Kimberly L. Cooper

Multiple Phases of Chondrocyte Enlargement Underlie Differences in Skeletal Proportions

The wide diversity of skeletal proportions in mammals is evident upon a survey of any natural history museums collections and allows us to distinguish between species even when reduced to their calcified components. Similarly, each individual is comprised of a variety of bones of differing lengths. The largest contribution to the lengthening of a skeletal element, and to the differential elongation of elements, comes from a dramatic increase in the volume of hypertrophic chondrocytes in the growth plate as they undergo terminal differentiation. However, the mechanisms of chondrocyte volume enlargement have remained a mystery. Here we use quantitative phase microscopy to show that mammalian chondrocytes undergo three distinct phases of volume increase, including a phase of massive cell swelling in which the cellular dry mass is significantly diluted. In light of the tight fluid regulatory mechanisms known to control volume in many cell types, this is a remarkable mechanism for increasing cell size and regulating growth rate. It is, however, the duration of the final phase of volume enlargement by proportional dry mass increase at low density that varies most between rapidly and slowly elongating growth plates. Moreover, we find that this third phase is locally regulated through a mechanism dependent on insulin-like growth factor. This study provides a framework for understanding how skeletal size is regulated and for exploring how cells sense, modify and establish a volume set point.

Initiation of proximal-distal patterning in the vertebrate limb by signals and growth.

Growth of limb cells in culture conditions with subsequent in vivo transplantation allows the dissection of limb patterning. Two broad classes of models have been proposed to explain the patterning of the proximal-distal axis of the vertebrate limb (from the shoulder to the digit tips). Differentiating between them, we demonstrate that early limb mesenchyme in the chick is initially maintained in a state capable of generating all limb segments through exposure to a combination of proximal and distal signals. As the limb bud grows, the proximal limb is established through continued exposure to flank-derived signal(s), whereas the developmental program determining the medial and distal segments is initiated in domains that grow beyond proximal influence. In addition, the system we have developed, combining in vitro and in vivo culture, opens the door to a new level of analysis of patterning mechanisms in the limb.

Patterning and post-patterning modes of evolutionary digit loss in mammals

A reduction in the number of digits has evolved many times in tetrapods, particularly in cursorial mammals that travel over deserts and plains, yet the underlying developmental mechanisms have remained elusive. Here we show that digit loss can occur both during early limb patterning and at later post-patterning stages of chondrogenesis. In the ‘odd-toed’ jerboa (Dipus sagitta) and horse and the ‘even-toed’ camel, extensive cell death sculpts the tissue around the remaining toes. In contrast, digit loss in the pig is orchestrated by earlier limb patterning mechanisms including downregulation of Ptch1 expression but no increase in cell death. Together these data demonstrate remarkable plasticity in the mechanisms of vertebrate limb evolution and shed light on the complexity of morphological convergence, particularly within the artiodactyl lineage.

Validating the Use of the Mimic dV-trainer for Robotic Surgery Skill Acquisition Among Urology Residents

OBJECTIVE To compare robotic surgery skill acquisition of residents trained with Mimic dVTrainer (MdVT) and da Vinci Surgical System (dVSS) console. No standardized curriculum currently exists for robotic surgical education. The MdVT is a compact hardware platform that closely reproduces the experience of the dVSS. METHODS Sixteen urology trainees were randomized into 3 groups. A baseline evaluation using dVSS was performed and consisted of 2 exercises requiring endowrist manipulation (EM), camera movement and clutching (CC), needle control (NC), and knot-tying (KT). Groups 1 and 2 completed a standardized training curriculum on MdVT and dVSS, respectively. Group 3 received no additional training. After completion of the training phase, all trainees completed a secondary evaluation on dVSS consisting of the same exercises performed during baseline evaluation. RESULTS There was no difference in baseline performance scores across the 3 groups. Although Group 3 showed no significant improvement in EM/CC domain (P = .15), Groups 1 and 2 had statistically significant improvement in EM/CC domain (P = .039 and P = .007, respectively). The difference in improvement between Groups 1 and group 2 was not statistically different (P = .21). Only Group 2 trainees showed significant improvement in the NC and KT domains during secondary evaluation (P = .02). CONCLUSION Curriculum-based training with MdVT or dVSS significantly improves robotic surgery aptitude. Similar improvements are seen for exercise domains shared between MdVT and dVSS groups. Follow-up studies are necessary to assess the efficacy of MdVT over a wider spectrum of domains.

Autonomous and nonautonomous functions for Hox/Pbx in branchiomotor neuron development

The vertebrate branchiomotor neurons are organized in a pattern that corresponds with the segments, or rhombomeres, of the developing hindbrain and have identities and behaviors associated with their position along the anterior/posterior axis. These neurons undergo characteristic migrations in the hindbrain and project from stereotyped exit points. We show that lazarus/pbx4, which encodes an essential Hox DNA-binding partner in zebrafish, is required for facial (VIIth cranial nerve) motor neuron migration and for axon pathfinding of trigeminal (Vth cranial nerve) motor axons. We show that lzr/pbx4 is required for Hox paralog group 1 and 2 function, suggesting that Pbx interacts with these proteins. Consistent with this, lzr/pbx4 interacts genetically with hoxb1a to control facial motor neuron migration. Using genetic mosaic analysis, we show that lzr/pbx4 and hoxb1a are primarily required cell-autonomously within the facial motor neurons; however, analysis of a subtle non-cell-autonomous effect indicates that facial motor neuron migration is promoted by interactions amongst the migrating neurons. At the same time, lzr/pbx4 is required non-cell-autonomously to control the pathfinding of trigeminal motor axons. Thus, Pbx/Hox can function both cell-autonomously and non-cell-autonomously to direct different aspects of hindbrain motor neuron behavior.

Impact of hydronephrosis and renal function on treatment outcome: antegrade versus retrograde endopyelotomy

OBJECTIVES To compare, in a single-surgeon, single-institution study, the efficacy of antegrade and retrograde endopyelotomy in terms of success rate and morbidity and to identify which risk factors affect treatment outcomes. METHODS The results were retrospectively reviewed for 88 patients with ureteropelvic junction obstruction treated with endopyelotomy. Antegrade endopyelotomy was performed with a hook knife, scissors, or cutting balloon device. Retrograde endopyelotomy was performed with a cutting balloon device. Objective results were based on intravenous urogram and/or diuretic nuclear renal scan findings, and subjective results were based on direct patient query and questionnaire. RESULTS Ninety-three endopyelotomy procedures, 64 antegrade and 29 retrograde, were performed. The mean follow-up was 37.0 months (range 5 to 76). The overall success rates between antegrade and retrograde endopyelotomy (81.3% versus 75.9%) were not statistically different (P = 0.553). Patients with massive hydronephrosis and poor initial renal function were less likely to have successful endopyelotomy. Antegrade endopyelotomy, however, was more successful than retrograde endopyelotomy in patients with massive hydronephrosis (66.7% versus 20.0%; P = 0.046). The average operative time for antegrade and retrograde endopyelotomy was 93.9 and 32.7 minutes (P <0.001), respectively. The average length of hospital stay after antegrade and retrograde endopyelotomy was 3.20 and 0.14 nights (P <0.001), respectively. CONCLUSIONS Both antegrade and retrograde endopyelotomy are effective treatments for ureteropelvic junction obstruction associated with minimal morbidity. Antegrade endopyelotomy appears to be more successful in patients with high-grade hydronephrosis. Retrograde endopyelotomy results in a shorter hospital stay, a shorter operative time, and less postoperative pain.

Unique expression patterns of multiple key genes associated with the evolution of mammalian flight.

Bats are the only mammals capable of true flight. Critical adaptations for flight include a pair of dramatically elongated hands with broad wing membranes. To study the molecular mechanisms of bat wing evolution, we perform genomewide mRNA sequencing and in situ hybridization for embryonic bat limbs. We identify seven key genes that display unique expression patterns in embryonic bat wings and feet, compared with mouse fore- and hindlimbs. The expression of all 5′HoxD genes (Hoxd9–13) and Tbx3, six known crucial transcription factors for limb and digit development, is extremely high and prolonged in the elongating wing area. The expression of Fam5c, a tumour suppressor, in bat limbs is bat-specific and significantly high in all short digit regions (the thumb and foot digits). These results suggest multiple genetic changes occurred independently during the evolution of bat wings to elongate the hand digits, promote membrane growth and keep other digits short. Our findings also indicate that the evolution of limb morphology depends on the complex integration of multiple gene regulatory networks and biological processes that control digit formation and identity, chondrogenesis, and interdigital regression or retention.

Multiple Phylogenetically Distinct Events Shaped the Evolution of Limb Skeletal Morphologies Associated with Bipedalism in the Jerboas

Recent rapid advances in experimental biology have expanded the opportunity for interdisciplinary investigations of the evolution of form and function in non-traditional model species. However, historical divisions of philosophy and methodology between evolutionary/organismal biologists and developmental geneticists often preclude an effective merging of disciplines. In an effort to overcome these divisions, we take advantage of the extraordinary morphological diversity of the rodent superfamily Dipodoidea, including the bipedal jerboas, to experimentally study the developmental mechanisms and biomechanical performance of a remarkably divergent limb structure. Here, we place multiple limb character states in a locomotor and phylogenetic context. Whereas obligate bipedalism arose just once in the ancestor of extant jerboas, we find that digit loss, metatarsal fusion, between-limb proportions, and within-hindlimb proportions all evolved independently of one another. Digit loss occurred three times through at least two distinct developmental mechanisms, and elongation of the hindlimb relative to the forelimb is not simply due to growth mechanisms that change proportions within the hindlimb. Furthermore, we find strong evidence for punctuated evolution of allometric scaling of hindlimb elements during the radiation of Dipodoidea. Our work demonstrates the value of leveraging the evolutionary history of a clade to establish criteria for identifying the developmental genetic mechanisms of morphological diversification.

Understanding of bat wing evolution takes flight

It has long been debated whether the processes and mechanisms responsible for phenotypic variation within a population or between closely related populations can be extrapolated to explain evolutionary trends over longer phylogenetic distances and especially the generation of novel structures. Although there has been great progress in recent years in addressing the genetic basis for microevolutionary changes, for the most part these efforts have done little to address this debate. Evolutionary genetic studies, by their nature, can only be applied to closely related groups. For example, quantitative trait mapping is limited to species with distinct morphological traits that are capable of producing viable progeny when crossed either naturally or artificially. There are only a few cases in which linkage mapping has thus far been applied even at the distance of interspecific hybrids to identify loci responsible for morphological differences between highly related species (Sucena et al. 2003; Shapiro et al. 2004; Colosimo et al. 2005; Kronforst et al. 2006; Protas et al. 2006; McGregor et al. 2007). While the genomes of more distantly related taxa can be compared, the effect of molecular drift over millions of years has generated a large noise of sequence variation that makes the identification of specific and functionally relevant changes analogous to finding a needle in a haystack. In this issue of Genes & Development, Cretekos et al. (2008) have found one such needle—an enhancer of the paired-box homeodomain transcription factor, Prx1, that accounts for at least part of the extension of the long bones in the wings of bats.

Zebrafish foggy/spt5 is required for migration of facial branchiomotor neurons but not for their survival

Transcript elongation is a critical step in the production of mature messenger RNAs. Many factors have been identified that are required for transcript elongation, including Spt5. Studies in yeast determined that spt5 is required for cell viability, and analyses in Drosophila indicate Spt5 is localized to sites of active transcription, suggesting it is required generally for transcription. However, the requirement for spt5 for cell viability in a metazoan organism has not been addressed. We determined that zebrafish foggy/spt5 is required cell‐autonomously for the posterior migration of facial branchiomotor neurons from rhombomere 4 (r4) into r6 and r7 of the hindbrain. These genetic mosaics also give us the unique opportunity to determine whether spt5 is required for mRNA transcription equivalently at all loci by addressing two processes within the same cell—neuronal migration and cell viability. In a wild‐type host, spt5 null facial branchiomotor neurons survive to at least 5 days postfertilization while failing to migrate posteriorly. This finding indicates that spt5‐dependent transcript elongation is required cell‐autonomously for a complex cell migration but not for the survival of these same cells. This work provides evidence that transcript elongation is not a global mechanism equivalently required by all loci and may actually be under more strict developmental regulation. Developmental Dynamics 234:651–658, 2005.