Development of Neocortical Areas: Molecular Signaling, Spontaneous Activity, Post-Natal Experience

Development of topographically organized maps : Development of Neocortical areas: Molecular signaling, spontaneous activity, post-natal experience

PART II 2021 Daniel J. Felleman

Department of Neurobiology & Anatomy Office: MSB 7.168 Telephone: 500-5629 [email protected] Objectives Genetics (molecular markers or gradients) Spontaneous (peri-natal) neuronal activity Experience-dependent circuit formation or refinement

• The optic tectum/ superior colliculus • Frog eye rotation • Growth cones and axon guidance • Chemo-affinity hypothesis of Sperry

• Ephrins / Ephs gradients; attraction, repulsion, terminal zone formation • Role of pre-natal spontaneous activity / retinal waves in map refinement • How does one fool map development? Roles of KO, Ki, conditional KO, pharmacology and what are the limitations? • What is left for post-natal experience? • Different maps (areas) different rules? • What are the ‘rules’ for cortical area development and cortical evolution? • NHP Face patch system • NHP cortical connections, streams, and functional specialization Visual areas in the brain are mapped topographically

How and when does this retinotopic mapping occur?

The primary function of the optic tectum is to localize the stimulus in space and to cause the animal to orient to the stimulus by moving its body and eyes. Retina-to-tectum: Eye rotation and re-wired (regenerated) projection Figure 23.4 Signals in embryo affect growth cones and growing axons (Part 1) Structure and action of growth cones Chemo-affinity hypothesis – by Roger Sperry-1963 Chemical markers on growing axons are matched with complementary chemical markers on their targets through attraction or repulsion to establish precise connections.

Membrane stripe assay

RGC from nasal retina show no preference for anterior or posterior colliculus RGC from temporal retina show preference for anterior colliculus

1990s Discovery of ELF-1 (Eph ligand family 1) and RAGS (repulsive axon guidance signal)

Expressed in low to high gradient in the OT and are ligands of the receptor tyrosine kinase

tectum EphA3 is expressed in a high to low temporal tectum -nasal gradient by RGCs)

Ephrin-A2 and Ephrin-A5 are axon repellants and serve as axonal guidance molecules.

tectum

tectum Mechanisms of topographic mapping in the vertebrate visual system

Expressed in low to high gradient in the OT and are ligands of the receptor tyrosine kinase

EphA3 is expressed in a high to low temporal -nasal gradient by RGCs)

Ephrin-A2 and Ephrin-A5 are axon repellants and serve as axonal guidance molecules. Developmental stages in mapping temporal-nasal retinal axis on anterior-posterior axis of colliculus/tectum in mice and chicks

• Initially RGC axons enter the SC at the anterior end and project to the posterior SC overshooting their topographically appropriate termination zone (dashed circles). • Then branching occurs from the axon at the topographically specific location followed by pruning of the posterior axon. • At later stages the branches are stabilized, and refined to form strong synapses. • In mice this process starts late in embryogenesis and lasts through the first week of life (E, embryonic day post conception; P, postnatal).

Feldheim D A , O’Leary D D M Cold Spring Harb Perspect Biol 2010;2:a001768 Triplett and Feldheim 2012 Seminar Cell Dev Biol. 23:7-15.

Expression patterns of Ephs and ephrins in the retino-collicular system A) The temporal-nasal (T-N) axis of the retina maps along the anterior-posterior (A-P) axis of the SC/OT. Graded expression of EphA and ephrin-A expression patterns in each structure in the mouse are indicated by blue and red bars, respectively. EphAs are expressed high temporally and anteriorly, while ephrin-As are expressed high nasally and posteriorly in complementary gradients.

B) The dorsal-ventral (D-V) axis of the retina maps along the lateral-medial (L-M) axis of the SC/OT. Graded EphB and ephrin-B expression patterns in each structure are indicated by blue and red bars, respectively. In the retina, EphBs are expressed in a V > D gradient, while ephrin-Bs are expressed in a complementary D > V gradient. In the SC, EphBs are expressed uniformly across the L-M axis, and ephrin-B1 expression is high at the midline, but is steeply reduced laterally. Regulation of topographic-specific interstitial branching along anterior–posterior axis of tectum/colliculus: the critical determinant of retinotopic mapping Graded repellent activity that preferentially affects temporal RGC axons is achieved by EphA forward signaling.

EphA is expressed in a high to low temporal (T) to nasal (N) gradient by RGCs, and ephrin-As in a low to high anterior (A) to posterior (P) gradient in the optic tectum (OT)/superior colliculus (SC).

This receptor-legend pairing generates through EphA forward signaling a high to low AP gradient of repellent activity that preferential affects temporal RGC axons This, in principle is sufficient to topographically guide RGC axonal growth cones along the AP axis to their appropriate terminal zone (TZ). This mapping mechanism is adequate in lower vertebrates.

However, a single repulsive gradient cannot generate the topographic interstitial branching of RGC axons along the AP axis observed in chicks and rodents, because branching would occur at a greater frequency more anterior to the TZ.

This is not observed in vivo—branching is focused on Feldheim D A , O’Leary D D M Cold Spring Harb Perspect the AP position of the TZ and is lower both anterior Biol 2010;2:a001768 and posterior it.

©2010 by Cold Spring Harbor Laboratory Press (C) Interstitial branching could be achieved by a model with a gradient of molecules with branch promoting activities that cooperate with the EphA forward signaling inhibition of branching.

Branching is prevented posterior to the correct TZ by ephrin-A repellent activity mediated by EphA forward signaling, and does not occur anterior to the correct TZ because the branch promoting activity is subthreshold.

Branching occurs at the AP position in which the branch promoter is suprathreshold and the branch inhibitor is subthreshold.

TrkB, in a similar distribution to EphAs in the retina and BDNF in the OT/SC have the appropriate activities to act as the graded branch promoter, and cooperate with the Branching occurs preferentially at a trough in the graded branch inhibitor generated by opposing repellent gradients that is subthreshold for EphA/ephrin-A forward signaling. branch inhibition and is located at the correct AP position of the TZ. (D) topographic-specific interstitial branching in the chick OT and mouse SC Topographic- At the position of this trough, the activity of a branch specific branching is generated by opposing promoter (such as BDNF signaling through TrkB) is gradients of branch inhibiting molecules suprathreshold for branch inhibition and generates combined with a branch promoting activity. branching at this topographically correct location for the TZ. Forward and reverse signaling through ephrin-As and Bs

Forward: in Eph containing cells—binding to EphA receptors results in activation of GTPase— leading to growth cone collapse (also regulation of Tsc2).

Reverse: in the ephrin-bearing cells—mediated by co-receptors TrkB and p75NTR –resulting in axonal branching and axon repulsion. Forward signaling through EphB –growth cone attraction or repulsion.

Reverse signaling through eprinBs is important for synapse maturation Duplicated maps Multiple aberrant terminal zones Structure of the retino-collicular map in EphA/ephrin-A knockout and transgenic mice A–B) In wild type mice, the retina is mapped topographically onto the SC. Temporal RGCs (blue dots) map to anterior SC, while nasal RGCs (red dots) project to posterior SC. Intrinsic optical imaging reveals topography by measuring the visual responses in the SC to a drifting bar along the N-T axis of the retina.

(C–D) Genetic deletion of ephrin-As or EphAs results in aberrant topographic map formation. Anatomical tracing reveals multiple termination zones along the A-P axis of the SC for both temporal and nasal RGCs Functionally, this results in areas of the SC with topographically inappropriate responses, with the general polarity of the map intact

(E–F) Relative levels of EphA expressed are used to map along the A-P axis. In Islet2-EphA3 knock-in mice, half of retinal ganglion cells express exogenous EphA3, resulting in neighboring cells with drastically different EphA expression levels. This results in a duplicated retino-collicular map as assessed by anatomical tracing (E) and functional imaging studies (F). Functional imaging of adult SC in wild type and mutant mice

Functional imaging of adult SC in wild type and mutant mice. The D-V retinal projection (elevation axis) onto the medial-lateral axis of the SC is largely normal in the mice shown.

The N-T projections (azimuth axis) map onto the anterior–posterior axis of the SC. The ephrin-A KOs have a patchy, disordered map (arrows), whereas ephrin-A2/A5/b2 tKOs have almost no map, specifically along the N-T axis.

The EphA3-ki mouse projections are duplicated within the SC (arrows mark regions of duplication). (Adapted from Cang et al. 2005b, 2008b; Triplett et al. 2009.)

Feldheim D A , O’Leary D D M Cold Spring Harb Perspect ©2010 by Cold Spring Harbor Laboratory Press Biol 2010;2:a001768 Figure 5 Alignment of the primary visual cortex (V1) and the primary somatosensory cortex (S1) inputs with retinocollicular maps in the superior colliculus (SC). (a–b) Models of visual map alignment in the SC. In the gradient-matching model (a), graded expression of EphA receptors in both the retina and V1 are used to guide topographic mapping in the SC, which expresses repulsive ephrin-A ligands in a gradient in both recipient layers. In the retinal-matching model (b), retinocollicular mapping is established first through the use of graded EphAs and ephrin-As. V1 projections then terminate in areas with similar activity patterns or with RGCs expressing complementary cell surface molecules. Abbreviations: N, nasal; T, temporal; A, anterior; P, posterior; D, dorsal; V, ventral; L, lateral; M, medial; uSGS, upper stratum griseum superficiale; lSGS, lower stratum griseum superficiale; WT, wild type. (c–d) Different outcomes of V1 and S1 inputs to the SC in EphA3ki/ki mice. (c) An injection of DiI into central V1 traces axons that project to the central SC (white arrow). A similar injection bifurcates and projects to each map in the EphA3ki/ki mouse (white arrows), showing that the retinal inputs to the SC direct V1 inputs; *, pretectal nucleus. (d) S1 axons do not alter their projection patterns in the EphA3ki/ki mouse, resulting in misalignment between retinal and SC maps. Diagrams and data are from Triplett et al. 2009, 2012. Interactions between molecular cues and structured retinal activity In the development of cortical topographic maps

Cang et al. (2008) Neuron 57: 511-523.

1. Disruption of azimuthal maps in Ephrin-A2/A5/beta2 KO 2. Receptive fields of cortical neurons—enlarged in the azimuthal direction 3. LGN RFs. 4. LGN topography Arborization phase: P0-P3-4 in mice SC Molecular guidance cues such as EphA/ ephrinA signaling

Stage III waves begin at approximately P10–P12 in mice and ferrets and are mediated by Dynamic phase: P3-4 to P8-9 glutamate released from retinal bipolar cells (Bansal et al. 2000, Wong et al. 2000, Remodeling of retino-collicular map by Zhou&Zhao 2000) (Figure 1). Stage III waves persist until around the time of eye-opening activity dependent arbor plasticity and (Bansal et al. 2000, Demas et al. 2003, Syed et al. 2004,Wong et al. 1993). competition for collicular resources Three Spontaneous Retinal Wave Epochs

Stage 1 Stage 2 Stage 3

AGE

Late gestation P0-P9 P10-eye opening

EXCITATORY DRIVE

Gap junction acetylcholine glutamate

IN VITRO WAVE CHARACTERISTICS Large brief waves Large and slow Small, brief waves waves with with repetitive random trajectories trajectories Retinal Waves: Development of orderly connections depends on activity before visual input

Correlated firing of retinal ganglion cells were recorded From isolated retinas of fetal cat. Neurons fired spikes in Synchronous bursts lasting a few seconds, often separated by 1-2 minutes. Bursts consisted of a wave of excitation, several hundred microns wide, sweeping across the retina at about 100 microns /sec. These are hypothesized to guide the refinement of connections in SC or layer segregation in LGN Defect in V1 azimuth maps in triple KO: Chang et al 2008

Figure 1. Cortical Azimuth Maps Are Severely Disrupted in Ephrin-A2A5-β2 Combination KOs A–C) Cortical azimuth maps of an A2−/−A5−/−β2+/− (A), an A2+/−A5+/−β2−/− (B), and an A2−/−A5−/−β2−/− combination KO (C). (A). Note that the lack of retinotopic organization in the map of A2−/−A5−/−β2−/− combination KO. (D) Quantification of map scatters for the azimuth maps of these genotypes. (E–H) Elevation maps of the same three mice and quantification of their map scatter. Error bars represent SEM. Substantial size differences in midbrain targets and distinct mechanisms of map development exhibited by model systems. (B) In mouse and chick RGC axons enter the OT/SC over a broad LM extent and significantly overshoot their future termination zone (TZ, circle). Interstitial branches form de novo from the axon shaft around the correct anterior–posterior (A–P) position of their future TZ.

Branches are directed along the lateral–medial (L–M) axis of the OT/SC toward the position of the future TZ, in which they arborize in a domain encompassing the forming TZ.

The broad, loose array of arbors is refined to a dense TZ in the topographically appropriate location.

(C) In frogs and fish the RGC growth cone extends to the TZ in which it forms a terminal arbor through a process of terminal branching of the growth cone and backbranching immediately behind the growth cone. The tectum expands as terminal arborizations Feldheim D A , O’Leary D D M Cold Spring Harb Perspect Biol elaborate and refine into a mature TZ 2010;2:a001768 Annual Reviews Schematic of (a) the mature retinotopic map in the SC, (b) the immature unrefined retinotopic map in the SC (c) Disruptions in retinotopic mapping in ephrin-A2/5−/− or EphA5−/− mice. Multiple dense termination zones are observed along the N-T axis of the target.

(d ) Disruptions in retinotopic mapping in EphB2/3−/− mice or in response to disrupting Wnt/ryk signaling; RGC terminals shift more medially (ryk disruption; or shift more laterally (EphB2/3 knockout;)

(e) The overall retinotopic map forms when stage II retinal waves are eliminated (because ephrin-A2/5 signaling is still intact), but RGC axons fail to form dense terminal arbors in their correct topographic locations and are abnormally broad.

( f )When stage II waves are prevented The same general defect pattern is observed in the in ephrin-A2/5−/− mice, N-T mapping of LGN and V1, where ephrin-As and Bs and retinal RGC projections is abolished waves regulate topographic map formation. Chang et al 2008 LGN-V1 projections: Disrupted order in azimuth but not elevation axis

Figure 3. Disruption of Geniculo-cortical Map in Ephrin-A2A5-β2 Combination KOs (A–D) Retrogradely labeled dLGN neurons of a WT (B), an A2−/−A5−/−β2+/− (C), and an A2 −/−A5 −/−β2 −/− (D). Neurons were labeled by injections of CTB-Alexa 488 (green) and CTB-Alexa 568 (red) at 500 μm apart in V1 along lateromedial axis (A). In all the panels, dotted lines mark the border of dLGN. Note the overlap between the green and red cells in (D). (E) Quantification of overlap between the two groups of labeled cells in the dLGN along the azimuth axis. (F–J) Retrograde labeling and quantification for dLGN neurons when the tracers were injected along elevation axis. Error bars represent SEM. Chang et al 2008 RF structure in V1 from 2 WT vs. 2 triple KO V1 neurons

Figure 4. Single-Unit Recording in Visual Cortex Demonstrates that the Receptive Fields of Cortical Neurons in Combination KOs Are Selectively Enlarged in the Azimuthal Direction (A and B) Representative receptive fields measured with moving short bars of two WT neurons (A1, 2) and two ephrin- A2A5-β2 combination KO neurons (B1, 2). Axes in degrees of visual space, color represents magnitude of response. (C) Receptive field radii in degrees, by Gaussian fit to sweeping short bar data, for all single units recorded. (D) Average receptive field size in azimuth and elevation from (C). (n = 31 units in control, 23 units combination KOs, from 5 animals each.) Error bars represent SEM. Tripplet et al 2009

Figure 2. EphA3ki/ki Mice Have Duplicated Functional Maps in the SC and a Single functional Map in V1 (A–D) Intrinsic optical imaging signal obtained from V1 (A and B) and SC (C and D) of WT adult mice presented a drifting bar stimulus along the azimuth (A and C) or elevation (B and D) axis. bar, 500 μm.

(E–H) Intrinsic optical imaging signal obtained from V1 (E and F) and SC (G and H) of EphA3ki/ki animals presented a drifting bar stimulus along the azimuth (E and G) or elevation (F and H) axis. bar, 500 μm. Gradient-matching model Retinal-matching model Arroyo and Feller 2016

FIGURE 1| Spontaneous retinal waves mediate eye-specific segregation and retinotopic refinement of retinofugal projections. The axons of retinal ganglion cells(RGCs)target the dorsal lateral geniculate nucleus (dLGN) of the thalamus and the superior colliculus (SC). RGC projections from opposite eyes are segregated into ipsilateral and contralateral regions (black/gray oppositions). Distinct spatiotemporal patterns of retinal waves instruct different features of retinofugal projections in mice.(A–D) Top: schematic of Retinal wave firing patterns. Larger dots represent RGCs with elevated firing rates during as in glutamate retinal wave. Bottom: schematic of Retino-geniculate wiring using same code as Figure1—colored hexagons represent retinotopy while gray/black regions represent eye-specific segregation.

Normally, RGCs exhibit high local correlations while activity of the two retinas is minimally correlated. This pattern supports both eye-specific segregation and retinotopic maps in both the dLGN and SC.

High correlations between retinal waves of opposite retinas (induced by optogenetic stimulation) is detrimental to eye-specific segregation while retinotopic maps are unaffected (Zhang et al., 2011).

(C) Disruption of local correlations either by an increase in uncorrelated firing or by abnormally elevated correlations between distant RGCs (global correlations), (ie. b2KO and Retb2-cKO mice), is detrimental to retinotopic map formation. (D) High global correlations paired with high inter-retina correlations (ie. observed Local correlations are sufficient for normal retinotopic in the b2KO mouse), is detrimental to both maps in Retb2-cKO, Rxb2-cKO and b2(TG) (Xu et al., eye-specific segregation and retinotopic 2011, 2015; Burbridge et al., 2014). maps (Xu et al., 2011; Burbridge et al., 2014). Xu et al Crair Neuron 2011: Binocular Competition Interferes with Retinotopic Map Refinement

(A and B) Focal DiI injections around the periphery of the retina results in focal target spots in the SC of WT mice, but much larger target zones in b2(KO) mice.

In b2(TG) mice, target zones are completely restored in regions of the SC that receive monocular input but remain enlarged in the regions that receive input from both eyes

(C and D) Focal DiI injections into ventral-temporal retina, which projects bilaterally, labels a spot in the rostro-medial portion of the contralateral SC in WT mice. A similar injection in b2(KO) and b2(TG) mice results in a much larger target zone.

(E and F) Enucleation of one eye at birth restores retinotopic refinement of ventral- temporal RGCs in b2(TG) mice, but not in b2(KO) mice. SUMMARY:

Whole animal b2KO mice lack expression of any b2-nAChRs, and RGC activity levels and retinal waves are severely perturbed in vivo

b2(TG) mice, which have b2-nAChR expression confined to the GCL but missing elsewhere in the retina and brain, have normal levels of RGC activity but truncated or “small” retinal waves

Eye segregation …globally but not strictly locally correlated activity across the retina appears necessary for eye-specific segregation, along with a minimum overall level of activity that is close to that normally found during development.

In Rx-b2cKO mice, retinal activity is locally but not globally correlated, like in b2(TG) mice, eye specific segregation is completely disrupted.

A minimum level of activity not too far below that found normally in the retina, along with correlated activity among RGCs across large swathes of the retina, but not between retinas, are necessary for the emergence of eye-specific segregation.

Topographic refinement: waves with a distinct wave front that propagates slowly across the retina and produces local correlations in activity between RGCs that are much stronger than global (long distance) correlations, appears necessary for normal retinotopic refinement.

Retinotopic refinement in ventral-temporal (“binocular zone”) RGCs in Rx-b2cKO is normal only when competition between the two eyes is removed by monocular enucleation.

Despite the fact that the development of refined retinotopy and eye-specific segregation are interdependent, each map is sensitive to distinct features of spontaneous retinal activity, including overall activity levels and RGC correlations. Developmental Specification of Neocortical Areas O’Leary et al., 2007

Mechanisms of Specification and Differentiation of Neocortical Areas The initial, tangential axial gradients of transcription factors (TFs) in the ventricular zone (VZ) are likely established by signaling molecules or morphogens (or both) secreted from localized patterning centers. • Fgf8 and Fgf17 are secreted from the anterior patterning center, the anterior neural ridge (ANR), which later becomes the commissural plate (CoP). • Wnts and Bmps are secreted from the posterior-medial-located cortical hem. • Sonic hedgehog (Shh) is secreted from a ventral domain. A lateral putative patterning center, termed the anti-hem, also might contribute to graded TF expression. • The graded expression of certain TFs, such as Pax6, Emx2, COUP-TFI, and Sp8, conveys positional or area identities to cortical progenitors, which form the cortical plate (CP). The CP also initially exhibits gradients of gene expression that are gradually converted to distinct patterns with sharp borders. • The distinct areas seen in the adult (M1, S1, A1, V1), differentiate from the CP. Genes that are differentially expressed across the cortex are often expressed in different patterns in different layers, suggesting that area-specific regulation of such genes is modulated by layer-specific properties. • The initial establishment of graded gene expression in the embryonic CP is controlled by mechanisms intrinsic to the telencephalon, the complex postnatal differentiation, develops in parallel with Thalamo-cortical axon input (extrinsic factors) A. Schematic views of Fgf8 action on area patterning. Roles for the CoP and Fgf8 in Cortical Fgf8 expressed in ANR/CoP controls the gradients of Area Patterning expression of key TFs Emx2 and Coup-TFI that have a low anterior and high posterior expression gradients.

Fgf8 is thought to suppress the expression of both genes in the anterior dTel . The combined actions of graded TFs are thought to establish gene expression with sharp boarders, resulting in anatomically and structurally defined areas.

(B) Fgf8 regulates gradients of Emx2 and Coup-TFI expression. Gradients of Emx2 and Coup-TF1 are shifted anteriorly in the dTel of E12/E12.5 Fgf8 hypomorphic (Fgf8neo/neo) embryos. (Left) Lateral and dorsal views of an Fgf8neo/neo embryo show the anterior shift in the anterior limit of this gradient (compare arrowheads). (Right) The anterior limit of high Coup-TFI expression (white arrowheads) is shifted anteriorly in Fgf8neo/neo embryos

(C) Anterior electroporation of Fgf8 or sFGFR3 causes opposite shifts of the S1 barrel fields. Tangential sections through layer 4 of flattened P6 cortices processed for cytochrome oxidase (CO) histochemistry are shown. White arrows mark the midpoint between anterior and posterior poles of the neocortex. Summary of Intrinsic Genetic Mechanisms of Graded expression of Emx2, Pax6, Coup-TFI, and Sp8 along anterior-posterior and lateral-medial axes. Area Patterning and Mutant Phenotypes • Emx2 is expressed in a high P-M to low A-L gradient. • Pax6 expression pattern is opposite to that of Emx2, with a high A-L to low P-M gradient. • Coup-TFI has a high P-L to low A-M gradient. Sp8 is expressed in a high A-M to low P-L gradient. • While the expression of Emx2, Pax6, and Coup- TFI is sustained in the VZ, Sp8 expression is quickly downregulated around the onset of cortical neurogenesis.

In the anterior signaling center, Fgf8 establishes the low anterior-graded expression of the TFs Emx2 and COUP-TFI by repression, and promotes the high anterior gradient of Sp8 expression.

• Putative posterior signaling molecules Bmps and Wnts, expressed in the cortical hem, positively regulate the high caudal gradient of Emx2 expression. • Genetic interactions between TFs also participate in the establishment of their graded expression. For example, Emx2 and Pax6 mutually suppress each other’s expression, Coup-TFI suppresses Pax6 expression and enhances Emx2 expression, and Sp8 suppresses Emx2 expression. Disproportionate Changes in Sizes of Primary Areas and Shifts in Their Locations Controlled by Emx2 in a Concentration-Dependent Manner

Serotonin immunostaining on tangential sections of flattened cortex of P7 Emx2 heterozygous knockout (Emx2+/, [A]), wild-type (B), heterozygous nestin-Emx2 transgenic (Ne- Emx2 het, [C]), and homozygous nestin-Emx2 transgenic (Ne-Emx2 homo, [D]) mice.

• Levels of Emx2 transcripts increase progressively in the genotypes from (A) to (D), with the lowest level in the Emx2+/ mice and the highest levels in the homozygous nestin-Emx2 transgenic mice.

• As levels of Emx2 increase, V1 expands, S1 is reduced in size and shifts anteriorly, and the domain remaining for frontal/motor areas (F/M) is reduced.

• The level of Emx2 in embryonic cortex determined by real-time PCR (Emx2; Leingartner et al., 2007), and the size of V1 (V1; Hamasaki et al., 2004), relative to that of wildtype (set at 100%), are indicated below each photo. The C3 barrel is marked with an asterisk (*), and the hind paw representation in S1 is marked with a white *, Figure is modified from Hamasaki et al. (2004). Expansion of the Frontal/Motor Areas and Posterior Compression of Sensory Areas in COUPTFI- Deficient Cortex

(A and B) Serotonin (5-HT) immunostaining of flattened cortices of P7 control (COUP-TFIfl/+) and conditional mutant (fl/fl; Emx1-Cre) mice.

(A) Serotonin staining reveals primary sensory areas, Frontal cortex (F) is located anterior to S1.

(B) In COUP-TFI fl/fl; Emx1-Cre conditional mutant brains, the primary sensory areas are much smaller than in controls and are compressed to ectopic positions at the posterior pole of the cortical hemisphere.

(C and D) In situ hybridization for Cad8 on whole mounts uniquely marks the frontal/motor areas (F/M) that massively expand following selective deletion of COUP-TFI.

(E and F) The S1-specific expression of MDGA1 in layers 4 and 6 confirms the reduced size and posterior shift of S1,

The majority of the cortex in the conditional mutants, including all of the neocortex anterior to the reduced, caudally shifted primary sensory areas, exhibits levels and patterns of serotonin staining and expression of MDGA1 that are characteristic of wild-type frontal/motor cortex (F/M). Scale bars, 1 mm. Summary of all reports of loss-of-function or gain-of-function mice mutant for TFs that regulate area patterning.

• Reducing Emx2 levels in the cortex of the heterozygote mutant mice results in posterior shifts of areas with shrinkage of V1, while overexpression of Emx2 under the control of the nestin promoter shifts areas anteriorly. • The small eye mutant without functional Pax6 shows anterior area shifts as judged by the gene expression patterns of cortical area marker genes. Unlike the Emx2 transgenic mice, YAC transgenic mice of Pax6 do not show area changes other than a slight, but significant, reduction in the size of S1 (asterisk). • Loss of COUP-TFI in cortical progenitors transforms the fate of primary sensory areas into frontal/motor areas; thus, COUP-TFI represses frontal/motor fates in sensory area domains. References

1. Neuroscience. 4th edition. Edited by D. Purves et al. Chapter 24. 2. Torborg, C.L and Feller, M.B. 2005 Spontaneous patterned retinal activity and the refinement of retinal projections. Prog. Neurobiology 76:213-235. 5. McLaughlin, T., Hinges, R., and O’Leary, D. 2003 Regulation of axial patterning of the retina and its topographic mapping in the brain. Current Opinion in Neurobiology 13; 57-69. 6. Feldheim, D.A. and O’Leary, D.M.D. (2010) Visual map development: bidirectional signaling, bifunctional guidance molecules, and competition. Cold Spring Harbor Perspect Biol. 2: a001768. 7. Klein R. (2012) Eph/ephrin signaling during development. Development 139: 4105-4109. 8. Cang, J, Niell CM, Liu X, Pfeiffenberger C, Feldheim DA, and Stryker MP (2008) Selective disruption of one Cartesian axis of cortical maps and receptive fields by deficiency in Ephrin-As and structured activity. Neuron 57: 511-523. 9. Grimbert F and Cang J (2012) New model of retino-collicular mapping predicts the mechanisms of axonal competition and explains the role of reverse molecular signaling during development. J. Neuroscience 32: 9755-9768. 10. Triplett JW and Feldheim DA (2012) Eph and ephrin signaling in the formation of topographic maps. Semin. Cell Dev Biol. 23: 7-15. 11. Arvanitis D and Davy A (2008) Eph/ephrin signaling networks. Genes and Development 22: 416-429. 12. Huberman, A.D., Feller, M.B., Chapman B. (2008) Mechanisms underlying development of visual maps and receptive fields. Ann. Rev. Neurosci. 31: 479-509. 13. Xu H-P. et al. Crair (2015) Spatial pattern of spontaneous retinal waves instructs retinotopic map refinement more than activity frequency. Developmental Neurobiology. 14. Xu H-P et al. Crair (2011) An instructive role for patterned spontaneous retinal activity in mouse visual map development. Neuron 70:1115-1127. 15. Godfrey, K.B. and Swindale, N.V. (2014) Modeling development in retinal afferents: retinotopy, segregation, and EphrinA/EphA mutant. PLOS ONE 9: 16. Sweeney, N.T. James, K.N., Sales, E.C., and Feldheim, D.A. (2015) Ephrin-As are required for the topographic mapping but not laminar choice of physiological distinct RGC types. Developmental Neurobiology: 584-593. 17. Arroyo, D.A. and Feller, M.B. 2016 Spatiotemporal features of retinal waves instruct the wiring of the visual circuitry. Front. Neural Cir. 10: 18. O’Leary, DDM, Chou S-J, and Sahara S. 2007 Area patterning of the mammalian cortex. Neuron 1. Batista-Brito, R. and Fishell, G. The developmental integration of cortical interneurons into a functional network. Current Topics in Developmental Biology 87: 82-118, 2009. 2. Cooper LN and Bear MF The BCM theory of synapse modification at 30: interaction of theory with experiment. Nature Reviews 13: 798-810, 2012. 3. Espinosa JS and Stryker MP Development and plasticity of the primary visual cortex Neuron 75: 230-249, 2012. 4. Kuhlman, SJ, Olivas, ND, Tring, E, Ikrar, T, Xu, X, Trachtenberg, JT. A disinhibitory microcircuit initiates critical-period plasticity in the visual cortex. Nature 501:543, 2013. 5. Leone, D. P., Srinivasan, K., Chen, B., Alcamo, E. and McConnell, S.K. The determination of projection neuron identity in the developing cerebral cortex. Current Opinion of Neurobiology 18: 28-35, 2008. 6. Priebe, NJ and McGee, AW. Mouse vision as a gateway for understanding how experience shapes neural circuits. Front. Neural Circuits. 8: 7. Rakic, P. A century of progress in cortico-neurogenesis: from silver impregnation to genetic engineering. Cerebral Cortex 16: i3-i17, 2006. 8. Srishasam, K., Vincent, JL, Livingstone, MS. Novel domain formation reveals proto-architecture in inferotemporal cortex. Nat. Neurosci. 17:1776-1783, 2014. 9. Stryker, MP. A neural circuit that controls cortical state, plasticity, and the gain of sensory responses in mouse. Cold Spring Harbor Symp. Quant. Biol. 79: 1-9, 2014. 10. White, LE and Fitzpatrick, D Vision and cortical map development. Neuron 56: 327-338, 2007. 11. Yu, H., Majewska AK, and Sur M. Rapid experience-dependent plasticity of synapse function and structure in ferret visual cortex in vivo PNAS 108: 21235-21240 2011. 12. Kaneko, M and Stryker, MP. Homeostatic plasticity mechanisms in mouse V1. Philos Trans R Soc. Lond B Biol. Sci. 2017 Mar 5;372(1715). 13. Livingstone et al. Development of the macaque face-patch system. Nat. Communications 2017. 14. Arcaro, MJ and Livingstone MS A hierarchical, retinotopic proto-organization of the primate visual system at birth. 2017. eLIFE. 15. Arcaro MJ et al. Seeing faces is necessary for face-domain formation. Nat. Neuroscience. 20: 1404. 2017 16. Gilbert and Li 2012 Adult visual cortical plasticity. Neuron 75: 250. 17. Keneko and Stryker 2014 Sensory experience during locomotion promotes recovery of function in adult visual cortex. eLIFE 3: Development of extrastriate functional specialization, processing streams and topography: Lessons learned from object category modules and functional connectivity Livingstone et al 2017 Development of the macaque face-patch system Nature Communications 8:

Figure 1 | Visual stimuli used for longitudinal scanning. The first three rows show a subset of the static images; the bottom row shows examples of the videos that were shown to the monkeys younger than 2 months. (Top row) Monkey faces. (Row 2) Scrambled monkey faces, scrambled using the method of Portilla and Simoncelli 51. (Row 3) Familiar rectilinear objects. (Row 4) The three categories of videos shown to monkeys 2 months old: L to R: monkeys grooming each other with prominent faces; same movies with the faces pixelated; traffic or time-lapse nature. Figure 2 | Development of face selectivity. Maps of activations (beta coefficients) for the contrast faces-minus- objects for each static- image scanning session for each of four young monkeys projected onto the standard F99 macaque brain flat map57,58. Where the age is indicated in black, the beta coefficients were thresholded at P<0.05, FDR corrected; No Faces > object activations were present in the STS at P<0.05, so the beta coefficients were thresholded at P<0.25, FDR corrected.

Seeing faces..Arcaro et al 2017

Figure 1 Faces>objects and hands>objects activations in control and face-deprived monkeys. Top, representative maps for the contrast faces-minus objects aligned onto the standard macaque F99 flattened cortical surface (light gray, gyri; dark gray, sulci). These examples show percent signal change (beta coefficients) thresholded at P < 0.01 (FDR corrected) for one session each for two control (B4 and B5; left) and two face-deprived (B6 and B9; right) monkeys at 252, 262, 295, and 270 days old, as indicated. Dotted white ovals indicate the STS region shown in the bottom half for all scan sessions for all seven monkeys. Dotted black outline in B4 map shows the STS ROI used for the correlation analysis described in the text.

Monkeys raised without exposure to faces did not develop face domains, but did develop domains for other categories and did show normal retinotopic organization, indicating that early face deprivation leads to a highly selective cortical processing deficit. Monkeys raised without exposure to faces did not develop face domains, but did develop domains for other categories and did show normal retinotopic organization, indicating that early face deprivation leads to a highly selective cortical processing deficit.

Figure 1 Faces>objects in control and face-deprived monkeys. aligned onto the standard macaque F99 flattened cortical surface (light gray, gyri; dark gray, sulci). These examples show percent signal change (beta coefficients) thresholded at P < 0.01 (FDR corrected) Dotted white ovals indicate the STS region shown in the bottom half for all scan sessions for all seven monkeys. Dotted black outline in B4 map shows the STS ROI used for the correlation analysis.

Activations to Bodies - Scenes Activations to Bodies - Scenes

Figure 3 Activations to bodies minus scenes. Top, subset of the images used for bodies-versus-scenes scans. Bottom, maps (beta values) for bodies minus scenes, thresholded at P < 0.01 (FDR-corrected), (2 controls and 3 FD)

The graph shows the number of voxels in each monkey in each session in each hemisphere in each ROI (outlined in the B4 maps) that were significantly more responsive to bodies than to scenes, or the reverse, as indicated (voxels selected at a P < 0.01 FDR-corrected threshold). Eccentricity organization in control and face-deprived monkeys.

Eccentricity, from 0° (red) to 10° (blue), Average eccentricity organization for two control monkeys (B4 and B5) (left) and two face-deprived monkeys (B6 and B9) (right). Thick black lines indicate areal borders of (left to right) V1, V2, V3, V4, and V4A, and the purple outline indicates the MT cluster, comprising areas MT, MST, FST, and V4t.

Across 19 retinotopic areas, the mean voxel-wise distance between eccentricity maps for the control and face-deprived monkeys was 0.9° ± 1° s.d. The correlation (r) between averaged maps for control and face-deprived monkeys was 0.82. Even considering only IT cortical areas, the mean difference between averaged maps for control and face-deprived monkeys was 1.4° ± 1.5° s.d., with a correlation of 0.66, which is comparable to the consistency between individual control monkeys (1.84° ± 1.76° s.d.; correlation = 0.54). Therefore, experience must be necessary for the formation (or maintenance) of face domains. Gaze tracking revealed that control monkeys looked preferentially at faces, even at ages prior to the emergence of face domains, but face-deprived monkeys did not, indicating that face looking is not innate.

A retinotopic organization is present throughout the visual system at birth, so selective early viewing behavior could bias category-specific visual responses toward particular retinotopic representations, thereby leading to domain formation in stereotyped locations in inferotemporal cortex, without requiring category-specific templates or biases.

Thus, we propose that environmental importance influences viewing behavior, viewing behavior drives neuronal activity, and neuronal activity sculpts domain formation. Figure 5 Looking behavior of control and face-deprived monkeys. (a) Normalized heat maps of fixations for nine of the images that the monkeys freely viewed while their gaze was monitored. Heat maps combine data from four control and three face-deprived monkeys overlaid onto a darkened version of the image viewed (left bottom photograph by A. Stubbs, courtesy of the Neural Correlate Society; middle middle photograph courtesy of D.M. Barron (oxygengroup); lower middle photograph by P. Opaska; all of these were used with permission; top middle image is a substitute for a configurally identical image that we do not have permission to reproduce). (b) Quantification of looking behavior for four control (red) and three face-deprived (green) monkeys from 90 d to 1 year of age. Large black-outlined symbols show means for each monkey. (c) Longitudinal looking behavior for four control monkeys from 2 d to >1 year of age at regions containing faces (filled symbols) or at regions containing hands (open symbols). Yellow bar indicates the range of ages at which control monkeys developed strong stable face patches20. (d) Same as in c for three face-deprived monkeys from 90 d to 383 d of age.

References

1. Neuroscience. 4th edition. Edited by D. Purves et al. Chapter 24. 2. Torborg, C.L and Feller, M.B. 2005 Spontaneous patterned retinal activity and the refinement of retinal projections. Prog. Neurobiology 76:213-235. 5. Feldheim, D.A. and O’Leary, D.M.D. (2010) Visual map development: bidirectional signaling, bifunctional guidance molecules, and competition. Cold Spring Harbor Perspect Biol. 2: a001768. 6. Cang, J, Niell CM, Liu X, Pfeiffenberger C, Feldheim DA, and Stryker MP (2008) Selective disruption of one Cartesian axis of cortical maps and receptive fields by deficiency in Ephrin-As and structured activity. Neuron 57: 511-523. 7. Triplett JW and Feldheim DA (2012) Eph and ephrin signaling in the formation of topographic maps. Semin. Cell Dev Biol. 23: 7-15. 8. Arvanitis D and Davy A (2008) Eph/ephrin signaling networks. Genes and Development 22: 416-429. 9. Huberman, A.D., Feller, M.B., Chapman B. (2008) Mechanisms underlying development of visual maps and receptive fields. Ann. Rev. Neurosci. 31: 479-509. 10.Xu H-P. et al. Crair (2015) Spatial pattern of spontaneous retinal waves instructs retinotopic map refinement more than activity frequency. Developmental Neurobiology. 11.Sweeney, N.T. James, K.N., Sales, E.C., and Feldheim, D.A. (2015) Ephrin-As are required for the topographic mapping but not laminar choice of physiological distinct RGC types. Developmental Neurobiology: 584-593. 12.Arroyo, D.A. and Feller, M.B. 2016 Spatiotemporal features of retinal waves instruct the wiring of the visual circuitry. Front. Neural Cir. 10: 13.O’Leary, DDM, Chou S-J, and Sahara S. 2007 Area patterning of the mammalian cortex. Neuron 14. Livingstone et al. Development of the macaque face-patch system. Nat. Communications 2017. 15.Arcaro, MJ and Livingstone MS A hierarchical, retinotopic proto-organization of the primate visual system at birth. 2017. eLIFE. 16.Arcaro MJ et al. Seeing faces is necessary for face-domain formation. Nat. Neuroscience. 20: 1404. 2017