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Gene expression can be regulated in many different ways. Alterations occurring at the level of chromatin without producing any change in DNA sequence can cause stable changes of gene transcription; these changes are called epigenetic modifications (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 cores (Figure 1A) and 147 based pair DNA nucleotides wrapped around them (MacDonald & Roskams, 2009; Shahbazain & Grunstein, 2007).
Histone modifications are a form of epigenetic modifications that generate changes in chromatin structure. Histones can be affected by acetylation and deacetylation of the lysine residues of their N-terminal tails. Such processes are carried out by histone acetyl transferases (HATs) and histone deacetylases (HDACs) respectively (MacDonald & Roskams, 2009; Abel & Zukin, 2008). When HATs acetylate histone protein tails the structure of chromatin relaxes. This relaxation facilitates the interaction between RNA polymerase with DNA enhancing transcription. In the other hand, when HDACs deacetylate histone tails, chromatin becomes more compact and RNA polymerase cannot bind to DNA resulting in transcription repression (Figure 1B) (Reviewed by De Ruijter, et al., 2003).
Many studies suggest that HATs activate gene expression, whereas HDACs function as gene repressors. However, recent research in yeast and cell cultures have demonstrated that Hdacs are present in chromatin that contains activated genes regulating the acetylation of histones to maintain a moderate level of acetylation which may allow efficient gene transcription (Reviewed by Shahbazain & Grunstein, 2007; Wang, et al., 2002; Vogelauer, et al., 2000; Wang, et al 2009).
Figure 1. Nucleosome structure and histone modifications 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 forming an octamer core. Lysine residues (K5, K8, K12, K16) of the amino-terminal tails of histone proteins can be acetylated by histone acetyl transferases (HATs). B) Transcription activation and repression by HATs and HDACs.
The participation of histone deacetylases in gene expression has made them an important topic of study to understand gene control caused by epigenetic modifications. Hdacs regulate transcription during development (Cunliffe, 2004; Cunliffe & Casaccia-Bonnefil, 2006; Yamaguchi, et al., 2005; Stadler, et al., 2005; Noël, et al., 2008; Tou; et al., 2004) ;but they have also been implicated in diseases like cancer (Reviewed by Timmermann, et al., 2001; Cress & Seto, 2000; Mariadason, 2008), cardiac hypertrophy (Trivedi, et al., 2004; Montgomery, et al., 2007) and neurodegenerative disorders (Abel & Zukin, 2008).
Two families of proteins with histone deacetylase activity have been described in yeast and mammals (reviewed by Mariadason, 2008). There are eighteen mammalian HDACs classified in these two families of proteins. The classical HDAC family is integrated by histone deacetylases from classes I (homologous to yeast Rpd3), II (homologous to yeast Had-1) and IV; and the NAD+-dependent family also named Sir2 (silent information regulator) family includes class III HDACs (Reviewed by De Ruijter, et al., 2003; Mariadason, 2008; MacDonald & Roskams, 2009).
Hdac1 in Zebrafish Central Nervous System
There is evidence to demonstrate that Hdac1, a class I histone deacetylase protein, is required in cellular differentiation of many zebrafish organs such as brain and retina (Cunliffe, 2004; Cunliffe & Casaccia-Bonnefil, 2006; Yamaguchi, et al., 2005; Stadler, et al., 2005), as well as in liver and exocrine pancreas development (Noël, et al., 2008).
Cunliffe (2004) observed that in zebrafish mutants lacking hdac1 function, brain structures were smaller than in its wild type siblings. In addition stably reduced expression of proneural genes ash1b and ngn1 was observed; as well as increased expression of the Notch target gene her6. Further working in this laboratory reveals that in the same zebrafish hdac1 mutants, the levels of additional proneural genes such as ascl1a and neurod4 show a dramatic decrease from 12 hours post fertilization onwards as compared with wild type embryos.
Taken together, these studies suggest that Hdac1 regulates proneural gene expression during central nervous system development in zebrafish from the earliest stages of neurogenesis onwards. The decrease expression of proneural genes asl1a, asl1b and neurod4 might be the reason of the reduction in neurogenesis in zebrafish hdac1 mutants.
Five families of genes involved in neurogenesis, which are classified as proneural genes, have been described initially in Drosophila and then in vertebrates (Reviewed by Powell, et al., 2004; Ledent & Vervoot, 2001). These families of proneural genes are achaete (ac), scute (sc), lethal of scute (lsc), asense (ase) families which form the achaete-scute gene system (achaete-scute complex or AS-C) (García-Bellido & Celis, 2009; Ghysen & Dambly-Chaudiere, 1988; Jan & Jan, 1994); and the most recently discovered family of preneural genes is the atonal (ato) family (Reviewed by Bertrand, et al., 2002). They encode transcription factors of the basic helix-loop-helix (bHLH) class (Reviewed by Ledent & Vervoot, 2001). Basic helix-loop-helix proteins are transcription factors involved in the control of gene expression during the development of invertebrates and vertebrates (Davis & Turner, 2001; Ledent & Vervoot, 2001). These transcription factors are formed by a DNA-binding basic domain and two α-helices separated by a loop (Figure 2) (Ledent & Vervoot, 2001). Proneural genes belong to class A bHLH proteins; this means that they bind to E-boxes which are DNA-sequences of 6 nucleotides. For their interaction with DNA they have to form heterodimeric complexes (Reviewed by Bertrand, et al., 2002; Ledent & Vervoot, 2001).
Figure 2. Basic helix-loop-helix protein structure (Taken from Betrand, et al., 2002). BHLH transcription factors such as proneural genes encoding proteins are formed by a basic domain that binds to the E-boxes in the DNA, and two helices separated by a loop.
Proneural genes promote neuronal development and differentiation from the ectoderm (Reviewed by Bertrand, et al., 2002). Neuronal differentiation is strictly regulated by proneural genes. They repress their own expression in adjacent cells by activating the Delta-Notch signalling pathway. Proneural gene repression regulated by the same proneural genes prevents cell differentiation in neighbouring cells; this is called lateral inhibition. They can also promote the expression of hairy and enhancer of split bHLH proteins (Espl in drosophila, Hes, Her, Esr in vertebrates) that in turn repress proneural gene expression (Figure 3) (Reviewed by Bertrand, et al., 2002; Ledent & Vervoot, 2001).
Proneural genes expressed in a neural progenitor promote the expression of Delta proteins that activate the Notch signalling pathway and repress proneural expression in neighbouring cells. But the expression of hairy and enhancer of split bHLH proteins induced by proneural gene expression can respress its expression.
The role of proneural genes during all the process of neuronal differentiation from cell cycle exit to the commitment of neural fate is still not well established.
Many studies have been carried out to investigate the roles of proneural genes in neuronal differentiation and cell fate. One of the best characterized members of the achaete-scute gene system is achaete-scute complex-like1, also known as ascl1 or ash1(mash1in mouse, cash1 in chicken, xash1 in xenopus and zash1 in zebrafish). In drosophila, the expression of achaete-scute complex is required for the development of central and peripheral nervous system (Jan & Jan, 1994). Since the discovery of the AS-C homologues in different organisms, the expression and function of these gene homologues have been studied in many models by loss of function (LOF) and gain of function (GOF) analysis (Bertrand, et al., 2002).
The cDNA of Xash1, the homologous gene of drosophila's achaete-scute complex in xenopus, was isolated by Ferreiro et al. in 1993. They studied the expression of this proneural gene in xenopus embryos by using in situ hibridization. They found that Xash1 mRNA was expressed in the anterior part of the neural tube. Xash1 expression increased and decreased in different brain regions during xenopus development; showing a defined pattern of expression (Ferreiro, et al., 1993). Thanks to Ferrerio's work we know the localization of Xash1 expression; but there has not been published research regarding to Xash1 function in neurogenesis. But in contrast with Xash1, the function of Mash1 has been extensively studied during mouse development.
Mash1 is a member of the achaete-scute family expressed in the nervous system of murines. Ma and co-workers (1997) investigated Mash1 and Ngn1 expression using in situ hybridization. Their results show that at embryonic day (E) 12.5 Mash1 mRNA was detected in sympathetic and enteric ganglia. They also observed that the regions in the forebrain where these two genes were expressed were adjacent but non-overlapping domains, showing a spatial complementarity of proneural genes. Interestingly in other regions of the central nervous system such as olfactory epithelium, midbrain and ventral spinal cord, the expression of both proneural genes is overlapped, but they can be implicated in different stages of neuronal differentiation (Ma, et al., 1997).
Several studies have found that the function of Mash1 is required for the differentiation of neurons of both peripheral and central nervous systems. The nervous cells that require Mash1 for their differentiation are sympathetic and parasympathetic neurons (Hirsh, et al., 1998; Parras, et al., 2006), olfactory sensory neurons (Cau, et al., 2002; Parras, et al., 2002), retinal neurons (Tomita, et al., 1996 cited by Pattyn, et al., 2002) and different kinds of neurons of the brain (Casarosa, et al., 199; Pattyn, et al., 2006; Nieto, et al., 2001; Parras et al., 2002).
When analysing mutant mice that did not express functional Mash1, Hirsh and others (1998) noticed that in autonomic peripheral nervous system, precursor neurons do not complete their differentiation process and degenerate. This group of researchers also found that in this Mash1 knocked down mice some noradrenergic and adrenergic centres of the hindbrain were missing. This suggests that Mash1 is required not only for the early stages of neurogenesis but it also has a role in the expression of noradrenergic phenotype (Hirsh, et al., 1998). A knock-in assay was made in order to find out if Ngn2, a proneural gene from the ato-family, was able to develop noradrenergic neurons in the absence of Mash1. The results show that noradrenergic neurons were differentiated but the levels of dopamine beta- hydroxylase (DBH) and Phox2a (normally expressed in these neurons) were severely reduced (Parras, et al., 2002).
In the olfactory epithelium (OE), Cau and co-workers (2002) observed that in the absence of Mash1 function there was a reduction of neurons in the rostral part of this epithelium, but in other zones of the OE neuronal differentiation remained unaltered in E12.5embryos. They also observed that Mash1 acts in combination with another proneural gene form the atonal family named Ngn1, where Mash1 acts in the generation of olfactory sensory neurons (OSN) basal precursors, whereas Ngn1 acts during OSN differentiation (Cau, et al., 2002). Another study of the role of Mash1 in the olfactory epithelium was carried out by Parras in 2002. Parras and co-workers used a knock-in strategy to study if Ngn2 was able to rescue neuronal differentiation in the absence of Mash1. Interestingly they found that the olfactory epithelium was normally developed when Ngn2 was ectopically expressed in the epithelium that did not express Mash1 (Parras, et al., 2002). Both studies confirmed the redundancy of Mash1 and other proneural genes in the development of olfactory sensory neurons.
In the brain, Mash1 is required for the differentiation of many types of neurons in the cerebral cortex and basal ganglia, mainly in the medial ganglionic eminence. In mice with Mash1 null mutation, the size of the ganglionic eminences was reduced. Besides, the specification of neuronal precursors of the medial ganglionic eminence was shown to be dependent of Mash1 expression and cell proliferation was decreased in the absence of this proneural gene (Casarosa, et al., 1999). The nucleus of solitary tract, located in the dorsal part of the hindbrain, is also dependent of Mash1 expression. In mutant mice lacking of the expression of functional Mash1, Pattyn and co-workers observed a 24 hour delay in the formation of the nucleus of the solitary tract as compared with wild type mice. The development of nTS precursors needs the function of Mash1. But when precursors accumulate when Mash1 is not functionally expressed, differentiation occurs at a slower pace. This reveals two different phases during nTS development in which Mash1 is implicated in different ways (Pattyn, et al., 2006). In the cerebral cortex the function of Mash1 was studied in combination with Ngn2 function. Both proneural genes are required for the differentiation of cortical neurons of different populations. They inhibit the glial fate and promote the neural fate in pluripotent progenitors (Nieto, et al., 2001). All this findings demonstrate that Mash1 is required for the generation of the precursors of different neural cells of the brain.
So far, all the studies mentioned above confirm that the expression and function of Mash1 is necessary for the differentiation of many neural cell types. However, to know if the activity of Mash1 is sufficient to determine the neuronal fate, tha activity of Mash1 in non-neuronal cells was investigated by Lo in 1997. Non-neuronal cells isolated by using an antibody against the receptor tyrosine kinase c-RET were subjected to a sustained expression of Mash1 induced by 50ng/ml BMP2 (bone morphogenetic protein). This mantained expression of Mash1 caused that 80% of the cells were differentiated in neuronal like cells expressing neuronal markers. But the sole expression of Mash1 was insufficient to commit cells to a neuronal fate. Mash1 mantained the competence for neurogenesis but differentiation is subjected to the exposure of other signals. (Lo, et al., 1997).
Taking together, these studies demonstrate that the aschaete-scute like-1 complex homologue in mouse (Mash1) is required for the differentiation process of several neuronal cell types. It has also been shown that Mash1 acts in a redundant way with other proneural genes of the atonal family working in combination with them to complete neuronal differentiation. 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 regions of zebrafish nervous system. Zash1a mRNAs can be found in the hindbrain and in rhombomere 1 in ventral cells, in the other hand zash1b is expressed in ventral cells of rhombomeres 2 to 6. Their expression in other brain structures occurs in different localizations as well (Allende & Weinberg, 1994).
There is few information about the functions of ascl1a and asl1b in different nervous system structures of zebrafish (Cau &Wilson, 2003). Cau and Wilson studied the function of ascl1a in the differentiation of epiphysial neurons. They reported that ascl1a is expressed intensively from 8s onwards in zebrafish embryos. Injection of a morpholino against ascl1a produced a reduction of isl1-positive cells (54.7% at 27 hpf compared with wildtype embryos). Islet1 (isl1) is a protein normally expressed in epihysial neurons that was used as a marker of neuronal differentiation. The reduction of isl1-positive cells showed a decrease number of neurons in the epiphysis (Cau & Wilson, 2003). They also studied if there was a crossed-regulation between asl1a and ngn1. They observed that neuronal differentiation in the epiphysis was impaired when the functional expression of both genes was absent. But the sole decrease of ngn1 expression did not cause a significant reduction of isl1-positive cells. To know the function of both genes during neuronal differentiation in epyphysis, they studied their role in the expression of other neurogenesis regulators such as delta proteins. The results obtained show that asl1a and ngn1 have redundant functions when regulating delta proteins, which are important in the notch signalling pathway (Cau & Wilson, 2003).
Neurod4 is a proneural gene of the atonal family. In mouse neurod4 is also known as Math3 and in zebrafish as Zath3. These proneural gene has not been as studied as achaete-scute complex like-1. In zebrafish Zath3 was isolated by Wang and col. in 2003 by polymerase chain reaction. They also examined Zath3 expression during zebrafish development. Its expression was detected at 11 hours post fertilization in the anterior portion of the neural plate and in the posterior neural plate in bilatera triple stripes. But at 22 hours post fertilization when secondary neurogenesis begins, zath3 expression becomes wider and more complicated in the neural tube (Wang, et al., 2003). The role of Zath3 in early neurogenesis was analysed by Park et al. in 2003. By using in situ hybridisation they observed that at 24 hpf, zath3 was expressed in eyes, forebrain and basal ganglions. When overexpressing zath3 in narrowminded zebrafish mutants that phenotypically lack of sensory neurons, this deficiency of sensory neurons was rescued by this overexpression. These results show that zath3 is sufficient for neuronal differentiation and neuronal fate comitment (Park, et al., 2003). As with the homologues of aschaete-scute, Zath3 was found to act redundantly with other genes such as ngn1 (Park et al., 2003).
Ascl1 and Neurod4 Redundancy
It would be interesting to know if ascl1a, ascl1b and neurod4 act in coordination during neuronal differentiation. Unfortunatelly there are not published studies that demonstrate the physiological interactions between this proneural genes in zebrafish. However there are few studies in mice that involve the co-ordinate function of Mash1 and Math3 (Tomita, et al., 2000; Oshawa, et al., 2005).
Tomita and colaborators observed that Mash1 and Math3 are coexpressed in many midbrain and hindbrain areas. This coexpression suggested a functional redundancy between them in these brain regions. To study if both genes act in combination they examined the neuronal differentiation in single and double mutant embryos. The absence of expression of both genes in double mutant mouse embryos caused a severe reduction of neuronal differentiation. This reduction suggests a blocked neuronal differentiation. They also observed that in double mutant embryos glial cells were formed instead of neurons implying that Mash1 and Math3 specify the neuronal fate (Tomita, et al., 2000). Similar data was obtain when analysing the differentiation of brachiomotor neurons in Mash1-Math3 double mutants. Where the development of brachiomotor neurons was more seriously impaired as compared with single null mutants. This confirms a redundant function of both proneural genes (Oshawa, et al., 2005).
Aims of the project
Taking all that into consideration, it would be interesting to know if the proneural genes whose expression is dependent on Hdac1 (asl1a, asl1b and neurod4) are required for neuronal differentiation. This information will give a new perspective of HDACs functions during neurogenesis.
The proneural genes ascl1a, ascl1b and neurod4 are essential for neuronal specification during CNS development in zebrafish embryos.
To achieve the aims of this project, the expression of the proneural genes: ascl1a, ascl1b and neurod4 will be knocked down using antisense morpholinos individually and in combinations of two, in order to see their effects in neuronal differentiation with neuronal markers such as HuD, HuC, erm and her6.