Evolutionary Transition in the Regulation of Vertebrate Pronephros Development: A New Role for Retinoic Acid.

The anterior-posterior (AP) axis in chordates is regulated by a conserved set of genes and signaling pathways, including Hox genes and retinoic acid (RA), which play well-characterized roles in the organization of the chordate body plan. The intermediate mesoderm (IM), which gives rise to all vertebrate kidneys, is an example of a tissue that differentiates sequentially along this axis. Yet, the conservation of the spatiotemporal regulation of the IM across vertebrates remains poorly understood. In this study, we used a comparative developmental approach focusing on non-conventional model organisms, a chondrichthyan (catshark), a cyclostome (lamprey), and a cephalochordate (amphioxus), to assess the involvement of RA in the regulation of chordate and vertebrate pronephros formation. We report that the anterior expression boundary of early pronephric markers (Pax2 and Lim1), positioned at the level of somite 6 in amniotes, is conserved in the catshark and the lamprey. Furthermore, RA, driving the expression of Hox4 genes like in amniotes, regulates the anterior pronephros boundary in the catshark. We find no evidence for the involvement of this regulatory hierarchy in the AP positioning of the lamprey pronephros and the amphioxus pronephros homolog, Hatschek's nephridium. This suggests that despite the conservation of Pax2 and Lim1 expressions in chordate pronephros homologs, the responsiveness of the IM, and hence of pronephric genes, to RA- and Hox-dependent regulation is a gnathostome novelty.

Comparative spatiotemporal analysis of Hox gene expression in early stages of intermediate mesoderm formation.

BACKGROUND: Hox genes are key players in AP patterning of the vertebrate body plan and are necessary for organogenesis. Several studies provide evidence for the role Hox genes play during kidney development and especially regarding metanephros initiation and formation. However, the role Hox genes play during early stages of kidney development is largely unknown. A recent study in our lab revealed the role Hoxb4 plays in conferring the competence of intermediate mesodermal cells to respond to kidney inductive signals and express early kidney regulators. RESULTS: As a first step in understanding the role Hox genes play in setting the formation of the pronephros morphogenetic field and the expression of early regulators of kidney development, we studied in detail the expression pattern of 10 Hox genes in relation to the 6th somite axial level, the anterior sharp border of the kidney field. Despite the idea of spatial co-linearity as exemplified in the Hox gene expression pattern in late developmental stages, a very dynamic spatio-temporal expression of these genes was found in early stages. CONCLUSIONS: Since mesodermal patterning occurs at gastrula stages, the relevance of a "Hox code" at early stages is questioned in this study.

Algorithm of myogenic differentiation in higher-order organisms.

Cell fate determination is governed by complex signaling molecules at appropriate concentrations that regulate the cell decision-making process. In vertebrates, however, concentration and kinetic parameters are practically unknown, and therefore the mechanism by which these molecules interact is obscure. In myogenesis, for example, multipotent cells differentiate into skeletal muscle as a result of appropriate interplay between several signaling molecules, which is not sufficiently characterized. Here we demonstrate that treatment of biochemical events with SAT (satisfiability) formalism, which has been primarily applied for solving decision-making problems, can provide a simple conceptual tool for describing the relationship between causes and effects in biological phenomena. Specifically, we applied the Lukasiewicz logic to a diffusible protein system that leads to myogenesis. The creation of an automaton that describes the myogenesis SAT problem has led to a comprehensive overview of this non-trivial phenomenon and also to a hypothesis that was subsequently verified experimentally. This example demonstrates the power of applying Lukasiewicz logic in describing and predicting any decision-making problem in general, and developmental processes in particular.

Interplay between activin and Hox genes determines the formation of the kidney morphogenetic field.

The kidney develops in a specific position along the anterior-posterior axis. All vertebrate kidney tissues are derived from the intermediate mesoderm (IM), and early kidney genes such as Lim1 and Pax2 are expressed in amniotes posterior to the sixth somite axial level. IM cells anterior to this level do not express kidney genes owing to changes in their competence to respond to kidney-inductive signals present along the entire axis. We aimed to understand the molecular mechanisms governing the loss of competence of anterior IM cells and the formation of the anterior border of the kidney morphogenetic field. We identified the dorsal neural tube as the potential kidney-inductive tissue and showed that activin, a secreted morphogen, is necessary but insufficient for Lim1 induction and establishment of the kidney field. Activin or activin-like and BMP signaling cascades are activated along the entire axis, including in anterior non-kidney IM, suggesting that competence to respond to these signals involves downstream or other components. Detailed expression pattern analysis of Hox genes during early chick development revealed that paralogous group four genes share the same anterior border as the kidney genes. Ectopic expression of Hoxb4 in anterior non-kidney IM, either by retinoic acid (RA) administration or plasmid-mediated overexpression, resulted in ectopic kidney gene expression. The anterior expansion of Lim1 expression was restrained when Hoxb4 was co-expressed with a truncated form of activin receptor. We suggest a model in which the competence of IM cells to respond to TGFbeta signaling and express kidney genes is driven by RA and mediated by Hoxb4.

Identification of the endostyle as a stem cell niche in a colonial chordate.

Stem cell populations exist in "niches" that hold them and regulate their fate decisions. Identification and characterization of these niches is essential for understanding stem cell maintenance and tissue regeneration. Here we report on the identification of a novel stem cell niche in Botryllus schlosseri, a colonial urochordate with high stem cell-mediated developmental activities. Using in vivo cell labeling, engraftment, confocal microscopy, and time-lapse imaging, we have identified cells with stemness capabilities in the anterior ventral region of the Botryllus' endostyle. These cells proliferate and migrate to regenerating organs in developing buds and buds of chimeric partners but do not contribute to the germ line. When cells are transplanted from the endostyle region, they contribute to tissue development and induce long-term chimerism in allogeneic tissues. In contrast, cells from other Botryllus' regions do not show comparable stemness capabilities. Cumulatively, these results define the Botryllus' endostyle region as an adult somatic stem cell niche.

Cell signaling and transcription factor genes expressed during whole body regeneration in a colonial chordate.

BACKGROUND: The restoration of adults from fragments of blood vessels in botryllid ascidians (termed whole body regeneration [WBR]) represents an inimitable event in the chordates, which is poorly understood on the mechanistic level. RESULTS: To elucidate mechanisms underlying this phenomenon, a subtracted EST library for early WBR stages was previously assembled, revealing 76 putative genes belonging to major signaling pathways, including Notch/Delta, JAK/STAT, protein kinases, nuclear receptors, Ras oncogene family members, G-Protein coupled receptor (GPCR) and transforming growth factor beta (TGF-beta) signaling. RT-PCR on selected transcripts documented specific up-regulation in only regenerating fragments, pointing to a broad activation of these signaling pathways at onset of WBR. The followed-up expression pattern of seven representative transcripts from JAK/STAT signaling (Bl-STAT), the Ras oncogene family (Bl-Rap1A, Bl-Rab-33), the protein kinase family (Bl-Mnk), Bl-Cnot, Bl-Slit and Bl-Bax inhibitor, revealed systemic and site specific activations during WBR in a sub-population of circulatory cells. CONCLUSION: WBR in the non-vertebrate chordate Botrylloides leachi is a multifaceted phenomenon, presided by a complex array of cell signaling and transcription factors. Above results, provide a first insight into the whole genome molecular machinery of this unique regeneration process, and reveal the broad participation of cell signaling and transcription factors in the process. While regeneration involves the participation of specific cell populations, WBR signals are systemically expressed at the organism level.

Evolutionary origin and development of snake fangs.

Many advanced snakes use fangs-specialized teeth associated with a venom gland-to introduce venom into prey or attacker. Various front- and rear-fanged groups are recognized, according to whether their fangs are positioned anterior (for example cobras and vipers) or posterior (for example grass snakes) in the upper jaw. A fundamental controversy in snake evolution is whether or not front and rear fangs share the same evolutionary and developmental origin. Resolving this controversy could identify a major evolutionary transition underlying the massive radiation of advanced snakes, and the associated developmental events. Here we examine this issue by visualizing the tooth-forming epithelium in the upper jaw of 96 snake embryos, covering eight species. We use the sonic hedgehog gene as a marker, and three-dimensionally reconstruct the development in 41 of the embryos. We show that front fangs develop from the posterior end of the upper jaw, and are strikingly similar in morphogenesis to rear fangs. This is consistent with their being homologous. In front-fanged snakes, the anterior part of the upper jaw lacks sonic hedgehog expression, and ontogenetic allometry displaces the fang from its posterior developmental origin to its adult front position-consistent with an ancestral posterior position of the front fang. In rear-fanged snakes, the fangs develop from an independent posterior dental lamina and retain their posterior position. In light of our findings, we put forward a new model for the evolution of snake fangs: a posterior subregion of the tooth-forming epithelium became developmentally uncoupled from the remaining dentition, which allowed the posterior teeth to evolve independently and in close association with the venom gland, becoming highly modified in different lineages. This developmental event could have facilitated the massive radiation of advanced snakes in the Cenozoic era, resulting in the spectacular diversity of snakes seen today.

Urochordate whole body regeneration inaugurates a diverse innate immune signaling profile.

The phenomenon of whole body regeneration (WBR) from minute soma fragments is a rare event in chordates, confined to the subfamily of botryllid ascidians and is poorly understood on the cellular and molecular levels. We assembled a list of 1326 ESTs from subtracted mRNA, at early stages of Botrylloides leachi WBR, and classified them into functional categories. Sixty-seven (15%) ESTs with roles in innate immunity signaling were classified into a broad functional group, a result supported by domain search and RT-PCR reactions. Gene ontology analysis for human homologous to the immune gene category, identified 22 significant entries, of which "peptidase activity" and "protease inhibitor activity", stood out as functioning during WBR. Analyzing expressions of serine protease Bl-TrSP, a representative candidate gene from the "peptidase activity" subgroup, revealed low transcript levels in naïve vasculature with upregulated expression during WBR. This was confirmed by in situ hybridization that further elucidated staining restricted to a circulating population of macrophage cells. Furthermore, Bl-TrSP was localized in regeneration niches within vasculature, in regenerating buds, and in buds, during blastogenesis. Functional inhibition of serine protease activity disrupts early remodeling processes of the vasculature microenvironment and hinders WBR. Comparison of genome-wide transcription of WBR with five other developmental processes in ascidians (including metamorphosis, budding and blastogenesis), revealed a broad conservation of immune signaling expressions, suggesting a ubiquitous route of harnessing immune-related genes within a broader range of tunicate developmental context. This, in turn, may have enabled the high diversity of life history traits represented by urochordate ascidians.

Sonic hedgehog promotes proliferation and differentiation of adult muscle cells: Involvement of MAPK/ERK and PI3K/Akt pathways.

Sonic hedgehog (Shh) has been reported to act as a mitogen and survival factor for muscle satellite cells. However, its role in their differentiation remains ambiguous. Here, we provide evidence that Shh promotes the proliferation and differentiation of primary cultures of chicken adult myoblasts (also termed satellite cells) and mouse myogenic C2 cells. These effects are reversed by cyclopamine, a specific chemical inhibitor of the Shh pathway. In addition, we show that Shh and its downstream molecules are expressed in adult myoblast cultures and localize adjacent to Pax7 in muscle sections. These gene expressions are regulated during postnatal muscle growth in chicks. Most importantly, we report that Shh induces MAPK/ERK and phosphoinositide 3-kinase (PI3K)-dependent Akt phosphorylation and that activation of both signaling pathways is essential for Shh's signaling in muscle cells. However, the effect of Shh on Akt phosphorylation is more robust than that on MAPK/ERK, and data suggest that Shh influences these pathways in a manner similar to IGF-I. By exploiting specific chemical inhibitors of the MAPK/ERK and PI3K/Akt signaling pathways, UO126 and Ly294002, respectively, we demonstrate that Shh-induced Akt phosphorylation, but not that of MAPK/ERK, is required for its promotive effects on muscle cell proliferation and differentiation. Taken together, we suggest that Shh acts in an autocrinic manner in adult myoblasts, and provide first evidence of a role for PI3K/Akt in Shh signaling during myoblast differentiation.

Systemic bud induction and retinoic acid signaling underlie whole body regeneration in the urochordate Botrylloides leachi.

Regeneration in adult chordates is confined to a few model cases and terminates in restoration of restricted tissues and organs. Here, we study the unique phenomenon of whole body regeneration (WBR) in the colonial urochordate Botrylloides leachi in which an entire adult zooid is restored from a miniscule blood vessel fragment. In contrast to all other documented cases, regeneration is induced systemically in blood vessels. Multiple buds appear simultaneously in newly established regeneration niches within vasculature fragments, stemming from composites of pluripotent blood cells and terminating in one functional zooid. We found that retinoic acid (RA) regulates diverse developmental aspects in WBR. The homologue of the RA receptor and a retinaldehyde dehydrogenase-related gene were expressed specifically in blood cells within regeneration niches and throughout bud development. The addition of RA inhibitors as well as RNA interference knockdown experiments resulted in WBR arrest and bud malformations. The administration of all-trans RA to blood vessel fragments resulted in doubly accelerated regeneration and multibud formation, leading to restored colonies with multiple zooids. The Botrylloides system differs from known regeneration model systems by several fundamental criteria, including epimorphosis without the formation of blastema and the induction of a "multifocal regeneration niche" system. This is also to our knowledge the first documented case of WBR from circulating blood cells that restores not only the soma, but also the germ line. This unique Botrylloides WBR process could serve as a new in vivo model system for regeneration, suggesting that RA signaling may have had ancestral roles in body restoration events.

Sef is synexpressed with FGFs during chick embryogenesis and its expression is differentially regulated by FGFs in the developing limb.

The signaling pathways leading to growth and patterning of various organs are tightly controlled during the development of any organism. These control mechanisms usually involve the utilization of feedback- and pathway-specific antagonists where the pathway induces the expression of its own antagonist. Sef is a feedback antagonist of fibroblast growth factor (FGF) signaling, which has been identified recently in zebrafish and mammals. Here, we report the isolation of chicken Sef (cSef) and demonstrate the conserved nature of the regulatory relationship with FGF signaling. In chick embryos, Sef is expressed in a pattern that coincides with many known sites of FGF signaling. In the developing limb, cSef is expressed in the mesoderm underlying the apical ectodermal ridge (AER) in the region known as the progress zone. cSef message first appeared after limb budding and AER formation. Expression was intense at stages of rapid limb outgrowth, and gradually decreased to almost undetectable levels when differentiation was clearly apparent. Gain- and loss-of-function experiments showed that FGFs differentially regulate the expression of cSef in various tissues. Thus, removal of the AER down-regulated cSef expression, and FGF2 but not FGF4 or FGF8 beads substituted for the AER in maintaining cSef expression. At sites where cSef is not normally expressed, FGF4 and FGF2, but not FGF8 beads, induced cSef expression. Our results demonstrate the complexity of cSef regulation by FGFs and point to FGF2 as a prime candidate in regulating cSef expression during normal limb development. The spatiotemporal pattern of cSef expression during limb development suggests a role for cSef in regulating limb outgrowth but not limb initiation.

Cell fate specification along the anterior-posterior axis of the intermediate mesoderm.

The vertebrate intermediate mesoderm (IM) is highly patterned along the anterior-posterior (A-P) axis. In the chick embryo, the kidney tissue, which is a derivative of the IM, is generated only from IM located posterior to the sixth somite axial level, which also marks the border between cranial and trunk segments. The cellular and molecular mechanisms that govern the formation of the anterior border of the kidney morphogenetic field are currently unknown. In this study, we asked whether specific A-P patterning information is conveyed by the movement of cells through the primitive streak (PS) at different time points that consequently affects the expression of kidney genes, or by the environment that these cells encounter during their migration to the IM. In this study, we show that kidney-inductive signals are present along the whole axis, including anterior non-kidney-generating regions. These inductive signals are generated by tissues that are located medial to the anterior IM. We also demonstrate that cells that migrate through the PS of early embryonic stages (Hamburger and Hamilton stage 3-4 and earlier), which will give rise to anterior nonkidney IM, are competent to respond to these inductive factors. This prospective anterior IM tissue loses its competence to respond to kidney inducing signals during its migration from the PS to its final location in the anterior IM. We present here a model in which changes in cell competence determine the formation of the anterior border of kidney gene expression and discuss the possible evolutionary implications of this developmental mechanism.

Pattern of Pax7 expression during myogenesis in the posthatch chicken establishes a model for satellite cell differentiation and renewal.

The paired-box transcription factor Pax7 plays a critical role in the specification of satellite cells in mouse skeletal muscle. In the present study, the position and number of Pax7-expressing cells found in muscles of growing and adult chickens confirm the presence of this protein in avian satellite cells. The expression pattern of Pax7 protein, along with the muscle regulatory proteins MyoD and myogenin, was additionally elucidated in myogenic cultures and in whole muscle from posthatch chickens. In cultures progressing from proliferation to differentiation, the expression of Pax7 in MyoD+ cells declined as the cells began expressing myogenin, suggesting Pax7 as an early marker for proliferating myoblasts. At all time points, some Pax7+ cells were negative for MyoD, resembling the reserve cell phenotype. Clonal analysis of muscle cell preparations demonstrated that single progenitors can give rise to both differentiating and reserve cells. In muscle tissues, Pax7 protein expression was the strongest by 1 day posthatch, declining on days 3 and 6 to a similar level. In contrast, myogenin expression peaked on day 3 and then dramatically declined. This finding was accompanied by a robust growth in fiber diameter between day 3 and 6. The distinctions in Pax7 and myogenin expression patterns, both in culture and in vivo, indicate that while some of the myoblasts differentiate and fuse into myofibers during early stages of posthatch growth, others retain their reserve cell capacity.