Cell and Developmental Biology Publications and Presentations

Variations in the inferior pelvic pathway of the lateral femoral cutaneous nerve: implications for laparoscopic hernia repair

Lynn M. DiBenedetto et al. — Fri, 17 May 2013 11:24:13 PDT

Laparoscopic repair of inguinal hernias is gaining acceptance in the repertoire of the general surgeon. However, nerve entrapment sequelae have been reported and appear to be higher with the laparoscopic approach. Contributing factors include pelvic variations in nerve pathways and the use of staples. We examined the pelvic relations of the lateral femoral cutaneous nerve (LFCN) to the anterior superior iliac spine (ASIS) and the iliopubic tract (IPT) because of the high morbidity of entrapment of this nerve, despite its low incidence. The LFCN, ASIS, and IPT were identified and their relationships measured in 48 male and 24 female cadavers ranging in age from 61 to 96 yr. The LFCN was located 1.7 (+/- 1.2) cm medial to the ASIS along the IPT and 1.4 (+/- 0.7) cm posterior (deep) to the IPT at this point, with no significant sex differences. The intrapelvic pathway of the LFCN, including its branches, varied widely so that in 18% of these specimens the LFCN was in either the vertical plane of the ASIS (13%) or in the plane of the IPT (5%). In 11% this nerve was within 1 cm of the ASIS. These data indicate that exclusive use of the ASIS as a guide for staple placement may result in entrapment of this nerve or its branches.

The clinical anatomy of laparoscopic inguinal hernia repair

Sandy C. Marks Jr. et al. — Fri, 17 May 2013 11:24:08 PDT

Laparoscopic approaches for abdominal surgery are being used with increasing frequency. Their advantages are sometimes negated by the disturbing incidence of postoperative sequelae. In the case of inguinal hernia repair, these are often the result of failing to understand that the anatomy of the anterior approach to the abdominal wall cannot necessarily be directly applied to laparoscopy. The inguinal ligament, easily identified in an anterior approach, is only seen laparoscopically after removal of the iliopubic tract, a key structure which lies in the plane of the original defect of most groin hernias. Thus, an understanding of the incompletely trilaminar anterior abdominal wall, including the iliopubic tract, is the foundation for effective inguinal hernia repair using any approach (anterior or posterior) or technique (sutures, mesh or staples). Laparoscopic inguinal hernia repair has produced an increase in the frequency of debilitating neuropathies, most notably of the lateral femoral cutaneous nerve (LFCN). This is directly related to the variable intrapelvic course of this nerve or its branches. In more than 13% of the 114 pelves we examined, the LFCN was within 0.5 cm of the iliopubic tract or in the vertical plane of the anterior superior iliac spine, key lateral landmarks and anchoring sites for mesh in laparoscopic hernia repairs. Medial landmarks also have variable features. These data indicate that the identity of anatomical landmarks and the variability of other structures will continue to be important in the successful development of new laparoscopic techniques.

Variability of the obturator vessels

Anne M. Gilroy et al. — Fri, 17 May 2013 11:24:06 PDT

The obturator artery and vein are usually described as branches or tributaries of the internal iliac vessels although variations with connections to the external iliac or inferior epigastric vessels have been reported. Because these anomalous vessels are at risk in groin or pelvic surgeries that require dissection or suturing along the pelvic rim, we measured the frequency of these variations in 105 pelvic walls (45 in the United States and 60 in China). Our data show that 70-82% of pelvic halves and 83-90% of whole pelves had an artery, vein, or both in the variant position. Arteries were most often found in the normal position only but normal and anomalous veins were most frequently found together. These data show that it is far more common to find a vessel coursing over the pelvic rim at this site than not and have implications for both pelvic surgeons and anatomists.

Antenatally diagnosed cloacal exstrophy variant with intravesical phallus in a twin pregnancy

Yegappan Lakshmanan et al. — Fri, 17 May 2013 11:24:04 PDT

We report a rare case of covered cloacal exstrophy variant with a hemiphallus trapped within partially closed bladder halves. The persistence of the cloacal membrane until at least 18 weeks' gestation, confirmed by antenatal ultrasound scanning, is discordant with existing theories of embryogenesis of cloacal exstrophy. The clinical presentation highlights the need for careful assessment, before and during surgery, to obtain a complete understanding of the anatomic defect before gender assignment and appropriate reconstructive efforts. A multispecialty approach and antenatal counseling are important, especially when only one fetus of twins has major birth defects.

Pregnancy modulates precursor cell proliferation in a murine model of focal demyelination

Shirin Haddady et al. — Fri, 17 May 2013 11:24:01 PDT

In mice, pregnancy has been shown to have a beneficial effect on the endogenous repair of focal lysolecithin-induced CNS demyelinative lesions, enhancing the genesis of new oligodendrocytes and the degree of remyelination. To identify local cells undergoing mitosis in response to such lesions, we examined the time course of phospho-histone H3 (PH3) and myelin basic protein (MBP) expression by immunohistochemistry. After lysolecithin injection into the corpus callosum of virgin female mice, the number of dividing cells peaked about 48 h after injection and declined gradually to baseline by day 7; in pregnant mice, this initial peak was unchanged, but a new delayed peak on day 4 was induced. Colocalization data using PH3 and NG2 proteoglycan, or bromodeoxyuridine (BrdU) and oligodendrocyte transcription factor 1 (Olig1), suggested that about 75% of the proliferating cells on day 2, and about 40% of the cells on day 4, were likely of oligodendrocyte lineage; these differential percentages were of the same magnitude in both virgin and pregnant animals. Notably, the heightened proliferative response to focal lysolecithin injection during pregnancy was specific to gestational stage (early, but not late) and to lesion location (in the corpus callosum of the periventricular forebrain, but not in the caudal cerebellar peduncle of the hindbrain).

Atlas of Anatomy

Anne M. Gilroy et al. — Mon, 29 Apr 2013 13:04:41 PDT

Atlas of Anatomy, Second Edition, is an essential resource for anyone studying gross anatomy. Containing over 2,400 full-color illustrations, this atlas guides you step-by-step through each region of the body, helping you master the details of anatomy.

Edited by Anne M. Gilroy, Brian R. MacPherson, Lawrence M. Ross ; based on the work of Michael Schuenke, Erik Schulte, Udo Schumacher ; illustrated by Markus Voll, Karl Wesker.

Citation: Gilroy, A. M., MacPherson, B. R., Ross, L. M., Schünke, M., Schulte, E., & Schumacher, U. (2012). Atlas of anatomy. New York: Thieme.

ISBN-10: 1604067454

ISBN-13: 978-1604067453

Partial preview available via Google Books.

Anatomy: An Essential Textbook

Anne M. Gilroy — Mon, 29 Apr 2013 13:04:39 PDT

Focuses on the targeted information about anatomy for USMLE success. Includes: a total of 400 USMLE-style review questions with explanatory answers; a streamlined, bullet point format to help you quickly learn essential facts and concepts; 70 tables for quick review of crucial information; more than 450 fully labeled color illustrations.

Citation: Gilroy, A. M. (2013). Anatomy: An essential textbook and review. New York: Thieme.

ISBN-10: 160406207X

ISBN-13: 978-1604062076

A partial preview is available via Google Books.

Serial Transplantation of Bone Marrow to Test Self-renewal Capacity of Hematopoietic Stem Cells In Vivo

Charusheila Ramkumar et al. — Thu, 25 Apr 2013 12:46:47 PDT

Hematopoietic stem cells (HSCs) have the ability to self-renew and replenish the blood and immune system for the life span of an individual. An age-associated decline in HSC function is responsible for the decreased immune function and increased incidence of myeloid diseases and anemia in the elderly. The changes in HSC function are thought to occur as the result of an intrinsic defect in the self-renewal potential of HSCs as they age. In this chapter, we describe a bone marrow serial transplantation protocol designed to test the self-renewal capacity of HSCs in vivo.

A FAP46 mutant provides new insights into the function and assembly of the C1d complex of the ciliary central apparatus

Jason Brown et al. — Thu, 25 Apr 2013 12:46:47 PDT

Virtually all motile eukaryotic cilia and flagella have a '9+2' axoneme in which nine doublet microtubules surround two singlet microtubules. Associated with the central pair of microtubules are protein complexes that form at least seven biochemically and structurally distinct central pair projections. Analysis of mutants lacking specific projections has indicated that each may play a unique role in the control of flagellar motility. One of these is the C1d projection previously shown to contain the proteins FAP54, FAP46, FAP74 and FAP221/Pcdp1, which exhibits Ca(2+)-sensitive calmodulin binding. Here we report the isolation and characterization of a Chlamydomonas reinhardtii null mutant for FAP46. This mutant, fap46-1, lacks the C1d projection and has impaired motility, confirming the importance of this projection for normal flagellar movement. Those cells that are motile have severe defects in phototaxis and the photoshock response, underscoring a role for the C1d projection in Ca(2+)-mediated flagellar behavior. The data also reveal for the first time that the C1d projection is involved in the control of interdoublet sliding velocity. Our studies further identify a novel C1d subunit that we term C1d-87, give new insight into relationships between the C1d subunits, and provide evidence for multiple sites of calmodulin interaction within the C1d projection. These results represent significant advances in our understanding of an important but little studied axonemal structure.

The role of retrograde intraflagellar transport in flagellar assembly, maintenance, and function

Benjamin D. Engel et al. — Thu, 25 Apr 2013 12:46:46 PDT

The maintenance of flagellar length is believed to require both anterograde and retrograde intraflagellar transport (IFT). However, it is difficult to uncouple the functions of retrograde transport from anterograde, as null mutants in dynein heavy chain 1b (DHC1b) have stumpy flagella, demonstrating solely that retrograde IFT is required for flagellar assembly. We isolated a Chlamydomonas reinhardtii mutant (dhc1b-3) with a temperature-sensitive defect in DHC1b, enabling inducible inhibition of retrograde IFT in full-length flagella. Although dhc1b-3 flagella at the nonpermissive temperature (34 degrees C) showed a dramatic reduction of retrograde IFT, they remained nearly full-length for many hours. However, dhc1b-3 cells at 34 degrees C had strong defects in flagellar assembly after cell division or pH shock. Furthermore, dhc1b-3 cells displayed altered phototaxis and flagellar beat. Thus, robust retrograde IFT is required for flagellar assembly and function but is dispensable for the maintenance of flagellar length. Proteomic analysis of dhc1b-3 flagella revealed distinct classes of proteins that change in abundance when retrograde IFT is inhibited.

Avalanche-like behavior in ciliary import

William B. Ludington et al. — Thu, 25 Apr 2013 12:46:45 PDT

Cilia and flagella are microtubule-based organelles that protrude from the cell body. Ciliary assembly requires intraflagellar transport (IFT), a motile system that delivers cargo from the cell body to the flagellar tip for assembly. The process controlling injections of IFT proteins into the flagellar compartment is, therefore, crucial to ciliogenesis. Extensive biochemical and genetic analyses have determined the molecular machinery of IFT, but these studies do not explain what regulates IFT injection rate. Here, we provide evidence that IFT injections result from avalanche-like releases of accumulated IFT material at the flagellar base and that the key regulated feature of length control is the recruitment of IFT material to the flagellar base. We used total internal reflection fluorescence microscopy of IFT proteins in live cells to quantify the size and frequency of injections over time. The injection dynamics reveal a power-law tailed distribution of injection event sizes and a negative correlation between injection size and frequency, as well as rich behaviors such as quasiperiodicity, bursting, and long-memory effects tied to the size of the localized load of IFT material awaiting injection at the flagellar base, collectively indicating that IFT injection dynamics result from avalanche-like behavior. Computational models based on avalanching recapitulate observed IFT dynamics, and we further show that the flagellar Ras-related nuclear protein (Ran) guanosine 5'-triphosphate (GTP) gradient can in theory act as a flagellar length sensor to regulate this localized accumulation of IFT. These results demonstrate that a self-organizing, physical mechanism can control a biochemically complex intracellular transport pathway.

The interrelationship between APC/C and Plk1 activities in centriole disengagement

Toshiyuki Hatano et al. — Thu, 25 Apr 2013 12:46:44 PDT

Mother-daughter centriole disengagement, the necessary first step in centriole duplication, involves Plk1 activity in early mitosis and separase activity after APC/C activity mediates securin degradation. Plk1 activity is thought to be essential and sufficient for centriole disengagement with separase activity playing a supporting but non-essential role. In separase null cells, however, centriole disengagement is substantially delayed. The ability of APC/C activity alone to mediate centriole disengagement has not been directly tested. We investigate the interrelationship between Plk1 and APC/C activities in disengaging centrioles in S or G2 HeLa and RPE1 cells, cell types that do not reduplicate centrioles when arrested in S phase. Knockdown of the interphase APC/C inhibitor Emi1 leads to centriole disengagement and reduplication of the mother centrioles, though this is slow. Strong inhibition of Plk1 activity, if any, during S does not block centriole disengagement and mother centriole reduplication in Emi1 depleted cells. Centriole disengagement depends on APC/C-Cdh1 activity, not APC/C-Cdc20 activity. Also, Plk1 and APC/C-Cdh1 activities can independently promote centriole disengagement in G2 arrested cells. Thus, Plk1 and APC/C-Cdh1 activities are independent but slow pathways for centriole disengagement. By having two slow mechanisms for disengagement working together, the cell ensures that centrioles will not prematurely separate in late G2 or early mitosis, thereby risking multipolar spindle assembly, but rather disengage in a timely fashion only late in mitosis.

beta3GnT2 null mice exhibit defective accessory olfactory bulb innervation

Timothy R. Henion et al. — Thu, 25 Apr 2013 12:46:43 PDT

Vomeronasal sensory neurons (VSNs) extend axons to the accessory olfactory bulb (AOB) where they form synaptic connections that relay pheromone signals to the brain. The projections of apical and basal VSNs segregate in the AOB into anterior (aAOB) and posterior (pAOB) compartments. Although some aspects of this organization exhibit fundamental similarities with the main olfactory system, the mechanisms that regulate mammalian vomeronasal targeting are not as well understood. In the olfactory epithelium (OE), the glycosyltransferase beta3GnT2 maintains expression of axon guidance cues required for proper glomerular positioning and neuronal survival. We show here that beta3GnT2 also regulates guidance and adhesion molecule expression in the vomeronasal system in ways that are partially distinct from the OE. In wildtype mice, ephrinA5(+) axons project to stereotypic subdomains in both the aAOB and pAOB compartments. This pattern is dramatically altered in beta3GnT2(-/-) mice, where ephrinA5 is upregulated exclusively on aAOB axons. Despite this, apical and basal VSN projections remain strictly segregated in the null AOB, although some V2r1b axons that normally project to the pAOB inappropriately innervate the anterior compartment. These fibers appear to arise from ectopic expression of V2r1b receptors in a subset of apical VSNs. The homotypic adhesion molecules Kirrel2 and OCAM that facilitate axon segregation and glomerular compartmentalization in the main olfactory bulb are ablated in the beta3GnT2(-/-) aAOB. This loss is accompanied by a two-fold increase in the total number of V2r1b glomeruli and a failure to form morphologically distinct glomeruli in the anterior compartment. These results identify a novel function for beta3GnT2 glycosylation in maintaining expression of layer-specific vomeronasal receptors, as well as adhesion molecules required for proper AOB glomerular formation.

A fast and sensitive alternative for beta-galactosidase detection in mouse embryos

Sakthi Sundararajan et al. — Thu, 25 Apr 2013 12:46:43 PDT

The bacterial lacZ gene is widely used as a reporter in a myriad of mouse transgenic experiments. beta-Galactosidase, encoded by lacZ, is usually detected using X-gal in combination with ferric and ferrous ions. This assay produces a blue indole precipitate that is easy to detect visually. Here, we show that Salmon-gal in combination with tetrazolium salts provides a more sensitive and faster staining reaction than the traditional beta-galactosidase assay in mouse embryos. Using a combination of Salmon-gal and tetranitroblue tetrazolium, we were able to visualize the activity of beta-galactosidase in embryos at stages when the customary X-gal reaction failed to detect staining. Our studies provide an enhanced alternative for beta-galactosidase detection in expression and cell fate studies that use lacZ-based transgenic mouse lines.

Regulation of mRNA Export by the PI3 kinase / AKT Signal Transduction Pathway

Alexandre Jose Christino Quaresma et al. — Thu, 25 Apr 2013 12:46:42 PDT

UAP56, ALY/REF, and NXF1 are mRNA export factors that sequentially bind at the 5' end of a nuclear mRNA, but are also reported to associate with the Exon Junction Complex (EJC). To screen for signal transduction pathways regulating mRNA export complex assembly we used Fluorescence Recovery after Photobleaching (FRAP) to measure the binding of mRNA export and EJC core proteins in nuclear complexes. The fraction of UAP56, ALY/REF, and NXF1 tightly bound in complexes was reduced by drug inhibition of the PI3 kinase / AKT pathway, as was the tightly bound fraction of the core EJC proteins eIF4A3, MAGOH, and Y14. Inhibition of the mTOR mTORC1 pathway decreased the tight binding of MAGOH. Inhibition of the PI3 Kinase/AKT pathway increased the export of poly(A) RNA and of a subset of candidate mRNAs. A similar effect of PI3 kinase/AKT inhibition was observed for mRNAs from both intron-containing and intron-less Histone genes. However, the nuclear export of mRNAs coding for proteins targeted to the Endoplasmic Reticulum or to Mitochondria was not affected by the PI3 kinase/AKT pathway. These results show that the active PI3 kinase/AKT pathway can regulate mRNA export and can promote the nuclear retention of some mRNAs.

Supervillin-mediated Suppression of p53 Protein Enhances Cell Survival

Zhiyou Fang et al. — Thu, 25 Apr 2013 12:46:41 PDT

Integrin-based adhesions promote cell survival as well as cell motility and invasion. We show here that the adhesion regulatory protein supervillin increases cell survival by decreasing levels of the tumor suppressor protein p53 and downstream target genes. RNAi-mediated knockdown of a new splice form of supervillin (isoform 4) or both isoforms 1 and 4 increases the amount of p53 and cell death, whereas p53 levels decrease after overexpression of either supervillin isoform. Cellular responses to DNA damage induced by etoposide or doxorubicin include down-regulation of endogenous supervillin coincident with increases in p53. In DNA-damaged supervillin knockdown cells, p53 knockdown or inhibition partially rescues the loss of cell metabolic activity, a measure of cell proliferation. Knockdown of the p53 deubiquitinating enzyme USP7/HAUSP also reverses the supervillin phenotype, blocking the increase in p53 levels seen after supervillin knockdown and accentuating the decrease in p53 levels triggered by supervillin overexpression. Conversely, supervillin overexpression decreases the association of USP7 and p53 and attenuates USP7-mediated p53 deubiquitination. USP7 binds directly to the supervillin N terminus and can deubiquitinate and stabilize supervillin. Supervillin also is stabilized by derivatization with the ubiquitin-like protein SUMO1. These results show that supervillin regulates cell survival through control of p53 levels and suggest that supervillin and its interaction partners at sites of cell-substrate adhesion constitute a locus for cross-talk between survival signaling and cell motility pathways.

Genetic modification of the association between peripubertal dioxin exposure and pubertal onset in a cohort of Russian boys

Olivier Humblet et al. — Thu, 25 Apr 2013 12:46:40 PDT

BACKGROUND: Exposure to dioxins has been associated with delayed pubertal onset in both epidemiologic and animal studies. Whether genetic polymorphisms may modify this association is currently unknown. Identifying such genes could provide insight into mechanistic pathways. This is one of the first studies to assess genetic susceptibility to dioxins.

OBJECTIVES: We evaluated whether common polymorphisms in genes affecting either molecular responses to dioxin exposure or pubertal onset influence the association between peripubertal serum dioxin concentration and male pubertal onset.

METHODS: In this prospective cohort of Russian adolescent boys (n = 392), we assessed gene-environment interactions for 337 tagging single-nucleotide polymorphisms (SNPs) from 46 candidate genes and two intergenic regions. Dioxins were measured in the boys' serum at age 8-9 years. Pubertal onset was based on testicular volume and on genitalia staging. Statistical approaches for controlling for multiple testing were used, both with and without prescreening for marginal genetic associations.

RESULTS: After accounting for multiple testing, two tag SNPs in the glucocorticoid receptor (GR/NR3C1) gene and one in the estrogen receptor-alpha (ESR1) gene were significant (q < 0.2) modifiers of the association between peripubertal serum dioxin concentration and male pubertal onset defined by genitalia staging, although not by testicular volume. The results were sensitive to whether multiple comparison adjustment was applied to all gene-environment tests or only to those with marginal genetic associations.

CONCLUSIONS: Common genetic polymorphisms in the glucocorticoid receptor and estrogen receptor-alpha genes may modify the association between peripubertal serum dioxin concentration and pubertal onset. Further studies are warranted to confirm these findings.

A multifaceted FISH approach to study endogenous RNAs and DNAs in native nuclear and cell structures

Meg Byron et al. — Thu, 25 Apr 2013 12:46:39 PDT

Fluorescence in situ hybridization (FISH) is not a singular technique, but a battery of powerful and versatile tools for examining the distribution of endogenous genes and RNAs in precise context with each other and in relation to specific proteins or cell structures. This unit offers the details of highly sensitive and successful protocols that were initially developed largely in our lab and honed over a number of years. Our emphasis is on analysis of nuclear RNAs and DNA to address specific biological questions about nuclear structure, pre-mRNA metabolism, or the role of noncoding RNAs; however, cytoplasmic RNA detection is also discussed. Multifaceted molecular cytological approaches bring precise resolution and sensitive multicolor detection to illuminate the organization and functional roles of endogenous genes and their RNAs within the native structure of fixed cells. Solutions to several common technical pitfalls are discussed, as are cautions regarding the judicious use of digital imaging and the rigors of analyzing and interpreting complex molecular cytological results.

Nuclear Shape Changes Are Induced by Knockdown of the SWI/SNF ATPase BRG1 and Are Independent of Cytoskeletal Connections

Karen M. Imbalzano et al. — Thu, 25 Apr 2013 12:46:38 PDT

Changes in nuclear morphology occur during normal development and have been observed during the progression of several diseases. The shape of a nucleus is governed by the balance of forces exerted by nuclear-cytoskeletal contacts and internal forces created by the structure of the chromatin and nuclear envelope. However, factors that regulate the balance of these forces and determine nuclear shape are poorly understood. The SWI/SNF chromatin remodeling enzyme ATPase, BRG1, has been shown to contribute to the regulation of overall cell size and shape. Here we document that immortalized mammary epithelial cells show BRG1-dependent nuclear shape changes. Specifically, knockdown of BRG1 induced grooves in the nuclear periphery that could be documented by cytological and ultrastructural methods. To test the hypothesis that the observed changes in nuclear morphology resulted from altered tension exerted by the cytoskeleton, we disrupted the major cytoskeletal networks and quantified the frequency of BRG1-dependent changes in nuclear morphology. The results demonstrated that disruption of cytoskeletal networks did not change the frequency of BRG1-induced nuclear shape changes. These findings suggest that BRG1 mediates control of nuclear shape by internal nuclear mechanisms that likely control chromatin dynamics.

A stereological study of the numbers of neurons and glia in the primary visual cortex across the lifespan of male and female rhesus monkeys

Eustathia Lela Giannaris et al. — Thu, 25 Apr 2013 12:46:37 PDT

Mild age-related declines in visual function occur in humans and monkeys, independent of ocular pathology, suggesting involvement of central visual pathways (Spear [1993] Vision Res 33:2589-2609). Although many factors might account for this decline, a loss of neurons in primary visual cortex (V1) could be a contributing factor. Previous studies of neuron numbers in V1 reported stability across age, but were limited in the ages and genders studied and sampled only limited parts of V1 or limited cell types, allowing for the possibility of a subtle loss of neurons. We pursued this question in 26 behaviorally tested adult male and female rhesus monkeys ranging from 7.4 to 31.0 years of age by using design-based stereology to estimate numbers of NeuN-labeled neurons and thionin-stained glia within three laminar zones, supragranular (layers II-IVB), granular (IVC), and infragranular (V-VI), across the entirety of V1. There were no significant differences between males and females on any measures, except for total brain weight (P = 0.0038). There was an average of 416,000,000 neurons in V1, but no effect of age on this total or numbers within any laminar zone. Similarly, there was an average of 184,000,000 glia in V1 (44% of the number of neurons), but no effect of age on this total. However, there was a significant age-related increase in numbers of glia in the infragranular zone, perhaps reflecting a glial response to pathology in myelinated projection fibers. This study provides further evidence that in normal aging neurons are not lost and hence cannot account for age-related dysfunction.