Overlapping function of Lmx1a and Lmx1b in anterior hindbrain roof plate formation and cerebellar growth.
ABSTRACT: The roof plate is an organizing center in the dorsal CNS that controls specification and differentiation of adjacent neurons through secretion of the BMP and WNT signaling molecules. Lmx1a, a member of the LIM-homeodomain (LIM-HD) transcription factor family, is expressed in the roof plate and its progenitors at all axial levels of the CNS and is necessary and sufficient for roof plate formation in the spinal cord. In the anterior CNS, however, a residual roof plate develops in the absence of Lmx1a. Lmx1b, another member of the LIM-HD transcription factor family which is highly related to Lmx1a, is expressed in the roof plate in the anterior CNS. Although Lmx1b-null mice do not show a substantial deficiency in hindbrain roof plate formation, Lmx1a/Lmx1b compound-null mutants fail to generate hindbrain roof plate. This observation indicates that both genes act in concert to direct normal hindbrain roof plate formation. Since the requirement of Lmx1b function for normal isthmic organizer at the mid-hindbrain boundary complicates analysis of a distinct dorsal patterning role of this gene, we also used a conditional knock-out strategy to specifically delete dorsal midline Lmx1b expression. Phenotypic analysis of single and compound conditional mutants confirmed overlapping roles for Lmx1 genes in regulating hindbrain roof plate formation and growth and also revealed roles in regulating adjacent cerebellar morphogenesis. Our data provides the first evidence of overlapping function of the Lmx1 genes during embryonic CNS development.
Project description:During development, the lumen of the neural tube develops into a system of brain cavities or ventricles, which play important roles in normal CNS function. We have established that the formation of the hindbrain (4th) ventricle in zebrafish is dependent upon the pleiotropic functions of the genes implicated in human Dandy Walker Malformation, Zic1 and Zic4. Using morpholino knockdown we show that zebrafish Zic1 and Zic4 are required for normal morphogenesis of the 4th ventricle. In Zic1 and/or Zic4 morphants the ventricle does not open properly, but remains completely or partially fused from the level of rhombomere (r) 2 towards the posterior. In the absence of Zic function early hindbrain regionalization and neural crest development remain unaffected, but dorsal hindbrain progenitor cell proliferation is significantly reduced. Importantly, we find that Zic1 and Zic4 are required for development of the dorsal roof plate. In Zic morphants expression of roof plate markers, including lmx1b.1 and lmx1b.2, is disrupted. We further demonstrate that zebrafish Lmx1b function is required for both hindbrain roof plate development and 4th ventricle morphogenesis, confirming that roof plate formation is a critical component of ventricle development. Finally, we show that dorsal rhombomere boundary signaling centers depend on Zic1 and Zic4 function and on roof plate signals, and provide evidence that these boundary signals are also required for ventricle morphogenesis. In summary, we conclude that Zic1 and Zic4 control zebrafish 4th ventricle morphogenesis by regulating multiple mechanisms including cell proliferation and fate specification in the dorsal hindbrain.
Project description:LMX1A and LMX1B encode two closely related members of the LIM homeobox family of transcription factors. These genes play significant, and frequently overlapping, roles in the development of many structures in the nervous system, including the cerebellum, hindbrain, spinal cord roof plate, sensory systems and dopaminergic midbrain neurons. Little is known about the cis-acting regulatory elements (REs) that dictate their temporal and spatial expression or about the regulatory landscape surrounding them. The availability of comparative sequence data and the advent of genomic technologies such as ChIP-seq have revolutionized our capacity to identify regulatory sequences like enhancers. Despite this wealth of data, the vast majority of loci lack any significant in vivo functional exploration of their non-coding regions. We have completed a significant functional screen of conserved non-coding sequences (putative REs) scattered across these critical human loci, assaying the temporal and spatial control using zebrafish transgenesis. We first identify and describe the LMX1A paralogs lmx1a and lmx1a-like, comparing their expression during embryogenesis with that in mammals, along with lmx1ba and lmx1bb genes. Consistent with their prominent neuronal expression, 47/71 sequences selected within and flanking LMX1A and LMX1B exert spatial control of reporter expression in the central nervous system (CNS) of mosaic zebrafish embryos. Upon germline transmission, we identify CNS reporter expression in multiple independent founders for 22 constructs (LMX1A, n = 17; LMX1B, n = 5). The identified enhancers display significant overlap in their spatial control and represent only a fraction of the conserved non-coding sequences at these critical genes. Our data reveal the abundance of regulatory instruction located near these developmentally important genes.
Project description:The dorsal midline structure is critical for patterning the developing central nervous system (CNS). We show here that Zfp423/OAZ, a multiple zinc-finger transcription factor involved in both OE/EBF and BMP-signaling pathways, is required for the proper formation of forebrain and hindbrain midline structures. During embryogenesis, OAZ is highly expressed at the dorsal neuroepithelium flanking the roof plate. OAZ-deficient mice are ataxic, attributed to the reduction of the cerebellar vermis and some regions of the hemispheres. Characterization of postnatal cerebellar development shows defects in Purkinje cell differentiation and granule cell proliferation. In the forebrain, dorsal telencephalic commissural neurons project axons, but these axons fail to cross the midline and midline glial cells are abnormally distributed. Moreover, there are malformations in midline structures including the septum, thalamus and hypothalamus, suggesting a pivotal role of OAZ in CNS midline patterning.
Project description:Serotonergic (5-HT) neurons in the brainstem modulate a wide range of physiological processes and behaviors. Two transcription factor genes, Pet-1 and Nkx2.2, are necessary but not sufficient to specify the 5-HT transmitter phenotype. Here we show that the Lim class homeobox gene Lmx1b is required for proper formation of the entire 5-HT system in the hindbrain, as indicated by the loss of expression of genes necessary for serotonin synthesis and transport in Lmx1b null mice. Lmx1b and Pet1 act downstream of Nkx2.2, and their expression is independently regulated at the time when 5-HT transmitter phenotype is specified. Ectopic expression of Lmx1b plus Pet-1 is able to induce formation of 5-HT cells in the most ventral spinal cord, where Nkx2.2 is normally expressed. Combined expression of all three genes, Lmx1b, Pet-1, and Nkx2.2, drives 5-HT differentiation in the dorsal spinal cord. Our studies therefore define a molecular pathway necessary and sufficient to specify the serotonergic neurotransmitter phenotype.
Project description:Bone morphogenetic proteins (BMPs) have been hypothesized to specify distinct dorsal neural fates. During neural development, BMPs are expressed in the roof plate and adjacent neuroepithelium. Because several hindbrain nuclei that form the proprioceptive/vestibular/auditory sensory network originate from the rhombic lip, near the roof plate, BMP signaling may regulate the development of these nuclei. To test this hypothesis genetically, we have examined the development of the hindbrain in BMP type I receptor knockout mice. Our results demonstrate that BMP signaling is involved in the formation of precerebellar mossy fiber nuclei, which give rise to cerebellar mossy fibers, but is not required for the development of the inferior olivary nucleus, which gives rise to cerebellar climbing fibers.
Project description:The cerebellar rhombic lip and telencephalic cortical hem are dorsally located germinal zones which contribute substantially to neuronal diversity in the CNS, but the mechanisms that drive neurogenesis within these zones are ill defined. Using genetic fate mapping in wild-type and Lmx1a(-/-) mice, we demonstrate that Lmx1a is a critical regulator of cell-fate decisions within both these germinal zones. In the developing cerebellum, Lmx1a is expressed in the roof plate, where it is required to segregate the roof plate lineage from neuronal rhombic lip derivatives. In addition, Lmx1a is expressed in a subset of rhombic lip progenitors which produce granule cells that are predominantly restricted to the cerebellar posterior vermis. In the absence of Lmx1a, these cells precociously exit the rhombic lip and overmigrate into the anterior vermis. This overmigration is associated with premature regression of the rhombic lip and posterior vermis hypoplasia in Lmx1a(-/-) mice. These data reveal molecular organization of the cerebellar rhombic lip and introduce Lmx1a as an important regulator of rhombic lip cell-fate decisions, which are critical for maintenance of the entire rhombic lip and normal cerebellar morphogenesis. In the developing telencephalon Lmx1a is expressed in the cortical hem, and in its absence cortical hem progenitors contribute excessively to the adjacent hippocampus instead of producing Cajal-Retzius neurons. Thus, Lmx1a activity is critical for proper production of cells originating from both the cerebellar rhombic lip and the telencephalic cortical hem.
Project description:The identification of small molecules capable of directing pluripotent cell differentiation towards specific lineages is highly desirable to both reduce cost, and increase efficiency. Within neural progenitors, LIM homeobox transcription factor 1 alpha (Lmx1a) is required for proper development of roof plate and cortical hem structures of the forebrain, as well as the development of floor plate and midbrain dopaminergic neurons. In this study we generated homologous recombinant cell lines expressing either luciferase or ?-lactamase under the control of the Lmx1a promoter, and used these cell lines to investigate kinase-mediated regulation of Lmx1a activity during neuronal differentiation. A screen of 143 small molecule tyrosine kinase inhibitors yielded 16 compounds that positively or negatively modulated Lmx1a activity. Inhibition of EGF, VEGF and DNA-dependent protein kinase (DNA-PK) signaling significantly upregulated Lmx1a activity whereas MEK inhibition strongly downregulated its activity. Quantitative FACS analysis revealed that the DNA-PK inhibitor significantly increased the number of Lmx1a+ progenitors while subsequent qPCR showed an upregulation of Notch effectors, the basic helix-loop-helix genes, Hes5 and Hey1. FACS further revealed that DNA-PK-mediated regulation of Lmx1a+ cells is dependent on the rapamycin-sensitive complex, mTORC1. Interestingly, this DNA-PK inhibitor effect was preserved in a co-culture differentiation protocol. Terminal differentiation assays showed that DNA-PK inhibition shifted development of neurons from forebrain toward midbrain character as assessed by Pitx3/TH immunolabeling and corresponding upregulation of midbrain (En1), but not forebrain (FoxG1) transcripts. These studies show that Lmx1a signaling in mouse embryonic stem cells contributes to a molecular cascade establishing neuronal specification. The data presented here identifies a novel regulatory pathway where signaling from DNA-PK appears to suppress midbrain-specific Lmx1a expression.
Project description:Mutations in the LIM-homeodomain transcription factor LMX1B cause nail-patella syndrome, an autosomal dominant pleiotrophic human disorder in which nail, patella and elbow dysplasia is associated with other skeletal abnormalities and variably nephropathy and glaucoma. It is thought to be a haploinsufficient disorder. Studies in the mouse have shown that during development Lmx1b controls limb dorsal-ventral patterning and is also required for kidney and eye development, midbrain-hindbrain boundary establishment and the specification of specific neuronal subtypes. Mice completely deficient for Lmx1b die at birth. In contrast to the situation in humans, heterozygous null mice do not have a mutant phenotype. Here we report a novel mouse mutant Icst, an N-ethyl-N-nitrosourea-induced missense substitution, V265D, in the homeodomain of LMX1B that abolishes DNA binding and thereby the ability to transactivate other genes. Although the homozygous phenotypic consequences of Icst and the null allele of Lmx1b are the same, heterozygous Icst elicits a phenotype whilst the null allele does not. Heterozygous Icst causes glaucomatous eye defects and is semi-lethal, probably due to kidney failure. We show that the null phenotype is rescued more effectively by an Lmx1b transgene than is Icst. Co-immunoprecipitation experiments show that both wild-type and Icst LMX1B are found in complexes with LIM domain binding protein 1 (LDB1), resulting in lower levels of functional LMX1B in Icst heterozygotes than null heterozygotes. We conclude that Icst is a dominant-negative allele of Lmx1b. These findings indicate a reassessment of whether nail-patella syndrome is always haploinsufficient. Furthermore, Icst is a rare example of a model of human glaucoma caused by mutation of the same gene in humans and mice.
Project description:Inductive factors are known to direct the regional differentiation of the vertebrate central nervous system (CNS) but their role in the specification of individual neuronal cell types is less clear. We have examined the function of GDF7, a BMP family member expressed selectively by roof plate cells, in the generation of neuronal cell types in the dorsal spinal cord. We find that GDF7 can promote the differentiation in vitro of two dorsal sensory interneuron classes, D1A and D1B neurons. In Gdf7-null mutant embryos, the generation of D1A neurons is eliminated but D1B neurons and other identified dorsal interneurons are unaffected. These findings show that GDF7 is an inductive signal from the roof plate required for the specification of neuronal identity in the dorsal spinal cord and that GDF7 and other BMP family members expressed by the roof plate have non-redundant functions in vivo. More generally, these results suggest that BMP signaling may have a prominent role in the assignment of neuronal identity within the mammalian CNS.
Project description:The roof plate is a specialized embryonic midline tissue of the central nervous system that functions as a signaling center regulating dorsal neural patterning. In the developing hindbrain, roof plate cells express Gdf7 and previous genetic fate mapping studies showed that these cells contribute mostly to non-neural choroid plexus epithelium. We demonstrate here that constitutive activation of the Sonic hedgehog signaling pathway in the Gdf7 lineage invariably leads to medulloblastoma. Lineage tracing analysis reveals that Gdf7-lineage cells not only are a source of choroid plexus epithelial cells, but are also present in the cerebellar rhombic lip and contribute to a subset of cerebellar granule neuron precursors, the presumed cell-of-origin for Sonic hedgehog-driven medulloblastoma. We further show that Gdf7-lineage cells also contribute to multiple neuronal and glial cell types in the cerebellum, including glutamatergic granule neurons, unipolar brush cells, Purkinje neurons, GABAergic interneurons, Bergmann glial cells, and white matter astrocytes. These findings establish hindbrain roof plate as a novel source of diverse neural cell types in the cerebellum that is also susceptible to oncogenic transformation by deregulated Sonic hedgehog signaling.