Intervertebral disc repair and regeneration: Insights from the notochord.

The vertebrate notochord plays an essential role in patterning multiple structures during embryonic development. In the early 2000s, descendants of notochord cells were demonstrated to form the entire nucleus pulposus of the intervertebral disc in addition to their key role in embryonic patterning. The nucleus pulposus undergoes degeneration during postnatal life, which can lead to back pain. Recently, gene and protein profiles of notochord and nucleus pulposus cells have been identified. These datasets, coupled with the ability to differentiate human induced pluripotent stem cells (iPSCs) into cells that resemble nucleus pulposus cells, provide the possibility of generating a cell-based therapy to halt and/or reverse disc degeneration.

Loss of BMP2 and BMP4 Signaling in the Dental Epithelium Causes Defective Enamel Maturation and Aberrant Development of Ameloblasts.

BMP signaling is crucial for differentiation of secretory ameloblasts, the cells that secrete enamel matrix. However, whether BMP signaling is required for differentiation of maturation-stage ameloblasts (MA), which are instrumental for enamel maturation into hard tissue, is hitherto unknown. To address this, we used an in vivo genetic approach which revealed that combined deactivation of the Bmp2 and Bmp4 genes in the murine dental epithelium causes development of dysmorphic and dysfunctional MA. These fail to exhibit a ruffled apical plasma membrane and to reabsorb enamel matrix proteins, leading to enamel defects mimicking hypomaturation amelogenesis imperfecta. Furthermore, subsets of mutant MA underwent pathological single or collective cell migration away from the ameloblast layer, forming cysts and/or exuberant tumor-like and gland-like structures. Massive apoptosis in the adjacent stratum intermedium and the abnormal cell-cell contacts and cell-matrix adhesion of MA may contribute to this aberrant behavior. The mutant MA also exhibited severely diminished tissue non-specific alkaline phosphatase activity, revealing that this enzyme's activity in MA crucially depends on BMP2 and BMP4 inputs. Our findings show that combined BMP2 and BMP4 signaling is crucial for survival of the stratum intermedium and for proper development and function of MA to ensure normal enamel maturation.

Cell fate specification in the lingual epithelium is controlled by antagonistic activities of Sonic hedgehog and retinoic acid.

The interaction between signaling pathways is a central question in the study of organogenesis. Using the developing murine tongue as a model, we uncovered unknown relationships between Sonic hedgehog (SHH) and retinoic acid (RA) signaling. Genetic loss of SHH signaling leads to enhanced RA activity subsequent to loss of SHH-dependent expression of Cyp26a1 and Cyp26c1. This causes a cell identity switch, prompting the epithelium of the tongue to form heterotopic minor salivary glands and to overproduce oversized taste buds. At developmental stages during which Wnt10b expression normally ceases and Shh becomes confined to taste bud cells, loss of SHH inputs causes the lingual epithelium to undergo an ectopic and anachronic expression of Shh and Wnt10b in the basal layer, specifying de novo taste placode induction. Surprisingly, in the absence of SHH signaling, lingual epithelial cells adopted a Merkel cell fate, but this was not caused by enhanced RA signaling. We show that RA promotes, whereas SHH, acting strictly within the lingual epithelium, inhibits taste placode and lingual gland formation by thwarting RA activity. These findings reveal key functions for SHH and RA in cell fate specification in the lingual epithelium and aid in deciphering the molecular mechanisms that assign cell identity.

Autonomous and nonautonomous roles of Hedgehog signaling in regulating limb muscle formation.

Muscle progenitor cells migrate from the lateral somites into the developing vertebrate limb, where they undergo patterning and differentiation in response to local signals. Sonic hedgehog (Shh) is a secreted molecule made in the posterior limb bud that affects patterning and development of multiple tissues, including skeletal muscles. However, the cell-autonomous and non-cell-autonomous functions of Shh during limb muscle formation have remained unclear. We found that Shh affects the pattern of limb musculature non-cell-autonomously, acting through adjacent nonmuscle mesenchyme. However, Shh plays a cell-autonomous role in maintaining cell survival in the dermomyotome and initiating early activation of the myogenic program in the ventral limb. At later stages, Shh promotes slow muscle differentiation cell-autonomously. In addition, Shh signaling is required cell-autonomously to regulate directional muscle cell migration in the distal limb. We identify neuroepithelial cell transforming gene 1 (Net1) as a downstream target and effector of Shh signaling in that context.

Sonic Hedgehog Signaling Is Required for Cyp26 Expression during Embryonic Development.

Deciphering how signaling pathways interact during development is necessary for understanding the etiopathogenesis of congenital malformations and disease. In several embryonic structures, components of the Hedgehog and retinoic acid pathways, two potent players in development and disease are expressed and operate in the same or adjacent tissues and cells. Yet whether and, if so, how these pathways interact during organogenesis is, to a large extent, unclear. Using genetic and experimental approaches in the mouse, we show that during development of ontogenetically different organs, including the tail, genital tubercle, and secondary palate, Sonic hedgehog (SHH) loss-of-function causes anomalies phenocopying those induced by enhanced retinoic acid signaling and that SHH is required to prevent supraphysiological activation of retinoic signaling through maintenance and reinforcement of expression of the Cyp26 genes. Furthermore, in other tissues and organs, disruptions of the Hedgehog or the retinoic acid pathways during development generate similar phenotypes. These findings reveal that rigidly calibrated Hedgehog and retinoic acid activities are required for normal organogenesis and tissue patterning.

Musculoskeletal integration at the wrist underlies the modular development of limb tendons.

The long tendons of the limb extend from muscles that reside in the zeugopod (arm/leg) to their skeletal insertions in the autopod (paw). How these connections are established along the length of the limb remains unknown. Here, we show that mouse limb tendons are formed in modular units that combine to form a functional contiguous structure; in muscle-less limbs, tendons develop in the autopod but do not extend into the zeugopod, and in the absence of limb cartilage the zeugopod segments of tendons develop despite the absence of tendons in the autopod. Analyses of cell lineage and proliferation indicate that distinct mechanisms govern the growth of autopod and zeugopod tendon segments. To elucidate the integration of these autopod and zeugopod developmental programs, we re-examined early tendon development. At E12.5, muscles extend across the full length of a very short zeugopod and connect through short anlagen of tendon progenitors at the presumptive wrist to their respective autopod tendon segment, thereby initiating musculoskeletal integration. Zeugopod tendon segments are subsequently generated by proximal elongation of the wrist tendon anlagen, in parallel with skeletal growth, underscoring the dependence of zeugopod tendon development on muscles for tendon anchoring. Moreover, a subset of extensor tendons initially form as fused structures due to initial attachment of their respective wrist tendon anlage to multiple muscles. Subsequent individuation of these tendons depends on muscle activity. These results establish an integrated model for limb tendon development that provides a framework for future analyses of tendon and musculoskeletal phenotypes.

BMPs are direct triggers of interdigital programmed cell death.

During vertebrate embryogenesis the interdigital mesenchyme is removed by programmed cell death (PCD), except in species with webbed limbs. Although bone morphogenetic proteins (BMPs) have long been known to be players in this process, it is unclear if they play a direct role in the interdigital mesenchyme or if they only act indirectly, by affecting fibroblast growth factor (FGF) signaling. A series of genetic studies have shown that BMPs act indirectly by regulating the withdrawal of FGF activity from the apical ectodermal ridge (AER); this FGF activity acts as a cell survival factor for the underlying mesenchyme. Other studies using exogenous factors to inhibit BMP activity in explanted mouse limbs suggest that BMPs do not act directly in the mesenchyme. To address the question of whether BMPs act directly, we used an interdigit-specific Cre line to inactivate several genes that encode components of the BMP signaling pathway, without perturbing the normal downregulation of AER-FGF activity. Of three Bmps expressed in the interdigital mesenchyme, Bmp7 is necessary for PCD, but Bmp2 and Bmp4 both have redundant roles, with Bmp2 being the more prominent player. Removing BMP signals to the interdigit by deleting the receptor gene, Bmpr1a, causes a loss of PCD and syndactyly, thereby unequivocally proving that BMPs are direct triggers of PCD in this tissue. We present a model in which two events must occur for normal interdigital PCD: the presence of a BMP death trigger and the absence of an FGF survival activity. We demonstrate that neither event is required for formation of the interdigital vasculature, which is necessary for PCD. However, both events converge on the production of reactive oxygen species that activate PCD.

An Lmx1b-miR135a2 regulatory circuit modulates Wnt1/Wnt signaling and determines the size of the midbrain dopaminergic progenitor pool.

MicroRNAs regulate gene expression in diverse physiological scenarios. Their role in the control of morphogen related signaling pathways has been less studied, particularly in the context of embryonic Central Nervous System (CNS) development. Here, we uncover a role for microRNAs in limiting the spatiotemporal range of morphogen expression and function. Wnt1 is a key morphogen in the embryonic midbrain, and directs proliferation, survival, patterning and neurogenesis. We reveal an autoregulatory negative feedback loop between the transcription factor Lmx1b and a newly characterized microRNA, miR135a2, which modulates the extent of Wnt1/Wnt signaling and the size of the dopamine progenitor domain. Conditional gain of function studies reveal that Lmx1b promotes Wnt1/Wnt signaling, and thereby increases midbrain size and dopamine progenitor allocation. Conditional removal of Lmx1b has the opposite effect, in that expansion of the dopamine progenitor domain is severely compromised. Next, we provide evidence that microRNAs are involved in restricting dopamine progenitor allocation. Conditional loss of Dicer1 in embryonic stem cells (ESCs) results in expanded Lmx1a/b+ progenitors. In contrast, forced elevation of miR135a2 during an early window in vivo phenocopies the Lmx1b conditional knockout. When En1::Cre, but not Shh::Cre or Nes::Cre, is used for recombination, the expansion of Lmx1a/b+ progenitors is selectively reduced. Bioinformatics and luciferase assay data suggests that miR135a2 targets Lmx1b and many genes in the Wnt signaling pathway, including Ccnd1, Gsk3b, and Tcf7l2. Consistent with this, we demonstrate that this mutant displays reductions in the size of the Lmx1b/Wnt1 domain and range of canonical Wnt signaling. We posit that microRNA modulation of the Lmx1b/Wnt axis in the early midbrain/isthmus could determine midbrain size and allocation of dopamine progenitors. Since canonical Wnt activity has recently been recognized as a key ingredient for programming ESCs towards a dopaminergic fate in vitro, these studies could impact the rational design of such protocols.

Dynamics of BMP signaling in limb bud mesenchyme and polydactyly.

Mutations in the Bone Morphogenetic Protein (BMP) pathway are associated with a range of defects in skeletal formation. Genetic analysis of BMP signaling requirements is complicated by the presence of three partially redundant BMPs that are required for multiple stages of limb development. We generated an inducible allele of a BMP inhibitor, Gremlin, which reduces BMP signaling. We show that BMPs act in a dose and time dependent manner in which early reduction of BMPs result in digit loss, while inhibiting overall BMP signaling between E10.5 and E11.5 allows polydactylous digit formation. During this period, inhibiting BMPs extends the duration of FGF signaling. Sox9 is initially expressed in normal digit ray domains but at reduced levels that correlate with the reduction in BMP signaling. The persistence of elevated FGF signaling likely promotes cell proliferation and survival, inhibiting the activation of Sox9 and secondarily, inhibiting the differentiation of Sox9-expressing chondrocytes. Our results provide new insights into the timing and clarify the mechanisms underlying BMP signaling during digit morphogenesis.

Notochord to Nucleus Pulposus Transition.

A tissue that commonly deteriorates in older vertebrates is the intervertebral disc, which is located between the vertebrae. Age-related changes in the intervertebral discs are thought to cause most cases of back pain. Back pain affects more than half of people over the age of 65, and the treatment of back pain costs 50-100 billion dollars per year in the USA. The normal intervertebral disc is composed of three distinct regions: a thick outer ring of fibrous cartilage called the annulus fibrosus, a gel-like material that is surrounded by the annulus fibrosus called the nucleus pulposus, and superior and inferior cartilaginous end plates. The nucleus pulposus has been shown to be critical for disc health and function. Damage to this structure often leads to disc disease. Recent reports have demonstrated that the embryonic notochord, a rod-like structure present in the midline of vertebrate embryos, gives rise to all cell types found in adult nuclei pulposi. The mechanism responsible for the transformation of the notochord into nuclei pulposi is unknown. In this review, we discuss potential molecular and physical mechanisms that may be responsible for the notochord to nuclei pulposi transition.

Let-7b/c enhance the stability of a tissue-specific mRNA during mammalian organogenesis as part of a feedback loop involving KSRP.

Gene silencing mediated by either microRNAs (miRNAs) or Adenylate/uridylate-rich elements Mediated mRNA Degradation (AMD) is a powerful way to post-transcriptionally modulate gene expression. We and others have reported that the RNA-binding protein KSRP favors the biogenesis of select miRNAs (including let-7 family) and activates AMD promoting the decay of inherently labile mRNAs. Different layers of interplay between miRNA- and AMD-mediated gene silencing have been proposed in cultured cells, but the relationship between the two pathways in living organisms is still elusive. We conditionally deleted Dicer in mouse pituitary from embryonic day (E) 9.5 through Cre-mediated recombination. In situ hybridization, immunohistochemistry, and quantitative reverse transcriptase-PCR revealed that Dicer is essential for pituitary morphogenesis and correct expression of hormones. Strikingly, αGSU (alpha glycoprotein subunit, common to three pituitary hormones) was absent in Dicer-deleted pituitaries. αGSU mRNA is unstable and its half-life increases during pituitary development. A transcriptome-wide analysis of microdissected E12.5 pituitaries revealed a significant increment of KSRP expression in conditional Dicer-deleted mice. We found that KSRP directly binds to αGSU mRNA, promoting its rapid decay; and, during pituitary development, αGSU expression displays an inverse temporal relationship to KSRP. Further, let-7b/c downregulated KSRP expression, promoting the degradation of its mRNA by directly binding to the 3'UTR. Therefore, we propose a model in which let-7b/c and KSRP operate within a negative feedback loop. Starting from E12.5, KSRP induces the maturation of let-7b/c that, in turn, post-transcriptionally downregulates the expression of KSRP itself. This event leads to stabilization of αGSU mRNA, which ultimately enhances the steady-state expression levels. We have identified a post-transcriptional regulatory network active during mouse pituitary development in which the expression of the hormone αGSU is increased by let7b/c through downregulation of KSRP. Our study unveils a functional crosstalk between miRNA- and AMD-dependent gene regulation during mammalian organogenesis events.

BMP2 activity, although dispensable for bone formation, is required for the initiation of fracture healing.

Adult bones have a notable regenerative capacity. Over 40 years ago, an intrinsic activity capable of initiating this reparative response was found to reside within bone itself, and the term bone morphogenetic protein (BMP) was coined to describe the molecules responsible for it. A family of BMP proteins was subsequently identified, but no individual BMP has been shown to be the initiator of the endogenous bone repair response. Here we demonstrate that BMP2 is a necessary component of the signaling cascade that governs fracture repair. Mice lacking the ability to produce BMP2 in their limb bones have spontaneous fractures that do not resolve with time. In fact, in bones lacking BMP2, the earliest steps of fracture healing seem to be blocked. Although other osteogenic stimuli are still present in the limb skeleton of BMP2-deficient mice, they cannot compensate for the absence of BMP2. Collectively, our results identify BMP2 as an endogenous mediator necessary for fracture repair.

Neurotransmitter map of the asymmetric dorsal habenular nuclei of zebrafish.

The role of the habenular nuclei in modulating fear and reward pathways has sparked a renewed interest in this conserved forebrain region. The bilaterally paired habenular nuclei, each consisting of a medial/dorsal and lateral/ventral nucleus, can be further divided into discrete subdomains whose neuronal populations, precise connectivity, and specific functions are not well understood. An added complexity is that the left and right habenulae show pronounced morphological differences in many non-mammalian species. Notably, the dorsal habenulae of larval zebrafish provide a vertebrate genetic model to probe the development and functional significance of brain asymmetry. Previous reports have described a number of genes that are expressed in the zebrafish habenulae, either in bilaterally symmetric patterns or more extensively on one side of the brain than the other. The goal of our study was to generate a comprehensive map of the zebrafish dorsal habenular nuclei, by delineating the relationship between gene expression domains, comparing the extent of left-right asymmetry at larval and adult stages, and identifying potentially functional subnuclear regions as defined by neurotransmitter phenotype. Although many aspects of habenular organization appear conserved with rodents, the zebrafish habenulae also possess unique properties that may underlie lateralization of their functions.

Ano1, a Ca2+-activated Cl- channel, coordinates contractility in mouse intestine by Ca2+ transient coordination between interstitial cells of Cajal.

Interstitial cells of Cajal (ICC) are pacemaker cells that generate electrical activity to drive contractility in the gastrointestinal tract via ion channels. Ano1 (Tmem16a), a Ca(2+)-activated Cl(-) channel, is an ion channel expressed in ICC. Genetic deletion of Ano1 in mice resulted in loss of slow waves in smooth muscle of small intestine. In this study, we show that Ano1 is required to maintain coordinated Ca(2+) transients between myenteric ICC (ICC-MY) of small intestine. First, we found spontaneous Ca(2+) transients in ICC-MY in both Ano1 WT and knockout (KO) mice. However, Ca(2+) transients within the ICC-MY network in Ano1 KO mice were uncoordinated, while ICC-MY Ca(2+) transients in Ano1 WT mice were rhythmic and coordinated. To confirm the role of Ano1 in the loss of Ca(2+) transient coordination, we used pharmacological inhibitors of Ano1 activity and shRNA-mediated knock down of Ano1 expression in organotypic cultures of Ano1 WT small intestine. Coordinated Ca(2+) transients became uncoordinated using both these approaches, supporting the conclusion that Ano1 is required to maintain coordination/rhythmicity of Ca(2+) transients. We next determined the effect on smooth muscle contractility using spatiotemporal maps of contractile activity in Ano1 KO and WT tissues. Significantly decreased contractility that appeared to be non-rhythmic and uncoordinated was observed in Ano1 KO jejunum. In conclusion, Ano1 has a previously unidentified role in the regulation of coordinated gastrointestinal smooth muscle function through coordination of Ca(2+) transients in ICC-MY.

The transmembrane protein 16A Ca(2+)-activated Cl- channel in airway smooth muscle contributes to airway hyperresponsiveness.

RATIONALE: Asthma is a chronic inflammatory disorder with a characteristic of airway hyperresponsiveness (AHR). Ca(2+)-activated Cl(-) [Cl((Ca))] channels are inferred to be involved in AHR, yet their molecular nature and the cell type they act within to mediate this response remain unknown. OBJECTIVES: Transmembrane protein 16A (TMEM16A) and TMEM16B are Cl((Ca)) channels, and activation of Cl((Ca)) channels in airway smooth muscle (ASM) contributes to agonist-induced airway contraction. We hypothesized that Tmem16a and/or Tmem16b encode Cl((Ca)) channels in ASM and mediate AHR. METHODS: We assessed the expression of the TMEM16 family, and the effects of niflumic acid and benzbromarone on AHR and airway contraction, in an ovalbumin-sensitized mouse model of chronic asthma. We also cloned TMEM16A from ASM and examined the Cl(-) currents it produced in HEK293 cells. We further studied the impacts of TMEM16A deletion on Ca(2+) agonist-induced cell shortening, and on Cl((Ca)) currents activated by Ca(2+) sparks (localized, short-lived Ca(2+) transients due to the opening of ryanodine receptors) in mouse ASM cells. MEASUREMENTS AND MAIN RESULTS: TMEM16A, but not TMEM16B, is expressed in ASM cells and its expression in these cells is up-regulated in ovalbumin-sensitized mice. Niflumic acid and benzbromarone prevent AHR and contraction evoked by methacholine in ovalbumin-sensitized mice. TMEM16A produces Cl((Ca)) currents with kinetics similar to native Cl((Ca)) currents. TMEM16A deletion renders Ca(2+) sparks unable to activate Cl((Ca)) currents, and weakens caffeine- and methacholine-induced cell shortening. CONCLUSIONS: Tmem16a encodes Cl((Ca)) channels in ASM and contributes to Ca(2+) agonist-induced contraction. In addition, up-regulation of TMEM16A and its augmented activation contribute to AHR in an ovalbumin-sensitized mouse model of chronic asthma. TMEM16A may represent a potential therapeutic target for asthma.

Hedgehog signaling is required for formation of the notochord sheath and patterning of nuclei pulposi within the intervertebral discs.

The vertebrae notochord is a transient rod-like structure that produces secreted factors that are responsible for patterning surrounding tissues. During later mouse embryogenesis, the notochord gives rise to the middle part of the intervertebral disc, called the nucleus pulposus. Currently, very little is known about the molecular mechanisms responsible for forming the intervertebral discs. Here we demonstrate that hedgehog signaling is required for formation of the intervertebral discs. Removal of hedgehog signaling in the notochord and nearby floorplate resulted in the formation of an aberrant notochord sheath that normally surrounds this structure. In the absence of the notochord sheath, small nuclei pulposi were formed, with most notochord cells dispersed throughout the vertebral bodies during embryogenesis. Our data suggest that the formation of the notochord sheath requires hedgehog signaling and that the sheath is essential for maintaining the rod-like structure of the notochord during early embryonic development. As notochord cells form nuclei pulposi, we propose that the notochord sheath functions as a "wrapper" around the notochord to constrain these cells along the vertebral column.

Phylogenetic, expression, and functional analyses of anoctamin homologs in Caenorhabditis elegans.

Ca(2+)-activated Cl(-) channels (CaCCs) are critical to processes such as epithelial transport, membrane excitability, and signal transduction. Anoctamin, or TMEM16, is a family of 10 mammalian transmembrane proteins, 2 of which were recently shown to function as CaCCs. The functions of other family members have not been firmly established, and almost nothing is known about anoctamins in invertebrates. Therefore, we performed a phylogenetic analysis of anoctamins across the animal kingdom and examined the expression and function of anoctamins in the genetically tractable nematode Caenorhabditis elegans. Phylogenetic analyses support five anoctamin clades that are at least as old as the deuterostome/protosome ancestor. This includes a branch containing two Drosophila paralogs that group with mammalian ANO1 and ANO2, the two best characterized CaCCs. We identify two anoctamins in C. elegans (ANOH-1 and ANOH-2) that are also present in basal metazoans. The anoh-1 promoter is active in amphid sensory neurons that detect external chemical and nociceptive cues. Within amphid neurons, ANOH-1::GFP fusion protein is enriched within sensory cilia. RNA interference silencing of anoh-1 reduced avoidance of steep osmotic gradients without disrupting amphid cilia development, chemotaxis, or withdrawal from noxious stimuli, suggesting that ANOH-1 functions in a sensory mode-specific manner. The anoh-2 promoter is active in mechanoreceptive neurons and the spermatheca, but loss of anoh-2 had no effect on motility or brood size. Our study indicates that at least five anoctamin duplicates are evolutionarily ancient and suggests that sensory signaling may be a basal function of the anoctamin protein family.

Shh pathway activation is present and required within the vertebrate limb bud apical ectodermal ridge for normal autopod patterning.

Expression of Sonic Hedgehog (Shh) in the posterior mesenchyme of the developing limb bud regulates patterning and growth of the developing limb by activation of the Hedgehog (Hh) signaling pathway. Through the analysis of Shh and Hh signaling target genes, it has been shown that activation in the limb bud mesoderm is required for normal limb development to occur. In contrast, it has been stated that Hh signaling in the limb bud ectoderm cannot occur because components of the Hh signaling pathway and Hh target genes have not been found in this tissue. However, recent array-based data identified both the components necessary to activate the Hh signaling pathway and targets of this pathway in the limb bud ectoderm. Using immunohistochemistry and various methods of detection for targets of Hh signaling, we found that SHH protein and targets of Hh signaling are present in the limb bud ectoderm including the apex of the bud. To directly test whether ectodermal Hh signaling was required for normal limb patterning, we removed Smo, an essential component of the Hh signaling pathway, from the apical ectodermal ridge (AER). Loss of functional Hh signaling in the AER resulted in disruption of the normal digit pattern and formation of additional postaxial cartilaginous condensations. These data indicate that contrary to previous accounts, the Hh signaling pathway is present and required in the developing limb AER for normal autopod development.

MicroRNA-responsive 'sensor' transgenes uncover Hox-like and other developmentally regulated patterns of vertebrate microRNA expression.

MicroRNAs (miRNAs) are a class of short ( approximately 22-nt) noncoding RNA molecules that downregulate expression of their mRNA targets. Since their discovery as regulators of developmental timing in Caenorhabditis elegans, hundreds of miRNAs have been identified in both animals and plants. Here, we report a technique for visualizing detailed miRNA expression patterns in mouse embryos. We elucidate the tissue-specific expression of several miRNAs during embryogenesis, including two encoded by genes embedded in homeobox (Hox) clusters, miR-10a and miR-196a. These two miRNAs are expressed in patterns that are markedly reminiscent of those of Hox genes. Furthermore, miR-196a negatively regulates Hoxb8, indicating that its restricted expression pattern probably reflects a role in the patterning function of the Hox complex.

Avian intervertebral disc arises from rostral sclerotome and lacks a nucleus pulposus: implications for evolution of the vertebrate disc.

Deterioration of the intervertebral discs is an unfortunate consequence of aging. The intervertebral disc in mammals is composed of three parts: a jelly-like center called the nucleus pulposus, the cartilaginous annulus fibrosus, and anterior and posterior endplates that attach the discs to vertebrae. To understand the origin of the disc, we have investigated the intervertebral region of chickens. Surprisingly, our comparison of mouse and chicken discs revealed that chicken discs lack nuclei pulposi. In addition, the notochord, which in mice forms nuclei pulposi, was found to persist as a rod-like structure and express Shh throughout chicken embryogenesis. Our fate mapping data indicate that cells originating from the rostral half of each somite are responsible for forming the avian disc while cells in the caudal region of each somite form vertebrae. A histological analysis of mammalian and nonmammalian organisms suggests that nuclei pulposi are only present in mammals.