Buckling without Bending: A New Paradigm in Morphogenesis.

A curious feature of organ and organoid morphogenesis is that in certain cases, spatial oscillations in the thickness of the growing "film" are out of phase with the deformation of the slower-growing "substrate," while in other cases, the oscillations are in phase. The former cannot be explained by elastic bilayer instability, and contradict the notion that there is a universal mechanism by which brains, intestines, teeth, and other organs develop surface wrinkles and folds. Inspired by the microstructure of the embryonic cerebellum, we develop a new model of 2D morphogenesis in which system-spanning elastic fibers endow the organ with a preferred radius, while a separate fiber network resides in the otherwise fluidlike film at the outer edge of the organ and resists thickness gradients thereof. The tendency of the film to uniformly thicken or thin is described via a "growth potential." Several features of cerebellum, +blebbistatin organoid, and retinal fovea morphogenesis, including out-of-phase behavior and a film thickness amplitude that is comparable to the radius amplitude, are readily explained by our simple analytical model, as may be an observed scale invariance in the number of folds in the cerebellum. We also study a nonlinear variant of the model, propose further biological and bioinspired applications, and address how our model is and is not unique to the developing nervous system.

Association Between Concussion and Lower Extremity Injuries in Collegiate Athletes.

BACKGROUND: Concussions have been associated with elevated musculoskeletal injury risk; however, the influence of unreported and unrecognized concussions has not been investigated. HYPOTHESIS: The purpose of this study was to examine the association between concussion and lower extremity musculoskeletal injury rates across a diverse array of sports among collegiate student-athletes at the conclusion of their athletic career. The hypothesis was that there will be a positive association between athletes who reported a history of concussions and higher rates of lower extremity injuries. STUDY DESIGN: Cross-sectional study. LEVEL OF EVIDENCE: Level 3. METHODS: Student-athletes (N = 335; 62.1% women; mean age, 21.2 ± 1.4 years) from 13 sports completed a reliable injury history questionnaire. Respondents indicated the total number of reported, unreported, and potentially unrecognized concussions as well as lower extremity injuries including ankle sprains, knee injuries, and muscle strains. Chi-square analyses were performed to identify the association between concussion and lower extremity injuries. RESULTS: There were significant associations between concussion and lateral ankle sprain ( P = 0.012), knee injury ( P = 0.002), and lower extremity muscle strain ( P = 0.031). There were also significant associations between reported concussions and knee injury ( P = 0.003), unreported concussions and knee injury ( P = 0.002), and unrecognized concussions and lateral ankle sprain ( P = 0.001) and lower extremity muscle strains ( P = 0.006), with odds ratios ranging from 1.6 to 2.9. CONCLUSION: There was a positive association between concussion history and lower extremity injuries (odds ratios, 1.6-2.9 elevated risk) among student-athletes at the conclusion of their intercollegiate athletic careers. CLINICAL RELEVANCE: Clinicians should be aware of these elevated risks when making return-to-participation decisions and should incorporate injury prevention protocols.

Scapular Upward-Rotation Deficits After Acute Fatigue in Tennis Players.

CONTEXT: Fatigue in overhead athletes reduces shoulder muscular contraction and proprioception. These deficits may lead to alterations in scapular upward rotation, which is associated with multiple chronic shoulder conditions prevalent in tennis players. OBJECTIVE: To identify the effect of a functional fatigue protocol on scapular upward rotation in collegiate male tennis players. DESIGN: Randomized controlled clinical trial. SETTING: Research laboratory. PATIENTS OR OTHER PARTICIPANTS: Twenty healthy male tennis players with no history of shoulder injury completed this study. Participants were divided into 2 groups, experimental (age = 19.4 ± 1.1 years, height = 180.1 ± 8.9 cm, weight = 72.7 ± 11.6 kg) and control (age = 19.6 ± 1.2 years, height = 181.1 ± 6.6 cm, weight = 81.6 ± 13.5 kg). INTERVENTION(S): Participants in the experimental group performed a tennis-serving protocol until the onset of fatigue. Fatigue was defined as a participant reporting a rating of 15 on the Borg Scale of Perceived Exertion and reaching a heart rate of 70% of maximum. Instead of completing the fatigue protocol, control participants rested for an interval time matched to the experimental group. MAIN OUTCOME MEASURE(S): Scapular upward rotation of the dominant arm was measured at rest and at 60°, 90°, and 120° of glenohumeral elevation in the scapular plane. Upward-rotation measurements were taken prefatigue, postfatigue, and at 24, 48, and 72 hours postexercise. Scapular upward-rotation values were calculated as change scores from baseline and analyzed using a 2 × 4 mixed-model analysis of variance. RESULTS: Significant group-by-time interaction effects were found in postfatigue change scores. The experimental group displayed scapular upward-rotation deficits at all testing positions postfatigue (rest: -2.1° ± 1.4°, 60°: -2.2° ± 2.2°, 90°: -3.2° ± 2.1°, 120°: -4.0° ± 1.3°). No differences were observed at 24, 48, or 72 hours after the fatigue protocol. CONCLUSIONS: Fatigue impaired scapular upward rotation in male tennis players, but values returned to baseline within 24 hours. Clinicians should monitor scapular upward rotation in tennis players returning to competition within a day after heavy serving activity.

Sideline Performance of the Balance Error Scoring System during a Live Sporting Event.

OBJECTIVE: The purpose was to examine the influence of a live sporting sideline environment on balance error scoring system (BESS) performance. DESIGN: Prospective longitudinal cohort study. SETTING: The BESS was performed by all participants at 3 locations: (1) quiet laboratory, (2) football stadium sidelines, and (3) basketball arena sidelines. PARTICIPANTS: The experimental group had 38 participants (age: 20.1 ± 1.1 years; height: 170.0 ± 7.7 cm; mass: 66.7 ± 9.5 kg) who were female intercollegiate student-athletes (SA). The control group consisted of 38 recreationally active female college students (age: 20.8 ± 1.1 years; height: 162.6 ± 6.0 cm; mass: 63.7 ± 10.6 kg). INTERVENTIONS: The 2 groups performed the tests at the same locations, the SA group during live sporting events and the control group when no event was occurring. MAIN OUTCOME MEASURES: The dependent variable was the total BESS score. Separate 2 × 3 mixed methods analyses of variance investigated the influence of the environment and practice effect. RESULTS: There was a significant interaction for group by environment (P = 0.004), and the SA group committed more errors at both the football and the basketball settings than the control group. The SA group also committed more errors at football (P = 0.028) than baseline. The control group demonstrated a likely practice effect with fewer errors during each administration. CONCLUSIONS: The BESS score deteriorated when performed on the sidelines of a live sporting event potentially challenging the clinical utility of the BESS. Clinicians need to consider the role of the local environment when performing the BESS test and should perform postinjury tests in the same environment as the baseline test. CLINICAL RELEVANCE: When performing balance testing of patients with suspected concussions, clinicians need to consider the environment in which the test is performed and attempt to match the preseason testing environment.

National Collegiate Athletic Association Division I athletic trainers' concussion-management practice patterns.

CONTEXT: A cornerstone of the recent consensus statements on concussion is a multifaceted concussion-assessment program at baseline and postinjury and when tracking recovery. Earlier studies of athletic trainers' (ATs') practice patterns found limited use of multifaceted protocols; however, these authors typically grouped diverse athletic training settings together. OBJECTIVE: To (1) describe the concussion-management practice patterns of National Collegiate Athletic Association (NCAA) Division I ATs, (2) compare these practice patterns to earlier studies, and (3) objectively characterize the clinical examination. DESIGN: Cross-sectional study. SETTING: Online survey. PATIENTS OR OTHER PARTICIPANTS: A total of 610 ATs from NCAA Division I institutions, for a response rate of 34.4%. MAIN OUTCOME MEASURE(S): The survey had 3 subsections: demographic questions related to the participant's experiences, concussion-assessment practice patterns, and concussion-recovery and return-to-participation practice patterns. Specific practice-pattern questions addressed balance, cognitive and mental status, neuropsychological testing, and self-reported symptoms. Finally, specific components of the clinical examination were examined. RESULTS: We identified high rates of multifaceted assessments (i.e., assessments using at least 3 techniques) during testing at baseline (71.2%), acute concussion assessment (79.2%), and return to participation (66.9%). The specific techniques used are provided along with their adherence with evidence-based practice findings. Respondents endorsed a diverse array of clinical examination techniques that often overlapped objective concussion-assessment protocols or were likely used to rule out associated potential conditions. Respondents were cognizant of the Third International Consensus Statement, the National Athletic Trainers' Association position statement, and the revised NCAA Sports Medicine Handbook recommendations. CONCLUSIONS: Athletic trainers in NCAA Division I demonstrated widespread use of multifaceted concussion-assessment techniques and appeared compliant with recent consensus statements and the NCAA Sports Medicine Handbook.

Concussion reporting rates at the conclusion of an intercollegiate athletic career.

OBJECTIVE: The purpose of this study was to explore the current reported, unreported, and potentially unrecognized concussion rates among collegiate student-athletes who have completed their collegiate athletic career. DESIGN: Retrospective survey. SETTING: College and University athletic training rooms. PARTICIPANTS: One hundred sixty-one collegiate student-athletes (56.5% women; aged 21.5 ± 1.3; 3.7 ± 1.0 years of collegiate athletic experience) from 10 institutions who had either completed their intercollegiate athletic eligibility or were no longer participating. MAIN OUTCOME MEASURES: The self-reported concussion rate, the unreported rate and reasons, and the potentially unrecognized concussion rate. RESULTS: The self-reported concussion rate was 33.5% (54/161), and 22.2% (12) self-reported at least 3 concussions. The unreported rate was 11.8% (19/161), and the potentially unrecognized rate was 26.1% (42/161) with the most common unrecognized symptom being "knocked silly/seen stars" (23.6% [38/161]). CONCLUSIONS: Overall, 49.7% of all respondents (80/161) reported 1 acknowledged, unreported, or potential concussion. The unreported rate was lower than previous high school studies; however, the potentially unrecognized rate remains high and should be clinically concerning. These findings suggest educational interventions targeting collegiate student-athletes should remain and continue to focus on identifying concussion symptoms and dispelling the common misconception that "bell ringers" and "dings" are not concussions.

Balance error scoring system performance changes after a competitive athletic season.

OBJECTIVE: To evaluate the change in Balance Error Scoring System (BESS) performance after an athletic season. DESIGN: A prospective longitudinal group study. SETTING: University biomechanics research laboratory. PARTICIPANTS: A total of 58 college-aged females (23 soccer student-athletes, 16 volleyball student-athletes, and 19 recreationally active healthy college students) participated in the study. INTERVENTIONS: The BESS test was administered on 2 occasions 90 days apart. For the student-athletes, the first test (PRE) was administered before the start of their athletic season and the second test (POST) was administered immediately after the season. For the recreationally active college students, the PRE test was at the beginning of the academic semester and the POST test exactly 90 days thereafter. MAIN OUTCOME MEASURES: Total BESS score at PRE and POST was compared with a 3 × 2 repeated measures analysis of variance. The overall change score and absolute value change score were also calculated and compared with a 1-sample t test to an expected change of zero errors. RESULTS: There was no group by time interaction; however, there was a main effect for time. There was a significant improvement (P = 0.003) between PRE (9.00 ± 2.97 errors) and POST (7.92 ± 2.78 errors) BESS performance. There were significant differences for both the overall change score (1.08 errors) and the absolute value change score (2.00 errors). CONCLUSIONS: A clinically and statistically significant difference in BESS performance was identified after a 90-day intercollegiate athletic season.

Embryonic origins of ZebrinII parasagittal stripes and establishment of topographic Purkinje cell projections.

The establishment of neural circuits involves both the precise positioning of cells within brain regions and projection of axons to specific target cells. In the cerebellum (Cb), the medial-lateral (M-L) and anterior-posterior (A-P) position of each Purkinje cell (PC) and the topography of its axon can be defined with respect to two coordinate systems within the Cb; one based on the pattern of lobules and the other on PC gene expression in parasagittal clusters in the embryo (e.g. Pcp2) and stripes in the adult (e.g. ZebrinII). The relationship between the embryonic clusters of molecularly defined PCs and particular adult PC stripes is not clear. Using a mouse genetic inducible fate mapping (GIFM) approach and a Pcp2-CreER-IRES-hAP transgene, we marked three bilateral clusters of PC clusters with myristolated green fluorescent protein (mGfp) on approximately embryonic day (E) 15 and followed their fate into adulthood. We found that these three clusters contributed specifically to ZebrinII-expressing PCs, including nine of the adult stripes. This result suggests that embryonic PCs maintain a particular molecular identity, and that each embryonic cluster can contribute PCs to more than one adult M-L stripe. Each PC projects a primary axon to one of the deep cerebellar nuclei (DCN) or the vestibular nuclei in the brainstem in an organized fashion that relates to the position of the PCs along the M-L axis. We characterized when PC axons from the three M-L clusters acquire topographic projections. Using a combination of GIFM to mark the PC clusters with mGfp and staining for human placental alkaline phosphatase (hAP) in Pcp2-CreER-IRES-hAP transgenic embryos we found that axons from each embryonic PC cluster intermingled with neurons within particular DCN or projected out of the Cb toward the vestibular nuclei by E14.5. These studies show that PC molecular patterning, efferent circuitry, and DCN nucleogenesis occur simultaneously, suggesting a link between these processes.

An examination of Euro-American and African-American differences in social physique anxiety among college women.

Many studies have examined sex differences in social physique anxiety; however, few researchers have examined possible perceptual differences in such anxiety based on ethnicity. The present purpose was to examine social physique anxiety among college-age women of Euro-American and African-American descent. Participants (N = 91) from physical activity classes at a university located in the southeastern United States completed the Social Physique Anxiety Scale. The participants were 67 Euro-Americans and 24 African Americans. An independent t test yielded a significant difference (p =.01) between groups on Eklund's scale, which supports the hypothesis.

Comparison of Cervical Spine Motion During Application Among 4 Rigid Immobilization Collars.

OBJECTIVE: To quantify the cervical spine range of motion that occurred during application of 4 rigid cervical immobilization collars, the time of application, and the amount of active range of motion available after application. DESIGN AND SETTING: We evaluated the amount of cervical motion that occurred during application of 4 commonly used collars: NECLOC (NL), StifNeck (SN), StifNeck Select (SNS), and Rapid Form Vacuum Immobilizer (VI). Each clinician applied a properly sized collar to both a small- and medium-size model 3 times. After application, active range-of-motion testing was completed with the subject in the supine and seated positions. SUBJECTS: A total of 17 certified athletic trainers participated. MEASUREMENTS: We used 3-dimensional kinematic head and thorax data to calculate peak angular displacement, total linear distance, and total angular distance during application and peak angular displacement during supine and seated range-of-motion testing. Application time was calculated during each trial. RESULTS: Significant differences between collars were noted for application time, total linear distance, and total angular distance (P<.01). The SN and SNS were applied significantly faster and with significantly less total linear distance and total angular distance than the NL and the VI collars. The NL was applied significantly faster and with significantly less total linear distance and total angular distance than the VI. During supine and seated active range-of-motion tests, the SN and SNS permitted significantly less cervical flexion-extension, rotation, and lateral flexion than the NL and VI. CONCLUSIONS: Of the collars tested, the SN and SNS appear to be the optimal collars for use by certified athletic trainers. They were applied with the least motion in the fastest time and provided superior restriction during active range-of-motion testing.

A novel gene, GliH1, with homology to the Gli zinc finger domain not required for mouse development.

The Sonic hedgehog (Shh)-Gli signaling pathway regulates development of many organs, including teeth. We cloned a novel gene encoding a transcription factor that contains a zinc finger domain with highest homology to the Gli family of proteins (61-64% amino acid sequence identity) from incisor pulp. Consistent with this sequence conservation, gel mobility shift assays demonstrated that this new Gli homologous protein, GliH1, could bind previously characterized Gli DNA binding sites. Furthermore, transfection assays in dental pulp cells showed that whereas Gli1 induces a nearly 50-fold increase in activity of a luciferase reporter containing Gli DNA binding sites, coexpression of Gli1 with Gli3 and/or GliH1 results in inhibition of the Gli1-stimulated luciferase activity. In situ hybridization analysis of mouse embryos demonstrated that GliH1 expression is initiated later than the three Gli genes and has a more restricted expression pattern. GliH1 is first detected diffusely in the limb buds at 10.0 days post coitus and later is expressed in the branchial arches, craniofacial interface, ventral part of the tail, whisker follicles and hair, intervertebral discs, teeth, eyes and kidney. LacZ was inserted into the GliH1 allele in embryonic stem cells to produce mice lacking GliH1 function. While this produced indicator mice for GliH1-expression, analysis of mutant mice revealed no discernible phenotype or required function for GliH1. A search of the Celera Genomics and associated databases identified possible gene sequences encoding a zinc finger domain with approximately 90% homology to that of GliH1, indicating there is a family of GliH genes and raising the possibility of overlapping functions during development.

Otx2 and Gbx2 are required for refinement and not induction of mid-hindbrain gene expression.

Otx2 and Gbx2 are among the earliest genes expressed in the neuroectoderm, dividing it into anterior and posterior domains with a common border that marks the mid-hindbrain junction. Otx2 is required for development of the forebrain and midbrain, and Gbx2 for the anterior hindbrain. Furthermore, opposing interactions between Otx2 and Gbx2 play an important role in positioning the mid-hindbrain boundary, where an organizer forms that regulates midbrain and cerebellum development. We show that the expression domains of Otx2 and Gbx2 are initially established independently of each other at the early headfold stage, and then their expression rapidly becomes interdependent by the late headfold stage. As we demonstrate that the repression of Otx2 by retinoic acid is dependent on an induction of Gbx2 in the anterior brain, molecules other than retinoic acid must regulate the initial expression of Otx2 in vivo. In contrast to previous suggestions that an interaction between Otx2- and Gbx2-expressing cells may be essential for induction of mid-hindbrain organizer factors such as Fgf8, we find that Fgf8 and other essential mid-hindbrain genes are induced in a correct temporal manner in mouse embryos deficient for both Otx2 and Gbx2. However, expression of these genes is abnormally co-localized in a broad anterior region of the neuroectoderm. Finally, we find that by removing Otx2 function, development of rhombomere 3 is rescued in Gbx2(-/-) embryos, showing that Gbx2 plays a permissive, not instructive, role in rhombomere 3 development. Our results provide new insights into induction and maintenance of the mid-hindbrain genetic cascade by showing that a mid-hindbrain competence region is initially established independent of the division of the neuroectoderm into an anterior Otx2-positive domain and posterior Gbx2-positive domain. Furthermore, Otx2 and Gbx2 are required to suppress hindbrain and midbrain development, respectively, and thus allow establishment of the normal spatial domains of Fgf8 and other genes.

Gli1 can rescue the in vivo function of Gli2.

In mice, three Gli genes are thought to mediate sonic hedgehog (Shh) signaling collectively. Mis-expression studies and analysis of null mutants for each gene have indicated that the Gli proteins have different functions. In particular, Gli1 appears to be a constitutive activator, and Gli2 and Gli3 have repressor functions. To determine the precise functional differences between Gli1 and Gli2, we have expressed Gli1 in place of Gli2 from the endogenous Gli2 locus in mice. Strikingly, a low level of Gli1 can rescue all the Shh signaling defects in Gli2 mutants; however, only in the presence of a wild-type Shh gene. These studies demonstrate that only the activator function of Gli2 is actually required, and indicates that in specific situations, Shh can modulate the ability of Gli1 to activate target genes. Furthermore, expression of both copies of Gli1 in place of Gli2 does not disrupt spinal cord patterning, but does result in new gain-of-function defects that lead to lethality. We show that the defects are enhanced when Gli3 function is reduced, demonstrating that an important difference between Gli1 and Gli2 is the ability of Gli1 to antagonize Gli3 function.

The HD mutation causes progressive lethal neurological disease in mice expressing reduced levels of huntingtin.

Huntingtin is an essential protein that with mutant polyglutamine tracts initiates dominant striatal neurodegeneration in Huntington's disease (HD). To assess the consequences of mutant protein when huntingtin is limiting, we have studied three lines of compound heterozygous mice in which both copies of the HD gene homolog (Hdh) were altered, resulting in greatly reduced levels of huntingtin with a normal human polyglutamine length (Q20) and/or an expanded disease-associated segment (Q111): Hdh(neoQ20)/Hdh(neoQ20), Hdh(neoQ20)/Hdh(null) and Hdh(neoQ20)/Hdh(neoQ111). All surviving mice in each of the three lines were small from birth, and had variable movement abnormalities. Magnetic resonance micro-imaging and histological evaluation showed enlarged ventricles in approximately 50% of the Hdh(neoQ20)/Hdh(neoQ111) and Hdh(neoQ20)/Hdh(null) mice, revealing a developmental defect that does not worsen with age. Only Hdh(neoQ20)/Hdh(neoQ111) mice exhibited a rapidly progressive movement disorder that, in the absence of striatal pathology, begins with hind-limb clasping during tail suspension and tail stiffness during walking by 3-4 months of age, and then progresses to paralysis of the limbs and tail, hypokinesis and premature death, usually by 12 months of age. Thus, dramatically reduced huntingtin levels fail to support normal development in mice, resulting in reduced body size, movement abnormalities and a variable increase in ventricle volume. On this sensitized background, mutant huntingtin causes a rapid neurological disease, distinct from the HD-pathogenic process. These results raise the possibility that therapeutic elimination of huntingtin in HD patients could lead to unintended neurological, as well as developmental side-effects.

Early anterior/posterior patterning of the midbrain and cerebellum.

Transplantation studies performed in chicken embryos indicated that early anterior/posterior patterning of the vertebrate midbrain and cerebellum might be regulated by an organizing center at the junction between the midbrain and hindbrain. More than a decade of molecular and genetic studies have shown that such an organizer is indeed central to development of the midbrain and anterior hindbrain. Furthermore, a complicated molecular network that includes multiple positive and negative feedback loops underlies the establishment and refinement of a mid/hindbrain organizer, as well as the subsequent function of the organizer. In this review, we first introduce the expression patterns of the genes known to be involved in this patterning process and the quail-chick transplantation experiments that have provided the foundation for understanding the genetic pathways regulating mid/hindbrain patterning. Subsequently, we discuss the molecular genetic studies that have revealed the roles for many genes in normal early patterning of this region. Finally, some of the remaining questions and future directions are discussed.

Identification and characterization of Lbh, a novel conserved nuclear protein expressed during early limb and heart development.

We report the cloning, protein characterization, and expression of a novel vertebrate gene, termed Lbh (Limb-bud-and-heart), with a spatiotemporal expression pattern that marks embryologically significant domains in the developing limbs and heart. Lbh encodes a highly conserved nuclear protein, which in tissue culture cells possesses a transcriptional activator function. During limb development, expression of Lbh initiates in the ectoderm of the presumptive limb territory in the lateral body wall. As the limb buds appear, Lbh expression is restricted primarily to the distal ventral limb ectoderm and the apical ectodermal ridge, and overlaps in these ectodermal compartments with En1 and Fgf8 expression. During heart formation, Lbh is expressed as early as Nkx2.5 and dHand in the bilateral heart primordia, with the highest levels in the anterior promyocardium. After heart tube fusion and looping, Lbh expression is confined to the ventricular myocardium, with the highest intensity in the right ventricle and atrioventricular canal, as well as in the sinus venosus. Based on the molecular characteristics and the domain-specific expression pattern, it is possible that Lbh functions in synergy with other genes known to be required for heart and limb development.

EN and GBX2 play essential roles downstream of FGF8 in patterning the mouse mid/hindbrain region.

Fgf8, which is expressed at the embryonic mid/hindbrain junction, is required for and sufficient to induce the formation of midbrain and cerebellar structures. To address through what genetic pathways FGF8 acts, we examined the epistatic relationships of mid/hindbrain genes that respond to FGF8, using a novel mouse brain explant culture system. We found that En2 and Gbx2 are the first genes to be induced by FGF8 in wild-type E9.5 diencephalic and midbrain explants treated with FGF8-soaked beads. By examining gene expression in En1/2 double mutant mouse embryos, we found that Fgf8, Wnt1 and Pax5 do not require the En genes for initiation of expression, but do for their maintenance, and Pax6 expression is expanded caudally into the midbrain in the absence of EN function. Since E9.5 En1/2 double mutants lack the mid/hindbrain region, forebrain mutant explants were treated with FGF8 and, significantly, the EN transcription factors were found to be required for induction of Pax5. Thus, FGF8-regulated expression of Pax5 is dependent on EN proteins, and a factor other than FGF8 could be involved in initiating normal Pax5 expression in the mesencephalon/metencephalon. The En genes also play an important, but not absolute, role in repression of Pax6 in forebrain explants by FGF8. Previous Gbx2 gain-of-function studies have shown that misexpression of Gbx2 in the midbrain can lead to repression of Otx2. However, in the absence of Gbx2, FGF8 can nevertheless repress Otx2 expression in midbrain explants. In contrast, Wnt1 is initially broadly induced in Gbx2 mutant explants, as in wild-type explants, but not subsequently repressed in cells near FGF8 that normally express Gbx2. Thus GBX2 acts upstream of, or parallel to, FGF8 in repressing Otx2, and acts downstream of FGF8 in repression of Wnt1. This is the first such epistatic study performed in mouse that combines gain-of-function and loss-of-function approaches to reveal aspects of mouse gene regulation in the mesencephalon/metencephalon that have been difficult to address using either approach alone.

Otx2, Gbx2 and Fgf8 interact to position and maintain a mid-hindbrain organizer.

A decade ago, chick-quail transplantation studies demonstrated that the junction between the midbrain and hindbrain has the properties of an organizing center capable of patterning the midbrain and cerebellum. Many of the genes that function to pattern these tissues have been identified and extensively studied. Recent experiments have shown that Otx2, Gbx2 and Fgf8 genes play a major role in the positioning and functioning of this organizing center.

Establishment and chimera analysis of 129/SvEv- and C57BL/6-derived mouse embryonic stem cell lines.

Hundreds of new mutant mouse lines are being produced annually using gene targeting and gene trap approaches in embryonic stem (ES) cells, and the number is expected to continue to grow as the human and mouse genome projects progress. The availability of robust ES cell lines and a simple technology for making chimeras is more attractive now than ever before. We established several new ES cell lines from 129/SvEv and C57BL/6 mice and tested their ability to contribute to the germline following blastocyst injections and/or the less expensive and easier method of morula-ES cell aggregation. Using morula aggregation to produce chimeras, five newly derived 129/SvEv and two C57BL/6 ES cell lines tested at early passages were found to contribute extensively to chimeras and produce germline-transmitting male chimeras. Furthermore, the two 129S/vEv ES cell lines that were tested and one of the C57BL/6 ES cell lines were able to maintain these characteristics after many passages in vitro. Our results indicate that the ability of ES cells to contribute strongly to chimeras following aggregation with outbred embryos is a general property of early passage ES cells and can be maintained for many passages. C56BL/6-derived ES cell lines, however, have a greater tendency than 129-derived ES cell lines to lose their ability to colonize the germline.

Members of the bHLH-PAS family regulate Shh transcription in forebrain regions of the mouse CNS.

The secreted protein sonic hedgehog (Shh) is required to establish patterns of cellular growth and differentiation within ventral regions of the developing CNS. The expression of Shh in the two tissue sources responsible for this activity, the axial mesoderm and the ventral midline of the neural tube, is regulated along the anteroposterior neuraxis. Separate cis-acting regulatory sequences have been identified which direct Shh expression to distinct regions of the neural tube, supporting the view that multiple genes are involved in activating Shh transcription along the length of the CNS. We show here that the activity of one Shh enhancer, which directs reporter expression to portions of the ventral midbrain and diencephalon, overlaps both temporally and spatially with the expression of Sim2. Sim2 encodes a basic helix-loop-helix (bHLH-PAS) PAS domain containing transcriptional regulator whose Drosophila homolog, single-minded, is a master regulator of ventral midline development. Both vertebrate and invertebrate Sim family members were found sufficient for the activation of the Shh reporter as well as endogenous Shh mRNA. Although Shh expression is maintained in Sim2(-)(/)(-) embryos, it was determined to be absent from the rostral midbrain and caudal diencephalon of embryos carrying a dominant-negative transgene that disrupts the function of bHLH-PAS proteins. Together, these results suggest that bHLH-PAS family members are required for the regulation of Shh transcription within aspects of the ventral midbrain and diencephalon.