ZBED6 expression pattern during embryogenesis and in the central nervous system
Axel Ericsson 2010
Department of Neuroscience – Developmental genetics Uppsala University
Supervisors: Klas Kullander and Martin Larhammar
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Table of contents
Abstract...3
Abbreviations ...3
Aim of study ...4
Introduction ...4
ZBED6...4
Development of central nervous system ...4
Materials and methods...5
Immunohistochemistry...5
Imaging...6
Results and discussion ...7
Conclusion...12
Acknowledgement ...12
References ...13
Abstract
ZBED6 is a recently discovered repressor protein, which was found due to a Quantative trait locus (QTL)‐mapping study comparing wild boar with domesticated pigs. A single nucleotide polymorphism which disrupted the ZBED6 interaction with the Insulin like growth factor‐II (igf‐II) gene resulted in an up regulated gene expression and increased muscle mass. The binding site for ZBED6 has been found in numerous growth factors, which indicates an important role for gene regulation. In this study we investigated the ZBED6 protein expression during embryonic development and in adult Central nervous system (CNS) in mouse. Here we show that ZBED6 is expressed by differentiated neurons and starts
approximately at embryonic day 10.5, with no expression observed in the proliferation zone.
From the expression pattern ZBED6 do not appear to be linked to any specific regions in the spinal cord, rather a general expression in differentiated neurons. The protein expression was mapped in the adult brain showing that ZBED6 is widely distributed in many regions and is expressed in both astrocytes and neurons, however the proportion of ZBED6 expressing cells varies between different brain regions.
Abbreviations
BMP ‐ Bone morphogenetic protein bHLH ‐ Basic helix loop helix
CP ‐ Caudate putamen CNS ‐ Central nervous system DLHP ‐ Dorsolateral hinge points FGF ‐ Fibroblast growthfactors GFAP ‐ Glial fibrillaary acidic protein HD ‐ Homeodomain
Igf ‐ Insulin like growth factors MHP ‐ Medium hinge points
NeuN ‐ Neuron nuclei specific antigen NSC ‐ Neural stem cells
QTL ‐ Quantative trait locus
RA ‐ Retinoid acid
Shh ‐ Sonic hedgehog
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Fig.1 Formation of the neural tube.
Lägg in A, B, C I bilden oxå så man kan följa.
Formation of neural plate (A)‐ bends and folds (B) closure of the neural tube migrating crest cells (C).
Aim of study
The aim of this study was to investigate ZBED6 expression pattern in mouse embryonic development and adult CNS using immunohistochemistry to assess its potential role as a regulator of proliferation.
Introduction
The understanding of gene regulation is important in many aspects since the complexity of an organism is not based on the number of genes but rather the regulation of them. How specific transcription factors interact and regulate gene expression is of interest for
understanding and screening for developmental diseases. Point mutations in specific regions can have severe consequences and lead to specific phenotypes. A previously unknown repressor protein was discovered using a QTL‐Mapping, by comparing European wild boar with Large White domestic pigs1. It was found that the domesticated pigs had accumulated a single nucleotide substitution and the favored allele was well conserved due to strong selection for meat production over the last 60 years1. This G‐A transition was located in a CpG island in intron 3 of the insulin like growth factor‐II (igf‐II) gene. The mutation in igf‐II disrupted the interaction with a repressor protein, named ZBED6, and led to an elevated gene expression of Igf‐II resulting in an increased muscle mass. ZBED6 contains two DNA binding BED domains (named after two chromatin binding proteins BEAF and DREAF) which can modify the chromatin structure and regulate the transcription of genes, and a hATC‐
dimerization domain which is related to the hATC superfamily of DNA transposons. The binding site has been found in over 200 genes and many of them associated with developmental disorders and neurological diseases1. In this study we investigated the ZBED6 protein expression during embryonic development using immunohistochemistry and compared it with neuronal and proliferation markers. We have also characterized the expression pattern in adult CNS to examine the importance and potential function of this repressor protein.
Background
Development of central nervous system
Neurulation is the embryonic process when the neural tube is formed, and starts with the formation of the neural plate2. The cells in the lateral ectoderm
differentiate into the neural plate, which then lengthens and narrows and the neuroepithelial cells migrates towards the midline and intercalate.3 The neurulation can be divided into primary and secondary neurulation.
The primary neurulation form the brain and most of the spinal cord while the secondary starts at more caudal part of the spinal cord including sacral and coccygeal
regions4. Furthermore can the primary neurulation be divided into four phases; formation, shaping, bending and closure of the neural tube (fig.1). The neural plate bend at two specific sites5, at the ventral midlines on the median hinge points (MHP) and near the junction of the neural plate, on the dorsolateral hinge points (DLHP). These two sites are involved in different stages of the bending and closing of the neural tube. In mouse neurulation the neural tube starts bending at MHP (E8.5), proceeds bending at MHP and DLHP (E9.5) and finally the lower neural tube only bends at DLHP (E10)6. The closure of the neural tube is initiated in the hindbrain/cervical parts and then proceeds unidirectional towards future brain and spinal regions, additionally two closure sites occurs at forebrain/midbrain and at the rostral end of forebrain. The spinal cord closure continues to the posterior neuropore in which the secondary neurulation starts7.
Arrangement of neurons in the spinal cord
From eleven progenitor domains align along the midline of the developing spinal cord all the spinal cord neuronal subtypes arise8. The arrangement of these progenitor cells and the differentiation to distinct neuronal subtypes is dependent on a large number of intrinsic and extrinsic factors. In the ventral part the important patterning protein sonic hedgehog (Shh) is active which is expressed from the notochordan9. A gradient of Shh regulates the
homeodomain (HD) transcription factors class I and II, by repressing class 1 and promoting class 210. In addition the bone morphogenic protein (BMP) is secreted from the roof plate and inhibits Shh effect in the dorsal parts of the neural tube11. Fibroblast growth factors (FGF) and WNT proteins contribute to proliferation of the neural stem cells (NSC) and are active in the ventricular zone of the neural tube12,13. The differentiation of progenitor cells are then dependent on retinoic acid 14, basic helix loop helix (bHLH) proteins and progenitor specific transcriptions factors, which by a synergic effect contributes to each distinct sub class of neuron15. In the ventral horn of the spinal cord five distinct subtypes of neurons are developed, four interneurons V0‐V3 and the motor neurons (MN)16 and in the dorsal part six cell types are formed, dl1‐3 are somatosensory relay interneurons and dl4‐6 associated interneurons8
Materials and methods Immunohistochemistry
The embryo samples at embryonic stages 9.5‐18, were collected from pregnant females mice sacrificed by cervical dislocation. Embryos were dissected and fixed in 4%
paraformaldehyde (PFA) in PBS for 10–60 min on ice, followed by cryo protection in 30%
sucrose. Tissue Tek O.C.T compound (A/S Chemi‐TEknikk) was used for embedding. The samples were cryo‐sectioned into 12‐16 µm slices using a Microm HM560 (MICROM International GmbH, Germany). Sections were collected on slides and dried, washed in PBS
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(3x10 min) and incubated in blocking solution (1x blocking reagent (Roche), 0.2% triton X‐
100 (Sigma, Germany), 0.02% Sodium azide diluted in PBS) for 2h. Primary antibodies against ZBED6 (rabbit, diluted 0.2 µg/ml) and Neuron‐specific nuclear antigen (NeuN, 1:400) diluted in blocking solution were incubated at 4°C over night. The following day, slides were washed in PBS (3x5 min) and incubated with a secondary antibody conjugated to Alexa 594
(Invitrogen, USA) and Alexa‐488 (Invitrogen, USA) , Alexa ‐647 (Invitrogen, USA) and DAPI (Sigma‐Aldrich, Germany) as a control for nuclear staining in PBS for 1h at room
temperature. Slides were washed in PBS and mounted with mowiol‐488 (ROTH, Germany).
Immunolabeling was analyzed in a fluorescence microscope (Olympus BX61WI, Japan), a fluorescence slide scanner microscope (3DHistech Pannoramic midi, HUNGARY) and a Confocal microscope‐( Zeiss LSM 510 META, Germany)
Imaging
Images were processed in adobe Photoshop CS5 (Adobe Systems, USA) and ImageJ1.43U using; FFT bandpass filter, despreckle function and arranged together in adobe Illustrator CS3
Figure. 3.ZBED6 expression in sargital section E12. Stained with ZBED6 (red) and NeuN (green) (A‐D).
Sargital (A) overview of embryo. Higher magnification images indicated in(B‐D) with ZBED(B) and NeuN(C) and co localization of ZBED6 and NeuN(D).
Images taken with 20x magnification images taken with (3DHistech Pannoramic midi) Scale bar indicates 100um
Results and discussion
The binding site for ZBED6 has been located in over 200 genes, many associated to developmental disorders and neurological diseases. In this study we investigate its expression pattern during the embryonic development by comparing ZBED6 with markers for proliferating and differentiated neurons, furthermore a ZBED6 protein expression screen was performed in the adult mouse brain. Immunohistochemistry on a sagittal Embryo at stage E12.5 stained with antibodies against neuronal marker NeuN17 and ZBED6 can be seen in figure 3. We observed staining and co‐localization of ZBED6 and NeuN in both sub ventricular zone and in the spinal cord but likely not in the ventricular and proliferation zones.
Images were taken with a Pannoramic midi scanner, which was useful in whole embryo imaging and giving multiple images in high magnification and resolution arranged into one picture. The major drawback with the Pannoramic midi
scanner is the sensitivity for crumples which results in out of focus images. To further characterize and establish when the expression of ZBED6 starts we performed an immunohistochemistry assay on coronal sections of whole embryos E9.5‐ E18.5, focusing on the development of the spinal cord due to its well‐known progenitor domains and many neuronal markers are availible. Co‐
labeling was performed with antibodies against ZBED6, NeuN and Ki67 (a general marker for proliferating cells)18. (fig.4), the protein expression onset was observed at
E10.5 in the ventral horn. ZBED6 was co‐localized with NeuN but no overlap was observed between ZBED6 and Ki67 at embryonic stage E10.5 (fig.4). This indicates that ZBED6 does not affect the early specification and is not expressed until the progenitor cells have
differentiated to neurons (fig. 5). The embryonic expression pattern indicates that ZBED6 is not linked to any specific region in the dorsal‐ventral patterning of the spinal cord and is also expressed in the dorsal root ganglion (DRG) (fig 5 M‐P). The number of ZBED6 expressing cells increased during development in the spinal cord in the same pattern as NeuN, and in the adult spinal cord ZBED6 expression was widely distributed over the entire spinal cord (fig.6). ZBED6 is widely expressed in other tissues during embryogenesis, however a more intense staining was observed in CNS. Due to the widespread expression during early development and in the adult spinal cord no co‐staining was performed with immuno
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markers for specific neurons. Regarding the proliferation marker Ki67, this antibody was problematic to get to bind and several antigen retrievals protocols were tried without success. The main parameter for ki67 antibody binding was the tissue fixation, often short fixation time was necessary to receive an analyzable signal.
Figure 5. ZBED6 expression pattern during early neuronal development.
ZBED6 and NeuN immunoflourescence analysis of coronal mouse E9.5‐E12.5 (A‐P)
Image of Spinal cord E9.5(A‐D), E10.5(E‐H) E11.5, (I‐L), 12.5 (M‐P), stained with DAPI (A,E,I,and M), ZBED6 (B,F,J,N), NeuN (C,G,K,O) and the co‐localization of ZBED6 and NeuN (D,H,L and P) indicated by arrowheads, double arrowheads indicates DRG. Scale bar indicates 100µm. Images taken with 20x magnification (Olympus BX61WI)
Figure 4. ZBED6 is co‐expressed with NeuN but not Ki67 in the spinal cord E10.5.
A) Ki67 as proliferation marker, (B) ZBED6 staining in the ventral horn neuraltube, (C) NeuN staining in ventral parts, (D) Yellow staining shows the overlap between ZBED6 and NeuN with no overlap with ki67 the proliferation marker.
Scale bar indicates 100 µm, images taken with 20x magnification (Olympus BX61WI).
ZBED6 expression is widely distributed in the spinal cord (fig 6.). To assess if ZBED6
expression was linked to specific brain regions we mapped the expression in the adult brain and measured the relative protein expression signal. Coronal brain sections were stained with antibodies against ZBED6, NeuN and glial fibrillary acidic protein (GFAP), together with DAPI. The expression of ZBED6 in different brain regions was analyzed and marked with either + (0‐25% of the cells), ++ (25‐50% of the cells), +++ (50‐75% of the cells). ZBED6 is widely distributed and has a protein expression range from 0‐80% of the cells (fig.6 and table.1). Regions with a low expression of ZBED6 appears to be white matter areas such as colossal commissure and the dorsal hippocampal commisure, and regions with high
expression density was for example multiple regions in amygdalaA high overlap with NeuN was observed in most brain regions. No statistical quantification of ZBED6 was done due to the widespread expression. To further investigate if ZBED6 expression in other cell types in CNS we used GFAP a marker for an intermediate filament protein expressed in astrocytes19. A small population of GFAP positive cells co‐localize with ZBED6 expression was observed (fig.8) the proportion of GFAP positive cells expressing ZBED6 was not measured.
Fig.6 ZBED6 expression is widespread in the adult spinal cord
Lumbar spinal cord labeled with DAPI (A) and ZBED6(B). ZBED6 is widely distributed over the entire spinal cord, mainly in the grey matter. Images taken with 4x magnification (Olympus BX61WI)
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Figure.6 The ZBED6 expression varies between different brain regions
Immunofluorescence analysis of Amygdala, secondary visual cortex, caudate putamen, olfactory bulb and DHC.
Coronal overview of brain (P,Q and R) assembled from 4x magnification images, boxed areas indicates higher magnified areas:
Cortex(A‐D), Caudate and putamen ( E‐G), Amygdala (H‐K), Olfactory bulb(M‐O) and DHC(Q‐S) stained with DAPI (A‐M) ZBED6
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Conclusion
During embryogenesis, ZBED6 protein expression starts before E10.5 in differentiated neurons. ZBED6 does not appear to be expressed in proliferating progenitor cells. In the adult brain and spinal cord ZBED6 is expressed in neurons and to some extent astrocytes. A widespread expression was observed in most parts of the brain, while a few regions did not express ZBED6 (e.g. dorsal hippocampal commisure and colossal commisure). The
widespread distribution of ZBED6 and the large number of binding sites indicates a
fundamental role in gene regulation. It has still yet to be determined which genes and how ZBED6 are regulating those in the CSN. How crucial ZBED6 is for normal development and its potential role in neurodevelopmental disorders has yet to be determined. A conditional ZBED6 knock out that allows deletion of Zbed6 in specific celltypes and cell populations will give us a deeper understanding of this protein’s gene regulating abilities.
Acknowledgement
I would like to thank Martin Larhammar which have been a great supervisor guiding me through this project always answering my questions. I’m also grateful to Klas Kullander and Leif Andersson who has given me the chance to do such an interesting project.
Table.1
Figure 7. ZBED6 is expressed by astrocytes
Immunofluorescence analysis of Hippocampus stained with anti‐ZBED6 ,anti‐ NeuN and anti‐GFAP(B‐E) antibodies.
Coronal overview of brain (A) assembled of 4x magnification images. Boxed area indicates higher magnification region.
Hippocampus stained with anti‐GFAP(B), anti‐ZBED6 (C) and anti‐NeuN (D). Arrows indicates co‐localization of GFAP and ZBED6(E), Double headed arrows indicates overlap between ZBED6 and NeuN(E) and arrow heads indicates GFAP positive cell lacking ZBED6. Images taken with 40x magnification (Confocal microscope‐ Zeiss LSM 510 META) Scale bar : 44µm
Olfactory bulb Hypothalamus
Anterior commissure intrabulbar part +++ latoanterior hypothalamus ++
Anterior olfactory area external part +++ anterior hypothalamus area ++
Anterior olfactory area lateral part +++ paraventriculus,med magnocell ++
Dorsal lateral olfactory tract +++ lateral septal nucleus ++
Ependymal subendymal layer +++ tringualar septal nucleus +++
External plexiform layer of the olfactory bulb +++ septofimbrial nucleus + External plexiform layer of the accessory olfactory bulb +++ ventral hippocampal comm +
Gloerular layer of the olfactory bulb +++ Thalamus +
Glomerular layer of the accesory olfactory bulb +++ Anterior commisure intrabulbar
part +
Granuel cell layer of the accesory olfactory bulb +++ Agrunular cortex D Granuel cell layer of the olfactory bulb +++ Agrunular insular cortex V Internal plexiform layer of the olfactory bulb +++ Anterior olfactory media
lateral olfactory tract +++ Anerior olfactory posterior part ++
mitral cell layer of the olfactory bulb +++ cingulate cortex ++
mitral cell layer of the accessory olfactory bulb +++ dorsal endopiriform claustrum +
Hippocampus
pyrmidial cell hippocampus +++
Cortical regions polymorph dentat gyrus
retrospinal granular cortex ++ dentat gyrus
retrosplenial dysgranual cortex ++ granular dentat gyrus +++
primary motorcortex ++ lacunosum moleculare
secondary motorcortex ++ PAG +
primary somatosens ++ dorsal peduncular cortex ++
secondary somatosensory cortex ++ Superior colliculus ++
granualr insular ++ superficial gray sup coll ++
dysgranular insular ++ optic nerve layer ++
agranular insular ++ intermed gray layer ++
piriform cortex ++ intermediate white layer ++
primary visual cortex ++ dorsal tenia tecta +++
secondary visual cortex: lat,mediolat,mediomed ++ frontal cortex ++
primary primary auditory cortex ++
intermediate endopiriform
claustrum +
seconday auditory cortex ++ lateral olfactory tract +
temporal association cortex ++ lateral orbital cortex ++
perihinal cortex ++ medial orbital cortex ++
dorsolateral perinal cortex ++ piriform cortex +
prelimbic cortex
Amygdala rhinal fissura
cortex amygdala transition +++ olfactory tubecle +
anterior cortical amygdaloid nucleus +++ ventral orbital cortex ++
basomedial amygdala ++ ventral tenia tecta ++
anterior amygdala area ++ caudate putamen +
lateral olfactory tract ++ globus pallidus +
IPAC lateral ++ internal capsul +
IPAC medial ++
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