizer signal FGF8
abstract: Local expression of FGF8 at the mid/hindbrain boundary (MHB) governs the development of multiple neurons and support cells. Here we show that the paired-domain protein Pax2 is necessary and sufficient for the induction of FGF8 in part by regulating the expression of Pax5&8. A network of transcription and secreted factors, including En1, Otx2, Gbx2, Grg4 and Wnt1&4, that is established independently of Pax2, further refines the expression domain and level of FGF8 at the MHB through opposing effects on Pax2 activity. Our results indicate that the expression of local organizing factors is controlled by combinatorial interaction between inductive and modulatory factors.

title: Otx dose-dependent integrated control of antero-posterior and dorso-ventral patterning of midbrain
abstract: Organizing centers emit signaling molecules that specify different neuronal cell types at precise positions along the anterior–posterior (A–P) and dorsal–ventral (D–V) axes of neural tube during development. Here we report that reduction in Otx proteins near the alar–basal plate boundary (ABB) of murine midbrain resulted in a dorsal shift of Shh expression, and reduction in Otx proteins at the midbrain–hindbrain boundary (MHB) resulted in an anterior expansion of the Fgf8 domain. Our data thus indicate that an Otx dose-dependent repressive effect coordinates proper positioning of Shh and Fgf8 expression. Furthermore, this control is effective for conferring proper cell identity in the floor-plate region of midbrain and does not require an Otx2-specific property. We propose that this mechanism may provide both A–P and D–V positional information to neuronal precursors located within the midbrain.

title: mBtd is required to maintain signaling during murine limb development.
abstract: buttonhead (btd) encodes an SP1-like transcription factor required for the generation and specification of Drosophila head segments. We identified a murine btd homolog, termed mouse Btd (mBtd), which can support btd-dependent head development in transgenic fly embryos. Functional studies show that mBtd-deficient mice develop to term and die at birth. They exhibit brain malformations, posterior axial skeleton truncations, and shortened limbs. We present evidence that mBtd is required during early limb development to maintain, but not to initiate Wnt/beta-catenin-dependent FGF, Shh, and BMP-mediated signaling. The data indicate that mBtd represents a novel key player mediating proximodistal outgrowth of the limb.

title: Sp8 is crucial for limb outgrowth and neuropore closure
abstract: In this report we describe the developmental expression and function of Sp8, a member of the Sp family of zinc finger transcription factors, and provide evidence that the legless transgene insertional mutant is a hypomorphic allele of the Sp8 gene. Sp8 is expressed during embryogenesis in the forming apical ectodermal ridge (AER), restricted regions of the central nervous system, and tail bud. Targeted deletion of the Sp8 gene gives a striking phenotype, with severe truncation of both forelimbs and hindlimbs, absent tail, as well as defects in anterior and posterior neuropore closure leading to exencephaly and spina bifida. Outgrowth of the limb depends on formation of the AER, a signaling center that forms at the limb bud apex. In Sp8 mutants, the AER precursor cells are induced and initially express multiple appropriate marker genes, but expression of these genes is not maintained and progression to a mature AER is blocked. These observations indicate that Sp8 functions downstream of Wnt3, Fgf10, and Bmpr1a in the signaling cascade that mediates AER formation.

title: Sensitive Nonradioactive Detection of mRNA in Tissue Sections: Novel Application of the Whole-mount In Situ Hybridization Protocol
abstract: The relative insensitivity of nonradioactive mRNA detection in tissue sections compared to the sensitive nonradioactive detection of single-copy DNA sequences in chromosome spreads, or of mRNA sequences in whole-mount samples, has remained a puzzling issue. Because of the biological significance of sensitive in situ mRNA detection in conjunction with high spatial resolution, we developed a nonradioactive in situ hybridization (ISH) protocol for detection of mRNA sequences in sections. The procedure is essentially based on the whole-mount ISH procedure and is at least equally sensitive. Increase of the hybridization temperature to 70C while maintaining stringency of hybridization by adaptation of the salt concentration significantly improved the sensitivity and made the procedure more sensitive than the conventional radioactive procedure. Thicker sections, which were no improvement using conventional radioactive ISH protocols, further enhanced signal. Higher hybridization temperatures apparently permit better tissue penetration of the probe. Application of this highly reliable protocol permitted the identification and localization of the cells in the developing heart that express low-abundance mRNAs of different members of the Iroquois homeobox gene family that are supposedly involved in cardiac patterning. The radioactive ISH procedure scarcely permitted detection of these sequences, underscoring the value of this novel method.

title: Segment-specific expression of a zinc-finger gene in the developing nervous system of the mouse
abstract: The process of segmentation, in which repeated homologous structures are generated along the anterior–posterior axis of the embryo is a widespread mechanism in animal development. In vertebrates, segmentation is most apparent in the somites and the peripheral nervous system1,2, but the existence of repetitive bulges, termed neuromeres, in the early neural epithelium of vertebrates suggests that the CNS may also be segmented3–9. Consistent with this, cranial ganglia8 and certain neurons8,10 are associated with specific hindbrain neuromeres. Here, we report that Krox-20 a zinc-finger gene, is expressed in two alternate neuromeres in the mouse early hindbrain. This pattern subsequently decays and Krox-20 is transiently expressed in specific hindbrain nuclei. In addition, Krox-20 is expressed in early neural crest cells, and then in the neural crest-derived boundary caps, glial components of the cranial and spinal ganglia. The demonstration that neuromeres are domains of gene expression provides molecular evidence for the segmentation of the CNS.

title: The Eph Kinase Ligand AL-1 Is Expressed by Rostral Muscles and Inhibits Outgrowth from Caudal Neurons
abstract: In the peripheral nervous system, neurons derived from specific rostrocaudal levels of the neuraxis selectively synapse on targets that arise from corresponding body positions. To identify molecules involved in such position-dependent connectivity, we used subtractive hybridization to isolate genes selectively expressed in rostral or caudal skeletal muscle. One mRNA that was more abundant in neck than in hindlimb muscles encoded the mouse ortholog of human AL-1 and chick RAGS, membrane-associated ligands of Eph tyrosine kinases that have recently been implicated in cortical axon fasciculation and retinotectal connectivity, respectively. We show here that mouse AL-1 is expressed in discrete regions of the central and peripheral nervous systems and in a subset of developing skeletal muscles. The abundance of AL-1 RNA in immortalized myogenic cell lines derived from rostral muscles is higher than in caudally derived lines, suggesting that levels are heritably maintained. Growth of neurites from cultured sensory ganglia and spinal cords is specifically inhibited by cells expressing AL-1, suggesting that this molecule could serve to guide peripheral axons. The inhibitory effects of AL-1 are position dependent, such that axons derived from caudal (lumbar) ganglia are more affected than those derived from rostral (cervical) ganglia. Together, these results support the notion that Eph kinases and their ligands regulate topographically appropriate neural connectivity in the peripheral nervous system, as well as in the central nervous system.

title: Early developmental failure of substantia nigra dopamine neurons in mice lacking the homeodomain gene Pitx3
abstract: The mesencephalic dopamine (mesDA) system is involved in the control of movement and behavior. The expression of Pitx3 in the brain is restricted to the mesDA system and the gene is induced relatively late, at E11.5, a time when tyrosine hydroxylase (Th) gene expression is initiated. We show here that, in the Pitx3-deficient aphakia (ak) mouse mutant, the mesDA system is malformed. Owing to the developmental failure of mesDA neurons in the lateral field of the midbrain, mesDA neurons are not found in the SNc and the projections to the caudate putamen are selectively lost. However, Pitx3 is expressed in all mesDA neurons in control animals. Therefore, mesDA neurons react specifically to the loss of Pitx3. Defects of motor control where not seen in the ak mice, suggesting that other neuronal systems compensate for the absence of the nigrostriatal pathway. However, an overall lower activity was observed. The results suggest that Pitx3 is specifically required for the formation of the SNc subfield at the onset of dopaminergic neuron differentiation.

title: Specification of dopaminergic and serotonergic neurons in the vertebrate CNS
abstract: The early specification of dopaminergic and serotonergic neurons during vertebrate CNS development relies on signals produced by a small number of organizing centers. Recent studies have characterized these early organizing centers, the manner in which they may be established, the inductive signals they produce, and candidate signaling systems that control the later development of the dopaminergic system.

title: FGF and Shh Signals Control Dopaminergic and Serotonergic Cell Fate in the Anterior Neural Plate
abstract: During development, distinct classes of neurons are specified in precise locations along the dorso-ventral and anterior-posterior axes of the neural tube. We provide evidence that intersections of Shh, which is expressed along the ventral neural tube, and FGF8, which is locally produced at the mid/hindbrain boundary and in the rostral forebrain, create induction sites for dopaminergic neurons in the midbrain and forebrain. The same intersection, when preceded by a third signal, FGF4, which is expressed in the primitive streak, defines an inductive center for hindbrain 5-HT neurons. These findings illustrate that cell patterning in the neural plate is a multistep process in which early inducers, which initially divide the neural plate into crude compartments, are replaced by multiple local organizing centers, which specify distinct neuronal cell types within these compartments.

title: Changing Requirements for Gbx2 in Development of the Cerebellum and Maintenance of the Mid/Hindbrain Organizer
abstract: We examined whether Gbx2 is required after embryonic day 9 (E9) to repress Otx2 in the cerebellar anlage and position the midbrain/hindbrain organizer. In contrast to Gbx2 null mutants, mice lacking Gbx2 in rhombomere 1 (r1) after E9 (Gbx2-CKO) are viable and develop a cerebellum. A Gbx2-independent pathway can repress Otx2 in r1 after E9. Mid/hindbrain organizer gene expression, however, continues to be dependent on Gbx2. We found that Fgf8 expression normally correlates with the isthmus where cells undergo low proliferation and that in Gbx2-CKO mutants this domain is expanded. We propose that Fgf8 permits lateral cerebellar development through repression of Otx2 and also suppresses medial cerebellar growth in Gbx2-CKO embryos. Our work has uncovered distinct requirements for Gbx2 during cerebellum formation and provided a model for how a transcription factor can play multiple roles during development.

title: New regulatory interactions and cellular responses in the isthmic organizer region revealed by altering Gbx2 expression
abstract: The mouse homeobox gene Gbx2 is first expressed throughout the posterior region of the embryo during gastrulation, and becomes restricted to rhombomeres 1-3 (r1-3) by embryonic day 8.5 (E8.5). Previous studies have shown that r1-3 do not develop in Gbx2 mutants and that there is an early caudal expansion of the midbrain gene Otx2 to the anterior border of r4. Furthermore, expression of Wnt1 and Fgf8, two crucial components of the isthmic organizer, is no longer segregated to adjacent domains in Gbx2 mutants. In this study, we extend the phenotypic analysis of Gbx2 mutants by showing that Gbx2 is not only required for development of r1-3, but also for normal gene expression in r4-6. To determine whether Gbx2 can alter hindbrain development, we generated Hoxb1-Gbx2 (HG) transgenic mice in which Gbx2 is ectopically expressed in r4. We show that Gbx2 is not sufficient to induce r1-3 development in r4. To test whether an Otx2/Gbx2 interface can induce r1-3 development, we introduced the HG transgene onto a Gbx2-null mutant background and recreated a new Otx2/Gbx2 border in the anterior hindbrain. Development of r3, but not r1 and r2, is rescued in Gbx2–/–; HG embryos. In addition, the normal spatial relationship of Wnt1 and Fgf8 is established at the new Otx2/Gbx2 border, demonstrating that an interaction between Otx2 and Gbx2 is sufficient to produce the normal pattern of Wnt1 and Fgf8 expression. However, the expression domains of Fgf8 and Spry1, a downstream target of Fgf8, are greatly reduced in mid/hindbrain junction area of Gbx2–/–; HG embryos and the posterior midbrain is truncated because of abnormal cell death. Interestingly, we show that increased cell death and a partial loss of the midbrain are associated with increased expression of Fgf8 and Spry1 in Gbx2 conditional mutants that lack Gbx2 in r1 after E9.0. These results together suggest that cell survival in the posterior midbrain is positively or negatively regulated by Fgf8, depending on Fgf8 expression level. Our studies provide new insights into the regulatory interactions that maintain isthmic organizer gene expression and the consequences of altered levels of organizer gene expression on cell survival.

title: The isthmic organizer signal FGF8 is required for cell survival in the prospective midbrain and cerebellum
abstract: Numerous studies have demonstrated that the midbrain and cerebellum develop from a region of the early neural tube comprising two distinct territories known as the mesencephalon (mes) and rostral metencephalon (met; rhombomere 1), respectively. Development of the mes and met is thought to be regulated by molecules produced by a signaling center, termed the isthmic organizer (IsO), which is localized at the boundary between them. FGF8 and WNT1 have been implicated as key components of IsO signaling activity, and previous studies have shown that in Wnt1-/- embryos, the mes/met is deleted by the 30 somite stage (∼E10) (McMahon, A. P. and Bradley, A. (1990) Cell 62, 1073-1085). We have studied the function of FGF8 in mouse mes/met development using a conditional gene inactivation approach. In our mutant embryos, Fgf8 expression was transiently detected, but then was eliminated in the mes/met by the 10 somite stage (∼E8.75). This resulted in a failure to maintain expression of Wnt1 as well as Fgf17, Fgf18, and Gbx2 in the mes/met at early somite stages, and in the absence of the midbrain and cerebellum at E17.5. We show that a major cause of the deletion of these structures is ectopic cell death in the mes/met between the 7 and 30 somite stages. Interestingly, we found that the prospective midbrain was deleted at an earlier stage than the prospective cerebellum. We observed a remarkably similar pattern of cell death in Wnt1 null homozygotes, and also detected ectopic mes/met cell death in En1 null homozygotes. Our data show that Fgf8 is part of a complex gene regulatory network that is essential for cell survival in the mes/met.

title: Sp8 and Sp9, two closely related buttonhead-like transcription factors, regulate Fgf8 expression and limb outgrowth in vertebrate embryos
abstract: Initiation and maintenance of signaling centers is a key issue during embryonic development. The apical ectodermal ridge, a specialized epithelial structure and source of Fgf8, is a pivotal signaling center for limb outgrowth. We show that two closely related buttonhead-like zinc-finger transcription factors, Sp8 and Sp9, are expressed in the AER, and regulate Fgf8 expression and limb outgrowth. Embryological and genetic analyses have revealed that Sp8 and Sp9 are ectodermal targets of Fgf10 signaling from the mesenchyme. We also found that Wnt/β-catenin signaling positively regulates Sp8, but not Sp9. Overexpression functional analyses in chick unveiled their role as positive regulators of Fgf8 expression. Moreover, a dominant-negative approach in chick and knockdown analysis with morpholinos in zebrafish revealed their requirement for Fgf8 expression and limb outgrowth, and further indicate that they have a coordinated action on Fgf8 expression. Our study demonstrates that Sp8 and Sp9, via Fgf8, are involved in mediating the actions of Fgf10 and Wnt/β-catenin signaling during vertebrate limb outgrowth.

title: Effects of Wnt1 signaling on proliferation in the developing mid-/hindbrain region
abstract: The secreted glycoprotein WNT1 is expressed in the caudal midbrain and is essential for proper development of the entire mid-/hindbrain region. To get better insights into Wnt1 function in the mid-/hindbrain region, we ectopically expressed Wnt1 under the control of the endogenous En1 promoter, thereby extending Wnt1 expression rostrally into the anterior midbrain and caudally into rhombomere 1. In these transgenic mice, the position of the mid-/hindbrain organizer is not altered and pattern formation is not changed. During midgestation, ectopic Wnt1 induced strong overproliferation of precursor cells only in the caudal midbrain in a gene dosage-dependent manner. Enhanced proliferation is at least in part mediated by shortening of the cell cycle length. In adults, Wnt1 exhibited a cell size promoting effect specifically on neurons. We suggest that Wnt1 acts as a regulator of proliferation of specific precursor populations in the developing mid-/hindbrain region and is only secondarily involved in maintenance of the mid-/hindbrain organizer.

title: FGF8 induces formation of an ectopic isthmic organizer and isthmocerebellar development via a repressive effect on Otx2 expression.
abstract: Beads containing recombinant FGF8 (FGF8-beads) were implanted in the prospective caudal diencephalon or midbrain of chick embryos at stages 9-12. This induced the neuroepithelium rostral and caudal to the FGF8-bead to form two ectopic, mirror-image midbrains. Furthermore, cells in direct contact with the bead formed an outgrowth that protruded laterally from the neural tube. Tissue within such lateral outgrowths developed proximally into isthmic nuclei and distally into a cerebellum-like structure. These morphogenetic effects were apparently due to FGF8-mediated changes in gene expression in the vicinity of the bead, including a repressive effect on Otx2 and an inductive effect on En1, Fgf8 and Wnt1 expression. The ectopic Fgf8 and Wnt1 expression domains formed nearly complete concentric rings around the FGF8-bead, with the Wnt1 ring outermost. These observations suggest that FGF8 induces the formation of a ring-like ectopic signaling center (organizer) in the lateral wall of the brain, similar to the one that normally encircles the neural tube at the isthmic constriction, which is located at the boundary between the prospective midbrain and hindbrain. This ectopic isthmic organizer apparently sends long-range patterning signals both rostrally and caudally, resulting in the development of the two ectopic midbrains. Interestingly, our data suggest that these inductive signals spread readily in a caudal direction, but are inhibited from spreading rostrally across diencephalic neuromere boundaries. These results provide insights into the mechanism by which FGF8 induces an ectopic organizer and suggest that a negative feedback loop between Fgf8 and Otx2 plays a key role in patterning the midbrain and anterior hindbrain.

title: Signalling by FGF8 from the isthmus patterns anterior hindbrain and establishes the anterior limit of Hox gene expression.
abstract: Current evidence suggests that the anterior segment of the vertebrate hindbrain, rhombomere 1, gives rise to the entire cerebellum. It is situated where two distinct developmental patterning mechanisms converge: graded signalling from an organising centre (the isthmus) located at the midbrain/hindbrain boundary confronts segmentation of the hindbrain. The unique developmental fate of rhombomere 1 is reflected by it being the only hindbrain segment in which no Hox genes are expressed. In this study we show that ectopic FGF8 protein, a candidate for the isthmic organising activity, is able to induce and repress gene expression within the hindbrain in a manner appropriate to rhombomere 1. Using a heterotopic, heterospecific grafting strategy we demonstrate that rhombomere 1 is able to express Hox genes but that both isthmic tissue and FGF8 inhibit their expression. Inhibition of FGF8 function in vivo shows that it is responsible for defining the anterior limit of Hox gene expression within the developing brain and thereby specifies the extent of the rl territory. Previous studies have suggested that a retinoid morphogen gradient determines the axial limit of expression of individual Hox genes within the hindbrain. We propose a model whereby activation by retinoids is antagonised by inhibition by FGF8 in the anterior hindbrain to set aside the territory from which the cerebellum will develop.

title: Evidence that FGF8 signalling from the midbrain-hindbrain junction regulates growth and polarity in the developing midbrain.
abstract: The developing vertebrate mesencephalon shows a rostrocaudal gradient in the expression of a number of molecular markers and in the cytoarchitectonic differentiation of the tectum, where cells cease proliferating and differentiate in a rostral to caudal progression. Tissue grafting experiments have implicated cell signalling by the mesencephalic-metencephalic (mid-hindbrain) junction (or isthmus) in orchestrating these events. We have explored the role of Wnt-1 and FGF8 signalling in the regulation of mesencephalic polarity. Wnt-1 is expressed in the caudal mesencephalon and Fgf8 in the most rostral metencephalon. Wnt-1 regulates Fgf8 expression in the adjacent metencephalon, most likely via a secondary mesencephalic signal. Ectopic expression of Fgf8 in the mesencephalon is sufficient to activate expression of Engrailed-2 (En-2) and ELF-1, two genes normally expressed in a decreasing caudal to rostral gradient in the posterior mesencephalon. Ectopic expression of Engrailed-1 (En-1), a functionally equivalent homologue of En-2 is sufficient to activate ELF-1 expression by itself. These results indicate the existence of a molecular hierarchy in which FGF8 signalling establishes the graded expression of En-2 within the tectum. This in turn may act to specify other aspects of A-P polarity such as graded ELF-1 expression. Our studies also reveal that FGF8 is a potent mitogen within the mesencephalon: when ectopically expressed, neural precursors continue to proliferate and neurogenesis is prevented. Taken together our results suggest that FGF8 signalling from the isthmus has a key role in coordinately regulating growth and polarity in the developing mesencephalon.

title: Functions of FGF signalling from the apical ectodermal ridge in limb development
abstract: To determine the role of fibroblast growth factor (FGF) signalling from the apical ectodermal ridge (AER), we inactivated Fgf4 and Fgf8 in AER cells or their precursors at different stages of mouse limb development. We show that FGF4 and FGF8 regulate cell number in the nascent limb bud and are required for survival of cells located far from the AER. On the basis of the skeletal phenotypes observed, we conclude that these functions are essential to ensure that sufficient progenitor cells are available to form the normal complement of skeletal elements, and perhaps other limb tissues. In the complete absence of both FGF4 and FGF8 activities, limb development fails. We present a model to explain how the mutant phenotypes arise from FGF-mediated effects on limb bud size and cell survival.

title: Otx2 regulates the extent, identity and fate of neuronal progenitor domains in the ventral midbrain
abstract: The specification of distinct neuronal cell-types is controlled by inducing signals whose interpretation in distinct areas along the central nervous system provides neuronal progenitors with a precise and typical expression code of transcription factors. To gain insights into this process, we investigated the role of Otx2 in the specification of identity and fate of neuronal progenitors in the ventral midbrain. To achieve this, Otx2 was inactivated by Cre recombinase under the transcriptional control of En1. Lack of Otx2 in the ventrolateral and posterior midbrain results in a dorsal expansion of Shh expression and in a dorsal and anterior rotation of the midbrain-hindbrain boundary and Fgf8 expression. Indeed, in this mutant correct positioning of the ventral site of midbrain-hindbrain boundary and Fgf8 expression are efficiently controlled by Otx1 function, thus allowing the study of the identity and fate of neuronal progenitors of the ventral midbrain in the absence of Otx2. Our results suggest that Otx2 acts in two ways: by repressing Nkx2.2 in the ventral midbrain and maintaining the Nkx6.1-expressing domain through dorsal antagonism on Shh. Failure of this control affects the identity code and fate of midbrain progenitors, which exhibit features in common with neuronal precursors of the rostral hindbrain even though the midbrain retains its regional identity and these neuronal precursors are rostral to Fgf8 expression. Dopaminergic neurons are greatly reduced in number, red nucleus precursors disappear from the ventral midbrain where a relevant number of serotonergic neurons are generated. These results indicate that Otx2 is an essential regulator of the identity, extent and fate of neuronal progenitor domains in the ventral midbrain and provide novel insights into the mechanisms by which neuronal diversity is generated in the central nervous system.

title: A Wnt1-regulated genetic network controls the identity and fate of midbrain-dopaminergic progenitors in vivo
abstract: Midbrain neurons synthesizing the neurotransmitter dopamine play a central role in the modulation of different brain functions and are associated with major neurological and psychiatric disorders. Despite the importance of these cells, the molecular mechanisms controlling their development are still poorly understood. The secreted glycoprotein Wnt1 is expressed in close vicinity to developing midbrain dopaminergic neurons. Here, we show that Wnt1 regulates the genetic network, including Otx2 and Nkx2-2, that is required for the establishment of the midbrain dopaminergic progenitor domain during embryonic development. In addition, Wnt1 is required for the terminal differentiation of midbrain dopaminergic neurons at later stages of embryogenesis. These results identify Wnt1 as a key molecule in the development of midbrain dopaminergic neurons in vivo. They also suggest the Wnt1-controlled signaling pathway as a promising target for new therapeutic strategies in the treatment of Parkinson's disease.

title: The zebrafish buttonhead-like factor Bts1 is an early regulator of pax2.1 expression during mid-hindbrain development.
abstract: Little is known about the factors that control the specification of the mid-hindbrain domain (MHD) within the vertebrate embryonic neural plate. Because the head-trunk junction of the Drosophila embryo and the MHD have patterning similarities, we have searched for vertebrate genes related to the Drosophila head gap gene buttonhead (btd), which in the fly specifies the head-trunk junction. We report here the identification of a zebrafish gene which, like btd, encodes a zinc-finger transcriptional activator of the Sp-1 family (hence its name, bts1 for btd/Sp-related-1) and shows a restricted expression in the head. During zebrafish gastrulation, bts1 is transcribed in the posterior epiblast including the presumptive MHD, and precedes in this area the expression of other MHD markers such as her5, pax2.1 and wnt1. Ectopic expression of bts1 combined to knock-down experiments demonstrate that Bts1 is both necessary and sufficient for the induction of pax2.1 within the anterior neural plate, but is not involved in regulating her5, wnt1 or fgf8 expression. Our results confirm that early MHD development involves several genetic cascades that independently lead to the induction of MHD markers, and identify Bts1 as a crucial upstream component of the pathway selectively leading to pax2.1 induction. In addition, they imply that flies and vertebrates, to control the development of a boundary embryonic region, have probably co-opted a similar strategy: the restriction to this territory of the expression of a Btd/Sp-like factor.