Introduction: Neural tube defects (NTDs) are one of the most common birth defects worldwide (Dolk, Loane and Garne, 2010) with an incidence of about 0.5-2 in 1000 pregnancies (Mitchell, 2005), making them an important clinical problem. They are a set of congenital disorders that occur due to failure of neural tube closure and other disturbances to normal neurulation during gestation (Vogel et al, 2012). Common NTDs include exencephaly, which occurs due to failure of cranial neurulation, and spina bifida, which occurs due to failure of spinal neurulation (Murdoch and Copp, 2010) (Figure 1). The etiology of these defects is not known, however, several environmental and genetic factors are known to govern their development (Mitchell, 2005). Current measures of tackling NTDs include folic acid supplementation during pregnancy to prevent NTDs, and in utero correction of NTDs like spina bifida (Wallingford et al, 2013). Recent advancements in research have highlighted the roles played by primary cilia and cilia-dependent Hedgehog signalling pathways in neural tube development, and illustrated how disruptions in these lead to abnormal neuronal patterning and NTDs (Vogel et al, 2012). Developing a detailed understanding of the above is required in order to improve prognosis and develop more effective means of NTD prevention and repair. This essay thus focuses on understanding the role played by primary cilia and the hedgehog signalling cascade in the development and pathophysiology of birth defects such as NTDs.Figure 1: Progression of NTDs due to failed cranial (Figures A-C) and spinal (Figures D-F) neurulation. Anencephaly and Myelomeningocele are the most severe forms of exencephaly and spina bifida respectively (Copp and Greene, 2013).    An Introduction to Mammalian Neurulation, Primary Cilia and Hedgehog Signalling Hedgehog (Hh) signalling pathways and their roles in embryonic development and disease were first studied in Drosophila melanogaster, and most pathway components were observed to have remained conserved across species (Murdoch and Copp, 2010). Mammalian hedgehog-signalling pathways comprise of three major proteins out of which Sonic-hedgehog (Shh) is the most well-characterised (Briscoe and Therond, 2013). This essay focuses on the Shh signalling pathway and its role in NTDs. The Shh pathway is extremely complex and a detailed overview of the molecular mechanisms underlying Shh signalling is beyond the scope of this essay. In summary, once released from cells, the Shh ligand binds to its receptor (Ptch1/Patched 1) and activates a transmembrane protein (Smo/Smoothened), resulting in the stimulation of transcription factors (Gli proteins) through complex intracellular signalling pathways (Murdoch and Copp, 2010). In vertebrates, specialised microtubule-based, cell-surface organelles called primary cilia contain several of the above components (Ptch1, Smo, Gli proteins) of the Shh pathway and are thus critical for effective Shh signalling (Haycraft et al, 2005; Corbit et al, 2005; Rohatgi et al, 2007). These primary cilia lack protein-making machinery, and their construction and maintenance depends on a mechanism called Intra-Flagellar-Transport (IFT) which regulates the movement of proteins (IFT proteins) from cells to cilia and vice versa (Witman and Rosenbaum, 2002). This ciliary transport mechanism was found to be essential for vertebrate Shh signalling (Ocbina and Anderson, 2008), and mutations in IFT regulators and proteins were associated with Shh signalling defects (Anderson and Eggenschwiler, 2007; Huangfu et al, 2003).It is therefore important to note that mutations affecting ciliary functions/mechanisms and/or any of the above Shh pathway components, can directly (through pathway components) or indirectly (through primary cilia) lead to defective Shh signalling, thereby causing developmental disorders like NTDs.A brief understanding of mammalian neurulation is required to gain a better insight into the roles played by primary cilia and hedgehog signalling in the regulation of this process. The major events occurring during mammalian neurulation are depicted in Figure 2. Disruptions in any of these events can result in impaired neural tube closure, and lead to NTDs. One of the most crucial pre-requisites for effective neural tube closure is formation of the depicted hinge-regions (Figure 2), and this process needs to be tightly regulated. Primary cilia and Shh signalling are known to regulate hinge-region (Figure 2) formation and neural tube patterning/shaping by controlling cell-proliferation, differentiation, apoptosis etc. (Smith and Schoenwolf, 1997).Figure 2: Electron Micrographs (Figures A-D) and diagrammatic representations (Figures 1-4) of neurulation in chick embryos. Note that folding and elevation of neural folds occurs along the Medial Hinge Point (MHP), which is formed soon after shaping of the neural plate (Figures 1(b)-2). Similarly, convergence of folds leading to neural tube closure, occurs along Dorsolateral Hinge Points (DLHPs), which are formed after MHP-mediated elevation of neural folds (Figure 3) (Smith and Schoenwolf, 1997). The role played by Primary Cilia and Shh Signaling in Neural Tube Development and Defects Primary cilia play a key role in Shh signalling and neural tube patterning (Murdoch and Copp, 2010). The Shh ligand acts as a graded morphogen directing dorsoventral(DV) patterning of the neural tube by regulating the expression of Shh-dependent dorsal and ventral markers (Wilson and Maden, 2005). This Shh-mediated patterning is regulated by primary cilia, and mutations impairing ciliogenesis/cilial function are associated with a loss of ventral markers due to impaired Shh signalling (Huangfu and Anderson, 2005). Primary cilia also regulate neural-tube closure by controlling Shh pathway activation during neurulation (Murdoch and Copp, 2010).Mutations in over 200 genes, about 24 of which regulate ciliogenesis, are associated with the development of NTDs (Walingford et al, 2013). As previously mentioned, these mutations can directly or indirectly affect Shh signalling. The probability of occurrence of NTDs, their type and severity, and the incidence of other developmental defects in mutants entirely depends on the type, location (in Shh pathway components or primary cilia) and functional defects produced by these mutations.Mutations of Shh pathway components resulting in a direct loss/decreased activation of Shh signalling in the neural tube, are associated with disrupted dorsoventral patterning, and cause developmental disorders such as Holoprosencephaly(HPE), a disorder characterised by the failure of separation of cerebral hemispheres, Cyclopia, Left-right asymmetry etc. (Chiang et al, 1996; Reiter and Skarnes, 2006; Goodrich et al, 1999). The incidence of NTDs in these mutants is very low because neural tube closure, surprisingly, occurs normally (Murdoch and Copp, 2010). Decreased Shh signalling can also be achieved indirectly through mutations in genes affecting ciliogenesis and cilial architecture (Murdoch and Copp, 2010), which result in decreased number of cilia and disrupted cilial shape/structure; and in contrast to the previous case, the incidence of NTDs in these mutants is quite high due to disrupted neural tube closure (Caspary, Larkins and Anderson, 2007; Hoover et al, 2008).Reasons for higher incidence of NTDs in mutants with indirect effects (through cilia) on Shh signalling is not known, and could possibly be related to other, unstudied roles of cilia in mammalian neurulation.In contrast to the above decrease in Shh signalling, certain other mutations of Shh pathway components directly cause an increased activation of Shh signalling, and the incidence of NTDs (particularly exencephaly and spina bifida) in these mutants is remarkably high (Pan et al, 2009; Eggenschwiler et al, 2001; Cooper et al, 2005). This is due to the inhibition of neural tube closure by hyperactive Shh signaling (Ybot-Gonzalez et al, 2002). Shh signalling regulates the number of hinge-regions (mentioned above) formed by supressing formation of DLHPs, thereby functioning as an inhibitor of DLHP-mediated dorsolateral bending of neural tube (Refer to Figure 3) (Ybot-Gonzalez et al, 2002). Therefore, hyperactivation of Shh-signalling results in additional, unnecessary inhibition of this DLHP-mediated dorsolateral bending, thus disrupting normal neural tube closure (Figure 3). This Shh-mediated inhibition is dependent on primary cilia and hyperactivation of Shh signalling shows a particularly strong, indirect correlation with mutations disrupting ciliary IFT/IFT proteins (Tran et al, 2008). Interestingly, in contrast to the previous case, these cilial mutants show phenotypes very similar to Shh pathway mutants, and both types of mutations result in a high incidence of NTDs (Murdoch and Copp, 2010).Figure 3: Effect of Shh pathway hyperactivation on spinal neurulation (Murdoch and Copp, 2010).Conclusion:Clearly, the method of disruption of Shh signaling plays a key role in determining the incidence, type and severity of NTDs. Primary cilia, though simple organelles, function as complex signaling centers and their role in regulating mammalian neurulation is yet to be fully understood. It is however clear from the above discussion that mutations of primary cilia serve to be stronger predisposing factors for NTDs (in comparison to mutations of Shh pathway components). 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