Ann C. Burke

Morphogenesis of the turtle shell: the development of a novel structure in tetrapod evolution

SUMMARY The turtle shell is an evolutionary novelty that is synapomorphic for chelonians. The carapace is initiated by the entrapment of the ribs by the carapacial ridge (CR), a lateral bulge of the dorsal ectoderm and dermal mesoderm. The mechanisms by which the CR is initiated, the ribs entrapped and the dorsal dermis ossified, remains unknown. Similarly, the formation of the plastron remains unexplained. Here, we present a series of anatomical investigations into plastron and carapace formation in the red‐eared slider, Trachemys scripta, and the snapping turtle, Chelydra serpentina. We document the entrapment of the ribs by the CR and the formation of the plastron and carapacial bones by intramembranous ossification. We note the formation of the ossification centers around each rib, which suggest that the rib is organizing dermal ossification by secreting paracrine factors. The nuchal ossification center is complex and appears to involve multiple bone‐forming regions. Individual ossification centers at the periphery of the carapace form the peripheral and pygial bones. The intramembranous ossification of the plastron proceeds from nine distinct ossification centers, and there appear to be interactions between the spicules of apposing centers as they draw near each other.

A new view of patterning domains in the vertebrate mesoderm.

The musculoskeletal system of vertebrates is derived from the embryonic mesoderm. Its structures are categorized as epaxial or hypaxial based on their adult position and innervation. The epaxial/hypaxial terminology is also used to describe regions of the embryonic somites based on fate mapping of somitic derivatives. However, the adult, functional distinctions are not fully consistent with the changing embryonic environments of mesodermal populations during morphogenesis, and the traditional terminology loses accuracy when used to describe certain mutant phenotypes. Here we describe a new terminology naming two mesodermal environments defined by the lineage of the included cells. We discuss how mutant phenotypes may be better explained by consideration of the embryonic context in which genes take their effect and argue that the recognition of these embryonic territories clarifies description and discussion of the morphogenesis and patterning of the musculoskeletal system.

Development of the turtle carapace: Implications for the evolution of a novel bauplan

The chelonian carapace is composed of the endochondral ribs and vertebrae associated with a specialized dermis. The ribs are found in an aberrant position compared to those of all other tetrapods; they are superficial and dorsal to the limb girdles. This morphological arrangement, which constitutes the unique chelonian Bauplan, is examined from a developmental perspective. Embryos of Chelydra serpentina were studied during stages of carapace development. Tissue morphology, autoradiography, and indirect immunofluorescent localization of adhesion molecules indicate that the outgrowth of the embryonic carapace occurs as the result of an epithelial–mesenchymal interaction in the body wall. A carapacial ridge composed of mesenchyme of the dermis and overlying ectoderm is formed dorsal to the ectodermal boundary between somitic and lateral plate mesoderm. It is the anlage of the carapace margin, in which the ribs will eventually terminate. The ectoderm of the carapacial ridge is thickened into a pseudostratified columnar epithelium, which overlies a condensation in the mesenchyme of the dermis. Patterns of cell proliferation and the distribution of N‐CAM and fibronectin in the carapacial ridge are consistent with patterns seen in other structures initiated by epithelial–mesenchymal interactions such as feathers and limb buds.

The development and homology of the chelonian carpus and tarsus

The long‐standing controversies involving the number and homologies of the elements of the carpus and tarsus of turtles are reviewed from a developmental perspective. The analysis is based on a detailed description of the chondrogenesis of the carpus and tarsus in the species Chelydra serpentina and Chrysemys picta. The first stage described is the differentiation of a Y‐shaped chondrogenetic condensation involving the humerus (femur)‐radius/ ulna (tibia/fibula). This stage is followed by the early formation of a series of connected condensations off the distal end of the postaxial element (ulna or fibula). This linear array, which we refer to as the primary axis, comprises the ulnare‐distal carpal 4‐metacarpal 4 in the carpus and the fibulare‐distal tarsal 4‐metatarsal 4 in the tarsus. There are two precondensations that branch off the primary axis. The proximal one will soon form the intermedium while the distal one will generate a digital arch that will give rise sequentially to digits 3‐2‐1, in this order. Digit 5 is not part of the digital arch and forms as an independent condensation.

6 Hox Genes and the Global Patterning of the Somitic Mesoderm

Publisher Summary The nature of the information that distinguishes the behavior of somites at different axial levels (such as neck versus trunk) is understood with far less detail. This chapter reviews the data that relate to the translation of local information into global patterning of the somitic mesoderm derivatives. The types of morphological variation are reviewed that are seen among vertebrate taxa that result from evolutionary changes in global patterning in the mesoderm. The aspects of local, or primary patterning within individual somites are described that are apparently universal within vertebrates. The morphological fate of the somitic mesoderm is also reviewed that are determined by classic experiments on model systems that indicate which somites contribute to which adult structures. Finally, experimental and comparative studies are presented that have investigated molecular genetic factors that may control global patterning. These factors provide a means of understanding intrinsic proximal events in the evolution of animal form.

The lateral somitic frontier: dorso-ventral aspects of anterio-posterior regionalization in avian embryos.

Patterning events along the anterior-posterior (AP) axis of vertebrate embryos result in the distribution of muscle and bone forming a highly effective functional system. A key aspect of regionalized AP patterning results from variation in the migratory pattern of somite cells along the dorsal-ventral (DV) axis of the body. This occurs as somite cell populations expand around the axis or migrate away from the dorsal midline and cross into the lateral plate. The fate of somitic cells has been intensely studied and many details have been reported about inductive signaling from other tissues that influence somite cell fate and behavior. We are interested in understanding the specific differences between somites in particular AP regions and how these differences contribute to the global pattern of the organism. Using orthotopic transplants of segmental plate between quail and chick embryos, we have mapped the interface of the somitic and lateral plate mesoderm during the formation of the body wall in cervical and thoracic regions. This interface does not change dramatically in the mid-cervical region, but undergoes extensive changes in the thoracic region. Based on this regional mapping and consistent with the extensive literature, we suggest a revised method of classifying regions of the body wall that relies on embryonic cell lineages rather than adult functional criteria.

Visualizing the lateral somitic frontier in the Prx1Cre transgenic mouse

Changes in the organization of the musculoskeletal system have accounted for many evolutionary adaptations in the vertebrate body plan. The musculoskeletal system develops from two mesodermal populations: somitic mesoderm gives rise to the axial skeleton and all of the skeletal muscle of the body, and lateral plate mesoderm gives rise to the appendicular skeleton. The recognition of embryonic domains resulting from the dynamics of morphogenesis has inspired new terminology based on developmental criteria. Two mesodermal domains are defined, primaxial and abaxial. The primaxial domain includes musculoskeletal structures comprising just somitic cells. The abaxial domain contains somitic myoblasts in connective tissue derived from lateral plate mesoderm, as well as lateral plate‐derived skeletal structures. The boundary between these two domains is the lateral somitic frontier. Recent studies have described the developmental relationship between these two domains in the chick. In the present study, we describe the labelling pattern in the body of the Prx1/Cre/Z/AP compound transgenic mouse. The enhancer employed in this transgenic leads to reporter expression in the postcranial, somatic lateral plate mesoderm. The boundary between labelled and unlabelled cell populations is described at embryonic day (E)13.5 and E15.5. We argue that the distribution of labelled cells is consistent with the somatic lateral plate lineage, and therefore provides an estimate of the position of the lateral somitic frontier. The role of the frontier in both development and evolution is discussed.

The lateral somitic frontier in ontogeny and phylogeny.

The vertebrate musculoskeletal system comprises the axial and appendicular systems. The postcranial axial system consists of the vertebrae, ribs and associated muscles, and the appendicular system comprises the muscles and skeleton of the paired appendages and their respective girdles. The morphology, proportions, and arrangements of these parts have undergone tremendous variation during vertebrate history. Despite this vertebrate diversity, the cells that form all of the key parts of the musculoskeletal system during development arise from two populations of embryonic mesoderm, the somites and somatic lateral plate. Nowicki et al. (2003. Mech Dev 120:227-240) identified two dynamic domains in the developing chick embryo. The primaxial domain is populated exclusively by cells from the somites. The abaxial domain includes muscle and bone that develop within lateral plate-derived connective tissue. The boundary between the two domains is the lateral somitic frontier. We hypothesize that the primaxial and abaxial domains are patterned independently and that morphological evolution of the musculoskeletal system is facilitated by partially independent developmental changes in the abaxial and primaxial domain. Here we present our hypothesis in detail and review recent experimental and comparative studies that use the concept of the lateral somitic frontier in the analysis of the evolution of the highly derived chelonian and limbless squamate body plans.

Global Patterning of the Vertebrate Mesoderm

We describe recent advances in the understanding of patterning in the vertebrate post‐cranial mesoderm. Specifically, we discuss the integration of local information into global level information that results in the overall coordination along the anterioposterior axis. Experiments related to the integration of the axial and appendicular musculoskeletal systems are considered, and examples of genetic interactions between these systems are outlined. We emphasize the utility of the terms primaxial and abaxial as an aid to understanding development of the vertebrate musculoskeletal system, and hypothesize that the lateral somitic frontier is a catalyst for evolutionary change. Developmental Dynamics 236:2371–2381, 2007.

Hox5 Genes Regulate the Wnt2/2b-Bmp4-Signaling Axis during Lung Development

Hox genes are required for proper anteroposterior axial patterning and the development of several organ systems. Here, we show that all three Hox5 paralogous genes play redundant roles in the developing lung. Hoxa5;Hoxb5;Hoxc5 triple-mutant embryos develop severely hypoplastic lungs with reduced branching and proximal-distal patterning defects. Hox5 genes are exclusively expressed in the lung mesoderm; however, defects are observed in both lung mesenchyme and endodermally derived epithelium, demonstrating that Hox5 genes act to regulate mesodermal-epithelial crosstalk during development. We show that Hox5 loss of function leads to loss of Wnt2/2b expression in the distal lung mesenchyme and the downregulation of previously identified downstream targets of Wnt2/2b signaling, including Lef1, Axin2, and Bmp4. Wnt2/2b-enriched media rescue proper Sox2/Sox9 patterning and restore Bmp4 expression in Hox5 triple-mutant lung explants. Taken together, these data show that Hox5 genes are key upstream mesenchymal regulators of the Wnt2/2b-Bmp4-signaling axis critical for proper lung patterning.