Identifying the mechanisms of segmentation in worm physiology


Metameric segmentation or repeated segments at intervals along anterior-posterior body axis by definition can only be found within the radiation of described phyla which contributes such excellent diversity to respective organism, acquiring broad evolutionary advantages and in the same time, such design principle usually prone to evolution convergence. Hence, it is reasonable to say that the molecular and evolutionary segmentation origins were broadly diversified. In this review, a pool of segmented worms which are not classified as a "model" organism will be the subject of study where biological, metabolic, regulatory and developmental pathways phenomena of this particular species of worm unknown, to determine its developmental mechanisms and point out those genes that are responsible for their body axis segmentation.

The mechanisms of segmentation development have been extensively studied particularly in vertebrates and insects. In point of fact, their developmental mechanisms showed significant differences. In Drosophila for instance, segmentation takes place using mechanism known as top-down segmentation, where an initial vast cellular field subdivided into smaller units through a series of interaction of transcription factor gradients. Contradictorily in vertebrates, the development of segmentation occurs via a mechanism called bottom-up segmentation. This involved cyclical activation of Delta-Notch signalling cascade during somitogenesis where particular cellular field encounters continuity up and down-regulation before a new somite is formed. Another developmental mode of segmentation can be seen in annelids, for example leeches where smaller cells bud off from a pool of teloblastic cells. These are the major developmental mechanisms that can be taken into account in order to identify the segmentation of the so called non-model worm species although it would seem deceptive to find congruous segmentation origins.

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There are a few possible methods can be carried out in identifying genes that regulate segmentation in the subject of study. In previous non-model organism such as honeybees, focusing on the expression of Delta and Notch where RNA was isolated from embryo of honeybees and using RT-PCR with oligo-dT primer, partial or full-length coding sequences of respective genes were amplified. The products were then cloned. Meanwhile, phylogenetic analysis can be used in analysing morphological and molecular data such as comparisons of multiple sequences, functional predictions and to work out evolutionary relationship among organisms. In order to study possible functions of specific signalling pathway that may involves in segmentation processes, key components of suggested pathway can be cloned and their sequence can be compared with other homologues. For instance, it has been shown from phylogenetic analysis in Figure 2, comparison of one of the predicted genes in honeybees, GB12464-PA with other Delta homologues most likely encode a protein sequence of Delta orthologue and now referred to as Am-Delta and for predicted gene XP_396734.3 that closely resembles Drosophila Notch was designated as Am-Notch orthologue. At stages where segmentation process occurred, the expression of both Am-Delta and Am-Notch were then examined using in-situ hybridisation where a labelled complementary DNA or RNA probe was used to localise respective genes in the embryo.

In specific term, the role of Notch signalling within worm species of subject of study can be tested by inhibiting their γ-secretase. DAPT is a γ-secretase inhibitor where it can affect the cleavage process of Notch receptor, hence the Notch signalling. In Drosophila larvae, DAPT can cause embryonic-related defects as results of the blocking of Notch-signalling. Nevertheless, when DAPT was applied in honeybee's embryos prior to larval stage, observation of segmentation defects was impossible as it was invariably fatal. Survivors grew morphologically normal and developed up to stages 9-12 past gastrulation and segmentation processes. Alternatively, embryos were stained for Am-wingless or Am-wg RNA since the expression of wingless indicates the posterior section of each parasegmental region and can be used to analyse DAPT effect on determination of segments. This has been observed in Drosophila, Schistocerca and spiders. Around the site where DAPT was applied, it has been observed that Am-wg expression was affected and cell death occurred but normal stripes seemed to develop normally in other region of the embryo, or fusion of Am-wg stripes, or causing segments overgrowth. When the injection of DAPT was failed, all embryos showed normal development as in controls. Above all, treatments with DAPT may affect segmentation phenotypes involving Notch signalling, suggesting that this pathway maybe hold a significant role in the definition of segmentation.

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The survival rate of embryos treated with DAPT was reduced and it is difficult to interpret DAPT effects on segmentation pattern for long-term. Hence, another method has been introduced which is RNA interference against Am-Delta. Am-Delta gene where expression can be found in stripes early cells and in patterns following pair-rule genes expression was targeted to determine plausible role of Delta-Notch pathway in the definition of segments. It is indeed a possible ligand in regulating segmentation via Notch pathway. Moreover from RNA interference experiments in honeybee embryos, Am-Delta RNAi embryos produced uniform phenotypes that deviated from the control. Larvae lengths were greatly compressed and reduced up to 70% of control mean length. Despite unaffected number and overall pattern of segments, shorter and flatter with absence of normal patterning of segmental furrow in segment epidermis were observed in treated embryos. Similar in DAPT treatment, RNAi effects were not widely spread in treated embryos rather than causing major defects in segmental patterning. In order to check whether the phenotypes observed were effectively by knocking down Notch pathway in the subject of study, one can determine the morphology of nervous system in treated embryos by staining for another RNA namely Am-Krϋppel at stage 9 of the embryo as the number of neural progenitor was significantly increased with injection of Am-Delta dsRNA.

Cis-regulatory elements are essential key regulatory in metazoan development process which ensures precise spatial and temporal patterns of gene expression. By looking at their "pattern generating potential" using computational strategy, one will be able to describe transcriptional regulatory networks and this had been done in Drosophila where regulatory network of multiple species of Drosophila was integrated with specific binding of transcription factor to find out the frequency of conserved binding site across their genome. In order to determine activity pattern of modules, one can build up a network model by combining profiles of binding site with expression of transcription factors and which then can be used to scan the genome for possible generation of whole or partial nearby gene expression, identifying the functions and locations of cis-regulatory elements in the network of segmentation and make predictions based on the overlapping expression activities with reference to genomic expression databases. It is not a surprise that the frequencies of conserved transcription binding site are also very convincing in the prediction of spatial activity and its pattern factor determinants.

The foundation approach of this method is that conserved activity of cis-regulatory elements across the model of study will maintain some parts of its binding activity for each essential transcription factors. Meanwhile the binding site of non-functional regions will be less preserved. A number of profiles are generated using a program called Hidden Markov Model-based Stubb which function to pick up strong and weak motif matches in probabilistic framework. These motif profiles are then combined from a number of model genomes samples and work out the average scores of the orthologous regions in order to develop a profile containing multi-species motifs that integrates evolutionary conservation. As an alternative, using the dynamics of Brownian Motion, one can illustrate the motif score of particular region as a random protean along evolutionary branches via "upward-downward" algorithm that measures intermittently with species number. These profiles of binding site then can be used to make prediction of quiescent transcriptional regulatory network activity in respective genomic region. Taking Drosophila cis-regulatory modules regression model as a reference, weighted contribution of each transcription factors was worked out heuristically as concentration product and the affinity of its binding activity to cis-regulatory module where positive weight regulating role indicates of an activator and the negative weight indicates a repressor. This model has been used in predicting the profiles of anterior-posterior expression of previously described cis-regulatory modules in segmentation network by using different expression pattern profiles of transcription factors of varied species.

One specific experimental technique that can be considered in this case study is chromatin immunoprecipitation (ChIP) as it can locate DNA binding sites and the strength of transcription factor occupancy in a particular protein. CRM analysis of mesoderm development is important for interpreting ChIP data as the expression of transcription factors may vary spatially and temporally, plus it can be used to predict multiple patterns of CRM activity. Meanwhile, complementary computational methods are useful in general approach for predicting particular regulatory networks in tissues and cells regardless of the difficulties to characterise them experimentally. For ChIP assays involving CRM analysis, one can predict the expression patterns and the functional occupancy of a module. In context of expression patterns, it will be advantageous to combine the prediction data from computational binding site with comparative genomics. A method to predict functional occupancy on the occurrence of segmentation modules binding sites which assembles transcription factors motif scores of active CRMS lie within each position throughout anterior-posterior axis has been discovered. It is known that CRMs which expression are driven within similar domains as a transcription factors would results an over-representation of activators binding sites, together with under-representation of repressor binding sites. This concludes that motif scores comparison of cross-species will give better estimation of a factor regulatory effect than ChIP assays which measure the occupancy of modules biochemical, but lacking of regulatory function's evolutionary filter.

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Recently, it has been raised that the formation of coelom; a fluid filled secondary body cavity within mesoderm, is closely connected to segmentation definition where noticeable similarities can be found in patterning of mesodermal segments across bilaterian animals. The repetition of segments in coelom is said to contribute the dynamic swimming and digging through sediments of early worm-like animals such as hemichordates and echinoderms. What distinguishes between separate forms of segmented coelom is the development of growth zone in coelomic cavities. The term of primary segmentation does not involve the building of growth zone from the point where segments occurred. In opposite, secondary segmentation develops repeated identical units and links with a growth zone which can be associated to metameric segmentation process.

In Drosophila, segmentation developmental process can be monitored during the transition from syncytial blastoderm to cellular blastoderm as almost all segments are specified at this stage which allows the gradients of transcription factor to determine the positioning of segmental borders. Anterior-posterior regions are defined by maternal genes which eventually determine positional values of gap gene domains. The products of gap genes will then involve in setting up double-segmental units where pair-rule genes are expressed and combined, hence the definition of segment polarity gene expression domains with respect to specific positions of the embryo. These domains are then used in the formation of embryonic segmental units or known as parasegments. Finally, a gene called wnt signalling pathway (wingless) and the transcription factor engrailed, come into roles and interact with each other to determine parasegmental borders. It is worth to mention the fact that hairy, even-skipped and runt; primary pair-rule genes are specified by separate enhancer elements resulting smooth repeated pattern of pair-rule stripes during blastoderm stage embryo in Drosophila. On the other hand, the definition of pair-rule and polarity of stripes is said to be more rapid in more anterior region compared to those in posterior ones, giving the idea that secondary growth process occurs.

From genetic evolutionary point of view, maternal-specific genes are said to be the most diverse in segmentation gene hierarchy in Drosophila. For instance, bicoid which involves in defining the anterior region is added into the gene hierarchy much later compared to other genes, making the system more ancestral-based. Orthodenticle and empty spiracle, the gap genes in most anterior region are responsible in the definition of head regions. Hunchback is expressed in early stages of cleavage, together with kruppel and giant and these central gap genes are well-preserved in vertebrates. Tailless is expressed in the posterior region and the conservation of this terminal gene gap gene occurs between insects and vertebrates but only with the expression in the anterior region. However the expression of tailess in the posterior region in Drosophila and beetles Tribolium shows different functionality. In the mean time, partial divergence and conservation pattern also can be seen in pair-rule genes of alternating segments and in segment polarity genes regardless of their functional conservation. The beetle Tribolium would be an excellent model organism in determining the functional conservation, to see if the hierarchical subdivision mode is applicable in short germband embryos with respect to subsequent segment specification. Mutagenesis experiments can be used in indicating a top-down mechanism of the respective model organism. Conterminous expression domains common for gap genes occurs in all known gap gene homologs of beetle Tribolium. From the analysis of hairy pair-rule gene within promoter region, it has been proposed that stripe-specific enhancer elements are exist in Tribolium similar in Drosophila suggesting direct regulation of by gap genes.

The similarities found in the regulation of genes mentioned indirectly touches cellular development of short germband embryos, if it has anything to do with syncytial development of Drosophila. Given that there are mutuality in the segmentation of Drosophila and short germband segmentation, it is not likely that complete replacement of transcription factor driven system by ancestral cell-cell signalling in Drosophila takes place.

Looking at the somitogenesis process in chicken as model system, a homolog of hairy gene to be specific c-hairy1, this pair-rule gene homolog is expressed in pre-somitic mesoderm comprising the growth zone in chordates. Nonetheless, it turns out that the expression of respective pair-rule gene homolog is highly dynamic instead of pair-rule-like and it is now known that the waves of expression to develop a new somatic border are initiated at posterior end moving towards anterior end of pre-somitic mesoderm. Individual wave forms one new somatic border and most caudal cells will go through 12 cycles at minimum of c-hairy1 expression before giving full commitment to a somite and overall this process is called somitogenesis clock which also applies in other model vertebrates such as Xenopus, zebrafish and mouse.

A vital component of somitogenesis clock is Notch signalling and correspondingly other genes of Notch pathway including lunatic fringe and Delta homologs demonstrate cyclic expression. In mouse, Wnt3a has been illustrated to commence Notch oscillation and wnt pathway is said to be a crucial component to initiate the clock. In reference to promoter studies, it has been suggested that the whole mode of repeated structures development process in vertebrates completely varied with respect to Drosophila top-down segmentation. These studies suggested that there are specific enhancer elements which conduct the cyclically expressed genes although none of them claimed any evidence in generating unambiguous stripes in specific body regions.

Some data shows the occurrence of top-down component called fgf gradient within this bottom-up system which determine the field for cycling to take place. At the posterior tip of embryo, transcription of fgf RNA takes place which gradually degraded in newly formed tissue, leading to an fgf gradient. The formation of somites can only happen when fgf concentration is low. This shows that fgf gradient specifies a broad segmented field, similar to gap gene gradients in Drosophila. Regardless of its function in coupling and coordinating somite formation with posterior growth, fgf gradient roles are varied from gap and maternal genes of Drosophila. Moreover, the generation mode of fgf gradient can serve as a model in short germband embryos as mentioned in the segmentation mode of Drosophila and beetle Tribolium.

Using leeches as model organism, the segmentation mode is rather distinctive compared to model organisms above. The development of segments in leeches came from a pool of cells known as teloblastic stem cells that cleave asymmetrically which then form segmental founder cells and gradually form segments. More interestingly, specification of the segments is cell lineage dependent and experimental frameshifts of some of teloblastic cell lineages results in development which specific to lineage rather than segmental position. There are other experiments that suggest segmental identity is specified according to the number of stem cell divisions (Seaver and Shankland 2001). On the other hand, even-skipped and hairy RNA homologs affiliate together with mitotic chromatin in segmental founder cells and transcription of hairy pair-rule gene cycles in antiphase to its nuclear localisation protein. It is implausible for this cell-cycle dependent expression to be determined by wave front signalling system like Delta-Notch driven segmentation clock. Crustaceans also generate teloblasts that divide simultaneously and asymmetrically in generating new segmental founder cells although it is not similar to the N and Q teloblasts of annelids.

Different modes of segmentation in distinct model organisms suggest that even in taxa where the formation of segments are obviously homologous, there is flexibility with reference to exact subdivision mode. Plus, the knowledge of molecular segmentation processes is essentially deduced from model systems which are not the main representatives of basal groups of segmented taxa. One should not expect vast similarities from the comparison of these segmentation processes.

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