Functional analysis of hdac1-regulated proneural genes during development of the zebrafish cns Essay


Histone Acetylation

Gene look can be regulated in many different ways. Changes happening at the degree of chromatin without bring forthing any alteration in DNA sequence can do stable alterations of cistron written text ; these alterations are called epigenetic alterations ( Jiang, et al. , 2008 ) . The basic unit of chromatin is the nucleosome. Nucleosomes are made up of histone proteins ( H4, H3, H2A, H2B ) which form octameric nucleuss ( Figure 1A ) and 147 based brace DNA bases wrapped around them ( MacDonald & A ; Roskams, 2009 ; Shahbazain & A ; Grunstein, 2007 ) .

Histone alterations are a signifier of epigenetic alterations that generate alterations in chromatin construction. Histones can be affected by acetylation and deacetylation of the lysine residues of their N-terminal dress suits. Such procedures are carried out by histone ethanoyl group transferases ( HATs ) and histone deacetylases ( HDACs ) severally ( MacDonald & A ; Roskams, 2009 ; Abel & A ; Zukin, 2008 ) . When HATs acetylate histone protein tails the construction of chromatin relaxes. This relaxation facilitates the interaction between RNA polymerase with DNA heightening written text. In the other manus, when HDACs deacetylate histone dress suits, chromatin becomes more compact and RNA polymerase can non adhere to DNA ensuing in written text repression ( Figure 1B ) ( Reviewed by De Ruijter, et al. , 2003 ) .

Many surveies suggest that HATs activate cistron look, whereas HDACs map as cistron repressers. However, recent research in barm and cell civilizations have demonstrated that Hdacs are present in chromatin that contains activated cistrons modulating the acetylation of histones to keep a moderate degree of acetylation which may let efficient cistron written text ( Reviewed by Shahbazain & A ; Grunstein, 2007 ; Wang, et al. , 2002 ; Vogelauer, et al. , 2000 ; Wang, et al 2009 ) .

Figure 1. Nucleosome construction and histone alterations by HATs and HDACs. ( Taken from de Ruijter, 2003 ) A ) Nucleosomes are formed by two tetramers of the histone proteins H3-H4 and H2A-H2B organizing an octamer nucleus. Lysine residues ( K5, K8, K12, K16 ) of the amino-terminal dress suits of histone proteins can be acetylated by histone ethanoyl group transferases ( HATs ) . B ) Transcription activation and repression by HATs and HDACs.

The engagement of histone deacetylases in cistron look has made them an of import subject of survey to understand cistron control caused by epigenetic alterations. Hdacs regulate written text during development ( Cunliffe, 2004 ; Cunliffe & A ; Casaccia-Bonnefil, 2006 ; Yamaguchi, et al. , 2005 ; Stadler, et al. , 2005 ; Noel, et al. , 2008 ; Tou ; et al. , 2004 ) ; but they have besides been implicated in diseases like malignant neoplastic disease ( Reviewed by Timmermann, et al. , 2001 ; Cress & A ; Seto, 2000 ; Mariadason, 2008 ) , cardiac hypertrophy ( Trivedi, et al. , 2004 ; Montgomery, et al. , 2007 ) and neurodegenerative upsets ( Abel & A ; Zukin, 2008 ) .

Two households of proteins with histone deacetylase activity have been described in barm and mammals ( reviewed by Mariadason, 2008 ) . There are 18 mammalian HDACs classified in these two households of proteins. The classical HDAC household is integrated by histone deacetylases from categories I ( homologous to yeast Rpd3 ) , II ( homologous to yeast Had-1 ) and IV ; and the NAD+-dependent household besides named Sir2 ( soundless information regulator ) household includes category III HDACs ( Reviewed by De Ruijter, et al. , 2003 ; Mariadason, 2008 ; MacDonald & A ; Roskams, 2009 ) .

Hdac1 in Zebrafish Central Nervous System

There is grounds to show that Hdac1, a category I histone deacetylase protein, is required in cellular distinction of many zebrafish variety meats such as encephalon and retina ( Cunliffe, 2004 ; Cunliffe & A ; Casaccia-Bonnefil, 2006 ; Yamaguchi, et al. , 2005 ; Stadler, et al. , 2005 ) , every bit good as in liver and exocrine pancreas development ( Noel, et al. , 2008 ) .

Cunliffe ( 2004 ) observed that in zebrafish mutations missing hdac1 map, encephalon constructions were smaller than in its wild type siblings. In add-on stably reduced look of proneural cistrons ash1b and ngn1 was observed ; every bit good as increased look of the Notch mark cistron her6. Further working in this research lab reveals that in the same zebrafish hdac1 mutations, the degrees of extra proneural cistrons such as ascl1a and neurod4 show a dramatic lessening from 12 hours post fertilisation onwards as compared with wild type embryos.

Taken together, these surveies suggest that Hdac1 regulates proneural cistron look during cardinal nervous system development in zebrafish from the earliest phases of neurogenesis onwards. The lessening look of proneural cistrons asl1a, asl1b and neurod4 might be the ground of the decrease in neurogenesis in zebrafish hdac1 mutations.

Proneural cistrons

Five households of cistrons involved in neurogenesis, which are classified as proneural cistrons, have been described ab initio in Drosophila and so in craniates ( Reviewed by Powell, et al. , 2004 ; Ledent & A ; Vervoot, 2001 ) . These households of proneural cistrons are achaete ( Ac ) , scute ( Sc ) , lethal of scute ( lsc ) , asense ( ase ) households which form the achaete-scute cistron system ( achaete-scute composite or AS-C ) ( Garcia-Bellido & A ; Celis, 2009 ; Ghysen & A ; Dambly-Chaudiere, 1988 ; Jan & A ; Jan, 1994 ) ; and the most late discovered household of preneural cistrons is the unkeyed ( ato ) household ( Reviewed by Bertrand, et al. , 2002 ) . They encode transcription factors of the basic helix-loop-helix ( bHLH ) category ( Reviewed by Ledent & A ; Vervoot, 2001 ) . Basic helix-loop-helix proteins are transcription factors involved in the control of cistron look during the development of invertebrates and craniates ( Davis & A ; Turner, 2001 ; Ledent & A ; Vervoot, 2001 ) . These written text factors are formed by a DNA-binding basic sphere and two ?-helices separated by a cringle ( Figure 2 ) ( Ledent & A ; Vervoot, 2001 ) . Proneural cistrons belong to category A bHLH proteins ; this means that they bind to E-boxes which are DNA-sequences of 6 bases. For their interaction with DNA they have to organize heterodimeric composites ( Reviewed by Bertrand, et al. , 2002 ; Ledent & A ; Vervoot, 2001 ) .

Figure 2. Basic helix-loop-helix protein construction ( Taken from Betrand, et al. , 2002 ) . BHLH written text factors such as proneural cistrons encoding proteins are formed by a basic sphere that binds to the E-boxes in the Deoxyribonucleic acid, and two spirals separated by a cringle.

Proneural cistrons promote neural development and distinction from the exoderm ( Reviewed by Bertrand, et al. , 2002 ) . Neural distinction is purely regulated by proneural cistrons. They repress their ain look in next cells by triping the Delta-Notch signalling tract. Proneural cistron repression regulated by the same proneural cistrons prevents cell distinction in neighboring cells ; this is called sidelong suppression. They can besides advance the look of hairy and foil of split bHLH proteins ( Espl in Drosophila melanogaster, Hes, Her, Esr in craniates ) that in bend repress proneural cistron look ( Figure 3 ) ( Reviewed by Bertrand, et al. , 2002 ; Ledent & A ; Vervoot, 2001 ) .

Proneural cistrons expressed in a nervous primogenitor promote the look of Delta proteins that activate the Notch signalling pathway and repress proneural look in neighboring cells. But the look of hairy and foil of split bHLH proteins induced by proneural cistron look can respress its look.

The function of proneural cistrons during all the procedure of neural distinction from cell rhythm issue to the committedness of nervous destiny is still non good established.

Many surveies have been carried out to look into the functions of proneural cistrons in neural distinction and cell destiny. One of the best characterized members of the achaete-scute cistron system is achaete-scute complex-like1, besides known as ascl1 or ash1 ( mash1in mouse, cash1 in poulet, xash1 in Xenopus and zash1 in zebrafish ) . In Drosophila melanogaster, the look of achaete-scute composite is required for the development of cardinal and peripheral nervous system ( Jan & A ; Jan, 1994 ) . Since the find of the AS-C homologues in different beings, the look and map of these cistron homologues have been studied in many theoretical accounts by loss of map ( LOF ) and addition of map ( GOF ) analysis ( Bertrand, et al. , 2002 ) .


The complementary DNA of Xash1, the homologous cistron of Drosophila melanogaster ‘s achaete-scute composite in Xenopus, was isolated by Ferreiro et Al. in 1993. They studied the look of this proneural cistron in xenopus embryos by utilizing in situ hibridization. They found that Xash1 messenger RNA was expressed in the anterior portion of the nervous tubing. Xash1 look increased and decreased in different encephalon parts during xenopus development ; demoing a defined form of look ( Ferreiro, et al. , 1993 ) . Thankss to Ferrerio ‘s work we know the localisation of Xash1 look ; but there has non been published research sing to Xash1 map in neurogenesis. But in contrast with Xash1, the map of Mash1 has been extensively studied during mouse development.


Mash1 is a member of the achaete-scute household expressed in the nervous system of murines. Ma and colleagues ( 1997 ) investigated Mash1 and Ngn1 look utilizing in situ hybridisation. Their consequences show that at embryologic twenty-four hours ( E ) 12.5 Mash1 messenger RNA was detected in sympathetic and enteral ganglia. They besides observed that the parts in the prosencephalon where these two cistrons were expressed were next but non-overlapping spheres, demoing a spacial complementarity of proneural cistrons. Interestingly in other parts of the cardinal nervous system such as olfactive epithelial tissue, mesencephalon and ventral spinal cord, the look of both proneural cistrons is overlapped, but they can be implicated in different phases of neural distinction ( Ma, et al. , 1997 ) .

Several surveies have found that the map of Mash1 is required for the distinction of nerve cells of both peripheral and cardinal nervous systems. The nervous cells that require Mash1 for their distinction are sympathetic and parasympathetic nerve cells ( Hirsh, et al. , 1998 ; Parras, et al. , 2006 ) , olfactive centripetal nerve cells ( Cau, et al. , 2002 ; Parras, et al. , 2002 ) , retinal nerve cells ( Tomita, et al. , 1996 cited by Pattyn, et al. , 2002 ) and different sorts of nerve cells of the encephalon ( Casarosa, et al. , 199 ; Pattyn, et al. , 2006 ; Nieto, et al. , 2001 ; Parras et al. , 2002 ) .

When analyzing mutant mice that did non show functional Mash1, Hirsh and others ( 1998 ) noticed that in autonomic peripheral nervous system, precursor nerve cells do non finish their distinction procedure and pervert. This group of research workers besides found that in this Mash1 knocked down mice some noradrenergic and sympathomimetic Centres of the rhombencephalon were losing. This suggests that Mash1 is required non merely for the early phases of neurogenesis but it besides has a function in the look of noradrenergic phenotype ( Hirsh, et al. , 1998 ) . A knock-in check was made in order to happen out if Ngn2, a proneural cistron from the ato-family, was able to develop noradrenergic nerve cells in the absence of Mash1. The consequences show that noradrenergic nerve cells were differentiated but the degrees of dopamine beta- hydroxylase ( DBH ) and Phox2a ( usually expressed in these nerve cells ) were badly reduced ( Parras, et al. , 2002 ) .

In the olfactive epithelial tissue ( OE ) , Cau and colleagues ( 2002 ) observed that in the absence of Mash1 map there was a decrease of nerve cells in the rostral portion of this epithelial tissue, but in other zones of the OE neural distinction remained unchanged in E12.5embryos. They besides observed that Mash1 acts in combination with another proneural cistron form the unkeyed household named Ngn1, where Mash1 acts in the coevals of olfactive centripetal nerve cells ( OSN ) basal precursors, whereas Ngn1 acts during OSN distinction ( Cau, et al. , 2002 ) . Another survey of the function of Mash1 in the olfactive epithelial tissue was carried out by Parras in 2002. Parras and colleagues used a knock-in scheme to analyze if Ngn2 was able to deliver neural distinction in the absence of Mash1. Interestingly they found that the olfactive epithelial tissue was usually developed when Ngn2 was ectopically expressed in the epithelial tissue that did non show Mash1 ( Parras, et al. , 2002 ) . Both surveies confirmed the redundancy of Mash1 and other proneural cistrons in the development of olfactive centripetal nerve cells.

In the encephalon, Mash1 is required for the distinction of many types of nerve cells in the intellectual cerebral mantle and basal ganglia, chiefly in the median ganglionic distinction. In mice with Mash1 void mutant, the size of the ganglionic distinctions was reduced. Besides, the specification of neural precursors of the median ganglionic distinction was shown to be dependent of Mash1 look and cell proliferation was decreased in the absence of this proneural cistron ( Casarosa, et al. , 1999 ) . The karyon of lone piece of land, located in the dorsal portion of the rhombencephalon, is besides dependent of Mash1 look. In mutant mice lacking of the look of functional Mash1, Pattyn and colleagues observed a 24 hr hold in the formation of the karyon of the lone piece of land as compared with wild type mice. The development of nTS precursors needs the map of Mash1. But when precursors accumulate when Mash1 is non functionally expressed, distinction occurs at a slower gait. This reveals two different stages during nTS development in which Mash1 is implicated in different ways ( Pattyn, et al. , 2006 ) . In the intellectual cerebral mantle the map of Mash1 was studied in combination with Ngn2 map. Both proneural cistrons are required for the distinction of cortical nerve cells of different populations. They inhibit the glial destiny and advance the nervous destiny in pluripotent primogenitors ( Nieto, et al. , 2001 ) . All this findings demonstrate that Mash1 is required for the coevals of the precursors of different nervous cells of the encephalon.

So far, all the surveies mentioned above confirm that the look and map of Mash1 is necessary for the distinction of many nervous cell types. However, to cognize if the activity of Mash1 is sufficient to find the neural destiny, tha activity of Mash1 in non-neuronal cells was investigated by Lo in 1997. Non-neuronal cells isolated by utilizing an antibody against the receptor tyrosine kinase c-RET were subjected to a sustained look of Mash1 induced by 50ng/ml BMP2 ( bone morphogenetic protein ) . This mantained look of Mash1 caused that 80 % of the cells were differentiated in neural like cells showing neural markers. But the exclusive look of Mash1 was deficient to perpetrate cells to a neural destiny. Mash1 mantained the competency for neurogenesis but distinction is subjected to the exposure of other signals. ( Lo, et al. , 1997 ) .

Taking together, these surveies demonstrate that the aschaete-scute like-1 complex homologue in mouse ( Mash1 ) is required for the distinction procedure of several neural cell types. It has besides been shown that Mash1 acts in a excess manner with other proneural cistrons of the unkeyed household working in combination with them to finish neural distinction. A similar thing has been found with the achaete-scute homologues in zebrafish.


There are two homologues of AS-C in zebrafish, these are zash1a and zash1b. Zash1a or ascl1a is 94 % similar to Mash1 whereas zash1b or ascl1b is 80 % similar to its mouse homologue. They are expressed in different parts of zebrafish nervous system. Zash1a messenger RNA can be found in the rhombencephalon and in rhombomere 1 in ventral cells, in the other manus zash1b is expressed in ventral cells of rhombomeres 2 to 6. Their look in other encephalon constructions occurs in different localisations as good ( Allende & A ; Weinberg, 1994 ) .

There is few information about the maps of ascl1a and asl1b in different nervous system constructions of zebrafish ( Cau & A ; Wilson, 2003 ) . Cau and Wilson studied the map of ascl1a in the distinction of epiphyseal nerve cells. They reported that ascl1a is expressed intensively from 8s onwards in zebrafish embryos. Injection of a morpholino against ascl1a produced a decrease of isl1-positive cells ( 54.7 % at 27 hpf compared with wildtype embryos ) . Islet1 ( isl1 ) is a protein usually expressed in epihysial nerve cells that was used as a marker of neural distinction. The decrease of isl1-positive cells showed a lessening figure of nerve cells in the epiphysis ( Cau & A ; Wilson, 2003 ) . They besides studied if there was a crossed-regulation between asl1a and ngn1. They observed that neural distinction in the epiphysis was impaired when the functional look of both cistrons was absent. But the exclusive lessening of ngn1 look did non do a important decrease of isl1-positive cells. To cognize the map of both cistrons during neural distinction in epyphysis, they studied their function in the look of other neurogenesis regulators such as delta proteins. The consequences obtained show that asl1a and ngn1 have excess maps when modulating delta proteins, which are of import in the notch signalling tract ( Cau & A ; Wilson, 2003 ) .


Neurod4 is a proneural cistron of the unkeyed household. In mouse neurod4 is besides known as Math3 and in zebrafish as Zath3. These proneural cistron has non been every bit studied as achaete-scute complex like-1. In zebrafish Zath3 was isolated by Wang and col. in 2003 by polymerase concatenation reaction. They besides examined Zath3 look during zebrafish development. Its look was detected at 11 hours post fertilisation in the anterior part of the nervous home base and in the posterior nervous home base in bilatera three-base hit chevrons. But at 22 hours station fertilisation when secondary neurogenesis begins, zath3 look becomes wider and more complicated in the nervous tubing ( Wang, et al. , 2003 ) . The function of Zath3 in early neurogenesis was analysed by Park et Al. in 2003. By utilizing in situ hybridization they observed that at 24 hpf, zath3 was expressed in eyes, prosencephalon and basal ganglions. When overexpressing zath3 in narrowminded zebrafish mutations that phenotypically lack of centripetal nerve cells, this lack of centripetal nerve cells was rescued by this overexpression. These consequences show that zath3 is sufficient for neural distinction and neural destiny comitment ( Park, et al. , 2003 ) . As with the homologues of aschaete-scute, Zath3 was found to move redundantly with other cistrons such as ngn1 ( Park et al. , 2003 ) .

Ascl1 and Neurod4 Redundancy

It would be interesting to cognize if ascl1a, ascl1b and neurod4 act in coordination during neural distinction. Unfortunatelly there are non published surveies that demonstrate the physiological interactions between this proneural cistrons in zebrafish. However there are few surveies in mice that involve the co-ordinate map of Mash1 and Math3 ( Tomita, et al. , 2000 ; Oshawa, et al. , 2005 ) .

Tomita and colaborators observed that Mash1 and Math3 are coexpressed in many mesencephalon and rhombencephalon countries. This coexpression suggested a functional redundancy between them in these encephalon parts. To analyze if both cistrons act in combination they examined the neural distinction in individual and dual mutant embryos. The absence of look of both cistrons in dual mutation mouse embryos caused a terrible decrease of neural distinction. This decrease suggests a out of use neural distinction. They besides observed that in dual mutant embryos glial cells were formed alternatively of nerve cells connoting that Mash1 and Math3 specify the neural destiny ( Tomita, et al. , 2000 ) . Similar information was obtain when analyzing the distinction of brachiomotor nerve cells in Mash1-Math3 dual mutations. Where the development of brachiomotor nerve cells was more earnestly impaired as compared with individual void mutations. This confirms a excess map of both proneural cistrons ( Oshawa, et al. , 2005 ) .

Purposes of the undertaking

Taking all that into consideration, it would be interesting to cognize if the proneural cistrons whose look is dependent on Hdac1 ( asl1a, asl1b and neurod4 ) are required for neural distinction. This information will give a new position of HDACs maps during neurogenesis.


The proneural cistrons ascl1a, ascl1b and neurod4 are indispensable for neural specification during CNS development in zebrafish embryos.


To accomplish the purposes of this undertaking, the look of the proneural cistrons: ascl1a, ascl1b and neurod4 will be knocked down utilizing antisense morpholinos separately and in combinations of two, in order to see their effects in neural distinction with neural markers such as HuD, HuC, erm and her6.