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Zebrafish (Danio rerio) are tropical freshwater teleost fishes which can grow upto 2.5 inches and life-span in captivity is around 2-3 years. Zebrafish have been using as a model organism for study fundamental processes in embryonic development, vertebrate development, gene function, and genetic basic of several human diseases because of its several unique features like genetic, functional and morphological similarity with the human (Shin et al., 2002). Zebra fish embryos are ideal for the study of vertebrate embryonic development because it is a vertebrate with simple husbandry requirement, high number of progeny production (100-200 embryos weekly), short generation time (2-3 months) which helps to produce large number of mutants, fertilization and development are external, and extraordinary optical transparency that makes them easy to study as individual cell or tissues (Kudoh et al., 2001; Shin et al., 2002, Lieschke et al., 2007). Zebrafish are closely related to human than that of other invertebrate models and can offer benefits as all other vertebrate model organism such mice, chick so that they can be offer as an excellent system to study the human disease and developmental study. As the vertebrate model organism, it would bridge the gap between C. elegans and human/mouse genetics (Zon, 1999). Zebrafish would relate to human so that mutants of it could define the disease loci. For example Zebrafish mutant with cystic kidneys represents the polycystic kidney disease (Drummond et al., 1998) and patients with ALAS-2 mutations, known ascongenital sierblastic anaemia are similar to fish disease (Zon, 1999).
Figure 1 (you need to mention Fig 1 in the text). A Male [Left] and Female [Right] adult Zebrafish
Blastomeres and embryos
Embryos are defined as the earliest stage of the development of an organism which is being used as first choice for developmental biologist since several decades. The process of embryonic development is known as embryogenesis which is accompanied by the series of regulated changes in the gene expression and function (Kudoh, 2001). Blastomeres and embryos contain both maternal and paternal genes. According to Ho and kimmel (1993), like many higher animals, the earliest cell of the Zebrafish embryo contain a full genetic makeup and are developmentally unrestricted and capable of expressing a number of possible phenotypes throughout late blastula and early gastrula stages in Zebrafish. Because this of this unique characteristics, blastomeres and embryos have been using for the developmental study.
Zebrafish Embryogenesis stages
Embryogenesis is the process of cell division and differentiation of embryo which occurs during the early development. In the Zebrafish, there are Series of Seven stages which provide the accuracy of embryogenesis. The seven stages are Zygote period, cleavage period, blastula period, gastrula period, segmentation period, pharyngula period and the hatching period (Kimmel et al., 1995). The seven stages of embryogenesis is described as;
Zygotic Period: A first stage of developing embryo is known as Zygotic period that have one cell and appears like a half-bubble. In this period chorin (egg shell) swells and eventually lifts away from the newly fertilised egg. The diameter size at this stage is about 0.7 mm and it lasts for 45 minutes (Kimmel et al., 1995; Andrew, 1998).
Cleavage Period: This stage begins after the first cleavage of the zygotic cell. In this stage cells continuously divide in every 15 minutes and lasts for 45 minutes to 2.25 hours . So during this period 6 cleavages occur. Due to the presence of large amount of yolk in the fertilised egg cell, the cytoplasmic cleavages or divisions are incomplete known as meroblastic (Kimmel et al., 1995). The nuclei are visible during first half of each cycle (i.e. during interphase) and then the nuclear shapes changes consistently and remains interconnected by cytoplasmic bridges (Kimmel et al., 1995).
Blastrula Period: This stage lasts from 2.25 to 5.25 hours i.e after eighth zygotic cell cycle (128-cell) to onset of gastrulation (Kimmel et al., 1995; Andrew, 1998). In the early stage of blastrula, the cells continue to divide synchronously as before in cleavage period and these early cleavages known as 'metasynchronous' (Kimmel et al., 1995). In this stage developing embryo enters to midblastrula transition (MTB) at 512-cell stage, and the yolk syncytical layer (YSL) forms (Kimmel et al., 1995). After midblastrula transition cell divisions becomes asynchronous and margin reaches to 30% epiboly (Zebrafish K-12). Epiboly begins in the late blastrula period where the thinning and spreading of both YSL and blastodisc occurs over the yolk cell (Kimmel et al., 1995).
Gastrula Period: The gastrula period begins at the time of 5.25 hours and lasts until 10 hours. After the end of blastula period epiboly continues and morphogenetic cell movements of involution, convergence and extension occurs to form the primary germ layers ( epiblast & hypoblast) and embryonic axis by the end of 90% epiboly (Kimmel et al., 1995; Zebrafish K-12).
Segmentation Period: Segmentation period occurs between 10 hours to 22 hours during embryogenesis and commonly known as 'tail bud period' because the tail bud appears at the caudal end of lengthening axis (Kimmel et al., 1995). Due to the morphogenetic movement of cell, somites, pharyangeal arch primordial and neuromeres develop; Primary organs start to become visible (Primary organogenesis); earliest body movement appears movement; tale becomes more prominent and the embryo elongates (Kimmel et al., 1995; Andrew, 1998; Zebrafish K-12).
Pharyngula Period: The period extends from 24 hours to 48 hours of embryogenesis. This is phylotypic-stage embryo which is most suitable stage of development to compare the morphologies with embryo of diverse vertebrates (Kimmel et al., 1995; Andrew, 1998). During pharyngula period body axis straightens from its early cuvature (about the yolk sac); circulation, pigmentation and beginning of fins developments occurs (Kimmel et al., 1995; Zebrafish K-12).
Hatching Period: Hatching period is the final stage of embryogenesis and occurs between 48-72 hours. In this period embryo continues to grow as earlier. During this time completion of morphogenesis of primary organs systems; cartilage development in head as well as pectoral fins occurs which can be seen as they are developing very fastly (Kimmel et al., 1995; Zebrafish K-12).
Figure 2: Early Stages in the development of a embryo [Adapted from Webb & Miller, 2003)
Figure 3: Three different stages of embryogenesis that our study will focus
Hox gene family
Structure and evolution of Hox gene family
Hox genes are the genes related to homeobox gene superfamily and being used as a source of fascination since the discovery because they have the key role in diversifying morphology on the head-tail axis of animal embryo (Lemons and McGinnis, 2006). Homeobox genes are known as family of regulatory genes that contains homeobox, a 183 nucleotide sequence coding for specific nuclear proteins called as homeoproteins (Gehring and Hiromi, 1986). On the molecular level, Hox genes encode proteins with conserved 60- aminoacid DNA binding motif called homeodomain which functions as transcription factors (Garcia-Fernandez, 2005; Meyer, 1998; Foronda et al., 2009; Hueber and Lohmann, 2008) which specify the fate of anterior-posterior axis of bilaterian animals (Amores, et al., 1998). Body patterning of animals starts early in embryonic development that is defined by the functions and expressions of hox genes (Garcia-Fernandez, 2005). So that hox proteins plays a key role to drive the morphological diversification of body segments by controlling the expression of downstream genes (Hueber and Lohmann, 2008). Hox genes are arranged in chromosomal cluster where the different paralogues in the cluster are arranged in collinear manner relative to their distinct expression domains (Pearson et al., 2005) and expressed in a spatially collinear fashion where anterior genes are expressed early in development towards the front of body and posterior genes are expressed later in development towards the more distal part of body (Meyer, 1998). Invertebrate has single cluster but the vertebrates the 39 Hox homeobox-containing genes are organized into four clusters, HoxA-HoxD on different chromosomes, A-D, which are thought to come from the duplication of single cluster, accompanying the increasing complexity of body during the evolution (Amores et al., 1998; Meyer, 1998; Maconochie et al., 1996). According to Crawford (2002) the hoxc gene cluster is different from other three clusters in such a way that it lacks the first three genes, Hoxc1, Hoxc2 and Hoxc3. Amores et al. (1998) reported that zebrafish contains at least 48 genes which are arranged in at least seven chromosomal clusters and also mentioned that teleosts is the most species rich group of vertebrates which have more copies developmental regulatory hox genes than in animal though they have less complexity in the anterior posterior axis. Similarly MR et al. (2010) mentioned that zebrafish harbours 8 hox clusters and one of them is reduced to a single microRNA. Many study stated that the vertebrate hox cluster arose from an ancestral complex by two step duplication during evolution (Kappen et al, 1989; Garica-Fernandez, 2005; Prince et al, 1998-6?). In 1994 Krumlauf (19994) mentioned that some of these duplicated genes must have been lost because none of the complexes have members in all 13 paralogous group.
Figure 3 (you need to mention every Fig in the text before showing them!): Schematic Hox gene cluster organization in Zebrafish and mouse (Adapted from Hunter and Prince, 2002)
Expression, Function and Genetics of Hox gene
It is well known fact that expression of different genes varies according to the stage and cellular function during development. In normal Zebrafish embryogenesis, Prince et al (1998-14) reported the expression of hox genes in between 6 hours (50 % epiboly) and 30 using the whole-mount in situ hybridization method. Many early studies reported that the expression of hox gene starts at gastrulation stages with a temporal patterns more 3' genes in the complex (hoxa1,Hoxb1, hoxa2, hoxb2) turn on during early stage of gastrulation and 5' genes are expressed later in the tail bud, after anterior somites are formed (Wellik, 2007). According to Prince et al (1998-6 what do you mean 1998-6???), anterioposterior system of developing embryo, specifically central nervous system (CNS) and the paraxial mesoderm (sclerotomal component of the somite) are the main area of hox gene expression. Moreover, Zebrafish hox genes are found to be expressed in a range of tissues in addition to CNS and anterior paraxial mesoderm such as genes expression in segmental manner in the posterior mesoderm which plays a role in somitogenesis as it is limited to the forming somites that are moving posterorly with somitogenesis (Prince et al., 1998-6).
According to Pearson et al (2007) [13???] mutation in hox genes result in morphological abnormalities which are restricted to discrete segmental zones along the anterior- posterior axis in a wide variety of animals. The hox gene controls the cell death in spinal cord of mouse which prevents apoptosis in cell resulting the formation of vulva so that hox gene regulates apoptosis which affect on the morphology of several animals (Foronda et al., 2009). Pearson et al (2009) reported that hox protein regulate cell adhesion, division, death, shape and migration in order to mould morphology of animals. Foronda et al. (2009) reviewed that Hox genes interact with signalling pathways in variety of ways such as; hox gene alters the expression of different factors in the signalling pathways, hox gene activity governed by signalling pathway and hox gene cooperates in signalling pathway inducing the expression of target genes. Lopez et al. (2006) studied the expression and localization of hox gene transcript in normal as well as cancerous human cervical tissues and found several hox genes like HOXB1, B2, B5, and B8 were found to expressed in normal as well as cervical epitheleium and sqamous cervical carcinoma. But HOXB2, HOXB4 AND HOXB13 genes were expressed only in tumour so that study suggested the role of hox genes in cervical cancer. To date several roles of hox genes described in organ formation such as the control of cell death, control of cell proliferation, control of pattern and cell specification (Foronda et al., 2009).
Regulation of Hox gene
Activity of hox gene can be regulated either at the transcriptional level or at Post-transcriptional level (Martinez and Amemiya, 2002). Till date several regulatory mechanisms have been confirmed via different regulatory factors including different cofactors, proteins and genes. In 1998, Isaacs et al. reported the role of caudal-related protein (cdx) in the regulation of posterior Hox gene where Xcad3 acts as transcription activator via fibrioblast growth factor (FGF) signalling pathway. Alexander et al. (2009) reviewed that the early expression of hox genes are started by retinoic acid (RA) and fibroblast growth factor (fgf). Retionic acid plays a crucial role in the vertebral patterning by regulating the hox gene expression (Alexander, et al., 2009). Krumlauf, 1994 reviewed that retinoic acid (RA) induce hox gene expression and there is colinear sensitivity in the level and time of response of hox genes to RA in the embryos and cell lines or cell cultures. RA plays important role in the development and patterning of spinal cord via Hoxc transcription factors (Maden, 2006) and patterning of hindbrain (Glover et al., 2006). Roy and Sagerstrom (2004) found that blocking of RA or Fgf pathway affects the hindbrain gene expression at gastrula stage. In contrast to that they also found that both RA and Fgf signalling pathways must be blocked to distrup the gene expression in hindbrain at segmentation stage which suggests the importance of both pathways at this stage. Also the factors like TGF-B, wnts, vhnf1, Meis3, Pbx4 appears to play the important role in establishing the expression of hox gene during embryonic development (Alexander et al, 2009; Hueber and Lohmann, 2008; Wiellette and Sive, 2003; Vlachakis, 2001). Foronda et al (2009) purposed two models for the regulation of hox protein activity due to the activity of different cofactors. Firstly in selective binding model, Extradenticle (exd)-hox combination discriminate among the similar sequences of approximately 10 bp. Secondly, the activity regulation model tells the hox proteins have promiscuous binding so that the difference in binding to a composite hox/Exd binding site are not crucial in conferring activity.
Hoxb1b is one of the several homeobox hox genes related to first paralogous group of Hox B cluster and located in a cluster on chromosome 12 of zebrafish genome and one of two Orthologs gene of mammalian HOXB1 and hoxb1 genes of human and mice respectively. It is belongs to first paralogous group among the 13 paralogous group located at second dublicate of Hox A cluster. Hoxb1b encodes the protein called homeobox protein hox-b1b and function as sequence specific factor in developmental regulatory system providing the cell with specific positional identities on the anterior posterion axis (Alexandre et al, 1996). Subcellular location of this gene is nucleus that and found to expressed first at 50 % epiboly stage [ZFIN]. Prince et al (1998-14) mentioned that the onset of zebrafish expression is durint gastrulation between 80% and 100 % epiboly with an anterior limit at level that will give rise to r4. It belongs to antp homeobox superfamily containing 1 homeobox DNA binding domain [ZFIN] and transcription of gene is known to be induced by retinoic acid (RA) (Kudoh et al, 2002). Similarly, hoxb1b proteins are transcription regulator located in nucleus and acts as developmental protein. Biological process of protein involves hindbrain development; regulation of transcription, DNA dependent and it has sequence specific DNA binding and transcription factor activity as a molecular level function (ZFIN].
Like other paralogous group 1 (PG1) genes, hoxb1 play multiple roles in hindbrain development in mouse where it shows regulatory relationship because hoxa1 helps to activate the hoxb1 in r4 (Alexander et al., 2009). Prince et al (1998-7) reported the expression of Hoxb1 within the developing notochord and in the anteroposterior region of developing notochord of zebrafish embryo where the onset of expression is about 10.5 h of development [ Kimmel et a.l, 1995 cited by prince et al., 1998-7]. From the mutational study on mouse Hoxb1 (Hozb1b ortholgs in Zebrafish ) gene was found to be essential for patterning of cells in dorsoventral axis of rhombomere via interacting with two principal signalling pathways mediating neuronal subtype specification, sonic hedgehogs (Ssh) and Mash (Gaufo et al., 2000). Ecoptic expression of Zebrafish hoxb1b found to cause transformation of rhombomere 2 (r2) towards an r4 identity (McClintock et al., 2001) where similar effect was shown in non-orthologous Hoxa1 gene in mice (Zhang et al., 1994). Similarly, from the mutational study on mice, (Goddard et al., 1996)] reported hoxb1 gene disruption leads to the formation of defective VIIth nerve (facial nerve). From that result they proposed that the primarly role of hoxb1 might be to specify the neurons which form the somatic component of VIIth nerve. In zebrafish embryo, when the function of knocked out the r3/r4 boundary was shifted towards the posterior leading to a reduction in AP extent of r4 and an increase in extent in AP of r3 and similar effect was found in mouse Hoxa1 (functional equivalent of Hoxb1b) knockout (McClintock et al., 2002). Moens and Prince (2002) reviewed that the role of hoxb1a and hoxb1b in patterning the hindbrain appears to be conserved so both gene work together to specify the r4 identity. Similarly, Triple knock down i.e. complete knockdown of PG1 including hoxb1b gene in Zebrafish embryo showed the development of reduced hindbrain and lack of segmentation (McNulty et al., 2005). In addition to that they reported to have a novel role of Hox PG1 in migration and cranial neural crest and development of pharyngeal arches. More recently MR???, et al., (2010), by using gain of function approach and in-situ hybridization identified that the Hoxb1b genes plays the roles in patterning as well as in regulation of cell movement during earliest stages of vertebrate development.
Similar to Hoxb1b, hoxc8a is one of the several homeobox hox genes located in eighth paralogous position a Hox C cluster on chromosome 23 of zebrafish genome. This gene expressed at the 10 somite stage in the paraxial mesoderm with an anterior limit at somite 7 and at the 20 somite stage in developing CNS with an anterior expression limit adjacent to somite 4 (Prince et al., 1998-6; ZFIN]. Retinoic acid was found to induce the expression of hox c8a in normal as well as leukemic myeloid cells. Hoxc8a encodes the protein called homeoox protein hoxc8a that are sequence specific factor in developmental regulatory system providing the cell with specific positional identities on the anterior posterior axis (Prince et al., 1998-6). Hoxc8a proteins are developmental protein located in nucleus and acts as a transcription regulator. In biological process, they plays a role in embryonic pectoral fin morphogenesis, endocrine development, exocrine pancrease development, DNA dependent, regulation of transcription and transcription Molecularly they are sequence-specific DNA binding and transcription activity (ZFIN). In zebrafish, Hoxc8 gene has an anterior expression limit adjacent to somite 4 in CNS (Prince et al., 1998-6?) and found at somite 7 in the paraxial mesoderm (Prince et al., 1998-7). On the other side, in 1995, Burke and collegues found that hoxc-8 axial expression in the trunk region, posterior to forelimb in both chick and mice. The anterior limit of expression was somite level 23-24 i.e the fifth vertebra in chick where the anterior-most region showed the expression in somite 17-18, corresponding to sixth thoracic vertebra in mice.
From the different level of mutational study of mice paralogous group 8 Hox genes, Akker et al, (2001) found that the involvement of hoxc gene in poisoning and morphogenesis of hindlimb. According to Anand et al. (2003) the spatial domain of hoxc8 expression extends anteriority in zebrafish and transcription of hoxc8 is controlled by separate cis-regulatory modules [figure below Figure ???]. Similarly, prince et al. (1998-7), reported that hoxc8 are expressed along the anteroposterior extent of developing notochord so that zebrafish is believed to be an ideal system to study notochord chord expression because of its relatively large notochord and transparent embryo. Similarly in transgenic mice, overexpression of hoxc8 showed the cartilage effect and their cartilage is charterised by the lack of mature proliferating chondrocyte (cillo, 1999) So that hoxc8 seems to control cohonrocyte differentiation pathway along with other homeoproteins.
Figure (number?) A Schematic of Hoxc8 genomic region showing the position of the early enhancer. E, EcoRI, B, BspEI, H, HindIII. Black boxes indicate two exons of Hoxc8. Black sphere is the position of the early enhancer located _3 kb upstream of the translational start site. Arrowhead indicates transcriptional start site. Cis-acting elements, A-E (colored boxes), and potential transcription factors interacting with these elements are indicated (Anand et al, 2003).
Gene expression is the process by which the functional part of genome; gene is converted in to the functional gene products such as RNA or proteins depending upon the conditions like embryo development, exercise, injury etc. The gene expression of different genes can be measured by measuring the level of mRNA level in cell and tissue. In the developing embryo, many morphogenetic events are rely or initiated by the expression of gene within specific, defined area of embryo so that Study of gene expression in the embryo is crucial for the understanding of mechanisms which are responsible for morphogenesis as well as cell differentiation (Sanzo and Tuan., 1998).
Figure 5: Overview of gene expression (Richard Twyman, 2003]
Methods to study gene expression
Several methods are being used to study the difference in gene expression such as semi-quantitative and quantative reverse transcriptase Polymerase chain reaction (RT-PCR), northern blot analysis subtractive hybridization, differential display, and serial analysis of gene expression (SAGE), expressed sequence tag and cDNA microarray hybridization (Moody, 2001). Gene related to specific function is expressed in particular part and stage of development where only the housekeeping genes are expressed always because they produced the protein which are crucial for the cell function. So studies of gene expression using different techniques provide the mechanism for the identification and clustering of novel gene sequences with related function (Moody, 2001). Generally, the methods for the Study of gene expression rely on two basic conditions; sufficient tissue and gene expressed must be high enough to detect by the methods (Koopman, 1993). So that where the gene expressed in small tissues and small group of cells, for example, embryos and blastomeres where the gene transcript are in very low amount, PCR is the solution because of its exquisite sensitivity (Koopman, 1993). Reverse Transcriptase-Polymerase chain reaction (RT-PCR) is a modern low throughout approach for measuring the presence of mRNA in sample.
Reverse transcription-Polymerase chain reaction (RT-PCR)
Reverse transcription-Polymerase chain reaction (PCR) is a Rapid, highly sensitive and widely used method in molecular biology and genetics for analysing the specific gene expression (by determining the presence or absence of transcript) in small tissue samples, such as a single embryonic somite (Koopman, 1993; Sanzo and Tuan, 1998). RT-PCR is divided in to two parts; Reverse transcription of mRNA and Polymerase Chain Reaction. In first part RT-PCR uses enzyme called as reverse transcriptase and primer to anneal and synthesize a desired mRNA sequence. If there is presence of mRNA of particular gene in the tissue or cell extract then primer will anneals to mRNA sequence and converts into cDNA using the enzyme reverse transcriptase. Reverse transcription-polymerase chain reaction (RT-PCR) uses one of housekeeping gene as an internal standard to analyse the expression of specific gene and EF-1Î± is one of the commonly used housekeeping gene as an internal control in RT-PCR (Dean, 2002).
The resulting cDNA from mRNA is used as template for the subsequent PCR reaction. PCR involves the cycle of three consecutive steps; denaturation of double stranded DNA (dsDNA), annealing of primer to their complementary ssDNA sequence and extension or synthesis which are repeated for the exponentially amplification of DNA molecules [see figure below]. The amplified DNA molecules act as the template for next cycle so that PCR can exponentially amplify the DNA during the PCR. The series of reaction are programmed in a closed PCR machine where each three step can be proceeds in orderly manner. Denaturation of double strand DNA occurs when the reaction is heated to 90-95oC. After denaturation short oligonucleotide primer binds to the to their complementary sequence of the template DNA at 50- 65oC and extension of primer is carried out by the use of tag DNA polymerase at 72oC for 2-3 minutes which depends upon the length of PCR product (Apte and Daniel, 2003; Burpo, 2001; Whitney, 2004).
Fig 2(??? number not correct!): Overview of three steps of Polymerase Chain Reaction (PCR)
Figure 3 (???): schematic representation of temperature cycle of polymerase chain reaction
Figure 4: Schematic overview of study of gene expression using Reverse Transcription-Polymerase Chain Reaction (you need to cite references for trhe figures)
PCR optimization and Primer design
Primer are small synthetic oligonucleotides (18-24 nucleotides) that hybridise to the complementary DNA sequence and function in pairs known as forward and reverse primer (Burpo, 2001). Primer acts as the starting point for synthesis of DNA or RNA using DNA polymerase enzyme during PCR reaction. PCR optimisation is usually carried out for the maximum specificity and product yield (Robertson and Walsh-Weller, 1998). Appropriate selection of primer's sequences and concentration are crucial for maximal specific and efficiency of PCR reaction (Loffert, 1997). Generally, GC content of primer should be between 40-60%. According to Loffert et al (1997), 3' terminal sequence of primer is more critical for specificity and sensitive for PCR. So that run triple run of G or C bases in 3' end should be avoided because it may stabilise the nonspecific annealing of the primer. Similarly, thymidine (T) also not allowed at 3' end because it is more prone to mispairing than other nucleotides (Loffert, 1997). To avoid the primer dimer formation, primer pairs should be checked for Complementarity because Complementarity at 3' end of primer sequences results dimer formation (Loffert, 1997). Usually, optimal length of primer is 18-30 nucleotide bases, an annealing temperature is 5oC below the Tm and primer concentration of 0.1-0.5 Î¼M can be used (Loffert, 1997).
Hox genes encode a conserver family of transcriptional factor implicated in conferring regional identity along anteroposterior (AP) axis of all bilateral animal embryos. Since there are very few studies available in the literature about these particular genes hoxb1b and hoxc8a, the studies of these genes are very important to understand developmental process of zebrafish as a whole. The data from these studies will also fill the gap in to draw complete picture of developmental process.