- Email: [email protected]
Co-ordinate regulation of angiogenesis and neurogenesis in the embryonic telencephalon
Poster abstracts / Int. J. Devl Neuroscience 24 (2006) 495–603
535
[P90]
[P92]
Regionalisation of Dlx-expressing progenitors in the medial ganglionic eminence gives rise to distinct subtypes of cortical interneurons in the adult brain
Transcriptional regulation of the mouse doublecortin gene in differentiating neurons
Ghanem 1,∗ ,
N. M. Ekker 1
1 University
J.
Long 2 ,
Hatch 1 ,
G.
of Ottawa, Canada; San Francisco, USA
J.
2 University
Rubenstein 2 , of California at
Dlx genes are required for the differentiation and migration of cortical GABAergic interneurons derived from progenitors located in the medial ganglionic eminence (MGE) and perhaps the caudal ganglionic eminence (CGE). It is not clear how the diversity of GABAergic interneuron subtypes is established and if it involves distinct progenitors in the MGE and CGE. We have identified three conserved enhancers, URE2, I12b and I56i in the Dlx gene loci of vertebrates. All three target expression in cells of the forebrain where endogenous Dlx genes are expressed but show differences in their regional and temporal activities. We examined if such differences in Dlx enhancer activity could be associated to different progenitor populations. We compared the activity of the three enhancers in the MGE and CGE, and in migrating neurons derived from these regions between E11.5 and E13.5 using: (1) reporter lines under the control of each enhancer; (2) double immunohistochemistry on lines with two reporter genes driven by different enhancers. We compared the migration potential of progenitors found in different ventral structures using DiI labelling and co-transplantation experiments in vitro. We also co-labelled adult cortical interneurons in the URE2 and I12b lines, separately. URE2, I12b and I56i displayed differential activities in the dorsal MGE, ventral MGE and CGE, and labelled distinct populations of tangentially migrating neurons at E12.5 and E13.5. We provide evidence that the dMGE and vMGE are distinct sources of tangentially migrating neurons at these ages. In the adult cortex, URE2 labelled the parvalbumin-, calretinin-, NPYand nNOS-positive interneurons, whereas I12b was active in subpopulations of these groups and marked specifically the somatostatin- and VIP-positive interneurons. Dlx gene regulation conferred by several enhancers provides molecular evidence that regional specification of progenitors located in subdivisions within the MGE and CGE generate different subtypes of cortical interneurons in the adult brain. Keywords: Migration
Dlx; GABAergic interneurons; Differentiation;
doi:10.1016/j.ijdevneu.2006.09.153
J.-C. Plumier, M. Muller, B. Rogister, M. Piens ∗ University of Li`ege, Belgium Doublecortin (DCX), a microtubule-associated protein, is transiently expressed in migrating and differentiating immature neurons in the developing and adult central nervous system. Whereas the temporal and spatial DCX expression patterns and DCX post-translational regulations are well known, dcx transcriptional regulation is still unclear. To determine and analyse important regulatory sequences of the dcx promoter, we generated different fragments and deletion constructs of regulatory sequences upstream from the mouse dcx coding region and used them to drive reporter gene expression. Transient expression experiments using these reporter vectors revealed that activation of reporter gene expression was restricted to cells expressing endogenous DCX and that dcx regulation differed in CNS versus PNS. Moreover, we characterized the DCX promoter activity at different time points during neuronal differentiation. Further sequence comparison of dcx promoters across several species revealed the presence of a highly conserved region in the proximal region of the promoter and of several conserved, putative transcription factor binding sites. Further experiments will focus on the identification of the transcription factors involved in dcx gene regulation. Keywords: Gene regulation; Doublecortin; Neuronal differentiation; Transcription factors doi:10.1016/j.ijdevneu.2006.09.154 [P93] Co-ordinate regulation of angiogenesis and neurogenesis in the embryonic telencephalon A. Vasudevan ∗ , P.G. Bhide Massachusetts General Hospital and Harvard Medical School, USA Introduction: The central nervous system (CNS) acquires its vasculature by angiogenesis, which begins early in CNS development and continues throughout life. Neurogenesis also begins early and continues throughout life—at least in some parts of the CNS. Majority of the research addressing interactions between CNS angiogenesis and neurogenesis has focused on the adult brain. However, angiogenesis and neurogenesis are significantly more robust in the embryonic CNS than in the adult. Indeed, the embryonic CNS is perhaps the only site (barring brain tumors) in which angiogenesis and neurogenesis are concurrently robust. Moreover, endothelial cell proliferation, migration and sprouting of new blood vessels occur in the embryonic CNS coincident and coordinate with neuroepithelial precursor cell proliferation
536
Poster abstracts / Int. J. Devl Neuroscience 24 (2006) 495–603
and elaboration of neuronal networks. Therefore, we chose the embryonic CNS as a model for examining mechanisms that coordinately regulate angiogenesis and neurogenesis. Methods: S-phase labeling methods were used to study cell proliferation; laser capture micro-dissection and real-time quantitative PCR to study gene expression profiles of angiogenesisrelated genes in neurogenic regions of the telencephalon. Results: Endothelial and neuroepithelial cell proliferation is concurrently active in the proliferative zones of the telencephalon early in embryonic development. However, later in embryonic development, endothelial cell proliferation continues in postmitotic domains independent of neuroepithelial cell proliferation. BrdU labeling indices in the ventricular and subventricular zones decline coordinately in endothelial and neuroepithelial cells from embryonic day 11 (E11) to E17. Differences between dorsal and ventral telencephalon were apparent in endothelial cell BrdU labeling patterns. Novel patterns of VEGF, Flt1, Flk1, Tie1 and Tie2 mRNA expression emerged in endothelial and non-endothelial cells in the proliferative zones from E11 to E17. Discussion: Our data lay the foundations for a comprehensive model of vascular and nervous system development and offer insights into molecules and mechanisms that integrate development and repair of both the systems. Keywords: Angiogenesis; Neurogenesis; Embryonic; Telencephalon doi:10.1016/j.ijdevneu.2006.09.155 [P94] Class 3 semaphorins expressed in motoneurons influence angiogenesis in the developing zebrafish R. Lamont ∗ , S. Childs Department of Biochemistry and Molecular Biology, Canada The mechanisms of neural and vascular patterning have many similarities including a reliance on similar sets of guidance molecules. Furthermore, physical interactions between the angioblasts/endothelial cells and neurons have been shown to pattern the vascular system during development. In zebrafish embryonic development, blood vessels in the trunk are guided to grow in a stereotypical pattern. The intersegmental vessels (ISV) of the trunk sprout from the dorsal aorta at approximately 19 h post fertilization (hpf) and migrate dorsally, restricted caudally, rostrally and laterally by the somites and medially by the notochord and neural tube. The out of bounds (obd) genetic zebrafish mutant has aberrant ISV patterning, such that angioblasts start migrating preciously from the dorsal aorta at approximately 17 hpf, and are no longer restricted by the somites caudally and rostrally. Positional cloning has identified endothelial-specific plexinD1 as the gene responsible for the obd phenotype. In the nervous system, plexins have been shown to serve as receptors for the semaphorin family of ligands. In order to further understand how ISV growth is guided, we have identified semaphorins
expressed by trunk motoneurons that influence endothelial cell migration in the developing zebrafish trunk. Keywords: Semaphorin; Zebrafish; Neurovascular patterning doi:10.1016/j.ijdevneu.2006.09.156 [P95] Analysis of the midbrain–hindbrain boundary cell fate using a boundary cell-specific Cre-mouse strain K. Kala 1,∗ , I. Pata 2 , J.M. Partanen 1 1 Institute
of Biotechnology, Finland; Technology, Estonia
2 Tallinn
University of
We describe here a novel transgenic mouse line Hoxb7Core5 expressing Cre recombinase in a group of cells at the midbrain and hindbrain boundary. We used this mouse line in a cell lineage study mapping brain areas subject to the contribution from the initial population of cells expressing Cre recombinase in the Hoxb7Core5 mouse line. The Cre-mediated recombination pattern is described from its emergence at the 44 somite stage until the adult stage, using a Cre reporter line Rosa26R. Initially, Cre expression coincides with the expression of PB-cadherin and p21 in a narrow population of cells overlapping wit the midbrain–hindbrain boundary (Otx2 expression border). In later stages of development, Cre expressing cells contribute to both adjacent brain areas: in the midbrain and hindbrain. In the adult stage, we have shown the presence of recombined cells in inferior colliculus and ventral tegmental area as well as to a small number of Purkinje cells in cerebellar vermis. However, the main population of recombined cells remain located close to the boundary between midand hindbrain. In summary, our results demonstrate the developmental fate of the newly described boundary cells between the midbrain and rhombomere 1 of the hindbrain. Keywords: Cell fate; Midbrain-hindbrain boundary; Cre recombinase doi:10.1016/j.ijdevneu.2006.09.157 [P96] Association and organization of cultured rat infantile pituitary cells after cell migration C. Solano-Agama 1 , B. Gonz´alez-Nava 1 , E. Azor´ın 1 , B. R´eyes-M´arquez 2 , M. Gonz´alez del Pliego 3 , E. Aguirre 3 , M. Eug Mendoza-Garrido 1,∗ 1 Department of Physiology, Biophysics and Neuroscience, CIN-
VESTAV, Mexico; 2 Department of Cellular Biology, CINVESTAV, Mexico; 3 Department of Embryology, Medicine Faculty, UNAM, Mexico The pituitary gland is organized in lobules or cell cords of granular or secretory cells (SC) and agranular or follicle-stellate
535
[P90]
[P92]
Regionalisation of Dlx-expressing progenitors in the medial ganglionic eminence gives rise to distinct subtypes of cortical interneurons in the adult brain
Transcriptional regulation of the mouse doublecortin gene in differentiating neurons
Ghanem 1,∗ ,
N. M. Ekker 1
1 University
J.
Long 2 ,
Hatch 1 ,
G.
of Ottawa, Canada; San Francisco, USA
J.
2 University
Rubenstein 2 , of California at
Dlx genes are required for the differentiation and migration of cortical GABAergic interneurons derived from progenitors located in the medial ganglionic eminence (MGE) and perhaps the caudal ganglionic eminence (CGE). It is not clear how the diversity of GABAergic interneuron subtypes is established and if it involves distinct progenitors in the MGE and CGE. We have identified three conserved enhancers, URE2, I12b and I56i in the Dlx gene loci of vertebrates. All three target expression in cells of the forebrain where endogenous Dlx genes are expressed but show differences in their regional and temporal activities. We examined if such differences in Dlx enhancer activity could be associated to different progenitor populations. We compared the activity of the three enhancers in the MGE and CGE, and in migrating neurons derived from these regions between E11.5 and E13.5 using: (1) reporter lines under the control of each enhancer; (2) double immunohistochemistry on lines with two reporter genes driven by different enhancers. We compared the migration potential of progenitors found in different ventral structures using DiI labelling and co-transplantation experiments in vitro. We also co-labelled adult cortical interneurons in the URE2 and I12b lines, separately. URE2, I12b and I56i displayed differential activities in the dorsal MGE, ventral MGE and CGE, and labelled distinct populations of tangentially migrating neurons at E12.5 and E13.5. We provide evidence that the dMGE and vMGE are distinct sources of tangentially migrating neurons at these ages. In the adult cortex, URE2 labelled the parvalbumin-, calretinin-, NPYand nNOS-positive interneurons, whereas I12b was active in subpopulations of these groups and marked specifically the somatostatin- and VIP-positive interneurons. Dlx gene regulation conferred by several enhancers provides molecular evidence that regional specification of progenitors located in subdivisions within the MGE and CGE generate different subtypes of cortical interneurons in the adult brain. Keywords: Migration
Dlx; GABAergic interneurons; Differentiation;
doi:10.1016/j.ijdevneu.2006.09.153
J.-C. Plumier, M. Muller, B. Rogister, M. Piens ∗ University of Li`ege, Belgium Doublecortin (DCX), a microtubule-associated protein, is transiently expressed in migrating and differentiating immature neurons in the developing and adult central nervous system. Whereas the temporal and spatial DCX expression patterns and DCX post-translational regulations are well known, dcx transcriptional regulation is still unclear. To determine and analyse important regulatory sequences of the dcx promoter, we generated different fragments and deletion constructs of regulatory sequences upstream from the mouse dcx coding region and used them to drive reporter gene expression. Transient expression experiments using these reporter vectors revealed that activation of reporter gene expression was restricted to cells expressing endogenous DCX and that dcx regulation differed in CNS versus PNS. Moreover, we characterized the DCX promoter activity at different time points during neuronal differentiation. Further sequence comparison of dcx promoters across several species revealed the presence of a highly conserved region in the proximal region of the promoter and of several conserved, putative transcription factor binding sites. Further experiments will focus on the identification of the transcription factors involved in dcx gene regulation. Keywords: Gene regulation; Doublecortin; Neuronal differentiation; Transcription factors doi:10.1016/j.ijdevneu.2006.09.154 [P93] Co-ordinate regulation of angiogenesis and neurogenesis in the embryonic telencephalon A. Vasudevan ∗ , P.G. Bhide Massachusetts General Hospital and Harvard Medical School, USA Introduction: The central nervous system (CNS) acquires its vasculature by angiogenesis, which begins early in CNS development and continues throughout life. Neurogenesis also begins early and continues throughout life—at least in some parts of the CNS. Majority of the research addressing interactions between CNS angiogenesis and neurogenesis has focused on the adult brain. However, angiogenesis and neurogenesis are significantly more robust in the embryonic CNS than in the adult. Indeed, the embryonic CNS is perhaps the only site (barring brain tumors) in which angiogenesis and neurogenesis are concurrently robust. Moreover, endothelial cell proliferation, migration and sprouting of new blood vessels occur in the embryonic CNS coincident and coordinate with neuroepithelial precursor cell proliferation
536
Poster abstracts / Int. J. Devl Neuroscience 24 (2006) 495–603
and elaboration of neuronal networks. Therefore, we chose the embryonic CNS as a model for examining mechanisms that coordinately regulate angiogenesis and neurogenesis. Methods: S-phase labeling methods were used to study cell proliferation; laser capture micro-dissection and real-time quantitative PCR to study gene expression profiles of angiogenesisrelated genes in neurogenic regions of the telencephalon. Results: Endothelial and neuroepithelial cell proliferation is concurrently active in the proliferative zones of the telencephalon early in embryonic development. However, later in embryonic development, endothelial cell proliferation continues in postmitotic domains independent of neuroepithelial cell proliferation. BrdU labeling indices in the ventricular and subventricular zones decline coordinately in endothelial and neuroepithelial cells from embryonic day 11 (E11) to E17. Differences between dorsal and ventral telencephalon were apparent in endothelial cell BrdU labeling patterns. Novel patterns of VEGF, Flt1, Flk1, Tie1 and Tie2 mRNA expression emerged in endothelial and non-endothelial cells in the proliferative zones from E11 to E17. Discussion: Our data lay the foundations for a comprehensive model of vascular and nervous system development and offer insights into molecules and mechanisms that integrate development and repair of both the systems. Keywords: Angiogenesis; Neurogenesis; Embryonic; Telencephalon doi:10.1016/j.ijdevneu.2006.09.155 [P94] Class 3 semaphorins expressed in motoneurons influence angiogenesis in the developing zebrafish R. Lamont ∗ , S. Childs Department of Biochemistry and Molecular Biology, Canada The mechanisms of neural and vascular patterning have many similarities including a reliance on similar sets of guidance molecules. Furthermore, physical interactions between the angioblasts/endothelial cells and neurons have been shown to pattern the vascular system during development. In zebrafish embryonic development, blood vessels in the trunk are guided to grow in a stereotypical pattern. The intersegmental vessels (ISV) of the trunk sprout from the dorsal aorta at approximately 19 h post fertilization (hpf) and migrate dorsally, restricted caudally, rostrally and laterally by the somites and medially by the notochord and neural tube. The out of bounds (obd) genetic zebrafish mutant has aberrant ISV patterning, such that angioblasts start migrating preciously from the dorsal aorta at approximately 17 hpf, and are no longer restricted by the somites caudally and rostrally. Positional cloning has identified endothelial-specific plexinD1 as the gene responsible for the obd phenotype. In the nervous system, plexins have been shown to serve as receptors for the semaphorin family of ligands. In order to further understand how ISV growth is guided, we have identified semaphorins
expressed by trunk motoneurons that influence endothelial cell migration in the developing zebrafish trunk. Keywords: Semaphorin; Zebrafish; Neurovascular patterning doi:10.1016/j.ijdevneu.2006.09.156 [P95] Analysis of the midbrain–hindbrain boundary cell fate using a boundary cell-specific Cre-mouse strain K. Kala 1,∗ , I. Pata 2 , J.M. Partanen 1 1 Institute
of Biotechnology, Finland; Technology, Estonia
2 Tallinn
University of
We describe here a novel transgenic mouse line Hoxb7Core5 expressing Cre recombinase in a group of cells at the midbrain and hindbrain boundary. We used this mouse line in a cell lineage study mapping brain areas subject to the contribution from the initial population of cells expressing Cre recombinase in the Hoxb7Core5 mouse line. The Cre-mediated recombination pattern is described from its emergence at the 44 somite stage until the adult stage, using a Cre reporter line Rosa26R. Initially, Cre expression coincides with the expression of PB-cadherin and p21 in a narrow population of cells overlapping wit the midbrain–hindbrain boundary (Otx2 expression border). In later stages of development, Cre expressing cells contribute to both adjacent brain areas: in the midbrain and hindbrain. In the adult stage, we have shown the presence of recombined cells in inferior colliculus and ventral tegmental area as well as to a small number of Purkinje cells in cerebellar vermis. However, the main population of recombined cells remain located close to the boundary between midand hindbrain. In summary, our results demonstrate the developmental fate of the newly described boundary cells between the midbrain and rhombomere 1 of the hindbrain. Keywords: Cell fate; Midbrain-hindbrain boundary; Cre recombinase doi:10.1016/j.ijdevneu.2006.09.157 [P96] Association and organization of cultured rat infantile pituitary cells after cell migration C. Solano-Agama 1 , B. Gonz´alez-Nava 1 , E. Azor´ın 1 , B. R´eyes-M´arquez 2 , M. Gonz´alez del Pliego 3 , E. Aguirre 3 , M. Eug Mendoza-Garrido 1,∗ 1 Department of Physiology, Biophysics and Neuroscience, CIN-
VESTAV, Mexico; 2 Department of Cellular Biology, CINVESTAV, Mexico; 3 Department of Embryology, Medicine Faculty, UNAM, Mexico The pituitary gland is organized in lobules or cell cords of granular or secretory cells (SC) and agranular or follicle-stellate