Ipsilateral lower limb motor performance and its association with gait after stroke.

BACKGROUND AND PURPOSE: Motor deficits of the ipsilateral lower limb could occur after stroke and may be associated with walking performance. This study aimed to determine whether the accuracy and movement path of targeted movement in the ipsilateral lower limb would be impaired in the chronic stage of stroke and whether this impairment would contribution to gait. METHODS: Twenty adults with chronic stroke and 20 age-matched controls went through Mini Mental Status Examination (MMSE), and a series of sensorimotor tests. The targeted movement tasks were to place the big toe ipsilateral to the lesion at an external visual target (EXT) or a proprioceptive target (PRO, contralateral big toe) with eyes open (EO) or closed (EC) in a seated position. A motion analysis system was used to obtain the data for the calculation of error distance, deviation from a straight path, and peak toe-height during the targeted movement tasks and gait velocity, step length, step width and step length symmetry of the lower limb ipsilateral to the brain lesion during walking. RESULTS: The stroke group had significantly lower MMSE and poorer visual acuity on the ipsilateral side, but did not differ in age or other sensorimotor functions when compared to the controls. For the targeted movement performance, only the deviation in PRO-EC showed significant between-group differences (p = 0.02). Toe-height in both EXT-EO and in PRO-EO was a significant predictor of step length (R2 = 0.294, p = 0.026) and step length symmetry (R2 = 0.359, p = 0.014), respectively. DISCUSSION AND CONCLUSIONS: The performance of ipsilateral lower limb targeted movement could be impaired after stroke and was associated with step length and its symmetry. The training of ipsilateral targeted movement with unseen proprioceptive target may be considered in stroke rehabilitation.

Walking performance of persons with chronic stroke changed when looking down but not in dimly lit environment.

Background and purpose: It is common to walk under different conditions, such as looking straight head, looking down at the feet or in dimly lit environment. The purpose of this study was to determine the impact of these different conditions on walking performance in persons with and without stroke. Methods: This was a case-control study. Persons with chronic unilateral stroke and age-matched control (n = 29 each) underwent visual acuity test, Mini Mental Status Examination (MMSE) and joint position sense test of the knee and ankle. The participants walked at their preferred speed under three walking conditions, looking ahead (AHD), looking down (DWN), and in dimly lit environment (DIM). A motion analysis system was used for the recording of the limb matching test and walking tasks. Results: Stroke participants differed from the control group in MMSE, but not in age, visual acuity or joint position sense. For the control group, the differences between the three walking conditions were nonsignificant. For the stroke group, DWN had significantly slower walking speed, greater step width and shorter single leg support phase, but not different symmetry index or COM location, compared to AHD. The differences between AHD and DIM were nonsignificant. Conclusion: Healthy adults did not change their gait patterns under the different walking conditions. Persons with chronic stroke walked more cautiously but not more symmetrically when looking down at the feet, but not in dimly lit environment. Ambulatory persons with stroke may need to be advised that looking down at the feet while walking could be more challenging.

Effects of Trunk Exercise on Unstable Surfaces in Persons with Stroke: A Randomized Controlled Trial.

Background: Improving balance-related ability is an important goal in stroke rehabilitation. Evidence is needed to demonstrate how this goal could be better achieved. Aim: Determine if trunk exercises on unstable surfaces would improve trunk control and balance for persons in the subacute stage of stroke. Design: An assessor-blind randomized controlled trial. Setting: Inpatients in the department of rehabilitation in a general hospital. Population: Patients who suffered a first-time stroke with onset from one to six months. Methods: Inpatients with stroke were assigned to upper limb exercises (control group, n = 17) or trunk exercises on unstable surfaces (experimental group, n = 18) to receive training twice a week for six weeks, in addition to their daily conventional stroke rehabilitation. Sensorimotor function tests, including hand grip, plantar sensitivity, stroke rehabilitation assessment of movement and Fugl-Meyer lower extremity motor scale, and clinical outcome assessments, including Trunk Impairment Scale and 6 m walk test, were conducted before and after six weeks of training. The center of the pressure area while maintaining static posture and peak displacement while leaning forward, as well as the average speed of raising the unaffected arm, were measured in sitting without foot support, sitting with foot support and standing to reflect trunk control, sitting balance and standing balance, respectively. Results: The between-group differences in the sensorimotor functions were nonsignificant before and after training. Compared with the control group, the experimental group had significantly greater forward leaning and faster arm raising in sitting without foot support, higher Trunk Impairment Scale total score, and shorter 6 m walking time after training, but not before training. Conclusion: Trunk exercises on unstable surfaces could further improve trunk control, the ability to raise the unaffected arm rapidly in sitting, and walking for persons in the subacute stage of stroke. This intervention may be considered to be included in stroke rehabilitation.

Balance performance when responding to visual stimuli in patients with Benign Paroxysmal Positional Vertigo (BPPV).

BACKGROUND: Patients with Benign Paroxysmal Positional Vertigo (BPPV) often report a sensation of disequilibrium before treatment with the canalith repositioning maneuvers. Apart from vestibular information, visual input also contributes to balance control. How visual stimuli influence balance control in BPPV patients is unclear. OBJECTIVE: To investigate the influence of visual stimuli on balance performance in BPPV patients. METHODS: Three groups of participants, patients with BPPV, and healthy young and older adults, were instructed to stand under three conditions: 1) eyes open (EO); 2) while watching a static picture via a video display; and 3) while watching a rotating visual scene. Antero-posterior (AP), lateral (ML), and total sway path during standing were analyzed. RESULTS: In all conditions, patients with BPPV showed significantly larger AP, ML and total sway path than young, whereas older adults only showed significantly larger AP and total sway path than young adults. During the visual stimuli, all participants exhibited significantly increased AP and total sway while watching a static image and a moving scene as compared with the EO condition. CONCLUSIONS: Patients with BPPV have similar balance control to older adults, but poorer balance control than young adults. The reduced lack of standing balance control in the coronal plane of patients with BPPV, might affect balance strategy after external perturbations.

Landing Kinematics, Sports Performance, and Isokinetic Strength in Adolescent Male Volleyball Athletes: Influence of Core Training.

CONTEXT: Core control and strength are important for reducing the risk of lower-extremity injury. Current evidence on the effect of core training in male adolescent athletes is limited, and other investigations into the effects of core training often emphasized core strength only. OBJECTIVE: To examine whether core training emphasizing both control and strength of the trunk and hip would improve joint kinematics during landing, sports performance, and lower-extremity muscle strength in adolescent male volleyball athletes. DESIGN: Single group pretest and posttest design. SETTING: University laboratory. PARTICIPANTS: Sixteen male participants (age: 13.4 [1] y, height: 167.8 [8.6] cm, mass: 58.6 [13.9] kg, and volleyball experience: 3.8 [1.5] y) from a Division I volleyball team at a junior high school. MAIN OUTCOME MEASUREMENTS: Kinematics of the trunk and lower-extremity during box landing and spike jump landing tasks, volleyball-related sports performance, and isokinetic strength of hip and knee muscles were assessed before and after a 6-week core training program. RESULTS: After training, the participants demonstrated decreased trunk flexion angle (P = .01, Cohen's d = 0.78) during the box landing task and reduced the maximum knee internal rotation angle (P = .04, Cohen's d = 0.56) during the spike jump landing task. The average isokinetic strength of hip flexors and external rotators, and knee flexors and extensors also significantly increased (P = .001, Cohen's d = 0.98; P = .04, Cohen's d = 0.57; P = .02, Cohen's d = 0.66; P = .003, Cohen's d = 0.87, respectively); however, sports performance did not show significant changes. CONCLUSIONS: A more erect landing posture following training suggests that the core training program may be beneficial for improving core stability. The long-term effect of core training for knee injury prevention needs further investigation.

Exploring the sea urchin genome with undergraduates using bioinformatic tools.

It is important to provide undergraduate students with research experiences so that they obtain essential problem-solving skills and come to appreciate the process of science whether or not they pursue graduate study. However, such experiences can be difficult to achieve at a primarily undergraduate institution where time and resources are limited. One strategy is to incorporate research into the laboratory component of courses, with students having input into the specific topic being investigated. In this chapter, we present a series of activities that can be brought together as a semester or year-long project after students select a gene with the potential to be analyzed in a novel species of echinoderm. Students become acquainted with important databases, software programs, and online tools as they clone their gene, confirm its identity through alignment with homologous sequences, and characterize its expression through both qPCR and WMISH. We provide streamlined protocols that allow the work to be accomplished in an efficient manner, and conclude with ideas for assignments that can be completed in parallel to improve students' writing and oral communication skills in preparation for any career.

Reactive balance control in older adults with diabetes.

Diabetes mellitus is a major health problem for older adults worldwide and could be associated with impaired ability to recover balance after postural disturbances. This study compared reactive balance control in three groups of adults, young (YA), healthy non-diabetes older (nonDM-OA) and diabetes older (DM-OA). Twenty participants in each group completed a series of vision, plantar cutaneous sensitivity, grip power and lower limb strength tests. In the reactive balance test, participants stood on a force platform and used the dominant hand to pull the handle of a cord that could be suddenly released to create an imbalancing force. The anteroposterior (AP) and mediolateral (ML) motion of the center of pressure (COP) immediately after the sudden release was calculated to represent the level of imbalance experienced by the participants. Regression analysis entering big toe plantar sensitivity and grip power as independent variable was conducted for COP range for the three groups separately. The results showed that, except for the knee extensor, DM-OA had significantly poorer muscle strength and plantar sensitivity, and greater COP ML motion than YA and nonDM-OA. DM-OA also had significantly greater COP AP motion than YA. Grip power alone and together with plantar sensitivity explained a significant amount of variance in the AP and ML COP motion respectively (r2 = 0.334 and 0.582, respectively) for DM-OA. These findings indicated that diabetes in older adults was associated with declines in reactive balance control, and these changes may be related to muscle weakness and plantar insensitivity.

Postural Responses to a Suddenly Released Pulling Force in Older Adults with Chronic Low Back Pain: An Experimental Study.

Chronic low back pain (CLBP), one of the most common musculoskeletal conditions in older adults, might affect balance and functional independence. The purpose of this study was to investigate the postural responses to a suddenly released pulling force in older adults with and without CLBP. Thirty community-dwelling older adults with CLBP and 26 voluntary controls without CLBP were enrolled. Participants were required to stand on a force platform while, with one hand, they pulled a string that was fastened at the other end to a 2-kg or to a 4-kg force in the opposite direction at a random order. The number of times the participants lost their balance and motions of center of pressure (COP) when the string was suddenly released were recorded. The results demonstrated that although the loss of balance rates for each pulling force condition did not differ between groups, older adults with CLBP had poorer postural responses: delayed reaction, larger displacement, higher velocity, longer path length, and greater COP sway area compared to the older controls. Furthermore, both groups showed larger postural responses in the 4-kg pulling force condition. Although aging is generally believed to be associated with declining balance and postural control, these findings highlight the effect of CLBP on reactive balance when responding to an externally generated force in an older population. This study also suggests that, for older adults with CLBP, in addition to treating them for pain and disability, reactive balance evaluation and training, such as reaction and movement strategy training should be included in their interventions. Clinicians and older patients with CLBP need to be made aware of the significance of impaired reactive balance and the increased risk of falls when encountering unexpected perturbations.

Functional Balance and Its Determinants in Older People with Diabetes.

OBJECTIVE: To determine functional balance abilities of older adults with diabetes, and identify determinants of these abilities. METHODS: Eighty diabetic and 67 healthy non-diabetic community-dwelling older adults completed the Mini Mental Status Examination (MMSE) and questionnaires about their medical and fall histories. Participants were also assessed for vision, plantar sensitivity, muscle strength, and functional balance, including Functional Reach (FR), Five Times Sit-to-Stand (FTSTS), and 180° turn (TURN). In addition to between-group comparisons, hierarchical regression analysis was conducted to identify the independent determinants for each of the individual balance tasks for the diabetes and control group separately. RESULTS: The diabetes group had significantly greater body mass index, higher rate of cardiac disease, and poorer plantar sensitivity, mental status, grip and lower limb strength. The diabetes group performed significantly poorer in FTSTS and TURN (both p<0.001), but not FR (p = 0.108). The significant determinants for the balance tasks varied substantially between tasks and groups. For the diabetes group, they included visual and plantar sensitivity and MMSE for FR (R2 = 0.39), ankle dorsiflexion strength for FTSTS (R2 = 0.377), and plantar sensitivity, knee extension strength and MMSE for TURN (R2 = 0.391). For the control group, knee extension strength emerged as the common and only significant determinant and only explained approximately 10% of the variance for FR and TURN. CONCLUSIONS: Impairments in functional balance abilities were evident for older adults with diabetes. Their underpinning functional limitations were different for different tasks and were also different from those of the control group. Screening of functional balance and mental status, lower limb strength and sensory function, and interventions to address these impairments may be important to maintain function, independence and safety for older clients with diabetes.

Steadiness of Spinal Regions during Single-Leg Standing in Older Adults with and without Chronic Low Back Pain.

The aims of this study were to compare the steadiness index of spinal regions during single-leg standing in older adults with and without chronic low back pain (LBP) and to correlate measurements of steadiness index with the performance of clinical balance tests. Thirteen community-dwelling older adults (aged 55 years or above) with chronic LBP and 13 age- and gender-matched asymptomatic volunteers participated in this study. Data collection was conducted in a university research laboratory. Measurements were steadiness index of spinal regions (trunk, thoracic spine, lumbar spine, and pelvis) during single-leg standing including relative holding time (RHT) and relative standstill time (RST), and clinical balance tests (timed up and go test and 5-repetition sit to stand test). The LBP group had a statistically significantly smaller RHT than the control group, regardless of one leg stance on the painful or non-painful sides. The RSTs on the painful side leg in the LBP group were not statistically significantly different from the average RSTs of both legs in the control group; however, the RSTs on the non-painful side leg in the LBP group were statistically significantly smaller than those in the control group for the trunk, thoracic spine, and lumbar spine. No statistically significant intra-group differences were found in the RHTs and RSTs between the painful and non-painful side legs in the LBP group. Measurements of clinical balance tests also showed insignificant weak to moderate correlations with steadiness index. In conclusion, older adults with chronic LBP demonstrated decreased spinal steadiness not only in the symptomatic lumbar spine but also in the other spinal regions within the kinetic chain of the spine. When treating older adults with chronic LBP, clinicians may also need to examine their balance performance and spinal steadiness during balance challenging tests.

Design and experimental evaluation of a 256-channel dual-frequency ultrasound phased-array system for transcranial blood-brain barrier opening and brain drug delivery.

Focused ultrasound (FUS) in the presence of microbubbles can bring about transcranial and local opening of the blood-brain barrier (BBB) for potential noninvasive delivery of drugs to the brain. A phased-array ultrasound system is essential for FUS-BBB opening to enable electronic steering and correction of the focal beam which is distorted by cranial bone. Here, we demonstrate our prototype design of a 256-channel ultrasound phased-array system for large-region transcranial BBB opening in the brains of large animals. One of the unique features of this system is the capability of generating concurrent dual-frequency ultrasound signals from the driving system for potential enhancement of BBB opening. A wide range of signal frequencies can be generated (frequency = 0.2-1.2 MHz) with controllable driving burst patterns. Precise output power can be controlled for individual channels via 8-bit duty-cycle control of transistor-transistor logic signals and the 8-bit microcontroller-controlled buck converter power supply output voltage. The prototype system was found to be in compliance with the electromagnetic compatibility standard. Moreover, large animal experiments confirmed the phase switching effectiveness of this system, and induction of either a precise spot or large region of BBB opening through fast focal-beam switching. We also demonstrated the capability of dual-frequency exposure to potentially enhance the BBB-opening effect. This study contributes to the design of ultrasound phased arrays for future clinical applications, and provides a new direction toward optimizing FUS brain drug delivery.

Forward-backward postural protective stepping responses in young and elderly adults.

OBJECTIVES: Protective steps are essential for fall avoidance. Most studies only examined forwards stepping despite considerable bio-mechanical and visual differences between the forwards and backwards directions. We assess forward-backward differences in protective steps in a young and elderly group. METHODS: Protective stepping responses were elicited by a platform moving unpredictably either forwards or backwards. For control purposes, voluntary steps, in response to vibration cues on the forehead or occiput were also recorded. Reaction time (RT), length and angular velocity of the steps were measured in 13 young (age 19-35years) and 13 elderly (age 58-86years) healthy volunteers. RESULTS: (i) Protective vs voluntary steps: protective steps were earlier, faster and longer than voluntary steps. (ii) Forwards-backwards differences: RT was quicker for backwards than forwards protective steps, in contrast to voluntary steps where RTs were similar in the two directions. (iii) Age difference: the elderly had universally slower steps and they generated shorter backwards than forwards protective steps. CONCLUSIONS: Protective steps appear more robust than voluntary steps - they are earlier (shorter RT), longer and faster than voluntary steps, indicating an automatic rather than a volitional reaction. Backwards protective steps occur earlier than forwards; such promptness may have evolved out of bio-mechanical features which make falling backwards easier. Since our elderly subjects had an average age <70years, their slower and shorter protective backwards steps may represent the first abnormality in this rescue postural response. The findings in the elderly may partly depend on dysfunction in fronto-basal ganglia postural loops.

Focused ultrasound-induced blood-brain barrier opening to enhance temozolomide delivery for glioblastoma treatment: a preclinical study.

The purpose of this study is to assess the preclinical therapeutic efficacy of magnetic resonance imaging (MRI)-monitored focused ultrasound (FUS)-induced blood-brain barrier (BBB) disruption to enhance Temozolomide (TMZ) delivery for improving Glioblastoma Multiforme (GBM) treatment. MRI-monitored FUS with microbubbles was used to transcranially disrupt the BBB in brains of Fisher rats implanted with 9L glioma cells. FUS-BBB opening was spectrophotometrically determined by leakage of dyes into the brain, and TMZ was quantitated in cerebrospinal fluid (CSF) and plasma by LC-MS\MS. The effects of treatment on tumor progression (by MRI), animal survival and brain tissue histology were investigated. Results demonstrated that FUS-BBB opening increased the local accumulation of dyes in brain parenchyma by 3.8-/2.1-fold in normal/tumor tissues. Compared to TMZ alone, combined FUS treatment increased the TMZ CSF/plasma ratio from 22.7% to 38.6%, reduced the 7-day tumor progression ratio from 24.03 to 5.06, and extended the median survival from 20 to 23 days. In conclusion, this study provided preclinical evidence that FUS BBB-opening increased the local concentration of TMZ to improve the control of tumor progression and animal survival, suggesting its clinical potential for improving current brain tumor treatment.

Protective stepping response in Parkinsonian patients and the effect of vibrotactile feedback.

The objectives of this study were (1) to characterize protective stepping responses to unpredictable forward/backward postural perturbation in Parkinson's disease (PD) and (2) to assess whether vibrotactile cues of the impending fall improve the stepping response. Twenty mild PD patients, 7 advanced PD patients, and 17 age-matched controls stood on a platform moving unpredictably forward and backward, requiring a protective step to maintain balance. Direction-coded vibrotactile cues, triggered by leg tilt, were provided to prompt step generation. All subjects showed quicker reaction time, shorter steps, and smaller total trunk displacement when stepping backward than when stepping forward. Advanced PD patients took shorter, slower, and an increased number of protective steps. The only abnormality observed in mild PD patients was slightly slower backward steps. Vibrotactile feedback reduced the amount of trunk displacement observed before taking a protective step but did not improve any abnormality in PD patients. Early PD patients had near-normal protective stepping responses to unpredictable perturbations, but advanced patients made slow and short steps both forward and backward. Given that latencies were preserved even in unpredictable conditions, step slowness and hypometria are the primary abnormalities of the stepping response in PD. As voluntary locomotor stepping in PD is reported to improve with sensory feedback, the lack of such improvement in our study implies that additional sensory cues cannot help automatic reflex-like stepping reactions in PD patients.

Agarose gel electrophoresis for the separation of DNA fragments.

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb(1). Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits(2). During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel's molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight(3). The leading model for DNA movement through an agarose gel is "biased reptation", whereby the leading edge moves forward and pulls the rest of the molecule along(4). The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation(5); 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: Understand the mechanism by which DNA fragments are separated within a gel matrix Understand how conformation of the DNA molecule will determine its mobility through a gel matrix Identify an agarose solution of appropriate concentration for their needs Prepare an agarose gel for electrophoresis of DNA samples Set up the gel electrophoresis apparatus and power supply Select an appropriate voltage for the separation of DNA fragments Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands Determine the sizes of separated DNA fragments.

Effect of supplementary carbon addition in the treatment of low C/N high-technology industrial wastewater by MBR.

The effect of supplementary carbon addition for the treatment of high-technology industrial wastewater in a membrane bioreactor (MBR) was investigated. The MBR was operated for 302 days under different C/N (BOD(L)/NH(4)(+)-N) ratios, i.e. 0.9-1 to 20 days, 1.6-21 to 42 days, 2.9-43 to 82 days, 3.6-83 to 141 days, 4.8-165 to 233 days and 9.3-240 to 302 days. Irrespective of the C/N ratios investigated, SS and BOD(5) removal efficiencies were above 95% and above 80% COD removal efficiency was observed. In addition, complete nitrification was observed throughout the investigation. However, denitrification and total nitrogen removal efficiencies reached their maximum values at the highest C/N ratio (9.3) investigated. Real-time PCR analysis revealed 10 times higher ammonia oxidizing bacteria to total bacteria ratio under the highest C/N ratio condition (9.3) compared to the low C/N ratio condition (1.6).

Exclusive developmental functions of gatae cis-regulatory modules in the Strongylocentrorus purpuratus embryo.

The gatae gene of Strongylocentrotus purpuratus is orthologous to vertebrate gata-4,5,6 genes. This gene is expressed in the endomesoderm in the blastula and later the gut of the embryo, and is required for normal development. A gatae BAC containing a GFP reporter knocked into exon one of the gene was able to reproduce all aspects of endogenous gatae expression in the embryo. To identify putative gatae cis-regulatory modules we carried out an interspecific sequence conservation analysis with respect to a Lytechinus variegatus gatae BAC, which revealed 25 conserved non-coding sequence patches. These were individually tested in gene transfer experiments, and two modules capable of driving localized reporter expression in the embryo were identified. Module 10 produces early expression in mesoderm and endoderm cells up to the early gastrula stage, while module 24 generates late endodermal expression at gastrula and pluteus stages. Module 10 was then deleted from the gatae BAC by reciprocal recombination, resulting in total loss of reporter expression in the time frame in which it is normally active. Similar deletion of module 24 led to ubiquitous GFP expression in the gastrula and pluteus. These results show that Module 10 is uniquely necessary and sufficient to account for the early phase of gatae expression during endomesoderm specification. In addition, they imply a functional cis-regulatory module exclusion, whereby only a single module can associate with the basal promoter and drive gene expression at any given time.

Cis-regulatory control of the nodal gene, initiator of the sea urchin oral ectoderm gene network.

Expression of the nodal gene initiates the gene regulatory network which establishes the transcriptional specification of the oral ectoderm in the sea urchin embryo. This gene encodes a TGFbeta ligand, and in Strongylocentrotus purpuratus its transcription is activated in the presumptive oral ectoderm at about the 30-cell stage. Thereafter Nodal signaling occurs among all cells of the oral ectoderm territory, and nodal expression is required for expression of oral ectoderm regulatory genes. The cis-regulatory system of the nodal gene transduces anisotropically distributed cytoplasmic cues that distinguish the future oral and aboral domains of the early embryo. Here we establish the genomic basis for the initiation and maintenance of nodal gene expression in the oral ectoderm. Functional cis-regulatory control modules of the nodal gene were identified by interspecific sequence conservation. A 5' cis-regulatory module functions both to initiate expression of the nodal gene and to maintain its expression by means of feedback input from the Nodal signal transduction system. These functions are mediated respectively by target sites for bZIP transcription factors, and by SMAD target sites. At least one SMAD site is also needed for the initiation of expression. An intron module also contains SMAD sites which respond to Nodal feedback, and in addition acts to repress vegetal expression. These observations explain the main features of nodal expression in the oral ectoderm: since the activity of bZIP factors is redox sensitive, and the initial polarization of oral vs. aboral fate is manifested in a redox differential, the bZIP sites account for the activation of nodal on the oral side; and since the immediate early signal transduction response factors for Nodal are SMAD factors, the SMAD sites account for the feedback maintenance of nodal gene expression.

Expression of Spgatae, the Strongylocentrotus purpuratus ortholog of vertebrate GATA4/5/6 factors.

Spgatae is the sea urchin ortholog of the vertebrate gata4/5/6 genes, as confirmed by phylogenetic analysis. The accumulation of Spgatae transcripts during embryonic development and the spatial pattern of expression are reported here. Expression was first detected in the 15 h blastula. The number of Spgatae RNA molecules increases steadily during blastula stages, with expression peaking during gastrulation. After gastrulation is complete, the level of expression decreases until the end of embryogenesis. Whole mount in situ hybridization showed that Spgatae transcripts were first detected in a ring of prospective mesoderm cells in the vegetal plate. Spgatae expression then expands to include the entire vegetal plate at the mesenchyme blastula stage. During gastrulation Spgatae is expressed at the blastopore, and at prism stage strongly in the hindgut and midgut but not foregut, and also in mesoderm cells at the tip of the archenteron. Towards the end of embryogenesis, expression in the hindgut decreases. The terminal pattern of expression is in midgut plus coelomic pouches.

A provisional regulatory gene network for specification of endomesoderm in the sea urchin embryo.

We present the current form of a provisional DNA sequence-based regulatory gene network that explains in outline how endomesodermal specification in the sea urchin embryo is controlled. The model of the network is in a continuous process of revision and growth as new genes are added and new experimental results become available; see http://www.its.caltech.edu/~mirsky/endomeso.htm (End-mes Gene Network Update) for the latest version. The network contains over 40 genes at present, many newly uncovered in the course of this work, and most encoding DNA-binding transcriptional regulatory factors. The architecture of the network was approached initially by construction of a logic model that integrated the extensive experimental evidence now available on endomesoderm specification. The internal linkages between genes in the network have been determined functionally, by measurement of the effects of regulatory perturbations on the expression of all relevant genes in the network. Five kinds of perturbation have been applied: (1) use of morpholino antisense oligonucleotides targeted to many of the key regulatory genes in the network; (2) transformation of other regulatory factors into dominant repressors by construction of Engrailed repressor domain fusions; (3) ectopic expression of given regulatory factors, from genetic expression constructs and from injected mRNAs; (4) blockade of the beta-catenin/Tcf pathway by introduction of mRNA encoding the intracellular domain of cadherin; and (5) blockade of the Notch signaling pathway by introduction of mRNA encoding the extracellular domain of the Notch receptor. The network model predicts the cis-regulatory inputs that link each gene into the network. Therefore, its architecture is testable by cis-regulatory analysis. Strongylocentrotus purpuratus and Lytechinus variegatus genomic BAC recombinants that include a large number of the genes in the network have been sequenced and annotated. Tests of the cis-regulatory predictions of the model are greatly facilitated by interspecific computational sequence comparison, which affords a rapid identification of likely cis-regulatory elements in advance of experimental analysis. The network specifies genomically encoded regulatory processes between early cleavage and gastrula stages. These control the specification of the micromere lineage and of the initial veg(2) endomesodermal domain; the blastula-stage separation of the central veg(2) mesodermal domain (i.e., the secondary mesenchyme progenitor field) from the peripheral veg(2) endodermal domain; the stabilization of specification state within these domains; and activation of some downstream differentiation genes. Each of the temporal-spatial phases of specification is represented in a subelement of the network model, that treats regulatory events within the relevant embryonic nuclei at particular stages.