Compositional Asymmetry Research Articles

Lateral organization, bilayer asymmetry, and inter-leaflet coupling of biological membranes

Understanding of cell membrane organization has evolved significantly from the classic fluid mosaic model. It is now recognized that biological membranes are highly organized structures, with differences in lipid compositions between inner and outer leaflets and in lateral structures within the bilayer plane, known as lipid rafts. These organizing principles are important for protein localization and function as well as cellular signaling. However, the mechanisms and biophysical basis of lipid raft formation, structure, dynamics and function are not clearly understood. One key question, which we focus on in this review, is how lateral organization and leaflet compositional asymmetry are coupled. Detailed information elucidating this question has been sparse because of the small size and transient nature of rafts and the experimental challenges in constructing asymmetric bilayers. Resolving this mystery will require advances in both experimentation and modeling. We discuss here the preparation of model systems along with experimental and computational approaches that have been applied in efforts to address this key question in membrane biology. We seek to place recent and future advances in experimental and computational techniques in context, providing insight into in-plane and transverse organization of biological membranes.

Use of high energy ball milling to study the role of graphene nanoplatelets and carbon nanotubes reinforced magnesium alloy

Graphene nanoplatelets (few layer graphene) and carbon nanotubes were used as reinforcement fillers to enhance the mechanical properties of AZ31 magnesium alloy through high energy ball milling, sintering, and hot extrusion techniques. Experimental results revealed that tensile fracture strain of AZ31 magnesium alloy was enhanced by +49.6% with 0.3 wt.% graphene nanoplatelets compared to −8.3% regression for 0.3 wt.% carbon nanotubes. The tensile strength of AZ31 magnesium alloy was decreased (−11.2%) with graphene nanoplatelets addition, while increased (+7.7%) with carbon nanotubes addition. Unlike tensile test, compression tests showed different trend. The compression strength of carbon nanotubes-AZ31 composite was +51.2% greater than AZ31 magnesium alloy as compared to +0.6% increase for graphene nanoplatelets. The compressive fracture strain of carbon nanotubes-AZ31 composite was decreased (−14.1%) while no significant change in fracture strain of graphene nanoplatelets-AZ31 composite was observed. The X-ray diffraction results revealed that addition of reinforcement particles weaken the basal textures which affect the composite's yield asymmetry. Microstructure evaluation revealed the absence of intermetallic phase formation between reinforcements and matrix. The carbon reinforcements in AZ31 magnesium alloy dissolve and isolate β phases throughout the matrix. The increased fracture strain and mechanical strength of graphene nanoplatelets and carbon nanotubes-AZ31 composites are attributed to large specific surface area of graphene nanoplatelets and stiffer nature of carbon nanotubes respectively.

Asymmetry in body composition in female hockey players

The aim of the study was to determine if a sport in which one side of the body is dominant, like field hockey, influences regional body composition and bone mineral density (BMD) distribution in particular body segments, and whether the sporting level is a determining factor. Dual energy X-ray absorptiometry (DXA) method (Lunar Prodigy Advance; General Electric, Madison, USA) with the whole body scan was used to measure bone mineral density, fat mass and lean mass in 31 female field hockey players divided according to their sporting level. The morphological asymmetry level was assessed between two body sides and body segments in athletes from the National Team (n=17) and from the Youth Team (n=14) separately and between groups.Bone mineral density in the lower extremity and of the trunk was significantly asymmetric in favor of the left side in the National Team. In the case of the Youth Team, only the trunk BMD indicated clear left–right difference with left side dominance. Both the lean mass and fat mass values were relatively higher on the left side of all body segments and it related to both analyzed groups of athletes.The present study shows that playing field hockey contributes to laterality in body composition and BMD and that the sporting level is a determining factor. In most cases the left side dominated. A greater asymmetry level was observed in more experienced female field hockey players.

Self-reported aggressiveness is not related to developmental instability.

In recent years, several researchers have focused on the relationship between fluctuating asymmetry and aggression. These studies reveal conflicting results. In this study, we examined the relationship between bodily symmetry and self-reported aggressive behavior in healthy young people living in Turkey. The sample comprised of 100 male and 102 female university students recruited from Cumhuriyet University, Sivas. The Buss and Perry aggressiveness questionnaire was used. Four bilateral traits were measured for fluctuating asymmetry analysis. For the subsequent statistical analyses, only the composite asymmetry index was used. There was no relationship between composite asymmetry and any component of aggressiveness for either sex. Our findings for people living in Turkey did not support the hypothesis that aggressiveness, as a signal of evolutionary fitness, is related to developmental instability.

Budding of domains in mixed bilayer membranes.

We propose a model that accounts for the budding behavior of domains in lipid bilayers, where each of the bilayer leaflets has a coupling between its local curvature and the local lipid composition. The compositional asymmetry between the two monolayers leads to an overall spontaneous curvature. The membrane free energy contains three contributions: the bending energy, the line tension, and a Landau free energy for a lateral phase separation. Within a mean-field treatment, we obtain various phase diagrams which contain fully budded, dimpled, and flat states. In particular, for some range of membrane parameters, the phase diagrams exhibit a tricritical behavior as well as a three-phase coexistence region. The global phase diagrams can be divided into three types and are analyzed in terms of the curvature-composition coupling parameter and domain size.

Phosphatidylglycerol Asymmetry and Translocation in Lipid Membranes

Our current understanding of the structure and dynamics of cellular membranes emerged in the early 1970 's. However, there is still much we do not know about the underlying lipid dynamics in cellular membranes, specifically the process of lipid translocation or flip-flop. The flip-flop rates of only a few lipid species, principally phosphatidylcholines (PCs), have been measured. Anionic lipids, such as phosphatidylseryine (PS), phosphatidylinositol (PI), and phosphatidylglycerol (PG), are known to be play active roles in membrane function, but almost nothing is known of the rates of translocation of these species. While PS and PI are the major anionic lipids of eukaryotic cellular membranes, PG is mainly found in prokaryotic membranes and comprises about 10% of the phospholipid content of the mitochondria membrane in eukaryotes. In the work presented here, the native flip-flop rates of 1,2-diasterol-sn-glycero-3-[phospho-(1′-rac-glycerol)] (DSPG) flip-flop in mixed DSPG: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) bilayers have been investigated. Using methods of classical surface chemistry coupled with nonlinear optical methods, we have developed a novel analytical approach, using sum-frequency vibrational spectroscopy (SFVS), to selectively probe lipid compositional asymmetry in a planar supported lipid bilayer. This new method allows for the detection of lipid flip-flop kinetics and compositional asymmetry without the need for a fluorescent or spin-labeled lipid species by exploiting the coherent nature of SFVS. Using SFVS, the rates of DSPG flip-flop in a DSPC matrix have been examined for the first time. Analysis of the dynamics provides an assessment of the underlying energetic barrier to PG translocation. The results will be discussed in the framework of the protein-free energetic barriers to PG flip-flop and the role of electrostatics in this process.

Measuring the composition-curvature coupling in binary lipid membranes by computer simulations.

The coupling between local composition fluctuations in binary lipid membranes and curvature affects the lateral membrane structure. We propose an efficient method to compute the composition-curvature coupling in molecular simulations and apply it to two coarse-grained membrane models-a minimal, implicit-solvent model and the MARTINI model. Both the weak-curvature behavior that is typical for thermal fluctuations of planar bilayer membranes as well as the strong-curvature regime corresponding to narrow cylindrical membrane tubes are studied by molecular dynamics simulation. The simulation results are analyzed by using a phenomenological model of the thermodynamics of curved, mixed bilayer membranes that accounts for the change of the monolayer area upon bending. Additionally the role of thermodynamic characteristics such as the incompatibility between the two lipid species and asymmetry of composition are investigated.

High-throughput formation of lipid bilayer membrane arrays with an asymmetric lipid composition.

We present a micro-device in which more than 10,000 asymmetric lipid bilayer membranes are formed at a time on micro-chamber arrays. The arrayed asymmetric lipid bilayers, where lipid compositions are different between the inner and outer leaflets, are formed with high efficiency of over 97% by injecting several types of liquids into a micro-device that has hydrophilic-in-hydrophobic surfaces. The lipid compositional asymmetry is an intrinsic property of bio-membranes, and therefore, this micro-device extends the versatility of artificial lipid-bilayer systems, which were previously limited to symmetric bilayer formation, and could contribute to the understanding of the role of lipid compositional asymmetry in cell physiology and also to further analytical and pharmacological applications.

Heterotypic gap junctions at glutamatergic mixed synapses are abundant in goldfish brain

Gap junctions provide for direct intercellular electrical and metabolic coupling. The abundance of gap junctions at “large myelinated club ending (LMCE)” synapses on Mauthner cells (M-cells) of the teleost brain provided a convenient model to correlate anatomical and physiological properties of electrical synapses. There, presynaptic action potentials were found to evoke short-latency electrical “pre-potentials” immediately preceding their accompanying glutamate-induced depolarizations, making these the first unambiguously identified “mixed” (i.e., chemical plus electrical) synapses in the vertebrate CNS. We recently showed that gap junctions at these synapses exhibit asymmetric electrical resistance (i.e., electrical rectification), which we correlated with total molecular asymmetry of connexin composition in their apposing gap junction hemiplaques, with connexin35 (Cx35) restricted to axon terminal hemiplaques and connexin34.7 (Cx34.7) restricted to apposing M-cell plasma membranes. We now show that similarly heterotypic neuronal gap junctions are abundant throughout goldfish brain, with labeling exclusively for Cx35 in presynaptic hemiplaques and exclusively for Cx34.7 in postsynaptic hemiplaques. Moreover, the vast majority of these asymmetric gap junctions occur at glutamatergic axon terminals. The widespread distribution of heterotypic gap junctions at glutamatergic mixed synapses throughout goldfish brain and spinal cord implies that pre- vs. postsynaptic asymmetry at electrical synapses evolved early in the chordate lineage. We propose that the advantages of the molecular and functional asymmetry of connexins at electrical synapses that are so prominently expressed in the teleost CNS are unlikely to have been abandoned in higher vertebrates. However, to create asymmetric coupling in mammals, where most gap junctions are composed of connexin36 (Cx36) on both sides, would require some other mechanism, such as differential phosphorylation of connexins on opposite sides of the same gap junction or on asymmetric differences in the complement of their scaffolding and regulatory proteins.

Asymmetric giant lipid vesicle fabrication.

Synthetic lipid bilayers have long been used as models of cell membranes. The compositional asymmetry in the eukaryotic plasma membrane is a key chemical characteristic of this membrane that has traditionally been difficult to reproduce in synthetic systems. In this chapter, we describe recent technologies for fabricating compositionally asymmetric giant unilamellar lipid vesicles (GUVs) and provide detailed protocols for a microfluidic-based fabrication technique.

Ori-Finder 2, an integrated tool to predict replication origins in the archaeal genomes.

DNA replication is one of the most basic processes in all three domains of cellular life. With the advent of the post-genomic era, the increasing number of complete archaeal genomes has created an opportunity for exploration of the molecular mechanisms for initiating cellular DNA replication by in vivo experiments as well as in silico analysis. However, the location of replication origins (oriCs) in many sequenced archaeal genomes remains unknown. We present a web-based tool Ori-Finder 2 to predict oriCs in the archaeal genomes automatically, based on the integrated method comprising the analysis of base composition asymmetry using the Z-curve method, the distribution of origin recognition boxes identified by FIMO tool, and the occurrence of genes frequently close to oriCs. The web server is also able to analyze the unannotated genome sequences by integrating with gene prediction pipelines and BLAST software for gene identification and function annotation. The result of the predicted oriCs is displayed as an HTML table, which offers an intuitive way to browse the result in graphical and tabular form. The software presented here is accurate for the genomes with single oriC, but it does not necessarily find all the origins of replication for the genomes with multiple oriCs. Ori-Finder 2 aims to become a useful platform for the identification and analysis of oriCs in the archaeal genomes, which would provide insight into the replication mechanisms in archaea. The web server is freely available at http://tubic.tju.edu.cn/Ori-Finder2/.

Droplet interface bilayer reconstitution and activity measurement of the mechanosensitive channel of large conductance from Escherichia coli.

Droplet interface bilayers (DIBs) provide an exciting new platform for the study of membrane proteins in stable bilayers of controlled composition. To date, the successful reconstitution and activity measurement of membrane proteins in DIBs has relied on the use of the synthetic lipid 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC). We report the functional reconstitution of the mechanosensitive channel of large conductance (MscL) into DIBs composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), a lipid of significantly greater biological relevance than DPhPC. MscL functionality has been demonstrated using a fluorescence-based assay, showing that dye flow occurs across the DIB when MscL is gated by the cysteine reactive chemical 2-(trimethylammonium)ethyl methane thiosulfonate bromide (MTSET). MscL has already been the subject of a number of studies investigating its interaction with the membrane. We propose that this method will pave the way for future MscL studies looking in detail at the effects of controlled composition or membrane asymmetry on MscL activity using biologically relevant lipids and will also be applicable to other lipid–protein systems, paving the way for the study of membrane proteins in DIBs with biologically relevant lipids.

Large replication skew domains delimit GC-poor gene deserts in human

Besides their large-scale organization in isochores, mammalian genomes display megabase-sized regions, spanning both genes and intergenes, where the strand nucleotide composition asymmetry decreases linearly, possibly due to replication activity. These so-called skew-N domains cover about a third of the human genome and are bordered by two skew upward jumps that were hypothesized to compose a subset of “master” replication origins active in the germline. Skew-N domains were shown to exhibit a particular gene organization. Genes with CpG-rich promoters likely expressed in the germline are over represented near the master replication origins, with large genes being co-oriented with replication fork progression, which suggests some coordination of replication and transcription. In this study, we describe another skew structure that covers ∼13% of the human genome and that is bordered by putative master replication origins similar to the ones flanking skew-N domains. These skew-split-N domains have a shape reminiscent of a N, but split in half, leaving in the center a region of null skew whose length increases with domain size. These central regions (median size ∼860kb) have a homogeneous composition, i.e. both a null and constant skew and a constant and low GC content. They correspond to heterochromatin gene deserts found in low-GC isochores with an average gene density of 0.81promoters/Mb as compared to 7.73promoters/Mb genome wide. The analysis of epigenetic marks and replication timing data confirms that, in these late replicating heterochomatic regions, the initiation of replication is likely to be random. This contrasts with the transcriptionally active euchromatin state found around the bordering well positioned master replication origins. Altogether skew-N domains and skew-split-N domains cover about 50% of the human genome.

Mixed lipid bilayers with locally varying spontaneous curvature and bending.

A model of lipid bilayers made of a mixture of two lipids with different average compositions on both leaflets, is developed. A Landau Hamiltonian describing the lipid-lipid interactions on each leaflet, with two lipidic fields ψ 1 and ψ 2, is coupled to a Helfrich one, accounting for the membrane elasticity, via both a local spontaneous curvature, which varies as C 0 + C 1(ψ 1 - ψ 2/2), and a bending modulus equal to κ 0 + κ 1(ψ 1 + ψ 2)/2. This model allows us to define curved patches as membrane domains where the asymmetry in composition, ψ 1 - ψ 2, is large, and thick and stiff patches where ψ 1 + ψ 2 is large. These thick patches are good candidates for being lipidic rafts, as observed in cell membranes, which are composed primarily of saturated lipids forming a liquid-ordered domain and are known to be thick and flat nano-domains. The lipid-lipid structure factors and correlation functions are computed for globally spherical membranes and planar ones and for a whole set of parameters including the surface tension and the coupling in the two leaflet compositions. Phase diagrams are established, within a Gaussian approximation, showing the occurrence of two types of Structure Disordered phases, with correlations between either curved or thick patches, and an Ordered phase, corresponding to the divergence of the structure factor at a finite wave vector. The varying bending modulus plays a central role for curved membranes, where the driving force κ 1 C 0 (2) is balanced by the line tension, to form raft domains of size ranging from 10 to 100 nm. For planar membranes, raft domains emerge via the cross-correlation with curved domains. A global picture emerges from curvature-induced mechanisms, described in the literature for planar membranes, to coupled curvature- and bending-induced mechanisms in curved membranes forming a closed vesicle.

Crystal structure and molecular packing of an asymmetric giant amphiphile constructed by one C60 and two POSSs

Based on our recent understanding and development of functionalized molecular nanoparticles (MNPs) as building blocks for constructing various giant molecules, we report our efforts on design and synthesis of an asymmetric giant amphiphile, diBPOSS–C60, composed of one [60]fullerene (C60) covalently linked with two isobutyl-functionalized polyhedral oligomeric silsesquioxane (BPOSS) MNPs. Its crystal structure and molecular packing were investigated. The compositional asymmetry between C60 and BPOSS led to a “sandwich-layered” molecular packing scheme, where a single layer of C60 is sandwiched between double BPOSS layers: a so-called “one-and-half-layered” structure. Within these layers, the molecules further organized into crystalline arrays. Interestingly, this compound can be viewed as size amplification of a class of atomically thin, two-dimensional layered transition metal dichalcogenides with the “one-and-half-layered” structure.

Asymmetric supported lipid bilayer formation via methyl-β-cyclodextrin mediated lipid exchange: influence of asymmetry on lipid dynamics and phase behavior.

Supported lipid bilayers (SLBs) are broadly used as minimal membrane models and commonly produced by vesicle fusion (VF) on solid supports. Despite its advantages, VF does not allow the controlled formation of bilayers that mimic the leaflet asymmetry in lipid composition normally found in biological systems. Here we present a simple, quick, and versatile method to produce SLBs with a desired asymmetric lipid composition which is stable for ca. 4 h. We apply methyl-β-cyclodextrin mediated lipid exchange to SLBs formed by VF to enrich the upper leaflet of the bilayer with sphingomyelin. The bilayer asymmetry is assessed by fluorescence correlation spectroscopy, measuring the lipid mobility separately in each leaflet. To check the compatibility of the method with the most common protein reconstitution approaches, we report the production of asymmetric SLBs (aSLBs) in the presence of a glycosylphosphatidylinositol-anchored protein, reconstituted in the bilayer both, via direct protein insertion, and via proteoliposomes fusion. We finally apply aSLBs to study phase separation and transbilayer lipid movement of raft-mimicking lipid mixtures. The observed differences in terms of phase separation in symmetric and asymmetric SLBs with the same overall lipid composition provide further experimental evidence that the transversal lipid distribution affects the overall lipid miscibility and allow to temporally investigate leaflet mixing.

The Drosophila melanogaster Phospholipid Flippase dATP8B Is Required for Odorant Receptor Function

The olfactory systems of insects are fundamental to all aspects of their behaviour, and insect olfactory receptor neurons (ORNs) exhibit exquisite specificity and sensitivity to a wide range of environmental cues. In Drosophila melanogaster, ORN responses are determined by three different receptor families, the odorant (Or), ionotropic-like (IR) and gustatory (Gr) receptors. However, the precise mechanisms of signalling by these different receptor families are not fully understood. Here we report the unexpected finding that the type 4 P-type ATPase phospholipid transporter dATP8B, the homologue of a protein associated with intrahepatic cholestasis and hearing loss in humans, is crucial for Drosophila olfactory responses. Mutations in dATP8B severely attenuate sensitivity of odorant detection specifically in Or-expressing ORNs, but do not affect responses mediated by IR or Gr receptors. Accordingly, we find dATP8B to be expressed in ORNs and localised to the dendritic membrane of the olfactory neurons where signal transduction occurs. Localisation of Or proteins to the dendrites is unaffected in dATP8B mutants, as is dendrite morphology, suggesting instead that dATP8B is critical for Or signalling. As dATP8B is a member of the phospholipid flippase family of ATPases, which function to determine asymmetry in phospholipid composition between the outer and inner leaflets of plasma membranes, our findings suggest a requirement for phospholipid asymmetry in the signalling of a specific family of chemoreceptor proteins.

The Influence of Natural Lipid Asymmetry upon the Conformation of a Membrane-inserted Protein (Perfringolysin O)

Eukaryotic membrane proteins generally reside in membrane bilayers that have lipid asymmetry. However, in vitro studies of the impact of lipids upon membrane proteins are generally carried out in model membrane vesicles that lack lipid asymmetry. Our recently developed method to prepare lipid vesicles with asymmetry similar to that in plasma membranes and with controlled amounts of cholesterol was used to investigate the influence of lipid composition and lipid asymmetry upon the conformational behavior of the pore-forming, cholesterol-dependent cytolysin perfringolysin O (PFO). PFO conformational behavior in asymmetric vesicles was found to be distinct both from that in symmetric vesicles with the same lipid composition as the asymmetric vesicles and from that in vesicles containing either only the inner leaflet lipids from the asymmetric vesicles or only the outer leaflet lipids from the asymmetric vesicles. The presence of phosphatidylcholine in the outer leaflet increased the cholesterol concentration required to induce PFO binding, whereas phosphatidylethanolamine and phosphatidylserine in the inner leaflet of asymmetric vesicles stabilized the formation of a novel deeply inserted conformation that does not form pores, even though it contains transmembrane segments. This conformation may represent an important intermediate stage in PFO pore formation. These studies show that lipid asymmetry can strongly influence the behavior of membrane-inserted proteins.

Asymmetric Supported Lipid Bilayer Formed via Methyl-β-Cyclodextrin Mediated Lipid Exchange: a Membrane Model System to Study Phase Separation and Transbilayer Lipid Movement

Supported lipid bilayers (SLBs) are broadly used as a membrane model system and commonly produced by vesicle fusion (VF). Despite its advantages, VF does not allow the controlled formation of asymmetric bilayers that mimic the asymmetry in lipid composition normally found in biological systems. Here we present a simple, quick and versatile method to produce stable SLBs with a desired asymmetric lipid composition. We apply a methyl-β-cyclodextrin (MβCD) mediated lipid exchange method to SLBs formed by VF to enrich the distal leaflet of the bilayer with sphingomyelin (SM). Bilayer asymmetry is assessed by fluorescence correlation spectroscopy (FCS), measuring lipid mobility separately in each leaflet. To check the compatibility of the method with the most common protein reconstitution approaches, we also report the production of asymmetric SLBs (aSLBs) in presence of a membrane protein, reconstituted both via direct protein insertion (i.e. directional insertion) and proteoliposomes fusion. aSLBs are a suitable membrane model system to study phase separation and transbilayer lipid movement of raft-mimicking lipid mixtures. Although their phase behavior was extensively investigated, the effect of compositional asymmetry on phase separation remains unclear. To address this question, we compared SLBs and aSLBs with the same overall lipid composition. The observed differences in terms of phase separation provide further experimental evidence that the transversal lipid distribution affect the overall lipid miscibility and allow to temporally investigate leaflet mixing (i.e. lipid flip-flop). Additionally, cholesterol distribution between cytosolic and extracellular side of the plasma membrane is of great importance in determining protein-membrane interaction and the formation of raft-like domains in vivo. Therefore, we use a combined FCS and mass spectrometric approach to investigate cholesterol distribution between inner and outer leaflet of SM-containing aSLBs.

Natural incubation patterns and the effects of exposing eggs to light at various times during incubation on post-hatch fear and stress responses in broiler (meat) chickens

Although environmental conditions during incubation can affect poultry welfare, one factor often overlooked in the commercial incubation of eggs is light. Light stimulation during embryonic development is now known to affect the physiology and behavior of broiler (meat) chickens post-hatch, but little is known about the pattern of exposure needed to produce these effects. We determined how often naturally incubated eggs were exposed to light by giving 10 broody Junglefowl×New Hampshire Red hens a nestbox containing five fertile eggs and a light sensitive data logger which recorded nest attendance by determining whether light was reaching the eggs. On average hens stayed on the nest without leaving for 14.3 of the days of the 21-day incubation cycle, although they did leave periodically, particularly during the last week. Utilizing this information, we then investigated how the timing of light exposure during incubation affected fear and stress responsiveness post-hatch in broiler chickens. Eggs were either not exposed to light (0DL), or exposed to light throughout incubation (21DL) or during only either the last 2 weeks (14DL) or the last week (7DL) of incubation. Lighting pattern had a significant effect on all parameters measured, with the most consistent differences found between 0DL and 21DL. For the fear measures, the 0DL birds had a longer latency to emerge during an emergence test (62.7 versus 37.2s), a longer latency to right during a tonic immobility test (223.8 versus 107.2s), wing-flapped more intensely during an inversion test (7.03 versus 6.4 flaps/s), and vocalized more during an isolation test (172.7 versus 127.1/3min) than the 21DL. For the stress measures, 0DL had a lower IgG titer (52,683 versus 97,375 units) and greater corticosterone response (1.18 versus 0.55ng/mL) to the crating stressor than 21DL, and showed more composite asymmetry (1.96 versus 1.49mm). The 14DL and 7DL groups were generally intermediate. Unlike dark-incubated chicks, all light-stimulated groups showed lateralization of escape direction during the emergence test, suggesting that light-induced cerebral laterality could play a role in the observed effects. However, the direction of lateralization differed depending upon timing of exposure. These results confirm the importance of light stimulation during incubation on the later behavior and physiology of broiler chickens, but also indicate that providing light only during the last week of incubation, which would coincide with the maximum light stimulation provided by hens’ excursions from the nest, is insufficient to produce these effects.