On-microscope staging of live cells reveals changes in the dynamics of transcriptional bursting during differentiation.

Determining the mechanisms by which genes are switched on and off during development is a key aim of current biomedical research. Gene transcription has been widely observed to occur in a discontinuous fashion, with short bursts of activity interspersed with periods of inactivity. It is currently not known if or how this dynamic behaviour changes as mammalian cells differentiate. To investigate this, using an on-microscope analysis, we monitored mouse α-globin transcription in live cells throughout erythropoiesis. We find that changes in the overall levels of α-globin transcription are most closely associated with changes in the fraction of time a gene spends in the active transcriptional state. We identify differences in the patterns of transcriptional bursting throughout differentiation, with maximal transcriptional activity occurring in the mid-phase of differentiation. Early in differentiation, we observe increased fluctuation in transcriptional activity whereas at the peak of gene expression, in early erythroblasts, transcription is relatively stable. Later during differentiation as α-globin expression declines, we again observe more variability in transcription within individual cells. We propose that the observed changes in transcriptional behaviour may reflect changes in the stability of active transcriptional compartments as gene expression is regulated during differentiation.

Characterization of the major regulatory element upstream of the human alpha-globin gene cluster.

The major positive regulatory activity of the human alpha-globin gene complex has been localized to an element associated with a strong erythroid-specific DNase I hypersensitive site (HS -40) located 40 kb upstream of the zeta 2-globin mRNA cap site. Footprint and gel shift analyses of the element have demonstrated the presence of four binding sites for the nuclear factor GATA-1 and two sites corresponding to the AP-1 consensus binding sequence. This region resembles one of the major elements of the beta-globin locus control region in its constitution and characteristics; this together with evidence from expression studies suggests that HS -40 is a primary element controlling alpha-globin gene expression.

A single beta-globin locus control region element (5' hypersensitive site 2) is sufficient for developmental regulation of human globin genes in transgenic mice.

The beta-globin gene complex is regulated by an upstream locus control region (LCR) which is responsible for high-level, position-independent, erythroid-cell-specific expression of the genes in the cluster. Its role in the developmental regulation of beta-like globin gene transcription remains to be established. We have examined the effect of a single LCR element, hypersensitive site 2 (HS2), on the developmental regulation of the human fetal gamma and adult beta genes in transgenic mice. In mice bearing HS2A gamma beta and HS2G gamma A gamma-117 delta beta human globin gene constructs, switching from gamma- to beta-gene expression begins at about day 13.5 of gestation and is largely completed shortly after birth. The larger construct also demonstrates a switch in G gamma- to A gamma-gene expression during the gamma-to-beta switch similar to that observed during normal human development. We conclude that HS2 alone is sufficient for developmental regulation of the human beta-globin genes.

Established epigenetic modifications determine the expression of developmentally regulated globin genes in somatic cell hybrids.

Somatic cell hybrids generated from transgenic mouse cells have been used to examine the developmental regulation of human gamma-to-beta-globin gene switching. In hybrids between mouse erythroleukemia (MEL) cells and transgenic erythroblasts taken at various stages of development, there was regulated expression of the human fetal gamma and adult beta genes, reproducing the in vivo pattern prior to fusion. Hybrids formed from embryonic blood cells produced predominantly gamma mRNA, whereas beta gene expression was observed in adult hybrids and a complete range of intermediate patterns was found in fetal liver hybrids. The adult environment of the MEL cells, therefore, did not appear to influence selective transcription from this gene complex. Irradiation of the embryonic erythroid cells prior to fusion resulted in hybrids containing only small fragments of donor chromosomes, but the pattern of gene expression did not differ from that of unirradiated hybrids. This finding suggests that continued expression of trans-acting factors from the donor erythroblasts is not necessary for continued expression of the human gamma gene in MEL cells. These results contrast with the lack of developmental regulation of the cluster after transfection of naked DNA into MEL cells and suggest that epigenetic processes established during normal development result in the gene cluster adopting a developmental stage-specific, stable conformation which is maintained through multiple rounds of replication and transcription in the MEL cell hybrids. On prolonged culture, hybrids that initially expressed only the gamma transgene switched to beta gene expression. The time period of switching, from approximately 10 to > 40 weeks, was similar to that seen previously in human fetal erythroblast x MEL cell hybrids but in this case bore no relationship to the time of in vivo switching. It seems unlikely, therefore, that switching in these hybrids is regulated by a developmental clock.

Understanding alpha globin gene expression: a step towards effective gene therapy.

During the past 20 years developments in molecular and cellular biology have kindled the hope that one might eventually ameliorate or even cure some serious genetic diseases by repairing or replacing the defective gene. Other articles deal with the formidable problems of isolating pluripotent hematopoietic stem cells; efficiently, safely, and stably transfecting them, and developing transplantation protocols to ensure that the corrected cells supplant the patient's abnormal stem cells after transplantation. Assuming that these hurdles can be overcome, it will also be important to establish the ideal segment of DNA to introduce into stem cells to ensure that, regardless of its position of integration in the genome, the gene in question will be appropriately regulated. In the case of the globin genes this is a particularly difficult task because in order to correct disorders of globin synthesis we need to obtain high levels of stable, tissue- and developmental-stage specific expression. Issues relevant to this problem arising from the analysis of the human beta globin cluster are discussed in the article in this issue by Grosveld. In this article we review our current understanding of how eukaryotic genes might be expressed from their normal chromosomal environment, using the human alpha globin cluster as a specific example. We also discuss how this information might be used in the development of strategies for gene therapy.

Convergence-evoked nystagmus. Congenital and acquired forms.

Pendular nystagmus occurring during binocualr fixation and pursuit of near objects is true convergence-evoked nystagmus. Two forms of this nystagmus are described, one congenital and the other acquired in the course of multiple sclerosis. The congenital form was conjugate and the acquired form disjunctive. Neuropathological examination provided no morphological explanation for the congenital pattern. These two unique forms of nystagmus are induced by one of the visuomotor subsystems controlling convergence.

Nystagmus of Pelizaeus-Merzbacher disease. A magnetic search-coil study.

Magnetic search-coil oculography of three brothers with clinically diagnosed Pelizaeus-Merzbacher disease disclosed the presence of binocular elliptical pendular nystagmus in two patients in whom the waveform of the nystagmus was not obvious on inspection. This study, the first reported application of high-resolution oculography to Pelizaeus-Merzbacher disease, also demonstrated primary position upbeat nystagmus in all three patients. The importance of finding this combination of elliptical pendular and upbeat nystagmus is that it is not described in any other childhood neurodegenerative states and, in combination with supportive clinical history and magnetic resonance imaging, may be so characteristic of Pelizaeus-Merzbacher disease that a strong presumptive diagnosis can be made.

Retinotopic and directional deficits of smooth pursuit initiation after posterior cerebral hemispheric lesions.

We investigated the initiation of ocular smooth pursuit with horizontal ramp targets in 16 patients with unilateral posterior cerebral lesions. Four of the 16 patients had directional pursuit asymmetry, in which smooth eye movement velocities were reduced toward the lesion, independent of target location on the retina. Of seven patients with normal visual fields for the target, two had a retinotopic eye movement deficit consisting of impaired smooth pursuit of targets moving in both horizontal directions in the hemifield contralateral to their lesion. Patients with retinotopic deficits and normal visual fields, and those with directional deficits, had impairment of smooth pursuit similar to that caused by unilateral lesions of cortical areas MT (middle temporal) and MST (medial superior temporal) in monkeys. All but one patient with either of these defects had a lesion near the junction of Brodmann's areas 19, 37, and 39, providing evidence that this region includes the human homologues of monkey areas MT and MST. One patient with a retinotopic pursuit defect and normal visual fields had a lesion of the rostral superior temporal sulcus, which may have included the homologue of the superior temporal polysensory area of monkeys.

Cerebral hemispheric localization of smooth pursuit asymmetry.

We recorded horizontal smooth pursuit in 23 patients with discrete unilateral cerebral hemispheric lesions and in 12 normal subjects. Most patients had bidirectional reduction of smooth pursuit gain, indicating that each cerebral hemisphere participates in smooth pursuit in both directions. Pursuit gain fell proportionately more with increasing target acceleration in patients than in normals. A normal phase relationship between eye and target motion in patients indicated an intact predictor mechanism for smooth pursuit. Ten patients had pursuit asymmetry with lower gain when tracking toward the side of cerebral damage; none had lower gain when tracking away. Two patients with lower ipsilateral gain had frontal lobe lesions. Areas of anatomic overlap of lesions associated with asymmetric pursuit in 8 patients provide evidence for a pursuit pathway that originates from Brodmann areas 19 and 39 and descends to the brainstem through the posterior limb of the internal capsule.

Tonic pupils in neurosyphilis.

Of 60 patients with tonic pupils, 29 had serologic tests for syphilis. Five patients had positive blood serology and confirmatory tests; four had other manifestations of neurosyphilis and positive CSF serology. All seropositive patients had bilateral tonic pupils with light-near dissociation and denervation hypersensitivity. Of the 10 tonic pupils, only 1 was miotic and 1 dilated. Although tonic pupils tend to become small and bilateral, they should be distinguished readily from Argyll Robertson pupils, which react briskly, not tonically, to near stimuli. Patients with bilateral tonic pupils should have serologic tests for syphilis.

Smooth pursuit dysfunction in Alzheimer's disease.

Smooth ocular pursuit was measured by magnetic search coil oculography in 13 patients with Alzheimer's disease and compared with control subjects. Smooth eye movement gain was uniformly reduced in Alzheimer's disease at all target velocities for several frequencies of sinusoidal target motion, signifying impairment of steady-state gain. Normal phase relationships between the target and eyes indicated an intact predictor mechanism for smooth pursuit. When peak target velocity was held constant, pursuit gain decreased markedly in response to small increments of target acceleration, indicating involvement of an acceleration saturating nonlinear element that limits smooth pursuit. Large-amplitude saccadic intrusions, in the direction of target motion, often disrupted pursuit; smooth eye movements continued in response to target velocity despite large position errors of the fovea from its target. These disorders of smooth eye movement control can quantify motor dysfunction in Alzheimer's disease.

Saccadic duration and intrasaccadic fatigue in myasthenic and nonmyasthenic ocular palsies.

We measured the duration and amplitude of saccades in three normal subjects, eight patients with myasthenia gravis, and eight patients with nonmyasthenic ocular palsies. Saccades were examined at the start of a repetitive saccade task, after 3 minutes of activity, and 1 minute after administration of edrophonium. The duration of saccades was prolonged initially in both myasthenic and nonmyasthenic palsies. Activity did not produce significant differences among the three groups in either the slope of the duration-amplitude relationship or the predicted durations of saccades of 5 degrees, 10 degrees, or 15 degrees. However, durations decreased in myasthenia but increased in nonmyasthenic palsies after edrophonium. Much of this decrease in myasthenic saccadic duration was due to reduction in deceleration time, indicating resolution of intrasaccadic fatigue after edrophonium administration. However, the relation of deceleration fraction (deceleration time divided by total duration) to total duration remained constant in all subject groups. Analysis of saccadic duration is a useful means of interpreting responses to edrophonium because it incorporates data from saccades of a wide range of amplitudes into a linear relation between duration and amplitude.

Palsy of upward and downward saccadic, pursuit, and vestibular movements with a unilateral midbrain lesion: pathophysiologic correlations.

Upward and downward gaze palsy was measured by a magnetic search coil technique and correlated with neuropathologic findings in a patient with a unilateral midbrain infarct. Oculography demonstrated (1) saccadic palsy above primary position and slow, limited vertical saccades below; (2) low-gain, restricted vertical pursuit; and (3) low-gain, abnormal phase lead, and restricted range of the vertical vestibulo-ocular reflex (VOR). Bidirectional palsy of vertical saccades is attributed to unilateral loss of burst cells in the rostral interstitial nucleus of the medial longitudinal fasciculus (riMLF) and interruption of burst cell fibers from the opposite riMLF. Pathways mediating vertical pursuit and integration of the vertical VOR also traversed the infarct, which included the interstitial nucleus of Cajal.

Superior oblique myokymia associated with a posterior fossa tumor: oculographic correlation with an idiopathic case.

Superior oblique myokymia (SOM) was the only neurologic sign in a patient with an astrocytoma involving the midbrain tectum. Oculography showed monocular bursts of tonic and phasic intorsion and depression and miniature oscillations identical to those of idiopathic SOM. SOM stopped after tumor resection.

On the cause of hyporeflexia in the Holmes-Adie syndrome.

Electrophysiologic studies were carried out on 11 patients with Holmes-Adie syndrome, 8 of whom had reduced or absent ankle jerks. Conduction velocities and evoked nerve and muscle compound action potentials in the peroneal, posterior tibial, and sural nerves were normal. The H reflex was absent (or virtually absent) in the patients with depressed reflexes. The amplitude of the composite Ia EPSP in single soleus motoneurons was estimated from changes in firing probability of voluntarily activated soleus motor units in response to stimulation of low threshold afferents in the tibial nerve. These amplitudes were used to test the afferent side of the reflex pathway. Composite group Ia EPSPs in Holmes-Aide patients with hyporeflexia were smaller than normal or absent, indicating that the areflexia in the Holmes-Aide syndrome is due to loss of large spindle afferents or reduced effectiveness of their monosynaptic connections to motoneurons.

Familial paralysis of horizontal gaze. Associated with pendular nystagmus, progressive scoliosis, and facial contraction with myokymia.

Paralysis of horizontal gaze, pendular nystagmus, and progressive scoliosis were manifestations of an autosomal recessive genetic disease in four siblings. Bilateral facial myokymia with continuous facial contraction developed in the oldest patient. Electromyographic examination of his facial muscles after facial nerve block at the stylomastoid foramen showed absence of all muscle potentials, consistent with a supranuclear origin of the myokymia. Normality of convergence, vertical gaze, and pupillary constrictor reflex activity assured integrity of midbrain ocular motor function. Absence of horizontal vestibulo-ocular reflexes signified involvement of the pontine tegmentum in this distinctive heredofamilial syndrome.

Cerebral square wave jerks.

Abnormal saccadic intrusions consisting of frequent sporadic horizontal saccades followed, after an interval, by corrective saccades occurred in 70% of 17 patients with acute or chronic focal cerebral lesions. These square wave jerks were significantly lower in amplitude than those in cerebellar system disease. The metrics of these jerks were uniform regardless of the site of cerebral damage. Mean durations approximated the reaction time for saccadic refixations triggered by visual feedback. Very short-latency corrective saccades in some patients are attributed to internal (nonretinal) feedback of eye position errors. Low-amplitude cerebral square wave jerks can be detected clinically by funduscopy.

Methanol optic neuropathy: a histopathological study.

The histopathologic effects of methanol on the optic nerve were studied in four patients. Circumscribed myelin damage occurred behind the lamina cribrosa in each nerve. Axons were preserved. Demyelination also occurred in cerebral hemispheric white matter in one patient. This selective myelinoclastic effect of methanol metabolism is probably caused by histotoxic anoxia in watershed areas of the cerebral and distal optic nerve circulations. Juxtabulbar demyelination may cause optic disk edema in methanol poisoning by compressive obstruction of orthograde axoplasmic flow. Visual loss may be due to disruption of saltatory conduction. Retrolaminar demyelinating optic neuropathy is an early morphologic correlate of visual loss in methanol intoxication.

Oblique misdirection and slowing of vertical saccades after unilateral lesions of the pontine tegmentum.

Three patients with unilateral lesions of the pontine tegmentum, identified by CT and MRI, had abnormal vertical saccades and slowed ipsilateral horizontal saccades. Attempted vertical saccades were misdirected obliquely, away from the side of the lesion, and their vertical components were prolonged. Oblique saccades had curved trajectories and prolonged durations of their vertical components. Unilateral damage to excitatory burst neurons and pause cells in the medial part of the caudal paramedian pontine reticular formation may cause these abnormal vertical and oblique saccades. Misdirection and slowing of vertical saccades can accompany the paralysis or slowing of ipsilateral horizontal saccades caused by pontine damage.

Smooth pursuit during dose-related on-off fluctuations in Parkinson's disease.

Smooth pursuit was studied during predictable L-dopa dose-related "off" periods of morning akinesia and wearing off and during "on" periods in eight patients with idiopathic Parkinson's disease. Smooth pursuit gain was significantly reduced in patients during both on and off phases. Despite marked fluctuations between parkinsonism and periods of near normal skeletal motion, there were no changes in smooth pursuit gain. We conclude that unvarying paresis of smooth pursuit in Parkinson's disease signifies involvement of neural circuits that are distinct from the dopaminergic mechanisms that mediate the on-off phenomenon of somatic motor control.