Timing the developmental origins of mammalian limb diversity.

Mammals have highly diverse limbs that have contributed to their occupation of almost every niche. Researchers have long been investigating the development of these diverse limbs, with the goals of identifying developmental processes and potential biases that shape mammalian limb diversity. To date, researchers have used techniques ranging from the genomic to the anatomic to investigate the developmental processes shaping the limb morphology of mammals from five orders (Marsupialia, Chiroptera, Rodentia, Cetartiodactyla, and Perissodactyla). Results of these studies suggest that the differential expression of genes controlling diverse cellular processes underlies mammalian limb diversity. Results also suggest that the earliest development of the limb tends to be conserved among mammalian species, while later limb development tends to be more variable. This research has established the mammalian limb as a model system for evolutionary developmental biology, and set the stage for more in-depth, cross-disciplinary research into the genetic controls, tissue-level cellular behaviors, and selective pressures that have driven the developmental evolution of mammalian limbs. Ideally, these studies will be performed in a diverse suite of mammalian species within a comparative, phylogenetic framework.

Transcriptomic insights into the genetic basis of mammalian limb diversity.

BACKGROUND: From bat wings to whale flippers, limb diversification has been crucial to the evolutionary success of mammals. We performed the first transcriptome-wide study of limb development in multiple species to explore the hypothesis that mammalian limb diversification has proceeded through the differential expression of conserved shared genes, rather than by major changes to limb patterning. Specifically, we investigated the manner in which the expression of shared genes has evolved within and among mammalian species. RESULTS: We assembled and compared transcriptomes of bat, mouse, opossum, and pig fore- and hind limbs at the ridge, bud, and paddle stages of development. Results suggest that gene expression patterns exhibit larger variation among species during later than earlier stages of limb development, while within species results are more mixed. Consistent with the former, results also suggest that genes expressed at later developmental stages tend to have a younger evolutionary age than genes expressed at earlier stages. A suite of key limb-patterning genes was identified as being differentially expressed among the homologous limbs of all species. However, only a small subset of shared genes is differentially expressed in the fore- and hind limbs of all examined species. Similarly, a small subset of shared genes is differentially expressed within the fore- and hind limb of a single species and among the forelimbs of different species. CONCLUSIONS: Taken together, results of this study do not support the existence of a phylotypic period of limb development ending at chondrogenesis, but do support the hypothesis that the hierarchical nature of development translates into increasing variation among species as development progresses.

A new developmental mechanism for the separation of the mammalian middle ear ossicles from the jaw.

Multiple mammalian lineages independently evolved a definitive mammalian middle ear (DMME) through breakdown of Meckel's cartilage (MC). However, the cellular and molecular drivers of this evolutionary transition remain unknown for most mammal groups. Here, we identify such drivers in the living marsupial opossum Monodelphis domestica, whose MC transformation during development anatomically mirrors the evolutionary transformation observed in fossils. Specifically, we link increases in cellular apoptosis and TGF-BR2 signalling to MC breakdown in opossums. We demonstrate that a simple change in TGF-ß signalling is sufficient to inhibit MC breakdown during opossum development, indicating that changes in TGF-ß signalling might be key during mammalian evolution. Furthermore, the apoptosis that we observe during opossum MC breakdown does not seemingly occur in mouse, consistent with homoplastic DMME evolution in the marsupial and placental lineages.

Cellular and molecular drivers of differential organ growth: insights from the limbs of Monodelphis domestica.

A fundamental question in biology is "how is growth differentially regulated during development to produce organs of particular sizes?" We used a new model system for the study of differential organ growth, the limbs of the opossum (Monodelphis domestica), to investigate the cellular and molecular basis of differential organ growth in mammals. Opossum forelimbs grow much faster than hindlimbs, making opossum limbs an exceptional system with which to study differential growth. We first used the great differences in opossum forelimb and hindlimb growth to identify cellular processes and molecular signals that underlie differential limb growth. We then used organ culture and pharmacological addition of FGF ligands and inhibitors to test the role of the Fgf/Mitogen-activated protein kinases (MAPK) signaling pathway in driving these cellular processes. We found that molecular signals from within the limb drive differences in cell proliferation that contribute to the differential growth of the forelimb and hindlimbs of opossums. We also found that alterations in the Fgf/MAPK pathway can generate differences in cell proliferation that mirror those observed between wild-type forelimb and hindlimbs of opossums and that manipulation of Fgf/MAPK signaling affects downstream focal adhesion-extracellular matrix (FA-ECM) and Wnt signaling in opossum limbs. Taken together, these findings suggest that evolutionary changes in the Fgf/MAPK pathway could help drive the observed differences in cell behaviors and growth in opossum forelimb and hindlimbs.

The Relationship between Gene Network Structure and Expression Variation among Individuals and Species.

Variation among individuals is a prerequisite of evolution by natural selection. As such, identifying the origins of variation is a fundamental goal of biology. We investigated the link between gene interactions and variation in gene expression among individuals and species using the mammalian limb as a model system. We first built interaction networks for key genes regulating early (outgrowth; E9.5-11) and late (expansion and elongation; E11-13) limb development in mouse. This resulted in an Early (ESN) and Late (LSN) Stage Network. Computational perturbations of these networks suggest that the ESN is more robust. We then quantified levels of the same key genes among mouse individuals and found that they vary less at earlier limb stages and that variation in gene expression is heritable. Finally, we quantified variation in gene expression levels among four mammals with divergent limbs (bat, opossum, mouse and pig) and found that levels vary less among species at earlier limb stages. We also found that variation in gene expression levels among individuals and species are correlated for earlier and later limb development. In conclusion, results are consistent with the robustness of the ESN buffering among-individual variation in gene expression levels early in mammalian limb development, and constraining the evolution of early limb development among mammalian species.

Exogenous retinoic acid induces digit reduction in opossums (Monodelphis domestica) by disrupting cell death and proliferation, and apical ectodermal ridge and zone of polarizing activity function.

BACKGROUND: Retinoic acid (RA) is a vitamin A derivative. Exposure to exogenous RA generates congenital limb malformations (CLMs) in species from frogs to humans. These CLMs include but are not limited to oligodactyly and long-bone hypoplasia. The processes by which exogenous RA induces CLMs in mammals have been best studied in mouse, but as of yet remain unresolved. METHODS: We investigated the impact of exogenous RA on the cellular and molecular development of the limbs of a nonrodent model mammal, the opossum Monodelphis domestica. Opossums exposed to exogenous retinoic acid display CLMs including oligodactly, and results are consistent with opossum development being more susceptible to RA-induced disruptions than mouse development. RESULTS: Exposure of developing opossums to exogenous RA leads to an increase in cell death in the limb mesenchyme that is most pronounced in the zone of polarizing activity, and a reduction in cell proliferation throughout the limb mesenchyme. Exogenous RA also disrupts the expression of Shh in the zone of polarizing activity, and Fgf8 in the apical ectodermal ridge, and other genes with roles in the regulation of limb development and cell death. CONCLUSION: Results are consistent with RA inducing CLMs in opossum limbs by disrupting the functions of the apical ectodermal ridge and zone of polarizing activity, and driving an increase in cell death and reduction of cell proliferation in the mesenchyme of the developing limb.

Foxa1 and Foxa2 are required for formation of the intervertebral discs.

The intervertebral disc (IVD) is composed of 3 main structures, the collagenous annulus fibrosus (AF), which surrounds the gel-like nucleus pulposus (NP), and hyaline cartilage endplates, which are attached to the vertebral bodies. An IVD is located between each vertebral body. Degeneration of the IVD is thought to be a major cause of back pain, a potentially chronic condition for which there exist few effective treatments. The NP forms from the embryonic notochord. Foxa1 and Foxa2, transcription factors in the forkhead box family, are expressed early during notochord development. However, embryonic lethality and the absence of the notochord in Foxa2 null mice have precluded the study of potential roles these genes may play during IVD formation. Using a conditional Foxa2 allele in conjunction with a tamoxifen-inducible Cre allele (ShhcreER(T2)), we removed Foxa2 from the notochord of E7.5 mice null for Foxa1. Foxa1(-/-);Foxa2(c/c);ShhcreER(T2) double mutant animals had a severely deformed nucleus pulposus, an increase in cell death in the tail, decreased hedgehog signaling, defects in the notochord sheath, and aberrant dorsal-ventral patterning of the neural tube. Embryos lacking only Foxa1 or Foxa2 from the notochord were indistinguishable from control animals, demonstrating a functional redundancy for these genes in IVD formation. In addition, we provide in vivo genetic evidence that Foxa genes are required for activation of Shh in the notochord.

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.

Nuclei pulposi formation from the embryonic notochord occurs normally in GDF-5-deficient mice.

STUDY DESIGN: The transition of the mouse embryonic notochord into nuclei pulposi was determined ("fate mapped") in vivo in growth and differentiating factor-5 (GDF-5)-null mice using the Shhcre and R26R alleles. OBJECTIVE: To determine whether abnormal nuclei pulposi formation from the embryonic notochord was responsible for defects present in adult nuclei pulposi of Gdf-5-null mice. SUMMARY OF BACKGROUND DATA: The development, maintenance, and degeneration of the intervertebral disc are not understood. Previously, we demonstrated that all cells in the adult nucleus pulposus of normal mice are derived from the embryonic notochord. Gdf-5-null mice have been reported to contain intervertebral discs in which the nucleus pulposus is abnormal. It is currently unclear if disc defects in Gdf-5-null mice arise during the formation of nuclei pulposi from the notochord during embryogenesis or result from progressive postnatal degeneration of nuclei pulposi. METHODS: Gdf-5 messenger RNA expression was examined in the discs of wild-type embryos by RNA in situ hybridization to determine when and where this gene was expressed. To examine nucleus pulposus formation in Gdf-5-null mice, intervertebral discs in which embryonic notochord cells were marked were analyzed in newborn and 24-week-old mice. RESULTS: Our Gdf-5 messenger RNA in situ experiments determined that this gene is localized to the annulus fibrosus and not the nucleus pulposus in mouse embryos. Notochord fate-mapping experiments revealed that notochord cells in Gdf-5-null mice correctly form nuclei pulposi. CONCLUSION: Our data suggest that the defects reported in the nucleus pulposus of adult Gdf-5-null mice do not result from abnormal patterning of the embryonic notochord. The use of mouse alleles to mark cells that produce all cell types that reside in the adult nucleus pulposus will allow for a detailed examination of disc formation in other mouse mutants that have been reported to contain disc defects.

Role of myofibril-inducing RNA in cardiac TnT expression in developing Mexican axolotl.

The Mexican axolotl, Ambystoma mexicanum, has been a useful animal model to study heart development and cardiac myofibrillogenesis. A naturally-occurring recessive mutant, gene "c", for cardiac non-function in the Mexican axolotl causes a failure of myofibrillogenesis due to a lack of tropomyosin expression in homozygous mutant (c/c) embryonic hearts. Myofibril-inducing RNA (MIR) rescues mutant hearts in vitro by promoting tropomyosin expression and myofibril formation thereafter. We have studied the effect of MIR on the expression of various isoforms of cardiac troponin T (cTnT), a component of the thin filament that binds with tropomyosin. Four alternatively spliced cTnT isoforms have been characterized from developing axolotl heart. The expression of various cTnT isoforms in normal, mutant, and mutant hearts corrected with MIR, is evaluated by real-time RT-PCR using isoform specific primer pairs; MIR affects the total transcription as well as the splicing of the cTnT in axolotl heart.

Development of new pyrrolopyrimidine-based inhibitors of Janus kinase 3 (JAK3).

A new class of bicyclic pyrrolopyrimidine-based Janus kinase 3 (JAK-3) inhibitors are described. Many of these inhibitors showed low nanomolar activity against JAK-3.

Development of N-4,6-pyrimidine-N-alkyl-N'-phenyl ureas as orally active inhibitors of lymphocyte specific tyrosine kinase.

A new class of lymphocyte specific tyrosine kinase (lck) inhibitors based on an N-4,6-pyrimidine-N-alkyl-N'-phenyl urea scaffold is described. Many of these compounds showed low-nanomolar inhibition of lck kinase activity as well as IL-2 synthesis from Jurkat cells. One of these analogs, 7i, was shown to be orally efficacious by in vivo testing in a rat adjuvant-induced arthritis study.

Development of N-2,4-pyrimidine-N-phenyl-N'-phenyl ureas as inhibitors of tumor necrosis factor alpha (TNF-alpha) synthesis. Part 1.

A new class of tumor necrosis factor alpha (TNF-alpha) synthesis inhibitors based on an N-2,4-pyrimidine-N-phenyl-N'-phenyl urea scaffold is described. Many of these compounds showed low-nanomolar activity against lipopolysaccharide stimulated TNF-alpha production. X-ray co-crystallization studies with mutated p38alpha showed that these trisubstituted ureas interact with the ATP-binding pocket in a pseudo-bicyclic conformation brought about by the presence of an intramolecular hydrogen bonding interaction.

Development of N-2,4-pyrimidine-N-phenyl-N'-alkyl ureas as orally active inhibitors of tumor necrosis factor alpha (TNF-alpha) synthesis. Part 2.

A new class of tumor necrosis factor alpha (TNF-alpha) synthesis inhibitors based on a N-2,4-pyrimidine-N-phenyl-N'-alkyl urea scaffold is described. Many of these compounds showed low-nanomolar activity against lipopolysaccharide stimulated TNF-alpha production. Two analogs were tested in an in vivo rat iodoacetate model of osteoarthritis and shown to be orally efficacious. X-ray co-crystallization studies with mutated p38alpha showed that these trisubstituted ureas interact with the ATP-binding pocket in a pseudo-bicyclic conformation brought about by the presence of an intramolecular hydrogen bonding interaction.