© 2015. Published by The Company of Biologists Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License

(http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium

provided that the original work is properly attributed.

A Novel Mouse Model of Tuberous Sclerosis Complex (TSC): Eye-specific Tsc1-

ablation Disrupts Visual Pathway Development.

Iwan Jones, Anna-Carin Hägglund, Gunilla Törnqvist, Christoffer Nord, Ulf Ahlgren & Leif

Carlsson.

Umeå Center for Molecular Medicine (UCMM), Umeå University, 901 87 Umeå, Sweden.

Corresponding author: leif.carlsson@umu.se

Keywords: TSC, retina, hamartoma

Summary Statement.

Conditional deletion of Tsc1 in the eye results in hamartoma formation and defects in retinal

ganglion cell development – a novel mouse model providing insights into visual pathway

involvement in TSC.

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

http://dmm.biologists.org/lookup/doi/10.1242/dmm.021972Access the most recent version at DMM Advance Online Articles. Posted 8 October 2015 as doi: 10.1242/dmm.021972http://dmm.biologists.org/lookup/doi/10.1242/dmm.021972Access the most recent version at

First posted online on 8 October 2015 as 10.1242/dmm.021972

Abstract.

Tuberous Sclerosis Complex (TSC) is an autosomal dominant syndrome that is best

characterised by neurodevelopmental deficits and the presence of benign tumours (called

hamartomas) in affected organs. This multiorgan disorder results from inactivating point

mutations in either the TSC1 or the TSC2 genes and consequent activation of the canonical

mammalian target of rapamycin complex 1 signalling (mTORC1) pathway. Since lesions to

the eye are central to TSC diagnosis, we report here the generation and characterisation of the

first eye-specific TSC mouse model. We demonstrate that conditional ablation of Tsc1 in eye

committed progenitor cells leads to the accelerated differentiation and subsequent ectopic

radial migration of retinal ganglion cells. This results in an increase in retinal ganglion cell

apoptosis and consequent regionalized axonal loss within the optic nerve and topographical

changes to the contra- and ipsilateral input within the dorsal lateral geniculate nucleus. Eyes

from adult mice exhibit aberrant retinal architecture and display all the classic

neuropathological hallmarks of TSC including an increase in organ and cell size, ring

heterotopias, hamartomas with retinal detachment and lamination defects. Our results provide

the first major insight into the molecular etiology of TSC within the developing eye and

demonstrate a pivotal role for Tsc1 in regulating various aspects of visual pathway

development. Our novel mouse model therefore provides a valuable resource for future studies

concerning the molecular mechanisms underlying TSC and also as a platform to evaluate new

therapeutic approaches for the treatment of this multiorgan disorder.

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

Introduction.

Genetic disruption during the formation of the central nervous system (CNS) is one of the

underlying causes of neurodevelopmental deficits whose incidence rate is prevalent within

multiorgan syndromes such as Tuberous Sclerosis Complex (TSC) (OMIM191100) (Han and

Sahin, 2011; Smalley, 1998; Thiele, 2004). TSC is an autosomal dominant disorder that is

caused by inactivating point mutations in either the TSC1 (9q34) or the TSC2 (16p13.3) genes.

The protein products of TSC1 and TSC2 (hamartin and tuberin, respectively) form a

heterodimeric complex that is stabilised by a third protein partner (TBC17D). Together this

complex negatively regulates cell growth and proliferation through a canonical signalling

pathway involving Ras homologue enriched in brain (Rheb) and the mammalian target of

rapamycin complex 1 (mTORC1). TSC is best characterised by the presence of benign tumours

(called hamartomas) in affected organs due to uncontrolled cell growth driven by mTORC1

hyperactivity. Hamartomas commonly present as cardiac rhabdomyomas, renal

angiomyolipomas and facial angiofibroma. At the neuropathological level, hamartomas take

the form of white matter radial migration lines (RML), subependymal nodules (SEN),

subependymal giant cell astrocytes (SEGA) and cortical tubers (Capo-Chichi et al., 2013;

Cheadle et al., 2000; Dibble et al., 2012; DiMario, 2004; Garami et al., 2003; Han and Sahin,

2011; Jones et al., 1999; Kwiatkowski and Manning, 2005; Samueli et al., 2015). TSC patients

also present with a myriad of complex neurological deficits with autism and epilepsy being

prevalent amongst affected individuals. These observations clearly demonstrate that TSC is a

multifaceted syndrome where multiple CNS regions contribute to both the neurological and

behavioural components (Costa-Mattioli and Monteggia, 2013; Han and Sahin, 2011; Jeste et

al., 2008; Smalley, 1998).

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

The generation of rodent models has proved to be a robust approach for establishing the

molecular etiology underlying TSC. Germline deletion of either Tsc1 or Tsc2 is embryonic

lethal due to organ dysgenesis while heterozygous animals develop a spectrum of phenotypes

with hepatic hemangiomas, renal carcinoma and renal cysts being prevalent (Kobayashi et al.,

2001; Kwiatkowski et al., 2002; Onda et al., 1999). Conditional Tsc1 and Tsc2 animal models

have also replicated some of the neuropathological features of TSC. Ablation of hamartin or

tuberin in cerebellar Purkinje cells leads to an increase in soma size and dendritic spine density

(Reith et al., 2013; Tsai et al., 2012). Moreover, loss of hamartin in the cerebral cortex and

hippocampus leads to dysplastic neurons, ectopic neurons and reduced myelination; while

astrocyte-specific deletion of Tsc1 initiates astrogliosis and the aberrant migration of

hippocampal pyramidal neurons (Meikle et al., 2007; Uhlmann et al., 2002). Such changes to

CNS architecture subsequently lead to functional and autistic-like behavioural deficits

(McMahon et al., 2014; Meikle et al., 2007; Reith et al., 2013; Tavazoie et al., 2005; Tsai et

al., 2012; Uhlmann et al., 2002). But while these previous conditional ablation studies have

generated significant insight into the neurological and behavioural aspects of TSC, it is still

imperative to generate innovative models that specifically address the roles of hamartin and

tuberin in other TSC-affected organs. This is especially true if animal models are to be used as

platforms to preclinically evaluate novel therapeutic approaches for the treatment of this

multiorgan disorder (Bissler et al., 2013; Franz et al., 2013; Napolioni et al., 2009; Samueli et

al., 2015).

An animal model that addresses the involvement of the eye and visual system in TSC is

currently overlooked. This is especially surprising since: (i) clinical examination of the eye is

one of the original diagnostic procedures used to demonstrate CNS involvement in TSC, (ii)

three distinct morphological groups of retinal hamartomas are routinely observed in TSC

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

patients and (iii) approximately 50% of all TSC patients present with eye involvement (Crino,

2013; Gomez, 1991; Mennel et al., 2007; Samueli et al., 2015; Sepp et al., 1996; Shields et al.,

2004). We report here the generation and characterization of an eye-specific TSC mouse model

that recapitulates the classic neuropathological hallmarks of this syndrome and also

demonstrate a pivotal role for Tsc1 in regulating various aspects of visual pathway

development. Our results provide the first major insight into the molecular etiology of TSC

within the developing eye.

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

Results.

Generation of an eye-specific Tsc1 conditional knockout mouse model.

To generate an eye-specific TSC animal model we used a loss-of-function approach by

breeding mice carrying a conditional Tsc1 allele (Tsc1tm1Djk, referred to as Tsc1+/f or Tsc1f/f)

with mice harbouring an Lhx2 promoter driven Cre-recombinase transgene (Tg(Lhx2-

Cre)1Lcar, referred to as Lhx2-Cre) (Hägglund et al., 2011; Uhlmann et al., 2002). This

transgene promotes recombination starting at embryonic day 8.25 (E8.25) and is expressed in

eye committed neural progenitor cells that give rise to the retinal pigment epithelium (RPE),

neural retina (NR) and optic stalk (Fig. S1A) (Hägglund et al., 2011). To confirm deletion of

Tsc1 we performed immunoblot analysis on eye homogenates (Fig. 1A). A significant

reduction in the amount of hamartin was detected in both Lhx2-Cre:Tsc1+/f and Lhx2-

Cre:Tsc1f/f mice compared with controls (n=3) suggesting that Tsc1 was ablated in an eye

specific manner (Fig. 1B). A major cellular consequence of Tsc1 loss is the activation of

mTORC1 kinase complex and subsequent increase in the levels of phospho-S6 ribosomal

protein (pS6) (Kwiatkowski et al., 2002; Meikle et al., 2007). We observed a significant

increase in pS6 (S235/236) and pS6 (S240/244) levels in Lhx2-Cre:Tsc1f/f mice when

compared to controls (Fig. 1A,B). Thus, conditional deletion of Tsc1 during eye development

results in a reduction of hamartin levels and subsequent hyperactivation of the mTORC1

pathway.

Conditional deletion of Tsc1 leads to enlarged eyes, hamartomas and loss of retinal

architecture.

We first examined the morphology of the eye in Lhx2-Cre:Tsc1f/f mice since hamartoma

formation is one of the pathological hallmarks of TSC (Samueli et al., 2015). Optical projection

tomography (OPT) analysis demonstrated that the eye of postnatal Lhx2-Cre:Tsc1f/f mice was

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

enlarged when compared to control animals (Fig. 2A,B). Most striking however was the

presence of severe retinal folding (Fig. 2B, white arrowhead) and the loss of ora serrata (ORS)

integrity (Fig. 2B, white arrow). These observations indicated that Tsc1 ablation had a profound

impact upon retinal morphology and that the Lhx2-Cre:Tsc1f/f mice were therefore a plausible

model to assess eye involvement in TSC.

The adult retina contains one glial cell type (Müller glia) and six classes of neurons (rod and

cone photoreceptors, horizontal, bipolar, amacrine and ganglion cells) whose cell bodies reside

within three distinct layers: photoreceptor cell bodies reside in the outer nuclear layer (ONL)

while the inner nuclear layer (INL) contains horizontal, bipolar, amacrine and Müller glia cell

bodies. The ganglion cell layer (GCL) contains ganglion cells, whose axons form the nerve

fiber layer (NFL) and displaced amacrine cells. Synapses between photoreceptors, bipolar and

horizontal cells occur in the outer plexiform layer (OPL) whereas connections between bipolar,

amacrine and ganglion cells occur in the inner plexiform layer (IPL) (Fig. 2Q) (Bassett and

Wallace, 2012; Livesey and Cepko, 2001).

Histological staining of coronal eye sections demonstrated that the retinal folding observed in

the OPT derived 3D volumes were hamartomas (Fig. 2C-F) that were commonly organised

into ring heterotopias (Fig. 2C-E) with retinal detachment also being occasionally observed in

areas of hamartoma formation (Fig. 2C,E, asterisks). The hamartomas were subsequently

determined to arise during late embryogenesis and were first consistently detectable at E17.5

(Fig. 2G). Moreover, these lesions were unlikely to arise as a secondary consequence to

aberrant proliferation at the early embryonic ages we analysed since the percentage of BrdU+

cells in the neural retina of control and Lhx2-Cre:Tsc1f/f mice did not differ (Fig. S2).

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

The hamartomas then became more pronounced during postnatal development (Fig. 2H-J) and

consisted of all the cellular classes that are contained within the retina. In addition, the

hamartomas were also enriched in pS6 (S235/236) and contained elevated levels of glial

fibrillary acidic protein (GFAP) that was indicative of reactive gliosis (Fig. 2K-P). Finally,

while laminar organisation of the retina in Lhx2-Cre:Tsc1f/f mice was grossly maintained (Fig.

2Q-T), several other abnormalities were readily apparent including an irregular ordering of the

INL and an IPL populated with ectopic cells (Fig. 2R,T, open black arrowheads). We also

observed a widening of the GCL and NFL (Fig. 2S,T, black brackets). This was corroborated

by neurofilament (NF) immunostaining, which demonstrated disorganised nuclei and aberrant

neurite trajectories in the GCL and NFL of Lhx2-Cre:Tsc1f/f mice (Fig. S3).

Conditional deletion of Tsc1 leads to aberrant neurite stratification of retinal

interneurons.

Conditional deletion of Tsc1 in the eye led to several morphological changes within the retina

that recapitulated many of the hallmarks of TSC. We therefore took advantage of this eye-

specific model to determine if the laminar disorganisation observed in Lhx2-Cre:Tsc1f/f mice

influenced neuronal circuitry. We first chose to focus on amacrine and bipolar neurons since

we observed a disorganised INL and IPL that contained cells with elevated levels of pS6

(S235/236) protein in Lhx2-Cre:Tsc1f/f mice (Fig. 3A,B). The IPL is subdivided into two

parallel circuits that respond to either a decrease (OFF pathway) or an increase (ON pathway)

in light intensity. These circuits are organised into distinct sublaminae (S) that allow for the

processing of visual information (OFF sublaminae: S1 and S2, ON sublaminae: S3, S4 and S5)

(Sanes and Zipursky, 2010).

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

To identify all amacrine cells we analysed for the presence of Pax6 protein (Ma et al., 2007).

The amacrine cell layer (Pax6+) was severely disrupted within the INL of Lhx2-Cre:Tsc1f/f

mice (Fig. 3C,D). A high number of Pax6+ cells were also observed within the IPL suggesting

that the majority of ectopic cells observed in our mutant mice are either amacrine or retinal

ganglion cells (RGCs) as this neuron class is also recognised by the Pax6 antibody (Fig. 3D,

yellow arrowheads) (Ma et al., 2007). To ask whether the disorganised cell body layers

influenced neurite positioning within the IPL we analysed the stratification patterns of

cholinergic (ChAT+, S2 and S4) and calretinin+ (S1/S2, S2/S3 and S3/S4) amacrine cell

subtypes (Fig. 3E-H). Ectopic ChAT+ neurites could be clearly seen extending into other

sublaminae (Fig. 3F, open yellow arrowheads) while a loss of specific sublamina stratification

(e.g. S2/S3) was observed for the calretinin+ cells (Fig. 3H, red arrowhead). Moreover, rod

bipolar cells (PKC+) elaborated distorted axonal projections through the INL (Fig. 3J, open

red arrowheads) and terminated with hypertrophic axon pedicles within enlarged S4 and S5

ON sublaminae (Fig. 3J, red arrow). The ectopic cell and dendritic stratification phenotype had

full penetrance as we observed at least one region in each eye analysed (n=18) that contained

ectopic cells, ectopic neurites or loss of stratification. The fact that we observed ectopic Pax6+

cells, aberrant neurite stratification and changes to sublaminae formation suggests profound

changes to the structure of the IPL in Lhx2-Cre:Tsc1f/f mice. We therefore performed scanning

electron microscopy (SEM) and observed that the IPL in Lhx2-Cre:Tsc1f/f mice was overgrown

and densely packed when compared to control animals (Fig. 3K,L). In conclusion, our

observations demonstrate that Tsc1 is involved in regulating cell body position within the INL

and stratification within the IPL. Moreover, the overgrown and densely packed IPL might form

a physical barrier that could partly explain the presence of ectopic cells within this region.

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

Conditional deletion of Tsc1 leads to accelerated RGC differentiation and radial

migration defects.

Our immunostaining analyses demonstrated that the disorganised cells in the GCL of Lhx2-

Cre:Tsc1f/f mice were enriched in pS6 (S235/236) protein (Fig. 3B). Moreover, aberrant and

defasciculated axonal projections were also evident in the NFL (Fig. S3B). These combined

observations suggest that Tsc1 is involved in regulating several aspects of RGC biology. We

therefore chose to examine RGC differentiation in our TSC mouse since this cell type is the

first neuronal class to differentiate in the retina and they appear following Tsc1 ablation. RGCs

are also an ideal model in which to study visual pathway development from the perspectives

of both neuronal circuitry and axonal guidance (Erskine and Herrera, 2007). Firstly, we

examined RGC differentiation during embryonic and postnatal ages and chose time points that

coincided with the important milestones of RGC development: neurogenesis (E12.5),

migration (E17.5) and target innervation (postnatal day 1 (P1) through P7) (Huberman et al.,

2008; Reese, 2011).

To follow RGC differentiation we analysed for the presence of Brn3 protein (Pan et al., 2005).

Initially, we found a few scattered Brn3+ RGCs at E11.5 in both control and Lhx2-Cre:Tsc1f/f

mice (Fig. 4A,B yellow arrowheads). The initial wave of RGCs born in the dorsocentral retina

was detected at E12.5 in control mice (Fig. 4C, yellow arrowhead). However, we found a

proportional increase in the number of Brn3+ cells in the Lhx2-Cre:Tsc1f/f mice that were

dispersed both dorsally and ventrally throughout the NR (Fig. 4D, yellow arrowheads). To

confirm this accelerated differentiation we analysed the expression patterns of Math5, Dlx1

and Dlx2 since these genes are essential for RGC development (de Melo et al., 2003; Yang et

al., 2003). While the expression of Math5, Dlx1 and Dlx2 in control mice was observed only

in the dorsocentral retina (Fig. 4E,G,I, open black arrowheads), the expression domains of these

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

genes in Lhx2-Cre:Tsc1f/f mice were expanded both dorsally and ventrally into the peripheral

NR as observed for Brn3 (Fig. 4F,H,J, open black arrowheads). Moreover, the maintenance of

dorsoventral identity based on the expression domains of Tbx5 and Vax2 demonstrates that the

accelerated differentiation of RGCs in the Lhx2-Cre:Tsc1f/f mice was not due to changes in

dorsoventral patterning (Fig. S4) (Behesti et al., 2006).

RGC differentiation in control mice proceeded in a wave from central to peripheral retina and

by E17.5 all the Brn3+ cells had migrated into their final laminar position within the future

GCL (Fig. 4K). The GCL then became more defined during the first postnatal week (P1, Fig.

4M and P7, Fig. 4O) and by the completion of neurogenesis at P14 it contained an ordered

radial distribution of Brn3+ RGCs (Fig. 4Q) (Young, 1985). In contrast, while the majority of

Brn3+ cells in the Lhx2-Cre:Tsc1f/f mice were observed to migrate into their final laminar

position, ectopic cells were detected beginning at E17.5 (Fig. 4L, red arrowheads) that became

readily apparent in all mice analysed at postnatal ages (P1, Fig. 4N; P7, Fig. 4P and P14, Fig.

4R; red arrowheads). Thus, conditional deletion of Tsc1 induces accelerated differentiation and

aberrant radial migration of RGCs.

Conditional deletion of Tsc1 leads to a reduction in RGC number with the remaining cells

exhibiting enlarged nuclei and asymmetric mosaic distribution.

The proportion of Brn3+ RGCs appeared to be reduced in postnatal Lhx2-Cre:Tsc1f/f mice. This

was confirmed by cell count analysis (Fig. 4S). Although the initial numbers of Brn3+ RGCs

was increased in the Lhx2-Cre:Tsc1f/f mice due to accelerated differentiation (E12.5), there was

a subsequent reduction in the population of Brn3+ cells through embryonic (E17.5) and

postnatal development (P3) compared to control animals. We therefore performed flat mount

analyses in order to further quantify the reduction in RGC numbers following the completion

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

of retinal neurogenesis (Fig. 5A,B) (Young, 1985). In control retinas, Brn3+ nuclei were

dispersed in regularly patterned mosaics. Contrastingly, loss of RGC nuclei in Lhx2-Cre:Tsc1f/f

mice resulted in asymmetric mosaic distribution. To confirm this irregularity we examined the

spatial properties using Voronoi analysis that computes the territory surrounding each cell

relative to its neighbours (Cantrup et al., 2012; Galli-Resta et al., 1997; Kay et al., 2012). The

Voronoi domain diagrams revealed an increase in cell territory occupied in Lhx2-Cre:Tsc1f/f

mice while the Voronoi regularity index demonstrated that these enlarged territories were

organised as irregular mosaics (Fig. 5C-E). To confirm RGC loss we performed cell count

analysis and observed a significant reduction in the number of Brn3+ cells in Lhx2-Cre:Tsc1f/f

mice compared to controls (Fig. 5F). We reasoned that an increased rate of apoptosis could

account for this observation and therefore quantified the number of activated caspase3+ cells

on serial sections taken from comparable regions of control and Lhx2-Cre:Tsc1f/f retinas. At all

ages analysed we observed a significant increase in the number of apoptotic cells in Lhx2-

Cre:Tsc1f/f mice (Fig. S5). Our observations therefore demonstrate that an increased level of

programmed cell death is one mechanism that contributes to the decreased numbers of RGCs

in Lhx2-Cre:Tsc1f/f mice. Finally, the remaining RGC nuclei appeared to be enlarged in Lhx2-

Cre:Tsc1f/f mice. To quantify this enlargement we measured the cross sectional area of Brn3+

cells in the same retinal flat mounts. We observed a significant increase in the size of the

remaining RGC nuclei within Lhx2-Cre:Tsc1f/f mice compared to controls (Fig. 5G). In

summary, conditional deletion of Tsc1 leads to a loss of RGCs at postnatal ages and this

reduction in cell number leads to asymmetric mosaics in Lhx2-Cre:Tsc1f/f mice. Of the RGCs

that survive, these cells exhibit increased nuclei size. Tsc1 is therefore critical for regulating

cell survival and size.

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

Conditional deletion of Tsc1 leads to a loss of retinogeniculate topography.

RGCs project their axons into the NFL which subsequently elaborate in a radial fashion to exit

the eye at the optic disc. From here, they enter the optic nerves (ON) and project through the

optic tracts (OT) to target the visual centres of the brain that includes the dorsal lateral

geniculate nucleus (dLGN) of the thalamus (Erskine and Herrera, 2007; Godement et al., 1984).

Analysis of RGC axon trajectory during embryonic development demonstrated a comparable

optic disc exit pattern in control and Lhx2-Cre:Tsc1f/f mice (Fig. S6). We therefore reasoned

that changes in ON composition would be observed during postnatal development in Lhx2-

Cre:Tsc1f/f mice since this time frame coincided with the greatest loss of RGC nuclei within

the retina (Figs. 4S, 5F and S5I). Transverse sections through the ON of control mice

demonstrated a clearly defined network of GFAP+ astrocytes that enmeshed the neurofilament

positive (NF+) RGC axons within the ON at both P7 and P18 (Fig. 6A,C,E,G). In contrast,

regionalized areas of reactive gliosis were clearly visible in several domains of the Lhx2-

Cre:Tsc1f/f ON at P7 (Fig. 6B,D, solid white arrowheads). Reactive gliosis in addition to

regionalized axonal loss became more apparent at P18 in the Lhx2-Cre:Tsc1f/f mice since we

observed cross sectional areas that were significantly enriched in GFAP+ astrocytes and

completely devoid of NF+ RGC axons (Fig. 6F,H, open white arrowheads). That these changes

were observed as patches and not randomly distributed across the ON clearly correlates with

the selective loss of RGC nuclei in discrete domains across the retina observed in our flat mount

analysis (Fig. 5B). These changes observed in the ON of Lhx2-Cre:Tsc1f/f mice were

reminiscent to those documented in a DBA/2J mouse model of glaucoma (Howell et al., 2007).

We therefore measured the intra ocular pressure (IOP) in control and Lhx2-Cre:Tsc1f/f mice

and found them to be comparable (Mean ± SEM of 18 ± 0.82 and 21 ± 1.9 mmHg for control

and Lhx2-Cre:Tsc1f/f mice, respectively).

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

We next proceeded to investigate whether the remaining axons in Lhx2-Cre:Tsc1f/f mice would

target the dLGN. RGC projections were labelled with an anterograde tracer to visualize the

binocular retinogeniculate projections within the dLGN (Fig. S7A). The inputs from each eye

in control mice were segregated with a single ipsilateral patch in the dorsomedial quadrant of

the dLGN being surrounded by the contralateral projection (Fig. 7A,B). As expected, a

significant reduction in the area occupied by both contralateral and ipsilateral projections were

observed in Lhx2-Cre:Tsc1f/f mice (Fig. 7C-E). This result clearly correlates with the dramatic

reduction in total RGC number and regionalised axonal loss within the ON. We also observed

a significant decrease in the percentage of the dLGN occupied by ipsilateral projections in the

Lhx2-Cre:Tsc1f/f mice (Fig. 7F-J). Also of note was the observation that the topography and

position of the ipsilateral patch within the dLGN of Lhx2-Cre:Tsc1f/f mice was altered in all

animals along the dorsoventral and lateromedial axis (Figs. 7H,I) and in some instances we

observed more than one ipsilateral patch (Fig. S7B-E, yellow arrows). Our data therefore

demonstrates that conditional deletion of Tsc1 has a profound influence on the development of

the visual pathway as demonstrated by a decrease in occupied contra- and ipsilateral territories

within the dLGN.

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

Discussion.

This study describes the generation of a novel eye-specific model of TSC that recapitulates

many of the neuropathological hallmarks of this multiorgan disorder: increase in organ and cell

size, ring heterotopias, hamartomas with retinal detachment and lamination defects (Crino,

2013; Shields et al., 2004). Moreover, conditional ablation of Tsc1 lead to severe disruptions

in visual pathway development as evidenced by: (i) accelerated differentiation of RGCs, (ii)

ectopic radial migration, (iii) neurite stratification deficits, (iv) an increase in apoptosis, (v) ON

degeneration and (vi) aberrant retinogeniculate topography (Fig. S8).

The mutant mice described in this report differ significantly from the majority of published

TSC models since we ablated Tsc1 during early embryonic development in a progenitor cell

population that generates both neural and glial lineages within the retina. This is in contrast to

previous studies where Tsc1 or Tsc2 genes have been ablated during later embryonic

development in either neuronal or glial lineages independently (Ehninger et al., 2008; Feliciano

et al., 2012; Meikle et al., 2007; Uhlmann et al., 2002; Way et al., 2009; Zeng et al., 2011;

Zhou et al., 2011). While these previous models documented some of the neuropathological

changes associated with TSC, none of them recorded the presence of hamartoma-like lesions.

This is presumably due to the fact that Cre-mediated recombination was initiated after tissue

architecture was already established. We therefore propose that hamartoma formation occurs

due to the fact that the Tsc1tm1Djk allele is recombined in a progenitor cell population prior to

the formation of any rudimentary eye structure (Hägglund et al., 2011) and that this might also

be a potential mechanism that drives hamartoma formation in TSC patients. Although it must

be noted that our fate mapping experiments demonstrated that the Lhx2-Cre transgene did not

recombine the ROSA26 allele in all parts of the retina in postnatal mice (Fig. S1B). We

therefore concede that some of the hamartomas formed in Lhx2-Cre:Tsc1f/f mice could be

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

mosaic in nature and composed of both Tsc1-null and normal cells. Non-cell autonomous

mechanisms could therefore also potentially contribute to hamartoma formation in our TSC

model as was previously demonstrated in zebrafish (Kim et al., 2011). We also cannot exclude

the possibility that reactive Müller glia cells are also involved in hamartoma formation in Lhx2-

Cre:Tsc1f/f mice as was recently demonstrated for other rodent models of retinal degeneration

(Hippert et al., 2015).

The eyes of Lhx2-Cre:Tsc1f/f mice were grossly indistinguishable from their littermates at birth.

The developmental processes involved in the formation of the rudimentary eye structure, i.e.

diencephalon evagination, optic vesicle formation, optic cup transformation and optic fissure

closing are therefore presumably independent of TSC-mTORC1 function (Adler and Canto-

Soler, 2007). OPT analysis demonstrated that the initial striking feature of Lhx2-Cre:Tsc1f/f

mice was that their eyes were enlarged compared to control littermates. Our observations

corroborate the original Drosophila melanogaster studies where an enlarged eye phenotype

was generated upon Tsc1 or Tsc2 ablation and further demonstrates that mTORC1 signalling

controls organ size within the nervous system (Anderl et al., 2011; Ehninger et al., 2008; Goto

et al., 2011; Ito and Rubin, 1999; Magri et al., 2011; Potter et al., 2001; Tapon et al., 2001;

Uhlmann et al., 2002; Way et al., 2009; Zeng et al., 2011; Zhou et al., 2011). Histological

analysis demonstrated the presence of ectopic cells in the IPL and adds further support to the

notion that TSC should be regarded as a neuronal migration syndrome (Feliciano et al., 2012;

Feliciano et al., 2011; Gomez, 1991; Magri et al., 2011; Way et al., 2009). Ectopic cells within

the IPL have also been observed upon deletion of other tumour suppressors such as Zac1 and

PTEN (Cantrup et al., 2012; Ma et al., 2007; Sakagami et al., 2012). The authors postulated

that both Zac1 and PTEN directly regulated cell migration by controlling the expression of cell

adhesion genes (Cantrup et al., 2012; Ma et al., 2007; Varrault et al., 2006). That the loss of

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

hamartin has also been documented to disrupt cell adhesion therefore provides a potential

mechanism for the aberrant radial migration and the appearance of ectopic cells observed in

this study (Lamb et al., 2000).

Neurites are segregated into distinct synaptic sublaminae within the IPL of the vertebrate retina

and this organisation establishes the basis for connectivity and function (Sanes and Zipursky,

2010). However, SEM analysis demonstrated that the IPL of Lhx2-Cre:Tsc1f/f mice was

overgrown and densely packed. This phenotype bears gross morphological similarity to

previous studies where Tsc1-ablation resulted in increased dendritic spine density in Purkinje

cells and an increase in spine length and width in hippocampal pyramidal neurons (Tavazoie

et al., 2005; Tsai et al., 2012). We therefore believe that conditional deletion of Tsc1 in the eye

leads to an increase in dendritic elaboration during development and that this aberrant neurite

outgrowth is responsible for the densely packed nature of the IPL seen in Lhx2-Cre:Tsc1f/f

mice. That rod bipolar cells were also observed to possess enlarged axon pedicles supports our

hypothesis. Moreover, this overgrowth could also function as a physical barrier and contribute

to the ectopic radial migration phenotype discussed above. Furthermore, the stratification

deficits seen for the amacrine cell subtypes may develop as a direct consequence of this

dendritic overgrowth since sublaminae organisation could be compromised in Lhx2-Cre:Tsc1f/f

mice. But what is also intriguing is that the aberrant IPL neurite targeting seen in our study is

also reminiscent of reports where transmembrane semaphorin or plexin signalling is perturbed

during retinal stratification (Matsuoka et al., 2011a; Matsuoka et al., 2011b; Sun et al., 2013).

Semaphorins mediate their effects through mTORC1 signalling (Campbell and Holt, 2001) and

therefore changes to semaphorin-plexin signalling as a direct result of Tsc1-ablation could also

be a contributing mechanism for the sublaminae deficits reported here.

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

We observed accelerated RGC differentiation that is reminiscent to a previous Drosophila

study (Bateman and McNeill, 2004). The authors demonstrated a loss of temporal neurogenic

control upon Tsc1 ablation that induced accelerated photoreceptor differentiation. Most

importantly, no changes to cell specification were observed. The authors therefore concluded

that loss of Tsc1 does not alter cell fates but has an impact upon the temporal control of when

cell fate decisions are made. It is therefore plausible that the accelerated differentiation of

RGCs observed in our study arises from a similar mechanism. That we observed the expanded

expression domains for several genes involved in RGC development (Math5, Dlx1 and Dlx2)

adds support to this hypothesis. Further studies into the mechanistic regulation of accelerated

RGC neurogenesis in Lhx2-Cre:Tsc1f/f mice are required. But the recent demonstration that

Unkempt and Headcase act in the same pathway to negatively regulate the temporal control of

Drosophila photoreceptor differentiation downstream of Tsc1-mTOR provides an intriguing

avenue for future investigation, particularly since both Unkempt and Unkempt-like are

expressed in the mouse retina (Avet-Rochex et al., 2014; Blackshaw et al., 2004).

RGCs project to the dLGN in a stereotypical manner with retinogeniculate topography being

established by a combination of EphA/ephrin-A signalling, patterned retinal activity-dependant

axonal refinement and competition (Feldheim et al., 1998; Penn et al., 1998; Torborg and

Feller, 2005). Notable differences were observed in retinogeniculate topography of the Lhx2-

Cre:Tsc1f/f mice. Firstly, an expected reduction in the total (contra- and ipsilateral) and

ipsilateral-only territories was evident. This was presumably due to RGC apoptosis and the

consequent disruption to mosaic regularity we observed in Lhx2-Cre:Tsc1f/f mice. Secondly,

the topography and position of the ipsilateral patch within the dLGN of Lhx2-Cre:Tsc1f/f mice

was altered and in some instances we observed more than one ipsilateral patch. This phenotype

is reminiscent to the dLGN ipsilateral projection phenotype reported for nicotinic acetylcholine

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

receptor 2 (nAChR2), ephrin-A2/A3/A5 and Phr1 mouse models (Culican et al., 2009;

Pfeiffenberger et al., 2005; Rossi et al., 2001). Three possible mechanisms could account for

this observed ipsilateral phenotype: (i) Firstly, Lhx2-Cre:Tsc1f/f mice could have defects in

axonal refinement. Previous studies have demonstrated that nAChR2-driven retinal waves

during the first postnatal week of development are critical for dLGN axonal refinement (Cang

et al., 2005; Grubb et al., 2003; Rossi et al., 2001). nAChR2-null mice have defects in axonal

refinement that are reminiscent to those observed in our study and therefore we cannot rule out

the possibility that Lhx2-Cre:Tsc1f/f mice display a similar loss of retinal activity within the

first postnatal week that contributes to the topographic phenotypes reported here. (ii) A second

possible mechanism is that Lhx2-Cre:Tsc1f/f mice could have defects in EphA/ephrin-A

dependent axon guidance mechanisms similar to that recently documented for Tsc2

heterozygous mice. The authors demonstrated that RGCs in Tsc2+/- mice had aberrant

ipsilateral projections and provided mechanistic data suggesting that TSC-mTORC1 signalling

cooperates with the ephrin-Eph receptor system to control projection topography within the

dLGN (Nie et al., 2010). Our observations expand on this previous report and clearly

demonstrate that the aberrant ipsilateral topography occurs through an RGC autonomous

mechanism since our fate mapping experiments verified that the Lhx2-Cre transgene was not

expressed in the dLGN (Fig. S7F) (iii) A third potential mechanism for the dLGN topographic

phenotype observed in Lhx2-Cre:Tsc1f/f mice is one that is independent of retinal activity or

EphA/ephrin-A signalling. A recent study reported that conditional deletion of a PHR protein

family member (Phr1) led to eye-specific domains within the dLGN being severely disturbed

in a manner similar to that observed in Lhx2-Cre:Tsc1f/f mice (Culican et al., 2009). The authors

did not propose a specific mechanism for the observed retinotopic changes upon Phr1 deletion,

but this third mechanism is intriguing since PHR proteins are enriched in the CNS and have

been demonstrated to interact and regulate hamartin:tuberin activity (Murthy et al., 2004).

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

In conclusion, we have generated a novel eye-specific model of TSC that recapitulates many

of the neuropathological hallmarks of this syndrome: an increase in organ and cell size, ring

heterotopias, hamartomas with retinal detachment and lamination defects. Moreover, our

results demonstrate a pivotal role for Tsc1 in regulating various aspects of visual pathway

development and demonstrate that TSC-mTORC1 pathway is critically involved in neuronal

differentiation, migration and circuit formation. Our mouse model will provide a valuable

resource for future studies concerning the molecular mechanisms underlying TSC and also as

a platform to evaluate new therapeutic approaches for the treatment of this multiorgan disorder.

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

Materials and Methods.

Animals.

All animal experiments were approved by the Animal Review Board at the Court of Appeal of

Northern Norrland in Umeå. The generation and genotyping of Tg(Lhx2-Cre)1Lcar transgenic

mice (referred to as Lhx2-Cre), Tsc1tm1Djk mice (referred to as Tsc1+/f or Tsc1f/f) and

Gt(ROSA)26Sortm1sor reporter mice (referred to as ROSA26R) have been described previously

(Hägglund et al., 2011; Soriano, 1999; Uhlmann et al., 2002). The genotype of all animals was

determined by PCR analysis of genomic DNA extracted from tail biopsies. Primers used to

identify Lhx2-Cre and ROSA26R mice have been described previously (Hägglund et al., 2011).

Primers used to identify Tsc1tm1Djk mice were IMR4008 5-GTCACGACCGTAGGAGAAGC-

3 and IMR4009 5´-GAATCAACCCCACAGAGCAT-3. Breeding Lhx2-Cre:Tsc1+/f and

Tsc1f/f mice generated experimental animals. Breeding Lhx2-Cre and ROSA26R mice generated

fate-mapping animals. The morning of the vaginal plug was considered as E0.5. Littermates

lacking the Lhx2-Cre transgene that were either heterozygous or homozygous for the Tsc1tm1Djk

allele were used as controls in all experiments. Lhx2-Cre:Tsc1f/f mice were born in Mendelian

ratios (19 of 74 pups were Lhx2-Cre:Tsc1f/f in one set of genotyping experiments) and no gross

eye defects were evident at birth. Lhx2-Cre:Tsc1f/f mice were indistinguishable from controls

until P5. Thereafter, they failed to thrive and no Lhx2-Cre:Tsc1f/f mice survived longer than

P21 (Fig. S9). The observed mortality presumably occurs due to the activity of the Lhx2-Cre

transgene and consequent ablation of the Tsc1tm1Djk allele in other domains outside the retina

(Hägglund et al., 2011).

Immunoblotting.

The cornea, lens, ON and blood vessels were removed from enucleated eyes and the retina/RPE

was snap frozen in liquid nitrogen. Tissues were homogenized in SDS lysis buffer (100 mM

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

Tris, pH 6.8; 2% (w/v) SDS), containing protease and phosphatase inhibitor cocktails

(Complete Mini & PhosSTOP, Roche) using a TissueLyser (Qiagen Inc.). The protein extracts

were centrifuged at 14000 x g for 5 min at 4oC and the concentration of the soluble fraction

was measured using the BCA Protein Assay Kit (Fisher Thermo Scientific). Western blotting

was performed on soluble protein extracts (10 g) using Criterion TGX gels (BioRad) and

nitrocellulose membranes (BioRad) as previously described (Mahmood and Yang, 2012).

Reactive proteins were visualized using SuperSignal West Dura Extended Duration Substrate

(Fisher Thermo Scientific). Imaging and quantification was performed using a ChemiDoc MP

Imaging System (Biorad). The following antibodies and dilutions were used: Hamartin (Cell

Signalling Technology, 1:1000), S6 (Cell Signalling Technology, 1:1000), pS6 (S235/236)

(Cell Signalling Technology, 1:1000), pS6 (S240/244) (Cell Signalling Technology, 1:1000)

and GAPDH (Cell Signalling Technology, 1:30000).

Optical Projection Tomography (OPT).

Enucleated eyes were pierced through the cornea with a 27-gauge needle to allow for fixative

infusion and prepared for OPT scanning as described previously (Alanentalo et al., 2007).

Specimens were scanned in transmission OPT mode on a Bioptonics 3001 OPT scanner

(Bioptonics) and the acquired data was processed as described previously (Cheddad et al.,

2012). 3D volumes based on the tomographic reconstructions were rendered in Drishti software

(Version 2.2).

Histology.

Heads or enucleated eyes were fixed in 2% (w/v) glutaraldehyde in PBS overnight at 4oC. Eyes

were immersed in 70% (v/v) ethanol and paraffin embedded. Paraffin sections (10 m) were

cleared by 2 x 5 min incubation in xylenes before rehydration though a graded series of ethanol

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

(99.5%, 95%, 90% and 80% (v/v) in PBS). Haematoxylin and eosin staining was performed as

previously described (Hägglund et al., 2011).

Immunohistochemistry.

For cryosection analysis, heads or enucleated eyes were fixed in 4% (w/v) PFA in PBS for up

to 2 h on ice, equilibrated overnight at 4oC in 30% (w/v) sucrose in PBS and embedded in OCT

compound (Sakura Finetek). For flat mount analysis, retinae were dissected out from

enucleated eyes and individually placed in separate wells of a 24-well plate and fixed in 4%

(w/v) PFA in PBS for 2 h on ice. Immunohistochemistry was performed on cryosections (10 –

20 m) or flat mount retinae as previously described (Cantrup et al., 2012; Ma et al., 2007).

The following antibodies and dilutions were used: pS6 (S235/236) (Cell Signalling

Technology, 1:100), Brn3 (Santa Cruz Biotechnology, 1:50), activated caspase3 (Abcam,

1:1000), NF (SMI31) (Covance, 1:1000), GFAP (Chemicon, 1:400), Pax6 (DSHB, 1:100),

ChAT (Millipore, 1:100), Calretinin (Swant, 1:1000), PKC (Santa Cruz Biotechnology,

1:100), Calbindin-D (Sigma-Aldrich, 1:100) and p27Kip1 (BD Biosciences, 1:750).

Proliferation Analyses.

Pregnant mice were injected intraperitoneally with 5-bromo-2′-deoxyuridine (BrdU) at 50 µg/g

body weight for 5 minutes before sacrifice and embryo collection. Embryonic heads were fixed

in 4% (w/v) PFA in PBS for up to 2 h on ice, equilibrated overnight at 4oC in 30% (w/v) sucrose

in PBS and embedded in OCT compound (Sakura Finetek). Cryosections (10 m) were washed

in PBS and then denatured in 2 M HCl for 30 minutes at 37°C. The slides were subsequently

neutralised with 0.1 M borate buffer (pH 8.5) for 10 minutes at RT followed by

immunohistochemistry as described above using an anti-BrdU antibody (BD Pharmingen,

1:20).

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

In Situ Hybridisation.

Heads or enucleated eyes were fixed in 4% (w/v) PFA in PBS for up to 2 h on ice, equilibrated

in 30% (w/v) sucrose in PBS overnight at 4oC and embedded in OCT compound (Sakura

Finetek). In situ hybridization on cryosections (10 m) was performed as previously described

(Schaeren-Wiemers and Gerfin-Moser, 1993). The following IMAGE clones (Source

Bioscience) were purchased to generate in situ probes: Crx (Clone #4527863), Dlx1 (Clone

#30360273), Dlx2 (Clone #5718422), Math5 (Clone #6824509), Tbx5 (Clone #30548234) and

Vax2 (Clone #40101825).

Intra Ocular Pressure (IOP) Measurement.

IOP measurements were performed using a Tonolab instrument (Icare Finland Oy) according

to the manufacturers instructions.

Lhx2-Cre:ROSA26R Fate-Mapping.

Embryonic heads or enucleated eyes were harvested from Lhx2-Cre:ROSA26 mice and fixed

in 4% (w/v) PFA in PBS for up to 2 h on ice. The tissues were subsequently equilibrated in

30% (w/v) sucrose in PBS overnight at 4oC, embedded in OCT compound (Sakura Finetek)

and coronal cryosections (10 m) were prepared. Adult Lhx2-Cre:ROSA26 mice (>6 weeks

old) were transcardially perfused with 30 ml of ice cold 4% (w/v) PFA in PBS. The brain was

subsequently removed and embedded in 4% (w/v) agarose in PBS. Coronal vibratome sections

(100 m) were prepared. -galactosidase staining was performed as described previously

(Hägglund et al., 2011).

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

Retinogeniculate Projection Analysis.

Heads were fixed in 4% (w/v) PFA in PBS for 24 h at 4oC. The cornea and lens were removed

from each eye and a 1-mm2 piece of Neurovue Red Plus or Neurovue Maroon (Polysciences

Inc.) was inserted into the optic disc (Fig. S7A). The heads were incubated in 4% (w/v) PFA

in PBS for 8 weeks at 37oC. The brain was subsequently removed and embedded in 4% (w/v)

agarose in PBS. Coronal vibratome sections (100 m) were counterstained with DAPI and

mounted with Aqua Polymount (Polysciences Inc.). Retinogeniculate analyses were performed

as described previously (Leamey et al., 2007) and were calculated across the entire rostrocaudal

extent of the dLGN. The total area (m2) of the dLGN (as determined by DAPI staining) was

first calculated using the freehand selection tool in Fiji (Schindelin et al., 2012). Background

was then subtracted using the rolling ball function and the area occupied by both the

contralateral and ipsilateral retinogeniculate projections was calculated using the same

approach as above for the total area analysis. The territory occupied by the ipsilateral input was

subsequently calculated as a percentage of the total dLGN area.

Scanning Electron Microscopy (SEM).

Enucleated eyes were pierced through the cornea with a 27-gauge needle to allow for fixative

infusion and fixed in 2% (w/v) glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) overnight

at 4oC. Eyes were subsequently immersed in 70% (v/v) ethanol and paraffin embedded.

Paraffin sections (10 m) were cleared by 2 x 5 min incubation in xylenes. Slides were

subsequently washed 2 x 5 min in 99.5% (v/v) ethanol and allowed to air dry. Dehydrated

samples were attached onto aluminium mounts using carbon adhesive tape and coated with 5

nm gold/palladium. Section morphology was examined using a Zeiss Merlin field emission

scanning electron microscope using a secondary electron detector at a beam accelerating

voltage of 4kV and probe current of 150pA.

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

Image analyses.

Immunohistochemistry and neurovue images were captured using a Zeiss LSM 710 confocal

microscope. Histology, in situ hybridisation and fate mapping images were captured using a

Nikon Eclipse E800 microscope fitted with a Nikon DS-Ri1 digital colour camera. All images

were compiled and analysed using Fiji (Schindelin et al., 2012), CellProfiler (Carpenter et al.,

2006; Kamentsky et al., 2011), Adobe Photoshop and/or Adobe Illustrator.

Statistical Analyses.

All statistical analyses were performed using Prism6 (GraphPad Software). Unpaired two-

tailed Student’s t-tests were used to determine statistical significances. All statistical analyses

were performed on data derived from at least 3 mice of each genotype. Error bars in all figures

represent the standard error of the mean. p-values are indicated as follows: ns, not significant;

*p 0.05, **p 0.01, ***p 0.001, ****p 0.0001.

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

Acknowledgements.

The authors would like to thank Sara Wilson, Camilla Holmlund and Carolina Wählby for

critical reading of the manuscript and technical assistance. The authors acknowledge the

facilities and technical assistance of the Umeå Core Facility Electron Microscopy (UCEM) at

the Chemical Biological Center (KBC), Umeå University. The Pax6 monoclonal antibody,

developed by Atsushi Kawakami, was obtained from the Developmental Studies Hybridoma

Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department

of Biology, Iowa City, IA 52242.

Competing Interests.

The authors declare no competing interests.

Author Contributions.

I.J., A-C.H., G.T., C.N., U.A. and L.C. designed and conceived the experiments.

I.J., A-C.H., G.T. and C.N. performed the experiments.

I.J., A-C.H., G.T., C.N., U.A. and L.C. analysed the data.

I.J. and L.C. wrote the paper.

Funding.

This work was supported by the Swedish Research Council, the Swedish Cancer Society and

the Medical Faculty at Umeå University.

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

References.

Adler, R. and Canto-Soler, M. V. (2007). Molecular mechanisms of optic vesicle

development: complexities, ambiguities and controversies. Dev Biol 305, 1-13.

Alanentalo, T., Asayesh, A., Morrison, H., Loren, C. E., Holmberg, D., Sharpe, J. and

Ahlgren, U. (2007). Tomographic molecular imaging and 3D quantification within adult

mouse organs. Nat Methods 4, 31-3.

Anderl, S., Freeland, M., Kwiatkowski, D. J. and Goto, J. (2011). Therapeutic value of

prenatal rapamycin treatment in a mouse brain model of tuberous sclerosis complex. Hum Mol

Genet 20, 4597-604.

Avet-Rochex, A., Carvajal, N., Christoforou, C. P., Yeung, K., Maierbrugger, K. T.,

Hobbs, C., Lalli, G., Cagin, U., Plachot, C., McNeill, H. et al. (2014). Unkempt is negatively

regulated by mTOR and uncouples neuronal differentiation from growth control. PLoS Genet

10, e1004624.

Bassett, E. A. and Wallace, V. A. (2012). Cell fate determination in the vertebrate retina.

Trends Neurosci 35, 565-73.

Bateman, J. M. and McNeill, H. (2004). Temporal control of differentiation by the insulin

receptor/tor pathway in Drosophila. Cell 119, 87-96.

Behesti, H., Holt, J. K. and Sowden, J. C. (2006). The level of BMP4 signaling is critical for

the regulation of distinct T-box gene expression domains and growth along the dorso-ventral

axis of the optic cup. BMC Dev Biol 6, 62.

Bissler, J. J., Kingswood, J. C., Radzikowska, E., Zonnenberg, B. A., Frost, M.,

Belousova, E., Sauter, M., Nonomura, N., Brakemeier, S., de Vries, P. J. et al. (2013).

Everolimus for angiomyolipoma associated with tuberous sclerosis complex or sporadic

lymphangioleiomyomatosis (EXIST-2): a multicentre, randomised, double-blind, placebo-

controlled trial. Lancet 381, 817-24.

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

Blackshaw, S., Harpavat, S., Trimarchi, J., Cai, L., Huang, H., Kuo, W. P., Weber, G.,

Lee, K., Fraioli, R. E., Cho, S. H. et al. (2004). Genomic analysis of mouse retinal

development. PLoS Biol 2, E247.

Campbell, D. S. and Holt, C. E. (2001). Chemotropic responses of retinal growth cones

mediated by rapid local protein synthesis and degradation. Neuron 32, 1013-1026.

Cang, J., Rentería, R. C., Kaneko, M., Liu, X., Copenhagen, D. R. and Stryker, M. P.

(2005). Development of precise maps in visual cortex requires patterned spontaneous activity

in the retina. Neuron 48, 797-809.

Cantrup, R., Dixit, R., Palmesino, E., Bonfield, S., Shaker, T., Tachibana, N., Zinyk, D.,

Dalesman, S., Yamakawa, K., Stell, W. K. et al. (2012). Cell-type specific roles for PTEN

in establishing a functional retinal architecture. PLoS One 7, e32795.

Capo-Chichi, J. M., Tcherkezian, J., Hamdan, F. F., Decarie, J. C., Dobrzeniecka, S.,

Patry, L., Nadon, M. A., Mucha, B. E., Major, P., Shevell, M. et al. (2013). Disruption of

TBC1D7, a subunit of the TSC1-TSC2 protein complex, in intellectual disability and

megalencephaly. J Med Genet 50, 740-4.

Carpenter, A. E., Jones, T. R., Lamprecht, M. R., Clarke, C., Kang, I. H., Friman, O.,

Guertin, D. A., Chang, J. H., Lindquist, R. A., Moffat, J. et al. (2006). CellProfiler: image

analysis software for identifying and quantifying cell phenotypes. Genome Biol 7, R100.

Cheadle, J. P., Reeve, M. P., Sampson, J. R. and Kwiatkowski, D. J. (2000). Molecular

genetic advances in tuberous sclerosis. Hum Genet 107, 97-114.

Cheddad, A., Svensson, C., Sharpe, J., Georgsson, F. and Ahlgren, U. (2012). Image

processing assisted algorithms for optical projection tomography. IEEE Trans Med Imaging

31, 1-15.

Costa-Mattioli, M. and Monteggia, L. M. (2013). mTOR complexes in neurodevelopmental

and neuropsychiatric disorders. Nat Neurosci 16, 1537-43.

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

Crino, P. B. (2013). Evolving neurobiology of tuberous sclerosis complex. Acta Neuropathol

125, 317-32.

Culican, S. M., Bloom, A. J., Weiner, J. A. and DiAntonio, A. (2009). Phr1 regulates

retinogeniculate targeting independent of activity and ephrin-A signalling. Mol Cell Neurosci

41, 304-12.

de Melo, J., Qiu, X., Du, G., Cristante, L. and Eisenstat, D. D. (2003). Dlx1, Dlx2, Pax6,

Brn3b, and Chx10 homeobox gene expression defines the retinal ganglion and inner nuclear

layers of the developing and adult mouse retina. J Comp Neurol 461, 187-204.

Dibble, C. C., Elis, W., Menon, S., Qin, W., Klekota, J., Asara, J. M., Finan, P. M.,

Kwiatkowski, D. J., Murphy, L. O. and Manning, B. D. (2012). TBC1D7 is a third subunit

of the TSC1-TSC2 complex upstream of mTORC1. Mol Cell 47, 535-46.

DiMario, F. J., Jr. (2004). Brain abnormalities in tuberous sclerosis complex. J Child Neurol

19, 650-7.

Ehninger, D., Han, S., Shilyansky, C., Zhou, Y., Li, W., Kwiatkowski, D. J., Ramesh, V.

and Silva, A. J. (2008). Reversal of learning deficits in a Tsc2+/- mouse model of tuberous

sclerosis. Nat Med 14, 843-8.

Erskine, L. and Herrera, E. (2007). The retinal ganglion cell axon's journey: insights into

molecular mechanisms of axon guidance. Dev Biol 308, 1-14.

Feldheim, D. A., Vanderhaeghen, P., Hansen, M. J., Frisen, J., Lu, Q., Barbacid, M. and

Flanagan, J. G. (1998). Topographic guidance labels in a sensory projection to the forebrain.

Neuron 21, 1303-13.

Feliciano, D. M., Quon, J. L., Su, T., Taylor, M. M. and Bordey, A. (2012). Postnatal

neurogenesis generates heterotopias, olfactory micronodules and cortical infiltration following

single-cell Tsc1 deletion. Hum Mol Genet 21, 799-810.

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

Feliciano, D. M., Su, T., Lopez, J., Platel, J. C. and Bordey, A. (2011). Single-cell Tsc1

knockout during corticogenesis generates tuber-like lesions and reduces seizure threshold in

mice. J Clin Invest 121, 1596-607.

Franz, D. N., Belousova, E., Sparagana, S., Bebin, E. M., Frost, M., Kuperman, R., Witt,

O., Kohrman, M. H., Flamini, J. R., Wu, J. Y. et al. (2013). Efficacy and safety of

everolimus for subependymal giant cell astrocytomas associated with tuberous sclerosis

complex (EXIST-1): a multicentre, randomised, placebo-controlled phase 3 trial. Lancet 381,

125-32.

Galli-Resta, L., Resta, G., Tan, S. S. and Reese, B. E. (1997). Mosaics of islet-1-expressing

amacrine cells assembled by short-range cellular interactions. J Neurosci 17, 7831-8.

Garami, A., Zwartkruis, F. J., Nobukuni, T., Joaquin, M., Roccio, M., Stocker, H.,

Kozma, S. C., Hafen, E., Bos, J. L. and Thomas, G. (2003). Insulin activation of Rheb, a

mediator of mTOR/S6K/4E-BP signaling, is inhibited by TSC1 and 2. Mol Cell 11, 1457-66.

Godement, P., Salaun, J. and Imbert, M. (1984). Prenatal and postnatal development of

retinogeniculate and retinocollicular projections in the mouse. J Comp Neurol 230, 552-75.

Gomez, M. R. (1991). Phenotypes of the tuberous sclerosis complex with a revision of

diagnostic criteria. Ann N Y Acad Sci 615, 1-7.

Goto, J., Talos, D. M., Klein, P., Qin, W., Chekaluk, Y. I., Anderl, S., Malinowska, I. A.,

Di Nardo, A., Bronson, R. T., Chan, J. A. et al. (2011). Regulable neural progenitor-specific

Tsc1 loss yields giant cells with organellar dysfunction in a model of tuberous sclerosis

complex. Proc Natl Acad Sci U S A 108, E1070-9.

Grubb, M. S., Rossi, F. M., Changeux, J. P. and Thompson, I. D. (2003). Abnormal

functional organization in the dorsal lateral geniculate nucleus of mice lacking the beta 2

subunit of the nicotinic acetylcholine receptor. Neuron 40, 1161-72.

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

Hägglund, A. C., Dahl, L. and Carlsson, L. (2011). Lhx2 is required for patterning and

expansion of a distinct progenitor cell population committed to eye development. PLoS One 6,

e23387.

Han, J. M. and Sahin, M. (2011). TSC1/TSC2 signaling in the CNS. FEBS Lett 585, 973-80.

Hippert, C., Graca, A. B., Barber, A. C., West, E. L., Smith, A. J., Ali, R. R. and Pearson,

R. A. (2015). Muller glia activation in response to inherited retinal degeneration is highly

varied and disease-specific. PLoS One 10, e0120415.

Howell, G. R., Libby, R. T., Jakobs, T. C., Smith, R. S., Phalan, F. C., Barter, J. W.,

Barbay, J. M., Marchant, J. K., Mahesh, N., Porciatti, V. et al. (2007). Axons of retinal

ganglion cells are insulted in the optic nerve early in DBA/2J glaucoma. J Cell Biol 179, 1523-

37.

Huberman, A. D., Feller, M. B. and Chapman, B. (2008). Mechanisms underlying

development of visual maps and receptive fields. Annu Rev Neurosci 31, 479-509.

Ito, N. and Rubin, G. M. (1999). gigas, a Drosophila homolog of tuberous sclerosis gene

product-2, regulates the cell cycle. Cell 96, 529-39.

Jeste, S. S., Sahin, M., Bolton, P., Ploubidis, G. B. and Humphrey, A. (2008).

Characterization of autism in young children with tuberous sclerosis complex. J Child Neurol

23, 520-5.

Jones, A. C., Shyamsundar, M. M., Thomas, M. W., Maynard, J., Idziaszczyk, S.,

Tomkins, S., Sampson, J. R. and Cheadle, J. P. (1999). Comprehensive mutation analysis of

TSC1 and TSC2-and phenotypic correlations in 150 families with tuberous sclerosis. Am J

Hum Genet 64, 1305-15.

Kamentsky, L., Jones, T. R., Fraser, A., Bray, M. A., Logan, D. J., Madden, K. L., Ljosa,

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

V., Rueden, C., Eliceiri, K. W. and Carpenter, A. E. (2011). Improved structure, function

and compatibility for CellProfiler: modular high-throughput image analysis software.

Bioinformatics 27, 1179-80.

Kay, J. N., Chu, M. W. and Sanes, J. R. (2012). MEGF10 and MEGF11 mediate homotypic

interactions required for mosaic spacing of retinal neurons. Nature 483, 465-9.

Kim, S. H., Speirs, C. K., Solnica-Krezel, L. and Ess, K. C. (2011). Zebrafish model of

tuberous sclerosis complex reveals cell-autonomous and non-cell-autonomous functions of

mutant tuberin. Dis Model Mech 4, 255-67.

Kobayashi, T., Minowa, O., Sugitani, Y., Takai, S., Mitani, H., Kobayashi, E., Noda, T.

and Hino, O. (2001). A germ-line Tsc1 mutation causes tumor development and embryonic

lethality that are similar, but not identical to, those caused by Tsc2 mutation in mice. Proc Natl

Acad Sci U S A 98, 8762-7.

Kwiatkowski, D. J. and Manning, B. D. (2005). Tuberous sclerosis: a GAP at the crossroads

of multiple signaling pathways. Hum Mol Genet 14 Spec No. 2, R251-8.

Kwiatkowski, D. J., Zhang, H., Bandura, J. L., Heiberger, K. M., Glogauer, M., el-

Hashemite, N. and Onda, H. (2002). A mouse model of TSC1 reveals sex-dependent lethality

from liver hemangiomas, and up-regulation of p70S6 kinase activity in Tsc1 null cells. Hum

Mol Genet 11, 525-34.

Lamb, R. F., Roy, C., Diefenbach, T. J., Vinters, H. V., Johnson, M. W., Jay, D. G. and

Hall, A. (2000). The TSC1 tumour suppressor hamartin regulates cell adhesion through ERM

proteins and the GTPase Rho. Nat Cell Biol 2, 281-7.

Leamey, C. A., Merlin, S., Lattouf, P., Sawatari, A., Zhou, X., Demel, N., Glendining, K.

A., Oohashi, T., Sur, M. and Fassler, R. (2007). Ten_m3 regulates eye-specific patterning in

the mammalian visual pathway and is required for binocular vision. Plos Biology 5, 2077-2092.

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

Livesey, F. J. and Cepko, C. L. (2001). Vertebrate neural cell-fate determination: lessons

from the retina. Nat Rev Neurosci 2, 109-18.

Ma, L., Cantrup, R., Varrault, A., Colak, D., Klenin, N., Gotz, M., McFarlane, S.,

Journot, L. and Schuurmans, C. (2007). Zac1 functions through TGFbetaII to negatively

regulate cell number in the developing retina. Neural Dev 2, 11.

Magri, L., Cambiaghi, M., Cominelli, M., Alfaro-Cervello, C., Cursi, M., Pala, M.,

Bulfone, A., Garcia-Verdugo, J. M., Leocani, L., Minicucci, F. et al. (2011). Sustained

activation of mTOR pathway in embryonic neural stem cells leads to development of tuberous

sclerosis complex-associated lesions. Cell Stem Cell 9, 447-62.

Mahmood, T. and Yang, P. C. (2012). Western blot: technique, theory, and trouble shooting.

N Am J Med Sci 4, 429-34.

Matsuoka, R. L., Chivatakarn, O., Badea, T. C., Samuels, I. S., Cahill, H., Katayama, K.,

Kumar, S. R., Suto, F., Chedotal, A., Peachey, N. S. et al. (2011a). Class 5 transmembrane

semaphorins control selective Mammalian retinal lamination and function. Neuron 71, 460-73.

Matsuoka, R. L., Nguyen-Ba-Charvet, K. T., Parray, A., Badea, T. C., Chedotal, A. and

Kolodkin, A. L. (2011b). Transmembrane semaphorin signalling controls laminar

stratification in the mammalian retina. Nature 470, 259-63.

McMahon, J. J., Yu, W., Yang, J., Feng, H., Helm, M., McMahon, E., Zhu, X., Shin, D.

and Huang, Y. (2014). Seizure-dependent mTOR activation in 5-HT neurons promotes

autism-like behaviors in mice. Neurobiol Dis 73C, 296-306.

Meikle, L., Talos, D. M., Onda, H., Pollizzi, K., Rotenberg, A., Sahin, M., Jensen, F. E.

and Kwiatkowski, D. J. (2007). A mouse model of tuberous sclerosis: neuronal loss of Tsc1

causes dysplastic and ectopic neurons, reduced myelination, seizure activity, and limited

survival. J Neurosci 27, 5546-58.

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

Mennel, S., Meyer, C. H., Peter, S., Schmidt, J. C. and Kroll, P. (2007). Current treatment

modalities for exudative retinal hamartomas secondary to tuberous sclerosis: review of the

literature. Acta Ophthalmol Scand 85, 127-32.

Murthy, V., Han, S., Beauchamp, R. L., Smith, N., Haddad, L. A., Ito, N. and Ramesh, V.

(2004). Pam and its ortholog highwire interact with and may negatively regulate the

TSC1.TSC2 complex. J Biol Chem 279, 1351-8.

Napolioni, V., Moavero, R. and Curatolo, P. (2009). Recent advances in neurobiology of

Tuberous Sclerosis Complex. Brain Dev 31, 104-13.

Nie, D., Di Nardo, A., Han, J. M., Baharanyi, H., Kramvis, I., Huynh, T., Dabora, S.,

Codeluppi, S., Pandolfi, P. P., Pasquale, E. B. et al. (2010). Tsc2-Rheb signaling regulates

EphA-mediated axon guidance. Nat Neurosci 13, 163-72.

Onda, H., Lueck, A., Marks, P. W., Warren, H. B. and Kwiatkowski, D. J. (1999). Tsc2(+/-

) mice develop tumors in multiple sites that express gelsolin and are influenced by genetic

background. J Clin Invest 104, 687-95.

Pan, L., Yang, Z., Feng, L. and Gan, L. (2005). Functional equivalence of Brn3 POU-domain

transcription factors in mouse retinal neurogenesis. Development 132, 703-12.

Penn, A. A., Riquelme, P. A., Feller, M. B. and Shatz, C. J. (1998). Competition in

retinogeniculate patterning driven by spontaneous activity. Science 279, 2108-12.

Pfeiffenberger, C., Cutforth, T., Woods, G., Yamada, J., Renteria, R. C., Copenhagen, D.

R., Flanagan, J. G. and Feldheim, D. A. (2005). Ephrin-As and neural activity are required

for eye-specific patterning during retinogeniculate mapping. Nat Neurosci 8, 1022-7.

Potter, C. J., Huang, H. and Xu, T. (2001). Drosophila Tsc1 functions with Tsc2 to

antagonize insulin signaling in regulating cell growth, cell proliferation, and organ size. Cell

105, 357-68.

Reese, B. E. (2011). Development of the retina and optic pathway. Vision Res 51, 613-32.

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

Reith, R. M., McKenna, J., Wu, H., Hashmi, S. S., Cho, S. H., Dash, P. K. and Gambello,

M. J. (2013). Loss of Tsc2 in Purkinje cells is associated with autistic-like behavior in a mouse

model of tuberous sclerosis complex. Neurobiol Dis 51, 93-103.

Rossi, F. M., Pizzorusso, T., Porciatti, V., Marubio, L. M., Maffei, L. and Changeux, J. P.

(2001). Requirement of the nicotinic acetylcholine receptor beta 2 subunit for the anatomical

and functional development of the visual system. Proc Natl Acad Sci U S A 98, 6453-8.

Sakagami, K., Chen, B., Nusinowitz, S., Wu, H. and Yang, X. J. (2012). PTEN regulates

retinal interneuron morphogenesis and synaptic layer formation. Mol Cell Neurosci 49, 171-

83.

Samueli, S., Abraham, K., Dressler, A., Groeppel, G., Jonak, C., Muehlebner, A., Prayer,

D., Reitner, A., Feucht, M. and Padiatrisches, T. S. C. Z. W. (2015). Tuberous Sclerosis

Complex: new criteria for diagnostic work-up and management. Wien Klin Wochenschr.

Sanes, J. R. and Zipursky, S. L. (2010). Design principles of insect and vertebrate visual

systems. Neuron 66, 15-36.

Schaeren-Wiemers, N. and Gerfin-Moser, A. (1993). A single protocol to detect transcripts

of various types and expression levels in neural tissue and cultured cells: in situ hybridization

using digoxigenin-labelled cRNA probes. Histochemistry 100, 431-440.

Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T.,

Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B. et al. (2012). Fiji: an open-source platform

for biological-image analysis. Nat Methods 9, 676-82.

Sepp, T., Yates, J. R. and Green, A. J. (1996). Loss of heterozygosity in tuberous sclerosis

hamartomas. J Med Genet 33, 962-4.

Shields, J. A., Eagle, R. C., Jr., Shields, C. L. and Marr, B. P. (2004). Aggressive retinal

astrocytomas in four patients with tuberous sclerosis complex. Trans Am Ophthalmol Soc 102,

139-47.

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

Smalley, S. L. (1998). Autism and tuberous sclerosis. J Autism Dev Disord 28, 407-14.

Soriano, P. (1999). Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat

Genet 21, 70-1.

Sun, L. O., Jiang, Z., Rivlin-Etzion, M., Hand, R., Brady, C. M., Matsuoka, R. L., Yau,

K. W., Feller, M. B. and Kolodkin, A. L. (2013). On and off retinal circuit assembly by

divergent molecular mechanisms. Science 342, 1241974.

Tapon, N., Ito, N., Dickson, B. J., Treisman, J. E. and Hariharan, I. K. (2001). The

Drosophila tuberous sclerosis complex gene homologs restrict cell growth and cell

proliferation. Cell 105, 345-55.

Tavazoie, S. F., Alvarez, V. A., Ridenour, D. A., Kwiatkowski, D. J. and Sabatini, B. L.

(2005). Regulation of neuronal morphology and function by the tumor suppressors Tsc1 and

Tsc2. Nat Neurosci 8, 1727-34.

Thiele, E. A. (2004). Managing epilepsy in tuberous sclerosis complex. J Child Neurol 19,

680-6.

Torborg, C. L. and Feller, M. B. (2005). Spontaneous patterned retinal activity and the

refinement of retinal projections. Prog Neurobiol 76, 213-35.

Tsai, P. T., Hull, C., Chu, Y., Greene-Colozzi, E., Sadowski, A. R., Leech, J. M., Steinberg,

J., Crawley, J. N., Regehr, W. G. and Sahin, M. (2012). Autistic-like behaviour and

cerebellar dysfunction in Purkinje cell Tsc1 mutant mice. Nature 488, 647-51.

Uhlmann, E. J., Wong, M., Baldwin, R. L., Bajenaru, M. L., Onda, H., Kwiatkowski, D.

J., Yamada, K. and Gutmann, D. H. (2002). Astrocyte-specific TSC1 conditional knockout

mice exhibit abnormal neuronal organization and seizures. Ann Neurol 52, 285-96.

Varrault, A., Gueydan, C., Delalbre, A., Bellmann, A., Houssami, S., Aknin, C., Severac,

D., Chotard, L., Kahli, M., Le Digarcher, A. et al. (2006). Zac1 regulates an imprinted gene

network critically involved in the control of embryonic growth. Dev Cell 11, 711-22.

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

Way, S. W., McKenna, J., 3rd, Mietzsch, U., Reith, R. M., Wu, H. C. and Gambello, M.

J. (2009). Loss of Tsc2 in radial glia models the brain pathology of tuberous sclerosis complex

in the mouse. Hum Mol Genet 18, 1252-65.

Yang, Z., Ding, K., Pan, L., Deng, M. and Gan, L. (2003). Math5 determines the competence

state of retinal ganglion cell progenitors. Developmental biology 264, 240-254.

Young, R. W. (1985). Cell proliferation during postnatal development of the retina in the

mouse. Brain Res 353, 229-39.

Zeng, L. H., Rensing, N. R., Zhang, B., Gutmann, D. H., Gambello, M. J. and Wong, M.

(2011). Tsc2 gene inactivation causes a more severe epilepsy phenotype than Tsc1 inactivation

in a mouse model of tuberous sclerosis complex. Hum Mol Genet 20, 445-54.

Zhou, J., Shrikhande, G., Xu, J., McKay, R. M., Burns, D. K., Johnson, J. E. and Parada,

L. F. (2011). Tsc1 mutant neural stem/progenitor cells exhibit migration deficits and give rise

to subependymal lesions in the lateral ventricle. Genes Dev 25, 1595-600.

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

Figures

Fig. 1. Conditional deletion of Tsc1 during eye development leads to an activation of the

mTORC1 pathway. (A) Immunoblot analysis of hamartin, total S6, pS6 (S235/236), pS6

(S240/244) and GAPDH from control, Lhx2-Cre:Tsc1+/f and Lhx2-Cre:Tsc1f/f eye extracts. (B)

Densitometry analysis demonstrates that conditional deletion of Tsc1 results in a reduction of

hamartin level and a parallel upregulation in pS6 (S235/236) and pS6 (240/244) levels.

GAPDH was used for normalization of hamartin densitometry data. Total S6 was used for

normalization of pS6 (S235/235) and pS6 (S240/244) densitometry data. All data represent the

mean SEM of three mice from each genotype. Abbreviations: GAPDH, Glyceraldehyde 3-

phosphate dehydrogenase; kDa, kilodalton. p values are denoted as follows: *p 0.05, ***p

0.001, ****p 0.0001.

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

Fig. 2. Conditional deletion of Tsc1 leads to enlarged eyes, hamartomas and loss of retinal

architecture. (A-B) OPT 3D volume rendering from control (A) and Lhx2-Cre:Tsc1f/f mice

(B) showing an enlargement of the eye, retinal folding (white arrowhead) and loss of ora serrata

(ORS) integrity (white arrow) in Lhx2-Cre:Tsc1f/f mice. (C-F) Histological analysis of coronal

eye sections demonstrating that the retinal folds observed during OPT analysis of Lhx2-

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

Cre:Tsc1f/f mice are hamartomas that were organised into ring heterotopias. Moreover, retinal

detachment was a common occurrence in areas of hamartoma formation (C, E, asterisks). (G-

J) Histological analysis of coronal eye sections demonstrating that hamartomas first become

evident during late embryogenesis (G, open black arrowhead) in Lhx2-Cre:Tsc1f/f mice. These

lesions then become more pronounced during postnatal development (H-J, open black

arrowheads). (K-P) Immunostaining and in situ hybridisation analyses demonstrating that

hamartomas in Lhx2-Cre:Tsc1f/f mice are enriched in pS6 (S235/236) protein (L) and consist

of all the cellular classes that populate the retina: Müller glia (K, GFAP+, p27Kip1+), RGCs (L,

Brn3+), bipolar cells (M, PKC+), horizontal cells (N, calbindin-D+), amacrine cells (O,

calretinin+) and photoreceptors (P, Crx+). (Q-T) Histological analysis of coronal eye sections

from control (Q, S) and Lhx2-Cre:Tsc1f/f mice (R, T) demonstrates the irregular ordering of the

INL, an IPL populated with ectopic cells (R, T, open black arrowheads) and a widening of the

GCL and NFL (S, T, brackets) in Lhx2-Cre:Tsc1f/f mice. Scale bars: (A-B, G-J), 500 m; (C-

F, Q-T), 50 m; (K) 100 m, (L-P), 200 m. Abbreviations: Brn, brain-specific homeobox;

Crx, cone-rod homeobox; D, dorsal; IS, inner segments; OS, outer segments; PKC, protein

kinase C; V, ventral.

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

Fig. 3. Conditional deletion of Tsc1 leads to aberrant neurite stratification of retinal

interneurons. (A-B) Coronal eye sections from control (A) and Lhx2-Cre:Tsc1f/f mice (B)

showing global upregulation of pS6 (S235/236) in the INL and GCL of Lhx2-Cre:Tsc1f/f mice.

(C-J) Coronal retinal sections from control (C, E, G, I) and Lhx2-Cre:Tsc1f/f mice (D, F, H, J).

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

Amacrine cells (D, Pax6+) have irregular cell body positions in the INL of Lhx2-Cre:Tsc1f/f

mice. Also evident are the presence of ectopic amacrine cells or RGCs (Pax6+) in the IPL (D,

yellow arrowheads). Stratification deficits were observed in the Lhx2-Cre:Tsc1f/f mice that

ranged from ectopic neurites extending into other sublaminae (F, ChAT+, open yellow

arrowheads) to a loss of specific sublamina stratification (H, calretinin+, red arrowhead).

Moreover, rod bipolar cells in Lhx2-Cre:Tsc1f/f mice have distorted axonal projections that

extend through the INL (J, PKC+, open red arrowheads) and terminate with enlarged axon

pedicles in the IPL (J, PKC+, red arrow). (K-L) Scanning electron microscopic (SEM)

analysis of the IPL in control (K) and Lhx2-Cre:Tsc1f/f mice (L). The density of the IPL in

Lhx2-Cre:Tsc1f/f mice was increased when compared to control mice. Scale bars: (A-J), 25 m,

(K-L), 1 m. Abbreviations: ChAT, choline acetyltransferase; Pax, paired box protein; PKC,

protein kinase C.

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

Fig. 4. Conditional deletion of Tsc1 leads to accelerated RGC differentiation and radial

migration defects. (A-D) Immunostaining analysis of Brn3+ RGCs (yellow arrowheads) in

control and Lhx2-Cre:Tsc1f/f mice at E11.5 (A, B) and E12.5 (C, D) demonstrates the

accelerated appearance of RGCs in Lhx2-Cre:Tsc1f/f mice. (E-J) In situ hybridisation analysis

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

at E12.5 demonstrates expanded expression domains for Math5 (E, F), Dlx1 (G, H) and Dlx2

(I, J) in Lhx2-Cre:Tsc1f/f mice (open black arrowheads). (K-R) Spatiotemporal analysis of RGC

position during migration (E17.5, K, L), target innervation (P1, M, N and P7, O, P) and

refinement (P14, Q, R) demonstrates that RGCs radially migrate to reach their final laminar

position within the GCL layer by early postnatal ages in control mice (K, M, O, Q). In contrast,

ectopic RGCs begin to appear at E17.5 (L, red arrowheads) and become more pronounced after

birth in Lhx2-Cre:Tsc1f/f mice (N, P, R, red arrowheads). (S) Quantification of Brn3+ RGC

number in control and Lhx2-Cre:Tsc1f/f mice during neurogenesis (E12.5), migration (E17.5)

and target innervation and synapse formation (P3). Although the initial numbers of Brn3+

RGCs was increased in the mutant mice (E12.5), there is a subsequent reduction in the number

of Brn3+ cells that continues through late embryonic (E17.5) and early postnatal development

(P3). The data represents the mean SEM of at least three mice from each genotype. Scale

bars: (A-D, E-J), 50 m; (K-R), 25 m. Abbreviations: Brn, brain-specific homeobox; D,

dorsal; Dlx, distal-less homeobox; INBL, inner neuroblastic layer; Math, mouse atonal; ONBL,

outer neuroblastic layer; V, ventral. p values are denoted as follows: *p 0.05, ****p 0.0001.

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

Fig. 5. Conditional deletion of Tsc1 leads to a reduction in RGC number and asymmetric

mosaic distribution. (A-B) Retinal flat mount analysis of Brn3+ RGCs in the tangential plane

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

of control (A) and Lhx2-Cre:Tsc1f/f mice (B) shows a reduction in the number of RGCs in

Lhx2-Cre:Tsc1f/f mice. (C-D) Voronoi domain analysis of the immunostaining images

demonstrates an increase in cell territories occupied in Lhx2-Cre:Tsc1f/f mice. (E) Voronoi

domain regularity indices for control and Lhx2-Cre:Tsc1f/f mice. A reduction in mosaic

regularity was observed for Lhx2-Cre:Tsc1f/f mice. The data represents the mean SEM of

three mice from each genotype. (F) Quantification of RGC number in the tangential plane of

control and Lhx2-Cre:Tsc1f/f mice. A decrease in the number of Brn3+ cells was observed in

Lhx2-Cre:Tsc1f/f mice. The data represents the mean SEM of three mice from each genotype.

(G) Quantification of the cross sectional RGC nucleus area in the tangential plane of control

and Lhx2-Cre:Tsc1f/f mice. An increase in the cross sectional area of Brn3+ cells was observed

in Lhx2-Cre:Tsc1f/f mice. The data represents the mean SEM of three mice from each

genotype. Scale bar: (A-B) 50 m. Abbreviations: Brn, brain-specific homeobox. VDRI,

Voronoi domain regularity index. p values are denoted as follows: **p 0.01, ****p 0.0001.

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

Fig. 6. Conditional deletion of Tsc1 leads to reactive gliosis and changes in optic nerve

morphology. (A-D) Transverse sections of the ON at P7 demonstrate a defined network of

GFAP+ astrocytes that enmesh the NF+ RGC axons in control mice (A, C). In contrast,

regionalized areas of reactive gliosis are clearly visible in the ON of Lhx2-Cre:Tsc1f/f mice (B,

D, solid white arrowheads). (E-H) Transverse sections of the ON at P18 demonstrate a defined

network of GFAP+ astrocytes that enmesh the NF+ RGC axons in control mice (E, G). In

contrast, axon loss is clearly visible in regionalized areas of the ON from Lhx2-Cre:Tsc1f/f

mice. Also evident are cross sectional areas that are significantly enriched in GFAP+ astrocytes

and completely devoid of NF+ RGC axons (F, H, open white arrowheads). Scale bars: (A-B,

E-F) 50 m; (C-D, G-H), 25 m.

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

Fig. 7. Conditional deletion of Tsc1 leads to aberrant retinogeniculate topography. (A-D)

Medial coronal sections showing retinogeniculate projections into the dLGN at P14 in control

(A, B) and Lhx2-Cre:Tsc1f/f mice (C, D). Dashed lines represent the borders of the dLGN. A

reduction in the area occupied by both contralateral and ipsilateral projections is evident in

Lhx2-Cre:Tsc1f/f mice. (E) Quantification of the total dLGN area (m2) in control and Lhx2-

Cre:Tsc1f/f mice at P14. The data represents the mean SEM of three mice from each genotype.

A significant reduction in the total area occupied by both contralateral and ipsilateral

projections is evident in Lhx2-Cre:Tsc1f/f mice. (F-I) Medial coronal sections showing the

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

ipsilateral retinogeniculate projections into the dLGN at P14 in control (F, G) and Lhx2-

Cre:Tsc1f/f mice (H, I). Dashed lines represent the borders of the dLGN. The topographical

appearance and position of the ipsilateral patch within the dLGN of Lhx2-Cre:Tsc1f/f mice was

altered along the dorsoventral and lateromedial axis. (K) Quantification of the percentage

ipsilateral projection area in control and Lhx2-Cre:Tsc1f/f mice at P14. The data represents the

mean SEM of three mice from each genotype. A significant reduction in the percentage area

occupied by the ipsilateral projection is evident in Lhx2-Cre:Tsc1f/f mice. Scale bars: (A-D, F-

I) 250 m. Abbreviations: D, dorsal; L, lateral. p values are denoted as follows: ****p

0.0001.

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Adv

ance

art

icle

Translational Impact.

(1) Clinical Issue: Tuberous Sclerosis Complex (TSC) is an inherited syndrome that is caused

by mutations in either the TSC1 or TSC2 genes. These mutations induce aberrant activation of

the mTORC1 pathway and TSC affected individuals display neurodevelopmental deficits and

the presence of benign tumours called hamartomas in affected organs. Lesions to the eye are

central to TSC diagnosis but the consequences of TSC for the visual system are not known.

Moreover, an animal model that addresses the involvement of the visual system in TSC is

currently lacking.

(2) Results: Here we report the generation of a novel eye-specific TSC mouse model by

conditional ablation of the Tsc1 gene. The mutant mice recapitulate many of pathological

hallmarks of TSC such as hamartomas with retinal detachment and an increase in organ and

cell size. Moreover, our mouse model demonstrates a pivotal role for Tsc1 in regulating various

aspects of visual system development such as neuronal differentiation, migration and circuit

formation.

(3) Implications and Future Directions: Our findings provide the first major insight into the

molecular etiology of TSC within the eye. Furthermore, our novel model establishes a

foundation for future studies that predict new clinical complications that could arise from TSC

in the visual system. In addition, our mouse model will also provide a platform for evaluating

new therapeutic approaches for the treatment of this multiorgan disorder. D

isea

se M

ode

ls &

Mec

hani

sms

• D

MM

• A

dvan

ce a

rtic

le