Conduction velocities in amphibian skeletal muscle fibres exposed to hyperosmotic extracellular solutions

Early quantitative analyses of conduction velocities in unmyelinated nerve studied in a constantly iso-osmotic volume conductor were extended to an analysis of the effects of varying extracellular osmolarities on conduction velocities of surface membrane action potentials in Rana esculenta skeletal muscle fibres. Previous papers had reported that skeletal muscle fibres exposed to a wide range of extracellular sucrose concentrations resemble perfect osmometers with increased extracellular osmolarity proportionally decreasing fibre volume and therefore diminishing fibre radius, a. However, classical electrolyte theory (Robinson and Stokes 1959, Electrolyte solutions 2nd edn. Butterworth & Co. pp 41–42) would then predict that the consequent increases in intracellular ionic strength would correspondingly decrease sarcoplasmic resistivity, Ri. An extension of the original cable analysis then demonstrated that the latter would precisely offset its expected effect of alterations in a on the fibre axial resistance, ri, and leave action potential conduction velocity constant. In contrast, other reports (Hodgkin and Nakajima J Physiol 221:105–120, 1972) had suggested that Riincreased with extracellular osmolarity, owing to alterations in cytosolic viscosity. This led to a prediction of a decreased conduction velocity. These opposing hypotheses were then tested in muscle fibres subject to just-suprathreshold stimulation at a Vaseline seal at one end and measuring action potentials and their first order derivatives, dV/dt, using 5–20 MΩ, 3 M KCl glass microelectrodes at defined distances away from the stimulus sites. Exposures to hyperosmotic, sucrose-containing, Ringer solutions then reversibly reduced both conduction velocity and maximum values of dV/dt. This was compatible with an increase in Ri in the event that conduction depended upon a discharge of membrane capacitance by propagating local circuit currents through initially passive electrical elements. Conduction velocity then showed graded decreases with increasing extracellular osmolarity from 250–750 mOsm. Action potential waveforms through these osmolarity changes remained similar, including both early surface and the late after-depolarisation events reflecting transverse tubular activation. Quantitative comparisons of reduced-χ 2 values derived from a comparison of these results and the differing predictions from the two hypotheses strongly favoured the hypothesis in which Riincreased rather than decreased with hyperosmolarity.
Introduction
A classic paper by Hodgkin (1954) (see also: Adrian 1975; Noble 1979) performed a quantitative cable analysis of the local circuit currents thought responsible for action potential propagation in unmyelinated nerve fibres studied in large volumes of constantly iso-osmotic extracellular fluid and demonstrated that their conduction velocities should vary as the square root of fibre radius, a. Action potential conduction velocity is also physiologically important in striated muscle: it ensures a rapid, sarcomeric activation leading to near-synchronous muscle contraction. The present paper accordingly extends these early analyses to effects of varying extracellular osmolarity on conduction velocities of surface membrane action potentials in Rana esculenta skeletal muscle. Skeletal muscle membrane differs from unmyelinated nerve in including an excitable transverse (T-) system whose activation initiates muscle contraction (Adrian et al. 1969, 1970; Huang 1993; Nielsen et al. 2004; Stephenson 2006). Nevertheless, it is likely that the initial sarcolemmal membrane activation precedes the subsequent inward tubular excitation and that the latter may take place partially independently of the propagation of the surface excitation wave. Sheikh et al. (2001) suggested a partially separable T-tubule excitation initiated by Na+ channels selectively clustered around the mouths of the T-tubular lumina: detubulation produced by osmotic shock left surface membrane conduction velocities unchanged. Furthermore, increases in extracellular osmolarity did not alter the tubular diameters at the necks of the T-tubular system important in electrical connectivity between T-tubule network and remaining extracellular space (Launikonis and Stephenson 2002, 2004).
However, variations in surface conduction velocities in skeletal muscle studied under varying osmolarities would be expected to differ from those of nerve fibres studied under constant, iso-osmotic conditions owing to the consequent changes in their cell volumes and fibre diameters. Nevertheless, skeletal muscle volume exhibits close to ideal osmotic behaviour, varying inversely with changes in extracellular osmolarity that would in turn predictably increase solute concentration. The Results section of this paper accordingly first develops the original treatment (Hodgkin 1954; Adrian 1975; Noble 1979) for situations in which fibre diameter is varied by alterations in extracellular osmolarity that would in turn alter intracellular ionic strength. This provided quantitative expectations for any resulting change in conduction velocity that corresponded to two contrasting situations. First, classical electrolyte theory (Robinson and Stokes 1959; Atkins 1998) would predict that increased intracellular ionic strength should proportionally decrease sarcoplasmic resistivity Ri. Our analysis then indicated that this would precisely offset any expected changes in fibre axial resistance, ri, produced by the osmotically induced alterations in fibre diameter and thereby leave conduction velocity unchanged. Secondly, Hodgkin and Nakajima (1972) reported that Ri increased with extracellular osmolarity, possibly due to increases in myoplasmic viscosity, although we certainly do not exclude possible factors arising from the more complex membrane structures found in muscle as opposed to nerve (see e.g. Sheikh et al. 2001). This led to predictions that conduction velocity would decrease with increasing extracellular osmolarity.
These predictions were then investigated by experimental determinations of conduction velocity obtained from action potential records derived from microelectrode measurements made at known distances from defined stimulation sites in surface muscle fibres of Rana esculenta studied at a fixed (∼7°C) temperature at varied extracellular osmolarities. These findings demonstrated reversible changes in the conduction velocities of action potentials the nature of whose waveforms, including early surface and later tubular components, were otherwise unchanged, and distinguished between hypotheses through the observed dependence of conduction velocity on extracellular osmolarity.
Materials and methods
Cold-adapted frogs, Rana esculenta, were killed by concussion followed by pithing (Schedule I: Animal Procedures Act, Home Office, UK). The skin was removed and the tendon of insertion of the sartorius into the patella ligated, cut distally and dissected along its borders up to and including its origin at the pelvic girdle and acetabulum. This was performed at room temperature (21.7 ± 0.51°C; mean ± standard error of the mean; n = 15) in standard Ringer solution consisting of (mM): 115 NaCl, 2.5 KCl, 1.8 CaCl2, 3 Hepes, titrated to pH 7, osmolarity 250 mOsm. The muscle was then stretched to 1.4 times its in situ length and secured in a transilluminated methacrylate polymer (Perspex) chamber by pinning the cleaned acetabulum and the ligature to minimize contractile artefact, which became less evident in solutions of higher osmolarities, and entirely absent with the higher (>350−400 mM) sucrose concentrations. The isolated sartorius muscles had an in situ length of 30.6 ± 0.94 mm and a length of 41.6 ± 1.15 mm (n = 15) when stretched. The muscle was laid over a supporting ramp with its dorsal surface uppermost.
A watertight Perspex partition, coated with a layer of Vaseline, with the muscle running through a notch at its lower surface electrically isolated the recording chamber into two compartments. The only path conducting the brief voltage stimuli applied from two platinum electrodes at opposite ends of the chamber was therefore through the muscle across the partition. The platinum electrode on the side of the chamber containing the acetabulum was the cathode. The temperature of the bath solution was lowered by circulating distilled water cooled in a magnetically stirred ice bath. The water was circulated through a glass coil placed in the chamber in close proximity to the muscle, using a Minipuls 3 peristaltic pump (Gilson, France). A digital thermometer incorporating a remote thermistor (P. Frost, Department of Physiology, Development and Neuroscience, Cambridge), previously linearised and calibrated against a platinum film resistor was placed in the bathing solution near the muscle to allow the temperature to be continuously monitored and adjusted to remain constant throughout the recordings. The temperature was kept constant at 7.0 ± 0.03°C (n = 15) (Sheikh et al. 2001). Action potentials are temperature-sensitive so this allowed conditions to be optimised. Cooling also prolonged fibre viability especially in the hyperosmotic conditions.
The muscle was studied in the range of extracellular osmolarities from 250 to 750 mOsm. Solutions of different osmolarities were made by adding to the standard Ringer solution the membrane-impermeant solute sucrose at varying (150, 250, 350, 400, 450, 500 mM respectively) concentrations to yield solutions whose increased osmolarities (mOsm) were calculated from their total solute concentrations, and checked against measurements using a standard vapour pressure osmometer. Hepes was obtained from Sigma-Aldrich (Gillingham, Dorset, UK) and all other reagents from BDH Limited (UK). Solutions were changed, avoiding contact with the muscle, between tests using a vacuum pump (Edwards, UK) after which approximately 5–10 min elapsed before recording to permit time for both temperature (7°C) and osmotic equilibration; previous reports (Ferenczi et al. 2004) have indicated that 200 s sufficed to permit complete volume changes in response to hyperosmotic solutions.
3 M KCl-filled glass capillary microelectrodes (resistances 5–20 MΩ; tip potentials <5 mV: Adrian 1956) drawn from 1.2 mm (inner diameter) glass tubing were used to obtain membrane potentials. These were mounted via Ag/AgCl junctions to a high impedance voltage follower balanced by matching Ag/AgCl junctions earthing the bath. Only surface fibres showing clear-cut penetrations and stable resting membrane potentials were studied. Rates of rise and fall, dV/dt, in the voltage traces, were obtained by electrical differentiation of the output, with voltage and dV/dt channels filtered between cutoffs of 0 Hz/5 kHz and 0 Hz/20 kHz respectively. Action potentials were elicited by direct just-suprathreshold stimulation which varied between 1.5 and 5 V, to minimise electrotonic spread of the stimulus voltage and the number of fibres in which electrical activity was initiated, across the Perspex partition, through two platinum stimulating electrodes.
Conduction velocity was calculated by dividing the distance from the site of action potential initiation to the position of the recording microelectrode tip, ascertained by a Vernier scale, which gave measurements in cm to two decimal places, by the latency given by the time from a clear stimulus artefact generated to the peak of the dV/dt input, which will be referred to as the maximum dV/dt. The corresponding resting membrane potential, action potential overshoot and the value of maximum dV/dt were also noted. These relatively early events in the timecourse of the regenerative response often could be measured even in records showing small contractile artefact, which often only became evident at later times in the recorded traces.
Results
The experiments utilized an extension of early quantitative analyses (Hodgkin 1954; Adrian 1975) of conduction velocity in unmyelinated nerve studied in a constantly iso-osmotic volume conductor to changes that would result from varying the extracellular osmolarities in Rana esculenta skeletal muscle fibres. The initial analysis used the cable equation for the current density through any patch of flat membrane, expressed as current per unit membrane area,
This describes conduction in a cable whose own finite internal volume is small compared to that of the extracellular conducting fluid in which it is studied to give large ratios between longitudinal intracellular, ri, and extracellular, ro, resistances to current flow per unit length, i.e., ro << ri, permitting extracellular terms relating to ro to be dropped.
Equation (1) is combined with the expression for conduction velocity,  using the chain rule of differentiation, to give:where Vm is the internal potential across the membrane at distance x along the fibre. Conduction at constant velocity through local circuit spread of excitation then requires Im to be a single valued function of Vm. The term containing the intrinsic membrane parameters consisting of the area of membrane per unit length of fibre s, and the axial cytoplasmic resistance per unit length ri, is then constant, whence , and where the constant k only depends on passive local membrane electrical properties. Thus:
For unmyelinated cylindrical fibres for which the axial cytoplasmic resistance per unit length, ri, is related to fibre radius a and sarcoplasmic resistivity Ri by Ri = riπ a2, the total fibre volume, vol, = π a2L where L is the length of the fibre and for which the total surface area of the fibre, A = sL:
In the case of skeletal muscle fibres exposed to solutions of different osmolarities, osm, the volume, vol, behaves as a perfect osmometer following the relationship: and reductions in fibre volume due to increased extracellular osmolarity would correspondingly increase solute concentration. Classical electrolyte theory (Robinson and Stokes 1959) then predicts that each participating ion contributes a specific conductance Ksp determined by its solute concentration ηC and a constant Λ defined for any given ion in any specified solute and referred to the conductivity of the ion at a 1 M concentration:
The overall conductivity of such an ideal solution is then the algebraic sum of the conductivities of individual component ions. Decreases in fibre volume resulting from increased extracellular osmolarities then would increase intracellular ionic strength and in turn increase Ksp and proportionally decrease Ri: Equation (7) then gives: Equations (5) and (8) then give,thereby predicting a constant conduction velocity in view of the fact that the terms k′, resulting from the additional proportionalities above, and A are both constants.
In contrast, Hodgkin and Nakajima (1972) suggested that sarcoplasmic resistivity, Ri, increased with increasing extracellular osmolarity, possibly owing to an increased myoplasmic viscosity, with reductions in fibre volume in hyperosmotic solutions, reporting a linear increase in Ri with increasing extracellular osmolarity:where D and E are constants.
From (5) and (10):
Because k′ and A are constants, we have:where D′ and E′ are also constants. This forms a contrasting expectation in which θ is expected to decrease with increasing extracellular osmolarity in a relationship modelled by Eq. (12) as a result of an increase in Ri. The alternative hypotheses could then be quantitatively tested by measurements of θ under different conditions of extracellular osmolarity.
Reversible effects of hyperosmotic extracellular solutions on action potential waveforms and latencies
Figure 1 shows typical action potential and dV/dt waveforms obtained at a temperature of ∼7°C before, during and after exposures to solutions with increased extracellular osmolarity with column A showing the action potential waveforms and column B their corresponding dV/dt records. Panel a shows typical results from fibres in iso-osmotic Ringer at 7°C. The muscle was then exposed to a hyperosmotic (600 mOsm) Ringer solution and recordings resumed 5–10 min following this solution change (Panel b). Finally, the muscle was then returned to iso-osmotic Ringer and recordings again resumed 5–10 min after the solution was restored (Panel c). Latencies were measured from the clear stimulus artefact to the maximum point of the dV/dt trace as seen in column B: this approach provided more consistent measurements of action potential latency than using arbitrarily chosen points on the action potential traces (see also Sheikh et al. 2001).Fig. 1Typical action potential (A) and dV/dt traces (B) obtained from muscle fibres exposed successively to (a) iso-osmotic Ringer (b) hyperosmotic (600 mOsm) Ringer and (c) returned to iso-osmotic Ringer, in the same sartorius muscle
The action potential traces showed prolonged positive after-depolarisation phases lasting well beyond 20 ms following the surface action potential deflections (Fig.1Aa), consistent with persistent excitation of an intact transverse (T-) tubular system (Adrian and Peachey 1973). These persisted both in the hyperosmotic solution (Fig. 1Ab) and the iso-osmotic solution to which the fibres were finally returned (Fig. 1Ac), confirming a persistence in transverse tubular excitation and its excitation following initiation of the surface component of the action potential through these manipulations.
Use of 600 mOsm as opposed to standard Ringer increased the action potential latencies and consequently the calculated values of conduction velocity. Thus, conduction velocities in the initial iso-osmotic Ringer solution, then at 600 mOsm-Ringer solution and finally, the returned iso-osmotic Ringer were 0.98 ± 0.092 m s−1 (n = 11 fibres), 0.58 ± 0.051 m s−1 (n = 10) and 0.72 ± 0.101 m s−1 (n = 6) respectively. The effect of hyperosmolarity on conduction velocity was thus at least partly reversible. However, this decrease in conduction velocity with increased osmolarity (from 250 to 600 mOsm) was not accompanied by qualitative changes in action potential or dV/dt waveform. Likewise, the waveforms showed no qualitative changes following return to the iso-osmotic Ringer solution after hyperosmotic exposure.
Grading of conduction velocity changes with extracellular osmolarity
Figure 2 summarises typical traces of action potentials (column A) and dV/dt (column B) for experiments that systematically investigated the effects of graded changes in extracellular osmolarity reflecting different extracellular sucrose concentrations on conduction velocity, action potential waveform, and dV/dt obtained at 7°C. Action potential waveforms again included both early rapid surface action potential deflections following the stimulus artefact and prolonged after-depolarisation phases that lasted well beyond 20 ms, reflecting T-tubular activation, at all the extracellular osmolarities studied (cf. Adrian and Peachey 1973). Measurements of latencies between the stimulus artefacts to the maximum value of the dV/dt traces then suggested a noticeable overall decrease in the calculated conduction velocities with increasing osmolarity. All these changes occurred in the absence of any excessive depolarization in resting membrane potential over the range of explored osmolarities. Thus resting potential at an osmolarity of 250 mOsm was −85.27 ± 1.05 mV (n = 87); that at an osmolarity of 700 mOsm was −75.94 ± 2.20 mV (n = 33). However, resting potential at an osmolarity of 750 mOsm was −62.71 ± 2.82 mV (n = 17) (cf. Fraser et al. 2006). At the latter extracellular osmolarity a small proportion of action potentials appeared to display markedly reduced amplitudes resulting in peak deflections that failed to show overshoots, and a firing of two consecutive action potentials after a single stimulation in the case of one fibre. Accordingly, the investigations were not performed at higher sucrose concentrations.Fig. 2Typical action potential (A) and dV/dt (B) traces recorded in the following extracellular osmolarities: (a) 250, (b) 400, (c) 500, (d) 600, (e) 650 and (f) 750 (all values given in mOsm). There is a notable overall increase in the latencies measured from (B)
Plots of conduction velocity and maximum dV/dt with increasing extracellular osmolarity
Figure 3 summarizes the results of systematic studies of the dependence of conduction velocity on extracellular osmolarity in a statistically larger number of muscle fibres. Action potentials and their corresponding dV/dt were measured systematically in n = 87, 18, 27, 11, 10, 33 and 13 fibres at sucrose concentrations of 0, 150, 250, 350, 400, 450 and 500 mM corresponding in turn to osmolarities of 250, 400, 500, 600, 650, 700 and 750 mOsm respectively. Figure 3 demonstrates that both conduction velocity (a) and maximum dV/dt (means ± standard errors of the mean) (b) monotonically decreased with extracellular osmolarity. Figure 3a then compares these experimental values of conduction velocity against predictions from the two hypotheses for the dependence of Ri on osmolarity. In both cases, θ was calculated from Ri and the extracellular osmolarity, osm, using the relationship derived from Eqs. (4) and (5) above that:Fig. 3Experimentally measured conduction velocities (a) and maximum dV/dt (mean ± standard error of the mean) plotted against extracellular osmolarity (◆). Dashed lines in (a): velocities predicted for a situation in which Ri decreases proportionally as a result of increased intracellular ionic strength produced by changes in cell volume brought about by the osmolarity changes. Continuous line: predictions when Ri increases with increasing extracellular osmolarity suggested by Hodgkin and Nakajima (1972). The changes in (a) were accompanied by decreases in maximum dV/dt (b) with increasing osmolarity, as expected for propagation brought about by local circuit currents
The dotted line denotes expectations from the first situation outlined above in which Ri decreases proportionally with increasing extracellular osmolarity: this predicts that conduction velocity does not alter with osmolarity from its control value in iso-osmotic solutions corresponding to a constant value of 0.85 m s−1 obtained in iso-osmotic solutions. The continuous line represents the second hypothesis in which Ri depends both on temperature and extracellular osmolarity as reported by Hodgkin and Nakajima (1972). The function displayed assumes the sarcoplasmic conductivity Gi(= 1/Ri) to have a Q10 = 1.37, and a value of Ri at 2°C, of 298.51 Ω cm in a muscle fibre within an extracellular solution of 250 mOsm osmolarity and of 390.63 Ω cm in a muscle fibre within an extracellular solution of 600 mOsm osmolarity, as suggested by Hodgkin and Nakajima (1970, 1972). Then, let θ1, Ri1 and osm1 correspond to conditions in iso-osmotic Ringer and θ2, Ri2 and osm2 correspond to conditions in any hyperosmotic solution. Thus:Since θ1 = 0.85 ± 0.029 m s−1 in iso-osmotic solution,where
The values generated by the above equations predicted a decline in conduction velocity with increasing extracellular osmolarity.
To test the statistical significance of the goodness-of-fit of the two contrasting predictions to the experimental data obtained, an Fx test that takes into account the difference of the two reduced-χ2 values in proportion to the first χ2 term was performed (Bevington 1969). This derived for each hypothesis a value of χ2 depicting the summed squared deviation of the original data, yi, to the predicted findings, y(xi), obtained at any extracellular osmolarity, xi, such that:
Values for χ2 obtained in the two cases, χ12 where the predicted conduction velocity is a constant value and χ22 where there is an increase in Ri with extracellular osmolarity were 21.360 and 15.141 respectively. These values were then used to compute a F-statistic, given by:
where n, the sample size = 200 and n−1 is the number of degrees of freedom.
The reduced-χ2 tests for goodness-of-fit yielded a F-statistic of 81.73 consistent with a significantly better fit (P << 0.001) to predictions from a situation in which Ri increased as described by Hodgkin and Nakajima (1972) rather than a decrease in Ri with increasing extracellular osmolarity.
Discussion
This paper begins from classic analyses (Hodgkin 1954; Adrian 1975) of the dependence of action potential conduction velocities upon the diameter of nerve fibres studied in large volume conductors under iso-osmotic extracellular conditions. This had employed cable analysis that attributed these propagation processes to local circuit currents driven by Na+ currents, INa, generating the rising phase of the action potential that in turn discharged initially passive circuit components (Valdiosera et al. 1974) equivalent to a membrane capacitance per unit area, Cm and resistance Rm of unit membrane area in series with an axial cytoplasmic resistance per unit length, ri. The latter is related to fibre radius a and sarcoplasmic resistivity Ri (kΩ cm) by Ri = riπa2 giving the original result that conduction velocity would vary as the square root of a.
The present paper extends this analysis to the effects of varying extracellular osmolarity on conduction velocities of surface membrane action potentials in Rana esculenta skeletal muscle, a situation that differed in a number of respects. First, skeletal muscle contains excitable transverse (T-) tubular membrane system responsible for initiating excitation-contraction coupling (Adrian et al. 1969, 1970; Huang 1993; Nielsen et al. 2004; Stephenson 2006). Nevertheless, the rapid initial sarcolemmal membrane activation that ensures rapid action potential propagation producing a synchronous sarcomeric activation likely largely precedes full tubular excitation (Adrian and Peachey 1973). Recent detubulation experiments left surface membrane conduction velocities unchanged suggesting a separation of surface and T-tubule excitation, the latter possibly initiated separately by Na+ channels localized around the T-tubular luminal mouths (Sheikh et al. 2001). Increasing extracellular osmolarity did not increase the diameter of the necks of the T-tubules important in electrical connectivity between T-tubule network and the fibre membrane (Launikonis and Stephenson 2002, 2004). These findings would permit muscle fibre conduction velocities to be analysed in terms of surface cylinders. Second, skeletal muscle volume alters inversely with extracellular osmolarity (Blinks 1965; Ferenczi et al. 2004) in turn correspondingly increasing solute concentration, in contrast to the situation represented by comparisons of nerve fibres of different diameters in similarly iso-osmotic extracellular solutions.
This paper then derived quantitative consequences from two possible hypotheses emerging from the above conditions. On the one hand, classical electrolyte theory (Robinson and Stokes 1959; Atkins 1998) predicts a specific conductance Ksp attributable to each intracellular ion species increasing proportionally with solute concentration following decreases in cell volume in hyperosmotic solution. This led to a prediction of a decrease in Ri precisely correcting out effects of any osmotically induced diameter change together leaving conduction velocity constant. On the other hand, empirical observations suggesting increases in Ri with extracellular osmolarity (Hodgkin and Nakajima 1972) permitted construction of a quantitative formula for the resulting variations in Ri with extracellular osmolarity as well as temperature. This led to predictions of a conduction velocity that decreased with increasing extracellular osmolarity.
The experiments described in this paper then sought to distinguish between the two hypotheses. It investigated the effects of extracellular osmolarity on conduction velocities of surface membrane action potentials in surface fibres, that would be maximally exposed to these solution changes, in frog skeletal muscle, following stimulation at a Vaseline seal at defined distances from the microelectrode recording site. Simultaneous records were made of the rate of change of membrane potential dV/dt that would be provided a consistent time point at which there would be a maximal action potential slope as well as rate of discharge of the membrane capacitance, Cm, by local currents driven by the Na+ current INa.
These studies showed that conduction velocity declined with increasing extracellular osmolarity along with maximum dV/dt as expected for a process dependent upon a local circuit current flow, despite relatively constant resting membrane potentials. These changes in conduction velocity in hyperosmotic solution were at least partially reversible. Furthermore, there were no changes in the nature of the action potential waveforms including early surface deflections and late after-depolarisation phases observed, consistent with a minimal change in the capacity for T-tubular excitation, through a still intact tubular system, that nevertheless followed generation of the initial surface component of the action potential wave.
Further systematic study in larger fibre numbers through a range of osmolarities (250–750 mOsm) demonstrated corresponding declines in conduction velocity and maximum dV/dt. At the highest osmolarity (750 mOsm), a small proportion of fibres variously showed low action potential overshoots which however did not correlate with the situations where there was a reduced conduction velocity, as well as multiple firing in a single muscle fibre. Nevertheless, further studies were not made at these and higher osmolarities; in any case, muscle fibres are thought only to act close to perfect osmometers at osmolarities up to around four times that of the iso-osmotic solution (Blinks 1965).
These findings are thus consistent with the second hypothesis in which sarcoplasmic resistivity, Ri, increases with the fibre shrinkage observed in hyperosmotic solutions, as suggested by Hodgkin and Nakajima (1972), as opposed to the first possibility outlined above in which conduction velocity should be constant. This was borne out by objective statistical analysis of the goodness-of-fit of the predictions derived from the two contrasting hypotheses as expressed in their resulting reduced-χ2 values, with the experimental values of conduction velocity generally assuming slightly higher values than predicted. The latter might reflect minor departures from a purely continuous conduction as postulated for unmyelinated cylindrical fibres, either due to contributions from peripheral regions of tubular membrane less isolated than the remaining tubular system (Hodgkin and Nakajima 1972), or the clustering of sodium channels around the tubular necks as reported by Sheikh et al. (2001) that may contribute to a more saltatory-like conduction in the muscle that would be expected to generally speed up propagation of electrical activity.
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Pathophysiology of focal segmental glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) is a major cause of idiopathic steroid-resistant nephrotic syndrome (SRNS) and end-stage kidney disease (ESKD). In recent years, animal models and studies of familial forms of nephrotic syndrome helped elucidate some mechanisms of podocyte injury and disease progression in FSGS. This article reviews some of the experimental and clinical data on the pathophysiology of FSGS.
Learning objectives Discuss the experimental and clinical data on the pathophysiology of FSGSReview the alterations in glomerular structure and function associated with FSGSTo identify potential mechanisms responsible for disease progression in FSGSDistinguish some targets for the future therapy of FSGS
Focal segmental glomerulosclerosis (FSGS) is a disease entity defined by findings on kidney biopsy [1, 2]. FSGS is the major cause of idiopathic steroid-resistant nephrotic syndrome (SRNS) in children and adults [3]. FSGS is the most common cause of acquired chronic renal insufficiency in children and frequently leads to progression to end-stage kidney disease (ESKD) [2]. FSGS may occur secondary to such disparate disease processes as HIV and obesity [1, 4]; this review focuses on the pathophysiology of primary FSGS (i.e., with no underlying illness).
Alterations of normal glomerular structure and function have been found in FSGS [5]. Normal function requires that the three major components of the glomerular filter (the endothelial cells, podocytes, and glomerular basement membrane) are intact and are able to provide a permselective filtration barrier (Fig. 1). Specialized tight junctions between podocyte foot processes create the slit diaphragm which is integral to preventing the loss of protein into the urinary space [6]. While the clinical presentation of FSGS is often heterogeneous, a characteristic feature of the disease is proteinuria, which implies the loss of this barrier [2, 7]. Indeed, electron microscopy has shown distortion of the normal architecture (or effacement) of the foot processes of podocytes in FSGS [1]. The connection between these projections of the epithelial cell and the underlying basement membrane can be disrupted, leading to the loss of nonspecific plasma proteins into the tubular filtrate [6].
Fig. 1A Components of the normal glomerular filtration barrier: (1) glomerular basement membrane (GBM); (2) podocyte foot process; (3) endothelial cell; B Progressive changes seen in focal segmental glomerulosclerosis (FSGS) leading to sclerosis: (1) foot process effacement; (2) podocyte apoptosis/loss and exposed glomerular basement membrane; (3) filtration of non-specific plasma proteins; (4) capillary expansion; (5) formation of synechiae; (6) misdirected filtration at points of synechiae; (7) mesangial matrix proliferation. Adapted from Kwoh et al. [9]
However, unlike other causes of proteinuria and nephrotic syndrome, such as minimal change disease (MCD), FSGS often progresses to ESKD. While foot process effacement is seen in MCD as well as FSGS, histologically, FSGS is characterized by increased extracellular matrix within the glomerular tuft with obliteration of the glomerular capillary lumen. These sclerotic lesions occur focally and in only some segments of glomeruli, and are typically not associated with immune complex deposition [1]. The location of sclerotic lesions by light microscopy defines the variants of FSGS: perihilar variant (with sclerosis of the vascular pole), cellular variant (associated with hypercellularity of the capillary space), tip variant (involving the part of the glomerulus near the origin of the proximal tubule), and collapsing variant (with one or more glomeruli with global or segmental collapse) [1]. Clinically, the variants of FSGS differ; for example, the collapsing variant tends to progress more rapidly to ESKD and commonly occurs in the setting of HIV [1]. It is possible that different mechanisms may play a role in the pathogenesis of each variant of FSGS [7, 8].
Insight into the pathogenesis of FSGS developed over the past decade from studies of genetic mutations in mice, models of progressive glomerulosclerosis (such as the rat remnant kidney model), and identification of gene mutations in some familial forms of nephrotic syndrome (including congenital nephrotic syndrome and familial and autosomal dominant FSGS) [7, 9, 10].
Key in the pathogenesis of FSGS is podocyte damage and loss [5, 6]. Injury to podocytes occurs by four major mechanisms: alteration of the components of the slit diaphragm or interference with its structure, dysregulation of the actin cytoskeleton, alteration of the glomerular basement membrane or its interactions with the podocyte, or alteration of the negative surface charge of the podocyte [6, 9]. Damage to podocytes triggers apoptosis and their detachment of podocytes from the glomerular basement membrane [6, 9]. The ensuing reduction in podocyte number is felt to play an important role in the pathogenesis of FSGS [7]. The podocyte is normally a terminally differentiated cell with limited proliferative capacity in response to injury [7]. The initial insult to the podocyte leads to further damage mediated by cytokine release, mechanical stress, and further loss of polarity, resulting in sclerosis and scarring of the glomerulus [7, 9].
Genetic mutations seen in congenital forms of nephrotic syndrome and FSGS enabled researchers to identify specific gene mutations involved in podocyte damage [10]. Mutations of the nephrin gene, a podocyte-specific transmembrane component of the slit diaphragm, are found in congenital Finnish-type nephrotic syndrome, and may lead to loss of normal caliber slit diaphragms [6, 9–11]. In mouse models, mutations of nephrin-like transmembrane genes (NEPH-1) which also localize to the slit diaphragm result in proteinuria and early death [6, 10].
It is unclear how alteration of the slit diaphragm results in podocyte loss. The slit diaphragm may be integral to maintaining cell polarity or its damage may alter the balance of cell signals, resulting in apoptosis. Mutations in a Fyn kinase (one of the src tyrosine kinases) that phosphorylates nephrin and may regulate cell cycle and apoptosis resulted in proteinuria and foot process effacement in a mouse model [9, 10].
Other proteins which are part of the slit diaphragm complex include: podocin, CD2-associated protein (CD2AP), FAT, ZO-1, P-cadherin, an LAP (leucine rich repeat and PDZ domain) protein, and MAGI-1 [6, 10]. Mutations in podocin (a transmembrane protein that interacts with nephrin, NEPH-1 and CD2AP) have been identified in familial FSGS [9, 10, 12]. Recently, mutations in CD2AP, an immunoglobulin-like protein that is involved in nephrin integration with the podocyte cytoskeleton, have also been linked to genetic forms of FSGS [10, 13, 14]. In mouse models, the loss of FAT1 and FAT2 (transmembrane proteins with cadherin-like repeats) results in the absence of slit diaphragms, proteinuria, and early death [10]. The role of the other components of the slit diaphragm in the pathophysiology of FSGS is not yet clear.
Alpha-actinin-4, an important structural component of the podocyte cytoskeleton, is mutated in some autosomal dominant forms of FSGS [10, 15–17]. Other mutations have been identified in association with FSGS in addition to abnormal structural proteins. For example, TRPC6 is a cation-selective ion-channel protein that mediates calcium signals and has also been associated with FSGS [18].
Certain clinical variants of FSGS are suggestive of different mechanisms of injury to the podocyte. For example, a circulating factor which leads to glomerular basement membrane injury has been proposed in the pathogenesis of some types of FSGS [19, 20]. For example, there appears to be a role of a circulatory factor in the recurrence of FSGS in transplanted kidneys [20]. In some patients with recurrent FSGS, proteinuria remits in response to plasmapheresis and the removal of serum proteins. In addition, injections of serum from patients with recurrent FSGS were capable of inducing proteinuria in rats [20].
Another example of alternative mechanisms of injury is collapsing FSGS, which occurs in the setting of viruses such as HIV. In collapsing FSGS, dysregulation of the podocyte cell cycle appears to result in immature, proliferative podocytes [21, 22]. Finally, recent work has focused on the role of the parietal epithelial cell in the pathophysiology of FSGS [23]. Proliferation of parietal epithelial cells was identified in both a transgenic model of FSGS and a biopsy from a patient with collapsing FSGS [23].
Of great clinical importance is the mechanism by which the initial podocyte injury progresses to the final sclerotic lesion (Fig. 1). As podocyte numbers decline, there is a relative exposure of the glomerular basement membrane. Maladaptive interactions develop between the glomerular basement membrane and the parietal epithelial cells. Expansion of synechiae and/or the leak of protein into Bowman’s space results in the deposition of collagen. Ultimately, this results in the collapse of the capillary loop and the loss of endothelial cells [5].
Factors resulting in the progression of FSGS to ESKD have also been the focus of recent research (Fig. 2). Cytokines and vasoactive factors are believed to play a major role in the progression of FSGS. The overexpression of transforming growth factor β (TGFβ) or its effector proteins, the Smads, leads to glomerulosclerosis in animal models [8, 24]. Activation of the renin-angiotensin system upregulates TGFβ and is felt to further lead to the progression of disease [7, 24]. Other angiogenic factors, such as platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) may also play a role in disease progression [24]. The evidence for this is primarily based on animal models of progressive glomerulosclerosis, such as the rat remnant kidney model. In this model, PDGF and VEGF are upregulated and the later loss of VEGF expression correlates with progression of the glomerulosclerosis [24, 25].
Fig. 2Factors involved in the progression of FSGS to end-stage kidney disease (ESKD): initial loss or injury to podocytes (related to defects in membrane proteins or cytoskeleton instability) leads to cytokine release, mechanical stress, hyperfiltration, and glomerular hypertrophy. These factors lead to upregulation of an inflammatory response mediated by monocytes, macrophages, and T-cells. The end result is collagen matrix deposition and fibrosis, and progression to ESKD
Mechanical stress is also believed to play a role in the progression of FSGS [9, 26]. Increased filtration due the defects of the filtration barrier results in increased single-nephron glomerular filtration rate (SNGFR). This hyperfiltration results in hypertrophy of glomeruli. The hypertrophy exacerbates the mismatch between the glomerular basement membrane and the decreased numbers of podocytes, resulting in further injury [9].
Another factor in the progression of FSGS is tubulointerstitial injury. Clinically, tubulointerstitial injury is a predictor of the loss of renal function in FSGS [1, 27]. The nonspecific entry of proteins into the tubular lumen is one potential source of damage to the interstitium. Indeed, persistence of nephrotic-range proteinuria is a negative prognostic factor for the progression of FSGS to ESKD [28]. While it is unclear if proteinuria itself is toxic to the tubulointerstitium, decreases in proteinuria achieved by angiotensin-converting enzyme (ACE) inhibitors and by angiotensin receptor blockers (ARB) appear to slow disease progression in some adults with FSGS [9, 29].
The presence of plasma proteins in the tubular filtrate may directly injure the tubulointerstitium. Cytokines (such as TGFβ), when present in the tubules, will recruit monocytes, macrophage, and T-cells. This stimulates other cytokines, including interleukin-1, tumor necrosis factor alpha, and other chemokines [24]. The inflammatory infiltrate leads to mesangial matrix deposition, promoting the collapse of glomeruli. The cellular infiltrate and cytokines also damage tubular epithelial cells, and some tubular epithelial cells may undergo transformation to mesenchymal cells (an epithelial-mesenchymal transition or EMT) [24]. These mesenchymal cells, as well as recruited and stimulated fibroblasts, result in collagen matrix deposition and tubulointerstitial fibrosis [24].
The beneficial effects of blocking the renin-angiotensin system may not be limited to their antiproteinuric or antihypertensive effects. As noted earlier, angiotensin stimulates TGFβ, contributing to fibrosis. It can also induce oxidative stress and it is stimulated by mechanical stress, such as hyperfiltration [24]. In addition, angiotensin affects intracellular calcium concentrations and the podocyte cytoskeleton [24]. Inhibition of angiotensin may slow progression by these local mechanisms [9, 29].
With the increasing incidence of FSGS in children [30], these pathways of podocyte injury and disease progression provide important targets for future intervention. Trials have already been initiated to antagonize cytokines, such as TGFβ. Future therapeutic targets may include factors involved in podocyte protection or tubulointerstitial injury.
Questions (Answers appear following the reference list)
Which of the following statements is TRUE regarding the current understanding of the pathogenesis of focal segmental glomerulosclerosis (FSGS)?
FSGS may result from immune-complex-mediated damage to endothelial cellsAlterations in components of the slit diaphragm may play a role in the pathogenesis of FSGSProliferation of podocytes leads to cytokine release and mechanical stress, resulting in scarring and sclerosis of the glomeruliMutations in a chloride channel have been associated with FSGS and may be pathogenicAll of the following are mutations of structural proteins that have been identified as pathogenic in FSGS EXCEPT:
Sodium channel mutationAlpha-actinin-4NephrinPodocinProgression of FSGS to end-stage kidney disease (ESKD) results from:
Downregulation of transforming growth factor β (TGFβ)Decreased glomerular filtrationTubulointerstitial injuryBlockade of the renin-angiotensin systemProteinuria in the setting of FSGS:
Has no effect on clinical courseMay be decreased by treatment with angiotensin-converting enzyme (ACE) inhibitorsResults from an increased number of glomerular foot processesLeads to the loss of mesangial matrixWhich of the following is FALSE:
A circulating factor may play a role in the pathogenesis of FSGSProliferation of parietal epithelial cells has been identified in collapsing FSGSPodocyte loss due to necrosis appears to play a role in the pathogenesis of FSGSCD2-associated protein, FAT, nephrin, and podocin are examples of slit diaphragm proteins

focal segmental glomerulosclerosis (fsgs)


Clinical and radiological features related to the growth potential of meningioma

Clinical and radiological features that help predict the growth potential of meningioma would be beneficial. The purpose of this study is to clarify the characteristics related to proliferating potential using the MIB-1 staining index. We analyzed the relationship of MIB-1 staining indices to characteristics of 342 consecutive patients with meningioma surgically removed between 1995 and 2004 by logistic regression analysis. One hundred and forty-nine of the patients with meningioma were ≥60 in age; 89 male; 48 recurrent; 203 symptomatic; 157 at the skull base; 124 over 20 cm3; 24 multiple; 136 with edema; 117 with calcification. The MIB-1 staining index in 56 of 296 grade I meningiomas in WHO classification was ≥ 3.0; in 27 of 28 grade II; and in 17 of 18 grade III, respectively. Logistic regression analysis demonstrated that male (odds ratio [OR], 2.374, p=0.003), recurrence (OR, 7.574, p=0.0001), skull base (OR, 0.540, p=0.035), calcification (OR, 0.498, p=0.019) were independent risk factors for a high MIB-1 staining index (≥3.0); age, symptomatic, volume, multiple, edema were not. Male, recurrence, non-skull base, absence of calcification are independent risk factors for a high proliferative potential. These should be taken into consideration when managing meningiomas.
Introduction
An estimated 2–3% of the population has an incidental asymptomatic meningioma in autopsy studies [14, 20]. With the wider use of CT and MRI, many meningiomas are discovered as incidental findings during investigation for unrelated symptoms [9, 13, 16, 17, 20, 21]. The growth potential of meningiomas varies. Some meningiomas remain unchanged in size for a long period, whereas others grow rapidly [15]. Sex, age, initial tumor size, and calcification were reported to be related to the tumor growth judging from follow-up scans [9, 13, 16, 17, 21].
The nuclear antigen Ki-67 expressed by proliferating cells has become available for routinely processed paraffin section. The MIB-1 antibody detects an epitope on the Ki-67 antigen, a nuclear protein present only during active phase of the cell cycle (G1, S, G2, and M) [2]. Several studies investigated how Ki-67 labelling indices could help to predict recurrence [1, 3, 4, 6–8, 11, 12, 18, 19]. An increased MIB-1 staining index was highly correlated with a shorter tumor volume doubling time [12]. In the cases that showed an MIB-1 of ≥ 3%, the tumor volume doubling time was <2 years. Nakaguchi et al. [12] found the formula which can calculate tumor doubling time (Td) from the MIB-1 staining index at surgery: log Td=31.4–0.14×MIB-1 SI (R2=0.556). The time interval to the next recurrence for recurrent meningiomas is associated with the MIB-1 staining index. Meningiomas with MIB-1 staining index of 3% or higher had a significantly higher tendency of recurrence [11]. Although these cell kinetics methods are valuable for growth potential, they can be applied only after the verification of pathology [15].
The purpose of this study was to clarify the clinical and radiological features related to meningioma proliferation using the MIB-1 staining index. Sex, age, calcification, edema, symptom, size, and shape of tumor were already reported to be related to the tumor growth and MIB-1 staining index [1, 3, 4–8, 11–13, 16–19, 21]. The results, however, were inconsistent because of a lack of sufficient case number and inadequate statistical analysis. Independent risk factors for high growth rate should be determined. We analyzed the relationship of MIB-1 staining indices to the characteristics of 342 consecutive patients with meningioma surgically removed between 1995 and 2004 by logistic regression analysis.
Materials and methods
Three hundred and forty-two patients with meningiomas were surgically treated in our department of neurosurgery between 1995 and 2004. Radiological features were analyzed by CT scans and/or MRI. Location of the tumor was classified as follows: convexity, falx, parasagittal, sylvian fissure, tentorial, ventricular, foramen magnum, olfactory groove, petroclival, petrous, sphenoid ridge, and tuberculum sellae. The latter six locations were considered as skull base. The tumor volume was calculated using the formula: length × depth × width × 0.5 [9]. When patients had multiple meningiomas, only the largest tumor was included. On the basis of conventional CT and bone window CT, patients were divided into two groups according to the low density area around the tumor and calcification in the tumor. A low density area due to surgical scar was not included in edema in patients with recurrent meningioma.
The tumors were histologically classified according to the World Health Organization classification of tumors [10]. An avidin-biotin immunoperoxidase or simple stain MAX-peroxidase (Nichirei, Tokyo) technique was used to perform MIB-1 monoclonal antibody (DAKO, Denmark) assay in selected sections of each case. All tissue sections were examined at high-power magnification (×400). The number of cells stained positively with MIB-1 and the total number of tumor cells were counted in several representative fields containing more than 1,000 cells. Their ratio was indicated as the MIB-1 staining index (%).
Statistical analysis
All data were stored on a personal computer and analyzed using commercially available statistical software (SPSS version 12.0, SPSS Inc.). Chi-squired analysis was used to compare the MIB-1 staining index to characteristics of patients with meningioma. All variables were included in a logistic regression model to determine which variables were independently associated with a high MIB-1 staining index (≥3.0). Significance was judged at a value of p<0.05 for all analyses.
Results
Table 1 shows the characteristics and MIB-1 staining index of the 342 patients. One hundred and forty-nine of patients with meningioma were ≥60 in age; 89 male; 48 recurrent; 203 symptomatic; 157 at the skull base; 124≥20 cm3 in volume; 24 multiple; 136 with edema; 117 with calcification. We compare these characteristics to the MIB-1 staining index. We divided them into two groups: <3.0 and ≥3.0 [11]. The MIB-1 staining index in 100 of 342 meningiomas was > 3.0. Sex (p=0.0001), recurrence (p=0.0001), symptomatic (p=0.013), volume (p=0.014), edema (p=0.001), and calcification (p=0.0001) were correlated with the MIB-1 staining index by chi-square test; age, skull base, and multiple were not.
Table 1Characteristics and MIB-1 staining index in 342 meningiomas MIB-1 staining index (%) Factor<3.0≥3.0P ValueAge (years)0.937-49652450-723260-793470-2610Sex (male/female)46/19643/570.0001Recurrence (yes/no)14/22834/660.0001Symptomatic (yes/no)133/10970/300.013Skull base (yes/no)119/12338/620.059Volume (cm3)0.014-9.91133710-531520-7648Multiple (yes/no)15/2279/910.244Edema (yes/no)82/16054/460.001Calcification (yes/no)97/14520/800.0001Total242100
Meningothelial, transitional, and fibrous meningiomas were the three major subtypes, and they accounted for about three fourth of the total. Two hundred and ninety-six meningiomas belonged to grade I; 28 grade II; and 18 grade III. MIB-1 staining index in 56 of 296 grade I meningiomas was ≥ 3.0; that in 27 of 28 grade II; and that in 17 of 18 grade III, respectively (Table 2).Table 2Histological subtypes and MIB-1 staining index of 342 meningiomasSubtypeMIB-1 staining index (%)Total<3.0≥3.0Grade IMeningothelial 10628134Fibrous671380Transitional431356Psammomatous606Angiomatous13215Microcystic101Secretory101Lymphoplasmacyte-rich101Metaplastic20224056296Grade IIAtypical12627Chordoid01112728Grade IIIRhabdoid011Papillary112Anaplastic0151511718Total242100342
Logistic regression analysis demonstrated that male (odds ratio [OR], 2.374, p=0.003), recurrence (OR, 7.574, p=0.0001), skull base (OR, 0.540, p=0.035), calcification (OR, 0.498, p=0.019) were independent risk factors for a high MIB-1 staining index (≥ 3.0); age, symptomatic, volume, multiple, and edema were not (Table 3).
Table 3Logistic regression analysis for factors independently related to MIB-1 staining indexFactorOdds ratio95%CIP ValueAge1.1090.841–1.4610.464Sex2.3741.336–4.2190.003Recurrence7.5743.558–16.1240.0001Symptomatic1.4680.774–2.7840.240Skull base0.5400.305–0.9560.035Volume1.3320.944–1.8790.103Multiple1.0270.398–2.6510.957Edema1.5080.838–2.7110.170Calcification0.4980.278–0.8920.019
Discussion
We analyzed the relationship of the MIB-1 staining indices to the characteristics of 342 consecutive patients with meningioma surgically removed between 1995 and 2004 by logistic regression analysis. Logistic regression analysis demonstrated that male (odds ratio [OR], 2.374, p=0.003), recurrence (OR, 7.574, p=0.0001), skull base (OR, 0.540, p=0.035), calcification (OR, 0.498, p=0.019) were independent risk factors for a high MIB-1 staining index (≥ 3.0); age, symptomatic, volume, multiple, and edema were not.
The relationship between the growth rate or MIB-1 staining index and age has been controversial: a higher MIB-1 staining index and higher growth rate were observed for younger patients [11, 13, 21]; but not in other reports [1, 12, 15, 19]. Our series of 342 patients with meningioma showed no relation.
It is well known that atypical and anaplastic meningiomas are predominant in males [10]. Matsuno et al. [11] reported that the mean MIB-1 staining index in 50 male patients was 5.5%, whereas that in 77 female patients was 2.7%. Our findings show that male (odds ratio [OR], 2.374, p=0.003) was an independent risk factor for a high MIB-1 staining index. We also found a higher MIB-1 staining index in males even in grade I meningioma (MIB-1 staining index in 32 of 226 females, and in 24 of 70 males was        ≥ 3.0, p=0.0001, chi-squire test).
Recurrence (OR, 7.574, p=0.0001) was the most significant independent risk factor for a high MIB-1 staining index (≥ 3.0). Therefore, we propose prompt management for recurrent meningiomas. In most of the recurrent meningiomas, the MIB-1 staining index was higher at the time of recurrence than at the time of initial surgery [1, 11, 19]. Changes in histological morphology and malignant transformation are also known in meningiomas.
Although there is a significant difference in the MIB-1 staining index between symptomatic and non-symptomatic meningiomas by chi-square test, symptomatic is not an independent risk factor for a high MIB-1 staining index. Meningiomas commonly present with seizure disorders, and are associated with location, perilesional edema, and convexity location. Symptoms and signs of elevated intracranial pressure could be due to the large size of meningioma itself, or to the pronounced cerebral swelling resulting from reactive vasogenic edema. Focal neurological deficits caused by meningiomas are generally related to direct local brain, cranial-nerve compression, and can be predicted from the site of origin of the tumor [20]. Thus, symptomatic meningioma may not be related to a high MIB-1 staining index. The growth rate of incidental meningioma may be similar to that of symptomatic meningioma.
Our results demonstrated that skull base (OR, 0.540, p=0.035) is an independent risk factor for a high MIB-1 staining index. No relationship has been reported between the MIB-1 staining index and the location of meningiomas [15, 19]. In general, the surgical risk for meningiomas is higher in skull base. A low proliferative potential in skull base meningiomas should be taken into consideration especially when treating elderly patients with asymptomatic meningiomas [9, 16].
Although there was a significant difference of the MIB-1 staining index in tumor size by chi-square test [15], tumor size is not an independent risk factor for a high MIB-1 staining index. The tumor volume is associated with the annual growth rate but not with doubling time [13, 16, 21]. Assuming that a tumor shows a constant relative volume increase, larger tumors will show higher growth rates if the annual volume increase is expressed in absolute values. Large tumors should be carefully observed even though the initial volume is not a risk factor for a high MIB-1 index, and it is not correlated with doubling time.
The etiology of peritumoral brain edema associated with meningiomas is multifactorial. Factors that may influence the etiology of peritumoral edema include tumor size, histological subtypes, vascularity, venous stasis, and brain invasion [1, 4]. Ide et al. [4] found a significant correlation of both the MIB-1 staining index and tumor size with the extent of edema. A high MIB-1 staining index itself did not seem to be directly responsible for perifocal edema, since our logistic regression analysis demonstrated that edema is not an independent risk factor for a high MIB-1 index [15].
Tumors with calcification grew significantly less than those without calcification [9, 13, 16]. Absence of calcification on CT correlated strongly with doubling time [5]. Diffusely calcified meningiomas had a low mean MIB-1 staining index of 0.57%. Focally calcified tumors showed a relatively low proliferative potential (0.92%) compared with that of noncalcified tumors (1.75%) [15]. The results are always consistent when the relationship between calcification and proliferative potential or growth rate is compared. We also confirmed this characteristic in meningioma (OR, 0.498, p=0.019).
In conclusion, male, recurrence, non-skull base, absence of calcification are independent risk factors for a high proliferative potential. These should be taken into consideration when managing meningiomas.


Polydactyly in the mouse mutant Doublefoot involves altered Gli3 processing and is caused by a large deletion in cis to Indian hedgehog

The mouse mutant Doublefoot (Dbf) shows preaxial polydactyly with 6–9 triphalangeal digits in all four limbs and additional abnormalities including a broadened skull, hydrocephalus, and a thickened, kinked tail. The autopod undergoes a characteristic expansion between late embryonic day (E) 10.5 and E11.5, following the onset of ectopic Indian hedgehog (Ihh) expression in the entire distal mesenchyme, except for the zone of polarising activity (ZPA), at E10.5. We show here that limb prepattern, as indicated by expression of Gli3 and Hand2 at E9.5 is unaffected by the mutation. As both Sonic hedgehog (Shh) and Ihh expression are present in Dbf limb buds at E10.5, we generated Dbf/+;Shh−/− mutants to analyse the effects of different patterns of Hedgehog activity on the limb phenotype and molecular differentiation. Dbf/+ embryos lacking Shh showed postaxial as well as preaxial polydactyly, and the Ihh expression domain extended posteriorly into the domain in which Shh is normally expressed, indicating loss of ZPA identity. Differences in gene expression patterns in wild type, single and compound mutants were associated with differences in Gli3 processing: an increased ratio of Gli3 activator to Gli3 repressor was observed in the anterior half of Dbf/+ limb buds and in both anterior and posterior halves of compound mutant limb buds at E10.5. To identify the cause of Ihh misregulation in Dbf/+ mutants, we sequenced ∼20 kb of genomic DNA around Ihh but found no pathogenic changes. However, Southern blot analysis revealed a ∼600 kb deletion disrupting or deleting 25 transcripts, starting 50 kb 5′ of Ihh and extending away from the gene. The large deletion interval may explain the wide range of abnormalities in Dbf/+ mutants. However, we did not detect anologous deletions in cases of Laurin–Sandrow syndrome, a human disorder that shows phenotypic similarities to Dbf.
1
Introduction
The Dbf mutant, which arose spontaneously in the 3H1 (C3H/HeH × 101/H F1 hybrid) genetic background at Harwell (UK), is a polydactylous mouse that exhibits semidominant inheritance. Mice heterozygous or homozygous for Dbf have 6–9 digits in all four limbs; the extra digits are all triphalangeal and arise preaxially (Lyon et al., 1996; Hayes et al., 1998a). Dbf/+ mice also show malformation of the tibia, a broadened skull, hydrocephalus, a thickened kinked tail, and reduced fertility and viability. Homozygotes additionally exhibit a midline facial cleft but cannot be recovered alive beyond embryonic day (E) 14.5.
Polydactyly has been described in many mouse mutants, all except two of which show a discrete anterior domain of Sonic hedgehog (Shh) expression (Masuya et al., 1995; Hill et al., 2003). The Extra-toes (XtJ) mutant has an extended Shh domain due to functional inactivation of Gli3 (Hui and Joyner, 1993), whereas Dbf mice exhibit ectopic Indian hedgehog (Ihh) expression in the distal limb bud mesenchyme (Yang et al., 1998). Ectopic Ihh upregulation is first detectable at E10.5 (Crick et al., 2003), the stage at which hyperexpansion of the autopod begins; downstream targets of Shh signalling are ectopically up-regulated (Hayes et al., 1998b; Yang et al., 1998). However, the molecular mechanism by which the polydactyly arises from ectopic Ihh expression has not been investigated.
The polydactylous phenotype of the XtJ mutant was originally thought to result from the enlarged Shh expression domain (Hui and Joyner, 1993). However, Shh−/−;Gli3−/− mutants exhibit polydactyly in a similar pattern to Gli3−/− mutants, suggesting that the polydactyly of Gli3-deficient mice is independent of Shh (te Welscher et al., 2002). In wild type (wt) limb buds, digital number and identity are regulated by interaction between Shh and Gli3 (Litingtung et al., 2002). In the presence of Shh, Gli3 remains as a 190 kDa activator species, Gli3A, that up-regulates Hedgehog (Hh)-responsive gene expression, while in the absence of Shh, Gli3A is processed to a smaller 83–86 kDa repressor form, Gli3R, which negatively regulates expression of Shh and its target genes (Dai et al., 1999; Shin et al., 1999; Sasaki et al., 1999). Litingtung et al. (2002) suggested that in wt limb buds the Gli3A:Gli3R ratio controlled by Shh limits the polydactylous potential of the autopod, imposing pentadactyl constraint. This is supported by the localization of Shh protein in wt limb buds, which extends anterior to the zone of polarising activity (ZPA) in a domain coincident with Patched1 (Ptc1) expression (Gritli-Linde et al., 2001), resulting in a posterior-to-anterior increase of the Gli3A:Gli3R ratio (Wang et al., 2000). Consistent with these observations, the Gli3 present throughout Shh−/− limb buds is mainly processed to Gli3R (Litingtung et al., 2002). Recently, the mutation underlying the polydactylous chicken talpid3 mutant has been reported to be in a novel gene and has also been shown to result in abnormal Gli3 processing (Davey et al., 2006). Given the evidence of involvement of abnormal Gli3 processing in the XtJ, Shh−/− and talpid3 mutants, it is possible that the polydactyly present in Dbf mice also results from aberrant Gli3 processing. This hypothesis is supported by evidence that Gli3 acts downstream of Ihh during endochondral skeletal development (Hilton et al., 2005; Koziel et al., 2005).
To investigate the mechanism by which polydactyly arises in Dbf we have analysed gene expression in Dbf/+ limbs, where there is an excess of Hedgehog (Hh) signalling, and compared this to Shh−/− limbs, where there is none. Since Shh and Dbf are located on different chromosomes (5 and 1, respectively) (Blake et al., 2003; Hayes et al., 2001), we have been able to generate mutant mice that carry two copies of the disrupted Shh allele and are heterozygous for the Dbf mutation. To further dissect the mechanisms underlying the limb malformations in both Shh and Dbf mutants, we have analysed the effects of the ectopic Ihh expression associated with Dbf limb abnormalities in the Shh-null background by correlating altered patterns of gene expression with the phenotype of single and double mutants. Differences in Gli3 processing between each genotype suggest that Hh-Gli3 interactions govern the observed differences in digital number, and that postaxial polydactyly results from expression of Ihh, but not Shh, in the posterior ZPA mesenchyme.
Previous attempts to identify the Dbf mutation have been unsuccessful. Hayes et al. (2001) constructed a high resolution genetic map and localized the mutation to a 0.4 cM interval on mouse chromosome 1. This region contained 35 genes including several plausible candidates for the Dbf mutation. However, despite the sequencing of three of these genes, the Dbf mutation remained unidentified. Based on the misregulation of Ihh expression in Dbf, we sequenced ∼20 kb of the surrounding genome but found no obvious pathogenic changes. To investigate whether a genomic rearrangement could be responsible, we used the mouse genome sequence to design a Southern blotting strategy to systematically screen the regions 5′ and 3′ of Ihh for copy number changes. We identified a ∼600 kb deletion starting ∼50 kb 5′ of Ihh, which removes or interrupts 25 known and predicted transcripts. This raises the possibility that additional abnormalities seen in Dbf/Dbf mice arise from loss of function of deleted genes, in addition to Ihh misregulation.
2
Results
2.1
The prepattern of Dbf limb buds is unaffected
Expression of Hand2 and Gli3 has been implicated in patterning the limb bud prior to Shh expression, and has been shown to be affected later by the absence of Shh (Chiang et al., 2001; te Welscher et al., 2002). We assayed expression of these two genes before (E9.5) and after (E11.5) the onset of ectopic Ihh at E10.5 in Dbf/+ mutant embryos (Fig. 1). Gli3 expression is restricted to the anterior portion of the limb bud in wt embryos at E9.5 (Fig. 1A) and this expression pattern is not altered in the limb buds of Dbf/+ mutants (Fig. 1B). Hand2 is expressed throughout the flank of wt embryos prior to formation of the limb bud, then becomes limited to the posterior region of the limb bud as it is initiated (Fig. 1C); this pattern is not altered in Dbf/+ embryos at E9.5 (Fig. 1D). At E11.5, expression of Gli3 in Dbf/+ limb buds differs from that in wt embryos in extending more distally; the domain is also broader although this probably simply reflects the greater breadth of the limb bud (Fig. 1F). Expression of Hand2 is limited to the proximal posterior margin in wt E11.5 limb buds (Fig. 1G); in contrast, the Hand2 domain in Dbf/+ limb buds extends anteriorly and distally (Fig. 1H). Hence the limb prepattern as indicated by the expression of Hand2 and Gli3 at E9.5 is unaffected in Dbf/+ limb buds, but the expression domains of both genes are altered in association with the presence of ectopic Ihh expression at E11.5 (Fig. 3H).
2.2
Altered limb phenotype of Dbf mutants in the absence of Shh
As Shh-null embryos die perinatally, gross morphological examination of wt, Shh−/−, Dbf/+ and Shh−/−;Dbf/+ embryos was conducted at E13.5 and alcian blue staining of the limb bones was carried out at E17.5 (Fig. 2). Both forelimb and hindlimb autopods of Shh−/−;Dbf/+ embryos resemble those of Dbf/+ except that the broadened digital plate is more regular, shows fewer bifurcations, and is more extensive posteriorly (compare Fig. 2B, F and J with D, H and L). At E13.5 the autopod forms a 180° fan, and the angle between the autopod and zeugopod on the postaxial side of the limb is decreased to 90° (Fig. 2D, arrow).
2.3
Ihh and Shh expression in compound mutant limbs is mutually exclusive
The expansion that characterizes the Dbf/+ autopod takes place from late E10.5 to E11.5. We therefore analysed the expression domains of Shh and Ihh in limb buds immediately prior to (E10.5) and after (E11.5) the period of expansion. In both wt and Dbf/+ limb buds at E10.5, Shh is expressed at the posterior margin (Fig. 3A and B), defining this region as the ZPA (Riddle et al., 1993). In wt mice Ihh is not expressed in limbs prior to E12.5 (St-Jacques et al., 1999) while in Dbf/+ mutant mice, Ihh expression is present in the distal mesenchyme of the limb bud at E10.5 (Fig. 3C). This ectopic Ihh domain extends throughout the area anterior to the ZPA and may correspond to the progress zone. Its absence from the ZPA was confirmed by double in situ hybridization to show nonoverlapping juxtaposed Shh and Ihh expression (Fig. 3D). Expression of Ihh in E10.5 Shh−/−;Dbf/+ mutant limb buds extends throughout the distal mesenchyme including the posterior margin, i.e. the domain in which Shh is expressed in Dbf/+ embryos (Fig. 3E).
At E11.5, Shh expression continues in the posterior margin of wt and Dbf/+ limb buds (Fig. 3F and G). Expression of Ihh in Dbf/+ mutant limb buds at E11.5 is progressively down-regulated from posterior to anterior, until it remains only in the anterior margin (Fig. 3H); in contrast, in Shh−/−;Dbf/+ mutant limbs, down-regulation of Ihh expression begins mid-distally, remaining strong in both the anterior and posterior mesenchyme (Fig. 3I).
2.4
Gene expression is altered in Dbf limb buds lacking Shh
To gain insight into the mechanisms underlying the different patterns of polydactyly generated in the presence of different sources of Hh signalling in Dbf/+ and Shh−/−;Dbf/+ limbs, we examined the expression of genes implicated in Shh signalling and limb patterning in wt, Dbf/+, Shh−/− and Shh−/−;Dbf/+ limb buds at E10.5 (Fig. 4); as shown in Fig. 3, this is the stage at which Ihh expression is first detected. Expression of the transcriptional targets of Hh signalling, Ptc1 and Gli1, is expanded anteriorly in Dbf/+ limbs; interestingly, expression of these genes is broader in the proximal mesenchyme of Dbf/+limbs lacking Shh, suggesting expansion of the domain of Hh signalling in these limb buds. Conversely expression of Gli3, which is thought to be repressed by Hh signalling (Takahashi et al., 1998), shows a reduced expression domain in Dbf/+ limbs. As expected, Gli3 is expressed throughout Shh−/− limbs at E10.5, but in the presence of Ihh in Shh−/−;Dbf/+ mutants it is dramatically down-regulated and required a prolonged colour development time for detection.
In wt and Dbf/+ limbs at E10.5 there is a strong expression of Hand2 in the posterior mesenchyme, with a graded lower expression anteriorly, similar expression is seen in Shh−/− limbs. However, in Shh−/−;Dbf/+ limbs there appears to be a second strong anterior domain of Hand2 expression, consistent with the extended expression seen at E11.5 (Fig. 1H). As reported previously (Hayes et al., 1998b; Yang et al., 1998), the Hoxd13 domain is expanded anteriorly in Dbf/+ limb buds; in Shh−/−;Dbf/+ limb buds, the domain shows even greater expansion, consistent with the more regular digital fan seen in these mutants. Expression of Fgf8 throughout the AER of expanded Dbf/+ and Shh−/−;Dbf/+ limbs indicates that in both mutants Hh signalling between the mesenchyme and ectodermal AER is intact. Ectopic anterior expression of Fgf4 in the expanded limb buds of both mutants is consistent with their ectopic Ihh expression. Bmp4 expression in the progress zone was slightly down-regulated in Dbf/+ limbs but up-regulated proximally; like wt limbs, it was absent from the AER. In contrast, Shh−/−;Dbf/+ limbs, which showed further down-regulation of Bmp4 in the mesenchyme of the progress zone, showed ectopic expression throughout the AER. Explanation for this pattern requires further investigation.
2.5
The Dbf mutation affects the limb bud Gli3 ratio
The action of Gli3 protein as a transcriptional activator relies on its maintenance as Gli3A, which requires Hh signalling (Dai et al., 1999; Sasaki et al., 1999; Shin et al., 1999). To determine the effect of differential Hh signalling on Gli3 processing in mutant limb buds, we used a Gli3 antibody combined with Western blot analysis to assess the comparative levels of Gli3A and Gli3R in the anterior and posterior halves of E10.5 limb buds of all four genotypes (Fig. 5). As reported previously, wt limbs have a higher ratio of Gli3R to Gli3A anteriorly than posteriorly (Wang et al., 2000, and Fig. 5B and C). Dbf/+ limb buds have a reduced level of the repressor relative to the activator, especially in the anterior half, where levels of the two forms of Gli3 are similar. In Shh−/− limb buds the difference between the anterior and posterior halves is greatly reduced with relatively high levels of Gli3R to Gli3A in both halves of the limb bud mesenchyme (Litingtung et al., 2002, and Fig. 5B and C). In Shh−/−;Dbf/+ mutants, both halves of the limb bud show a decreased ratio of Gli3R to Gli3A compared with the wt result; the ratio is similar in both halves of the limb bud, in contrast to the Dbf/+ result which shows an A–P asymmetry.
2.6
A ∼600 kb deletion underlies Dbf
To determine the cause of the ectopic Ihh expression in Dbf limbs, we initially searched for the genetic lesion by sequencing 20 kb of the region around Ihh in Dbf heterozygotes and both parental strains, but found no pathogenic changes (data not shown). Subsequently we sought genomic rearrangements using a systematic Southern blotting strategy to interrogate the mouse genome sequence (http://genome.ucsc.edu/), which initially identified the absence of a polymorphic 8 kb SpeI fragment in Dbf (see Section 4.5). Characterization of the breakpoint by Southern analysis and subsequently by inverse PCR led to identification of the centromeric breakpoint at position 75,098,488 bp on chromosome 1 (Fig. 6). Analysis of sequence 3′ to this in Dbf/+ DNA revealed the telomeric breakpoint to be at position 75,694,480 bp on chromosome 1. The deleted region therefore appears to be 595,992 bp; however this figure is not precise because the deletion encompasses a ∼16 kb gap in the current mouse genome sequence (mm9 assembly) present between 75,102,130 and 75,118,131 bp. We confirmed the deletion by PCR using primers flanking the breakpoint and further demonstrated that three different loci distributed within the putatively deleted region were present only in a single copy in Dbf/+ mutant DNA (see Section 4.5). Analysis of the wt sequences at the two breakpoints showed that the sequence at the centromeric breakpoint is unique, lying within the gene Non-homologous end joining factor 1 (Nhej1). However, a hexanucleotide motif CCAAAC present at the breakpoint is repeated 17 nucleotides upstream, separated by four copies of a trinucleotide CCT motif. The telomeric breakpoint resides within the 3′ terminal region of a B1 repetitive element at the endpoint of a very T-rich motif (35 thymine residues in 47 bases) which is likely to represent the complement of an ancestral poly(A) tract related to the B1 element and does not disrupt any known gene. There is a three nucleotide ambiguity in the position of the breakpoint as the sequence ACA is present on both sides of the deletion (Fig. 6). In addition to disrupting Nhej1, the deletion completely removes 24 known and predicted genes (Fig. 6, Supplementary Table 2 and Section 3).
2.7
Laurin–Sandrow syndrome does not result from large deletions 5′ of IHH
Laurin–Sandrow syndrome (LSS) (MIM 135750) is rare human developmental disorder characterized by triphalangeal preaxial polydactyly of the hands and feet, with variable involvement of the proximal limb elements. It has been previously suggested that LSS shares many similarities with Dbf and may also arise from ectopic IHH expression (Innis and Hedera, 2004). To investigate the possibility that Dbf and LSS share a common etiology, we screened five patients diagnosed with LSS for copy number variation at 23 sites between IHH and EPHA4 using multiplex ligation-dependent probe amplification (see Supplementary Information). No copy number variation was detected (data not shown).
3
Discussion
3.1
Ectopic Ihh expression in Dbf/+ is modified in the absence of Shh and is associated with loss of ZPA identity
Although we have previously shown that expression of ectopic Ihh in Dbf/+ limb buds coincides with the onset of limb bud expansion at E10.5 (Crick et al., 2003), it was not known whether the prepattern of Dbf limbs might be affected by the mutation prior to Ihh expression. However, no differences were detected in the expression of Gli3 or Hand2 in wt and Dbf/+ embryos at E9.5 or E10.5, consistent with the hypothesis that ectopic Ihh expression represents the primary pathogenic event. By E11.5, expression domains of both Hand2 and Gli3 were more extensive in Dbf/+ than wt limb buds, suggesting that Ihh signalling is able to modify their expression.
In Dbf/+ limb buds, Ihh and Shh are expressed in discrete adjacent domains. Exclusion of Ihh from the Shh domain is reminiscent of the exclusion of the Hh-inducible gene Gremlin from this domain; Scherz et al. (2004) suggested that the effect may be due to high levels of intracellular autocrine Shh signalling. The loss of identity of the ZPA resulted in a abnormal expansion of the posterior limb bud mesenchyme in Shh−/−;Dbf/+ mice leading to the additional postaxial polydactyly seen in these mutants.
3.2
Abnormal gene expression leading to an aberrant Gli3 ratio underlies Dbf polydactyly
To elucidate the limb patterning underlying Dbf/+ polydactyly and to investigate the generation of the broader, more regular fan of digits seen in Shh−/−;Dbf/+ mutants, we studied the expression of a range of limb patterning and development genes at E10.5. Dbf/+ mutant limbs show an anterior expansion of the positive regulators of Hh signalling Ptc1, Gli1 and the downstream targets Hoxd13 at E10.5 and Hand2 by E11.5. Dbf/+ limbs also show a reduction of Gli3 expression, which is thought to be negatively regulated by Hh signalling. Conversely, due to the complete lack of Hh activity in Shh−/−;Dbf/+ mutant limbs prior to E10.5, Gli3 is ubiquitously expressed in these limb buds until this stage, when it is down-regulated in Shh−/−;Dbf/+ but not Shh−/− mutants. Gli3R is thought to repress expression of Hoxd13 and Hand2 and Fgf4 in the anterior of wt limb buds while Gli3A induces the expression of Gli1 in the posterior region (reviewed in Tickle, 2006). Therefore, the postaxial polydactyly seen in Shh−/−;Dbf/+ mutants may be due to the loss of identity of the ZPA with concomitant posterior extension of the Ihh domain. In contrast, the preaxial polydactyly that is present in both Dbf/+ and Shh−/−;Dbf/+ mutants is correlated with ectopic Gli3A-induced Hh signal transduction together with lack of repression of posterior patterning genes by Gli3R in the anterior of the limb bud. We suggest that the discrepancy between the very low level of Gli3 mRNA (Fig. 4) and the Gli3 protein detected in Shh−/−;Dbf/+ limb buds at E10.5 (Fig. 5) indicates the perdurance of protein after the gene has been down-regulated.
3.3
Identification of the Dbf mutation
The interpretation of the mechanism of the Dbf mutation has been hampered previously by the failure of attempts to identify the underlying mutation. Using a Southern blotting and inverse PCR strategy we have demonstrated that a ∼600 kb deletion underlies the Dbf phenotype. The presence of simple sequences at both breakpoints may have predisposed them to breakage; the lack of significant similarity between the breakpoints (except for a 3 nucleotide identity at the breakpoints themselves) suggests that the rearrangement is likely to have involved nonhomologous end joining (NHEJ). Further analysis of sequence at the breakpoints revealed that the distal breakpoint resides within the degenerate poly(A)n tract of a short retrotransposon (SINE) of the rodent B1 family, which, like human Alu repeats originate from 7Sl RNA (Vassetzky et al., 2003).
The deleted region in Dbf is relatively gene-dense and completely deletes 24 known and predicted transcripts as well as interrupting Nhej1 at the centromeric breakpoint. Several of these genes have previously been implicated in abnormal mouse phenotypes or human disease; information on the known expression patterns and functions of these genes is summarized in Supplementary Table 2. Abnormalities associated with genes in the deleted region may contribute to additional aspects of the heterozygous Dbf phenotype such as the broadened skull, hydrocephalus, reduced viability and fertility, thickened tail and supernumerary hair follicles. However, none of the homozygous null phenotypes resulting from specific targeting of the Ptprn, Des, Inha or Slc4a3 genes is lethal in late embryogenesis so the cause of death at E14.5 in Dbf homozygotes remains unclear. This could be attributable to loss of function of any of the genes within the interval for which homozygous mice have not yet been described, and/or to homozygosity for the ectopic Ihh expression defect. Interestingly a recent study reported a human fetus with a balanced de novo translocation t(2;7)(q36;p22) with the chromosome 2 breakpoint interrupting the orthologue of Nhej1 at a position similar to the start of the Dbf deletion (Cantagrel et al., 2007). The consequence of this translocation, as in Dbf, would be to isolate the human IHH gene from possible regulatory sequences present on the opposite side of the NHEJ1 breakpoint. Although the terminated fetus exhibited syndactyly of all four limbs, polydactyly was not present, suggesting that the translocation did not result in ectopic IHH expression.
We have presented evidence that the prepattern of Dbf limb buds is unaffected and that the preaxial polydactyly is attributable to a reduction in Gli3R resulting from ectopic Ihh expression. It is interesting that preaxial polydactyly, the most striking aspect of the Dbf phenotype, is unlikely to result directly from haploinsufficiency of any of the genes in the deleted region. Rather, the deletion appears to affect a cis-acting regulatory element of Ihh, which could be a repressor located within the deletion, or an enhancer beyond the deleted region. Other examples of regulatory mutations acting at a distance have been reviewed by Kleinjan and van Heyningen (2005). Pinpointing the regulatory sequences involved remains a major challenge, one notable success being the identification of the ZPA sequence regulatory sequence (ZRS) which lies ∼1.0 Mb upstream of Shh and regulates its expression in the ZPA; mutations in the ZRS lead to ectopic Shh expression resulting in preaxial polydactyly (Lettice et al., 2002). However, owing to the large size of the Dbf deletion and the large number of genes and highly conserved non-coding elements within it, it will be challenging to delineate the precise mechanism underlying ectopic Ihh expression in the Dbf mouse.
4
Materials and methods
4.1
Generation and identification of mutant mice
Mice heterozygous for the Shh null allele (Chiang et al., 1996) on the C57BL/6J background were mated to Dbf/+ mice on the 3H1 background. The Shh mutant allele was detected as previously described (Chiang et al., 1996). Homozygous Shh−/− embryos were identified by their phenotype.
To genotype Dbf/+ embryos (prior to the identification of the causative deletion), Dbf/+ mice were crossed with wt Mus musculus castaneus and the Dbf F1 progeny were bred with C3H wt mice. Embryos were genotyped using primers which amplify the marker D1Mit46 located ∼2.3 cM from Ihh (P1 5′-AGTCAGTCAGGGCTACATGATG-3′, P2 5′-CACGGGTGCTCTATTTGGAA-3′). This produces amplification products of 276 bp and 320 bp on the C3H and Mus musculus castaneus backgrounds respectively.
4.2
Whole mount in situ hybridization
Doubly heterozygous Shh+/−;Dbf/+ were crossed with Shh+/− mice and embryos of all six possible genotypes were collected for analysis of gene expression domains and morphology: wt, Shh+/−, Shh−/−, Dbf/+, Shh+/−;Dbf/+ and Shh−/−;Dbf/+. To ensure consistency between developmental stages, only forelimb buds were analysed and a minimum of two samples were examined with each probe. Timing of embryos was by the vaginal plug method: 12.00 noon on the day on which the plug was observed was regarded as E0.5. Pregnant females were sacrificed on the appropriate day by cervical dislocation and the embryos were dissected from the uterus in ice cold phosphate buffered saline (PBS) (140 mM NaC1, 3 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4) followed by immersion in cold tissue fixative in accordance with the Animals (Scientific Procedures) Act, 1986. Where necessary, yolk sacs were removed for genotyping and embryos were fixed by immersion overnight at 4 °C in 4% paraformaldehyde in PBS. Embryos were dehydrated by sequential washing in 25%, 50%, 75% ethanol in PBT (PBS + 0.1% Tween 20) and finally by two washes in 100% ethanol. They were stored at −20 °C until required.
Single stranded digioxygenin-UTP labelled antisense riboprobes were generated from linearized plasmids containing cDNAs. Whole mount in situ hybridization was carried out essentially as described by Wilkinson (1992).
4.3
Skeletal preparations
Embryos for skeletal staining were dissected and fixed in 95% ethanol for 1–3 days. They were immersed in alcian blue stain (75% ethanol, 20% acetic acid, 3 mg/ml alcian blue) for 21–28 days at 37 °C. They were cleared in 0.8% KOH, 20% glycerol. Following clearing, they were sequentially dehydrated and stored in 50% ethanol/50% glycerol.
4.4
Western Blotting
The polyclonal antibody specific for the amino terminus of Gli3 was a gift from Dr. Chin Chiang (Litingtung et al., 2002). Three μg of protein lysate derived from the anterior and posterior halves of ∼6 E10.5 forelimb buds were resolved on 4–12% polyacrylamide gels. Gli3 protein was detected using anti-N-terminal Gli3 (1:300) primary antibody and biotinylated anti-rabbit immunoglobulin-γ secondary antibody (1:1000). Protein bands were visualized by incubation with a streptavidin-peroxidase conjugate followed by an enhanced chemiluminescence detection method (Amersham).
4.5
Characterization of the Dbf deletion
To determine whether genomic rearrangements were associated with Dbf, single copy probes labelled with α32P-dCTP were synthesized and used to hybridize Southern blots of DNA isolated from heterozygous 3H1 (C3H × H101 hybrid) Dbf mice and wt mice from both background strains. We used the mouse genome mm9 sequence release (July 2007) for all analyses presented in this paper. A probe corresponding to 75,099,314–75,099,651 bp revealed the absence of a polymorphic 8 kb SpeI fragment in Dbf, found in the C3H parental strain, suggesting the existence of a deletion. A further Southern blot using a probe corresponding to 75,097,987–75,098,255 bp revealed a 1.2 kb BspHI fragment present only in Dbf. This 1.2 kb breakpoint fragment was isolated by inverse PCR. Briefly, genomic DNA from Dbf was digested with BspHI, diluted to 10 ng/μl and T4 DNA ligase was added to promote intramolecular ligation. Religated DNA was used directly in an inverse PCR using the primer pair: 5′-GCATTTGAGATTGAGACAAGCACTCTCCACAC-3′ and 5′-ACAGCGCTAGACAGAAAGCCTGCTTGCT-3′. DNA sequencing revealed 228 bp of unknown sequence that was shown by BLAST analysis to originate from a region of chromosome 1, ∼596 kb telomeric from the breakpoint. PCR amplification with primers designed either side of the breakpoint (5′-TGGTCTGGAGAGACAGCTCGTCCAGAG-3′ and 5′-GAGTTGAAGAGTTGGCATAGTGGTGCACAC-3′) was employed to confirm the site of the deletion.
To confirm the Dbf lesion was a true deletion, primers were designed to amplify three regions within the deletion predicted to contain polymorphisms variable between the C3H and H101 background strains. These regions were located at ∼150 kb intervals within the deleted region. The primer pairs used were site 1, 5′-GCCCTCATGCTTGAGTACCTTGCCTGTGAT-3′ and 5′-GTCCTCCCAGGGGCTGAGCAGAGTG-3′; site 2, 5′-TAGACTGAGCACCCGGCCTAACATGCTC-3′ and 5′-TGTGTCATCCACCCGGTGCCTCTGACT-3′; site 3, 5′-TAGAATTCCCACTGGGTCCACCCACTC-3′ and 5′-CATACATCCGTGTACATGTACTGACTGTCACTG-3′. Amplification products were digested with appropriate restriction endonucleases to discriminate between the alleles. Site 1 contained a novel TACC insertion polymorphism and was digested with HphI, site 2 contained a known C/T polymorphism (rs31657679) and was digested with AvaI and site 3 contained a known polymorphism (rs3049959) and was digested with Hpy8I. In each case only the H101 allele was present indicating that the C3H chromosome carried the deletion. The presence of both background strains was confirmed on the centromeric side of the Dbf deletion by sequence polymorphisms observed during Southern blotting (data not shown). Both strain backgrounds were shown to be present on the telomeric side of the deletion by AseI restriction digest of a fragment containing a novel informative C/T polymorphism which was amplified using the primer pair 5′-CAACAAAGCCCACATCAATTCACTCAGGCCGTG-3′ and 5′-CACCCTGCCTCAACCTCTCACCTGCTAG-3′.