sall1 regulates cortical neurogenesis and laminar fate specification … · sall1 in neural pcs and...

INTRODUCTIONThe mature cortex is a six-layered structure that controls complexfunctions, including motor coordination, and auditory, visual andsomatosensory processing, as well as cognition (reviewed in Rosner,1970). Appropriate regulation of cell number, cell-type specification,laminar positioning and circuit formation is essential for normalfunctioning of the mature nervous system. Dysregulation of corticaldevelopment can lead to a variety of gross cortical malformationsand psychiatric disorders, including lissencephaly, periventricularheterotopia, microcephaly, epilepsy, autism and schizophrenia(reviewed in Arnold, 1999; Lian and Sheen, 2006; Pilz et al., 2002;Polleux and Lauder, 2004; Schwartzkroin and Walsh, 2000).

The type of division a progenitor cell (PC) makes is an importantmechanism in regulating cell number and fate in the cortex. Earlyin development, the PC population expands by symmetric divisions,resulting in the production of two progeny radial glial cells (RGCs)(Noctor et al., 2008; Takahashi et al., 1996b). At the onset of

neurogenesis (~E10.5 in mice), RGC asymmetric neurogenicdivisions result in the generation of a neuroblast and an RGC(Haubensak et al., 2004; Noctor et al., 2001; Noctor et al., 2008).By mid-neurogenesis (~E14.5 in mice) these divisions represent thepredominant division type in the ventricular zone (VZ) (Noctor etal., 2004). Subsequent asymmetric RGC divisions produce an RGCand an intermediate progenitor cell (IPC) (Haubensak et al., 2004;Miyata et al., 2004; Noctor et al., 2004; Noctor et al., 2008). IPCs(also referred to as basal progenitors) predominately undergosymmetric terminal neurogenic division at the basal side of the VZor within the subventricular zone (SVZ), resulting in the productionof two neurons (Attardo et al., 2008; Haubensak et al., 2004; Miyataet al., 2004; Noctor et al., 2004; Noctor et al., 2008). Although rare,symmetric proliferative IPC divisions have also been reported,resulting in the production of two daughter IPCs (Miyata et al.,2004; Noctor et al., 2004). Recent studies suggest that IPCs giverise to the majority of cortical neurons, so perturbing thispopulation during development has the potential to impactneuronal organization and ultimately behavior (Haubensak et al.,2004; Martinez-Cerdeno et al., 2006; Miyata et al., 2004; Noctor etal., 2004; Noctor et al., 2008; Pontious et al., 2008; Sessa et al., 2008).The molecular mechanisms regulating specification, maintenanceand fate of this population are just beginning to be understood.

This study investigated the role of a member of the Sall genefamily, Sall1, in the developing brain and identifies a unique rolefor the Sall1 gene in regulating PCs in the cerebral cortex. Sall1 isa C2H2 zinc-finger-containing putative transcription factor that ishighly expressed in the developing CNS and peripheral organs.Previous studies have shown that members of the Sall gene familyplay a role in cell cycle regulation, proliferation, neuronaldifferentiation, migration and cell adhesion in other species(Barembaum and Bronner-Fraser, 2004; Basson and Horvitz, 1996;

Disease Models & Mechanisms 351

Disease Models & Mechanisms 5, 351-365 (2012) doi:10.1242/dmm.002873

1Department of Neurobiology, 2Biochemistry and Molecular Genetics GraduateProgram and 5Department of Psychiatry, University of Pittsburgh, Pittsburgh, PA 15261, USA3Department of Kidney Development, Institute of Molecular Embryology andGenetics, Kumamoto University, Kumamoto 860-0811, Japan4Department of Molecular, Cellular and Developmental Biology, University ofColorado at Boulder, Boulder, CO 80309, USA*Present address: Department of Neurology, University of California, San Francisco,CA 94143, USA‡Author for correspondence ([email protected])

Received 15 July 2011; Accepted 15 December 2011

© 2012. Published by The Company of Biologists LtdThis is an Open Access article distributed under the terms of the Creative Commons AttributionNon-Commercial Share Alike License (http://creativecommons.org/licenses/by-nc-sa/3.0), whichpermits unrestricted non-commercial use, distribution and reproduction in any medium providedthat the original work is properly cited and all further distributions of the work or adaptation aresubject to the same Creative Commons License terms.

SUMMARY

Progenitor cells in the cerebral cortex undergo dynamic cellular and molecular changes during development. Sall1 is a putative transcription factorthat is highly expressed in progenitor cells during development. In humans, the autosomal dominant developmental disorder Townes-Brocks syndrome(TBS) is associated with mutations of the SALL1 gene. TBS is characterized by renal, anal, limb and auditory abnormalities. Although neural deficitshave not been recognized as a diagnostic characteristic of the disease, ~10% of patients exhibit neural or behavioral abnormalities. We demonstratethat, in addition to being expressed in peripheral organs, Sall1 is robustly expressed in progenitor cells of the central nervous system in mice. Bothclassical- and conditional-knockout mouse studies indicate that the cerebral cortex is particularly sensitive to loss of Sall1. In the absence of Sall1,both the surface area and depth of the cerebral cortex were decreased at embryonic day 18.5 (E18.5). These deficiencies are associated with changesin progenitor cell properties during development. In early cortical progenitor cells, Sall1 promotes proliferative over neurogenic division, whereas,at later developmental stages, Sall1 regulates the production and differentiation of intermediate progenitor cells. Furthermore, Sall1 influences thetemporal specification of cortical laminae. These findings present novel insights into the function of Sall1 in the developing mouse cortex andprovide avenues for future research into potential neural deficits in individuals with TBS.

Sall1 regulates cortical neurogenesis and laminar fatespecification in mice: implications for neuralabnormalities in Townes-Brocks syndromeSusan J. Harrison1,2,*, Ryuichi Nishinakamura3, Kevin R. Jones4 and A. Paula Monaghan1,2,5,‡

RESEARCH ARTICLED

iseas

e M

odel

s & M

echa

nism

s

DM

M

Cantera et al., 2002; de Celis et al., 1999; Elling et al., 2006; Harrisonet al., 2008a; Harrison et al., 2008b; Li et al., 2001; Sakaki-Yumotoet al., 2006; Toker et al., 2003). Mutation of SALL1 in humans resultsin the autosomal dominant developmental disorder Townes-Brockssyndrome (TBS). These mutations are predicted to produce atruncated protein retaining the transcriptional repression domainthat is hypothesized to function in a transdominant negativemanner (Kiefer et al., 2003; Kohlhase et al., 1998). TBS ischaracterized by multiple malformations with variable expression(Kohlhase et al., 1998; Townes and Brocks, 1972). The mostcommon diagnostic features include imperforate anus, polydactyly,outer ear anomalies with hearing loss, and kidney abnormalities(Kohlhase et al., 1998; Townes and Brocks, 1972). Althoughcognitive alterations are only occasionally reported in TBS, it hasnot been investigated in depth; however, significant evidence hasaccumulated that indicates a role for Sall1 in neural development.First, a variety of neural and behavioral abnormalities have beendescribed in individuals with TBS, including mental retardation,developmental delay, attention deficit hyperactivity disorder,hypoplasia of the corpus callosum, and seizures (Borozdin et al.,2006; Botzenhart et al., 2005; Cameron et al., 1991; Ishikiriyama etal., 1996). Also, studies in mice indicate that Sall1 is highly expressedin CNS PCs from early embryonic stages (Buck et al., 2000; Ott etal., 2001). Furthermore, mice expressing a truncated Sall1 proteinthat recapitulates the human mutation exhibit neural abnormalities,in addition to mimicking the human deficits observed in thegastrointestinal tract, limbs and kidneys (Kiefer et al., 2003). Thesestudies suggest that Sall1 might also regulate neural development.

Sall1–/– mice die at birth because of kidney abnormalities(Nishinakamura et al., 2001). In light of the robust expression ofSall1 in neural PCs and its role in neural specification in otherorganisms (de Celis et al., 1999; Ott et al., 2001; Rusten et al., 2001),we investigated the role of Sall1 in the development of the CNS.Using classical- and conditional-knockout studies, we demonstratea unique role for Sall1 in regulating cell cycle exit and neuronalspecification in the cerebral cortex. Our studies indicate that, atearly developmental stages, Sall1 promotes RGC cell cycle re-entry,whereas, from mid-neurogenesis, Sall1 promotes IPC cell cycle exit.Our analyses indicate that Sall1 promotes the terminaldifferentiation of IPCs and the production of superficial corticallayer neurons. These findings indicate that the cerebral cortex isparticularly sensitive to loss of Sall1 and that Sall1 is required toregulate progenitor cell maturation and neuronal specification inthe cerebral cortex. We have identified a cellular process that mightbe altered in individuals with TBS and could be used as a modelsystem to evaluate the benefits of future therapeutic intervention.

RESULTSSall1 is expressed by cortical progenitor cellsIn Drosophila, Sall has been shown to play a role in cell fatespecification and neuronal differentiation (de Celis et al., 1999;Rusten et al., 2001). We have previously shown that, in mice, Sall1mRNA is expressed in the CNS, including in the telencephalon,diencephalon, hindbrain and spinal cord, from E8.5 (Buck et al.,2000; Ott et al., 2001) (note: sall1 is termed msal3 in Ott et al.). Inlight of the reported neural abnormalities in some individuals withTBS, a detailed expression study of the Sall1 protein in thedeveloping forebrain of mice was performed. Expression of Sall1

protein in the early developing telencephalon mimicked thepreviously described Sall1mRNA patterns. At E9.5 and E10.5, Sall1protein is expressed in both dorsal and ventral PCs; expression ishighest in lateral and ventral regions (our unpublishedobservations). By E11.5, Sall1 is robustly expressed by PCs in theVZ of the dorsal cortex, with weaker expression in the medial wall(Fig. 1A,D). Ventrally, Sall1 is expressed by cells in the lateralganglionic eminence and the lateral-medial ganglionic sulcus, withweak expression in the medial ganglionic eminence (Fig. 1A). ByE13.5, Sall1 expression extends medially into the medial corticalwall and ventrally into the medial ganglionic eminence and preopticarea (Fig. 1B,E). Sall1 was not expressed by cells in the subicularneural epithelium (Fig. 1A,B). At E17.5, expression persisted in PCsand Sall1 expression was found in cells in the cortical wall (Fig.1C,F). Double labeling with neuronal markers indicated that thesecells are not neurons and are most probably glial cells (data notshown).

To determine whether Sall1 was expressed in all or in a subsetof PCs, we examined the colocalization of Sall1 with cell- and stage-specific markers. Double labeling with Sall1 and eitherbromodeoxyuridine (BrdU; pulsed for 1 hour; data not shown) orphosopho-histone H3 (PH3) indicated that Sall1 is expressed inthe S-phase and M-phase of the cell cycle in RGCs at all agesexamined (Fig. 2A-C). Sall1 is coexpressed in close to 100% ofnestin-positive (Fig. 2D,E) and of Pax6-positive (data not shown)cells in the VZ, and is absent from Tbr1-positive neurons fromE12.5 (Fig. 2G) until birth (data not shown). IPCs can be identifiedfrom E12.5 as basal PH3-positive or Tbr2-positive cells. Sall1 iscoexpressed in a subset of Tbr2-positive cells at the VZ-SVZboundary, but is downregulated in Tbr2-positive cells in the SVZ(Fig. 2F,H,I). These findings suggest that Sall1 is expressed in RGCsand is downregulated once these cells differentiate into IPCs orneurons.

The cerebral cortex is decreased in size at E18.5 in Sall1–/– animalsAs mentioned, Sall1-deficient animals die at birth owing to kidneydeficits (Nishinakamura et al., 2001). The gross anatomy of the brainand spinal cord of Sall1-mutant animals was therefore examined1 day before birth at E18.5 (Fig. 3A,B and our unpublishedobservations). In Sall1-deficient animals, the total length anddorsal surface area of the brain was decreased by 8% (P<0.01, n3)and 12% (P<0.01, n3), respectively, compared with controls (Fig.3A-D). This decrease was predominantly due to a decrease in thesize of the olfactory bulbs (Harrison et al., 2008a) and a decreasein the length (by 12%; P<0.05, n3) and dorsal surface area (by 17%;P<0.01, n3) of the cerebral cortex, compared with controls (Fig.3A-D). However, no alteration in the length (P0.9, n3) or dorsalsurface area (P0.5, n3) of the midbrain (Fig. 3A-D) or grossanatomical abnormalities in the rest of the brain or spinal cord (ourunpublished observations) were observed at all ages examined inSall1-mutant animals, compared with controls. These data suggestthat, although Sall1 is expressed in all CNS PCs, the developingcerebral cortex is particularly sensitive to the loss of Sall1.

To assess the basis of the decrease in cortical size in Sall1-mutantanimals, we histologically examined serial coronal sections of thecortex at E18.5 (n4; Fig. 3E-J). Deficiencies were observed in boththe dorsal and ventral telencephalon. The cortical plate wasdecreased in size in Sall1–/– animals at all rostral-caudal levels,

dmm.biologists.org352

Sall1 regulates cortical neurogenesisRESEARCH ARTICLED

iseas

e M

odel

s & M

echa

nism

s

DM

M

compared with controls (Fig. 3E-K). In ventral regions, the striatumwas smaller in Sall1-mutant animals compared with controls (Fig.3F). In addition, an apparent increase in the number of darklystained cells adjacent to the ventricles in both the dorsal cerebralwall and in the lateral and caudal ganglionic eminences wasobserved in Sall1-deficient animals compared with controls (Fig.3F,H), which suggests an increase in the number of PCs in thetelencephalon.

To verify these observations, the number of cells in each layerof the dorsal cerebral wall was quantified at E18.5. Total cell numberwas decreased by 9% in Sall1-deficient mice compared with controlsat E18.5 (supplementary material Table S1, Fig. S1). Although thenumber of cells in the cortical plate was decreased by 26% (P<0.01,n4), the number of PCs was increased by 14% (P<0.01, n4; Fig.3I-K). Together, these findings suggest that Sall1 is required forneurogenesis in both the dorsal and ventral telencephalon.

The decrease in the size of the cortical plate and changes in theproportion of different cell types could be due to abnormalities inproliferation, differentiation, migration or survival. We firstdetermined whether alterations in cell survival could lead to thechanges in cell number observed in the Sall1-deficient mouse

cerebral cortex at E18.5. The number of activated-caspase-3-positive cells in the dorsal cerebral wall was quantified at E12.5,E14.5 and E18.5. No difference in the number of activated-caspase-3-positive cells was observed between Sall1–/– and control mice(data not shown; E12: n2, P0.86; E14.5: n2, P0.99; E18.5: n4,P0.15), which suggests that Sall1 is not required for cell survival.

Early born neuronal populations are overproduced in Sall1-mutant animalsTo identify the primary cellular deficit caused by loss of Sall1, thedevelopment of the dorsal cerebral wall from E11.5 until birth wasexamined. Cortical neurogenesis initiates at E9.5 when asubpopulation of RGCs in the dorsal cortex differentiate, producingneurons primarily destined for the preplate. Preplate neurogenesiscontinues until E13.5 (Price et al., 1997; Sheppard and Pearlman,1997). To identify the onset of the deficits observed in Sall1–/–animals, the number of Tuj1-positive preplate neurons and Tuj1-negative RGCs was quantified in the dorsal cortex at E11.5 and E12.5(Fig. 4). At E11.5, no significant difference in the number of RGCsor neurons was evident in Sall1–/– mice compared with controls (n3,Fig. 4C). However, by E12.5, a 17.6% decrease (P<0.01, n4) in the


Sall1 regulates cortical neurogenesis RESEARCH ARTICLE

Fig. 1. Sall1 is expressed by cortical PCs. Sall1immunohistochemistry (red) at E11.5 (A,D), E13.5 (B,E)and E17.5 (C,F). Sections are counterstained with DAPI(blue). At E11.5, E13.5 and E17.5, strong Sall1expression is observed in the progenitor population inthe dorsal cortex and lateral ganglionic eminence (A-C). Weaker expression is observed in the medial cortexat E11.5 (*; A) and the medial ganglionic eminence atE11.5 and E13.5 (A,B). Sall1 is also expressed by asubpopulation of cells outside the progenitorpopulation (arrows, A,B,E,F). Sall1 expression is absentfrom the subicular neural epithelium (S; A,B). High-power examination of the dorsal cortex indicates thatSall1 was expressed by PCs (D-F) and, at E17.5, bysubsets of cells in the SVZ (F). Dorsal (DL) is up andmedial (M) is right, as indicated (A). LGE, lateralganglionic eminence; MGE, medial ganglioniceminence; VZ, ventricular zone; POA, pre-optic area; PP,preplate; CP, cortical plate; SVZ, subventricular zone; IZ,intermediate zone; SP, subplate; MZ, marginal zone.Scale bar: 75m (D); 125m (E); 200m (F); 300m(A); 600m (B); 1100m (C).

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

number of RGCs and a 24.6% increase (P<0.05, n4) in the numberof neurons in the preplate was observed in Sall1–/– mice comparedwith controls (Fig. 4A-C), suggesting that early cortical PCsdifferentiate at the expense of re-entering the cell cycle.

The preplate subsequently splits to form the subplate andmarginal zone. To determine whether structures derived from theenlarged preplate were altered in Sall1-mutant animals, weexamined molecular markers of the marginal zone and subplate atE14.5 and E18.5. Chondroitin sulphate proteoglycan (CSPG) is anextracellular matrix protein expressed by cells in the pia, subplateand marginal zone (Sheppard et al., 1991). In Sall1-mutant mice,an increase in CSPG staining was observed in the subplate andmarginal zone at E14.5 (n3; Fig. 4D,E) and E18.5 (data not shown),compared with controls. Quantification of Tbr1-immunopositivesubplate cells (see Methods) verified the increase in subplate cellnumber in Sall1-mutant animals at E18.5 (n3, P<0.01; Fig. 5D).Intense staining for CSPG in pial cells makes it difficult to quantifyCSPG-positive cells in the marginal zone; therefore, an independentmarker of marginal zone cells was examined. Reelin is a secretedmolecule expressed by Cajal-Retzius cells in the marginal zone(Ogawa et al., 1995; Schiffmann et al., 1997). In Sall1-deficient mice,more reelin-expressing cells were apparent in the marginal zone atE14.5 (Fig. 4F,G; n4) and E18.5 (n4; data not shown), comparedwith controls. Interestingly, these changes were accompanied by a28.9% decrease in the number of cells in the cortical plate at E14.5in Sall1-deficient animals compared with controls (supplementarymaterial Table S1; n3, P<0.05). This implies that, in the absenceof Sall1, more cells are committed to early born structures, thepreplate and its derivatives (the marginal zone and the subplate),and that fewer cells are committed to the later born structures suchas the cortical plate.

To verify this hypothesis, birth-dating studies were performedto determine whether the temporal order of neuronal specificationwas altered in the absence of Sall1. Pregnant dams were injected

with BrdU on E11.5, E12.5 or E14.5, and the number anddistribution of BrdU-labeled cells was examined at E18.5. The totalnumber of cells born on E11.5 and E12.5 and present at E18.5 wasincreased in Sall1-mutant animals compared with controls(supplementary material Fig. S2, Table S1; E11.5: 41% increase, n3,P<0.05; E12.5: 26.3% increase, n4, P<0.01); however, the totalnumber of cells born on E14.5 and present at E18.5 was similarbetween Sall1-deficient animals and controls (supplementarymaterial Fig. S2, Table S1; n3, P0.1). Interestingly, the proportionof labeled cells in the cortical plate was decreased (E11.5: –45.7%,n3, P<0.05; E14.5: –11.8%, n3, P<0.01), with a concomitantincrease in the subplate (E11.5: +47.8%, n3, P<0.05; E12.5: +15.8%,P<0.05, n4) or intermediate zone and PC populations (E14.5:+22.2%, n3, P<0.05, and +98.5%, n3, P<0.05, respectively) inSall1–/– versus control animals (Fig. 5A-C). The majority of cellsborn on E14.5 that were located within the intermediate zone atE18.5 expressed neuronal markers and were observed below thehistologically distinguished subplate in Sall1-deficient animals(supplementary material Fig. S2), which suggests that cells bornon E14.5 are not continuously committed to a subplate fate pasttheir normal birth-date in Sall1–/– animals but that neuronalmigration might be altered.

Sall1 regulates deep versus superficial laminar fateGiven the decreased size of the cortical plate and increased numberof cells committed to the subplate and marginal zone, and thechanges in PC number, we investigated whether the inside-outpattern of cortical neurogenesis and layer specification waspreserved in Sall1 mutant mice. The expression of four markersthat are expressed in sequentially more superficial layers wasexamined at E18.5. No alteration was observed in the position of:the layer VI marker Tbr1 (supplementary material Fig. S1A,B)(Bulfone et al., 1995; Hevner, 2007; Hevner et al., 2001); the layerV marker Ctip2 (Arlotta et al., 2005; Avram et al., 2000)


Sall1 regulates cortical neurogenesisRESEARCH ARTICLE

Fig. 2. Sall1 is differentially expressed by RGCsversus IPCs. (A-G)Expression of phospho-histoneH3 (PH), Sall1 (S1), nestin (N), Tbr2 (T2) and Tbr1(T1) in E12.5 embryos. Sall1 is in red and othermarkers are in green. Double labeling (arrows)with Sall1 and PH3 (A-C) or nestin (D,E) indicatesthat these markers are coexpressed with Sall1 inPCs. White arrows in B and C indicatecoexpression. The nestin-positive cell identified inD is magnified in E. At E12.5, Sall1 is absent fromTbr1-positive neurons (G) and present in a subsetof Tbr2-positive cells (F). (H,I)Sall1 is expressed in asubset of Tbr2-positive cells at E14.5 (H) and E18.5(I). The line in H,I represents the VZ-SVZ border.

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

(supplementary material Fig. S1C,D); Brn2, expressed by layer Vand layer II/III cells, as well as PCs and the intermediate zone (Heet al., 1989; McEvilly et al., 2002) (supplementary material Fig.S1E,F); or Cux1, which is expressed by cells in upper cortical layersII/III and IV, as well as cells in PCs and the intermediate zone(supplementary material Fig. S1G,H) (Nieto et al., 2004). Takentogether, these data suggest that loss of Sall1 does not alter cell-type specification of neurons destined for the cortical plate andthat the inside-out pattern of neurogenesis is preserved.

In light of the decrease in size of the cortical plate and changesin the properties of cells born on E14.5, when upper cortical layersare being specified, we investigated whether Sall1 regulated thetemporal production of deep versus superficial layer fate. Theproportion of cells born on E14.5 that gave rise to deep(BrdU+Tbr1+; layer VI) or superficial (BrdU+Cux1+; layers II/III,IV) cortical layer neurons was quantified at E18.5. In Sall1–/–animals, the proportion of cells born on E14.5 specified as deepcortical layer neurons was increased by 213.6% (P<0.05, n3;supplementary material Table S1), whereas the production ofsuperficial cortical layer neurons was decreased by 28.6% (P<0.01,n3; supplementary material Table S1), compared with controls.By E18.5, total cell number in layer VI and superficial cortical layers

were decreased by 24.5% (P<0.05, n3) and 35.9% (P<0.05, n4),respectively, in Sall1–/– animals versus controls (Fig. 5D).Interestingly, when these values were expressed as a percent of totalcortical plate cell number, no significant difference in the proportionof cells committed to layer VI was observed (n3, P0.4), whereasthe proportion of cells committed to superficial cortical layers wasdecreased by 19.1% (n4, P<0.01) in Sall1–/– animals compared withcontrols (Fig. 5E). Therefore, between E11.5 and E14.5, cells areselectively committed to a deep cortical layer fate at the expenseof a more superficial cortical layer fate in Sall1–/– animals comparedwith controls.

Sall1 regulates cell cycle exit and IPC number during developmentThe changes in the number of neurons in different cortical regionscould be a consequence of disruptions in PC proliferation,differentiation and/or neuronal migration. RGCs give rise to asecond proliferative population, the SVZ, which consists of IPCs,a PC population characterized by basal mitotic divisions. Recentdata suggest that the majority of neurons in the cortical plate areproduced via terminal neurogenic divisions of IPCs (Haubensak etal., 2004; Miyata et al., 2004; Noctor et al., 2004; Noctor et al., 2008).In light of the alterations in PC and neuronal number in Sall1–/–



Fig. 3. The cerebral cortex isdecreased in size at E18.5 in Sall1–/–

mice. The cerebral cortex isdecreased in size in Sall1-deficientanimals (B) compared with controls(A) at E18.5. Measurements of brainsurface area (C) and length (D) ofcontrol and Sall1–/– animals. (E-J)Nissl-stained coronal sections ofthe cortex at E18.5 (E-J) in control(E,G,I) and Sall1–/– animals (F,H,J). Thecortical plate and striatum aredecreased in size in Sall1-deficientanimals (E-J). *, ventral progenitorpopulation (E-H). (K)Quantification ofdorsal cell number in control andSall1-mutant animals. *, P<0.05; **,P<0.01 (C,D,K). OB, olfactory bulb; CC,cerebral cortex; MB, midbrain; CP,cortical plate; S, striatum; DG, dentategyrus; H, hippocampus; PC,progenitor population; IZ,intermediate zone; CP, cortical plate;MZ: marginal zone; D, dorsal; L,lateral. Scale bars: 2500m (in B forA,B); 100m (in J for I,J); 500m (in Jfor E-H).

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

animals, the proliferative properties of RGCs and IPCs wereexamined using short-term BrdU incorporation studies todetermine whether Sall1 regulates PC cell cycle kinetics. Thesestudies indicated that RGCs and IPCs were differentially alteredover time in Sall1–/– mice compared with controls (Fig. 6A). Timedpregnant animals were injected with BrdU on E11.5, E14.5 or E17.5,and embryos were collected 60 minutes later. The number of cellsin the VZ and SVZ was calculated and the proportion labeled withBrdU calculated. The labeling index was calculated for each ageand progenitor region (BrdU+/total VZ PC or BrdU+/total SVZPC; details of cell counts are provided in the Methods). Theselabeling indices were identical at all ages and regions examined incontrol and mutant animals, indicating that the proportion of cellsin S-phase of the cell cycle is not altered in the VZ or SVZ in the

absence of Sall1, and suggests that Sall1 does not regulate cell cyclekinetics or the rate of proliferation of cortical progenitors (Fig. 6B;E11.5: n3, P0.8; E14.5: n4, VZ P0.5, SVZ P0.4; E17.5: n4,VZ P0.5, SVZ P0.4). However, the total number of RGCs in theVZ was initially decreased at E12.5 (n4, P<0.01), increased by24.8% at E14.5 (n3, P<0.01), and unchanged at E17.5 and E18.5(n3, P0.7; n4, P0.2, respectively; supplementary material TableS1, Fig. S3; Fig. 6A) in Sall1–/– animals compared with controls.To determine whether early IPCs require Sall1 expression, weutilized PH3 staining to identify and quantify the number of basalmitotic divisions. No significant difference in the number of basalcortical mitotic divisions was observed at E12.5 between controland Sall1–/– mice (n4, P0.2; data not shown), which suggests thatearly cortical IPCs are not altered in the absence of Sall1. Expression



Fig. 4. Early born neuronal populations are overproduced in Sall1–/– mice. (A,B)Tuj1 (green) staining identified an increase in preplate size at E12.5 in Sall1-deficient animals (B) compared with controls (A). (C)Quantification of cortical cell number at E11.5 and E12.5. *, P<0.05; **, P<0.01. (D,E)CSPG (red)immunostaining of the subplate (bracketed region) and marginal zone identified an increase in these structures in Sall1–/– mice (E) compared with controls (D) atE14.5. Note: CSPG is also expressed by the meninges at the pial surface. (F,G)Reelin (red) immunostaining identified an increase in Cajal-Retzius cells in Sall1–/–

mice (G) compared with controls (F) at E14.5. (A,B,D-G) Sections are counterstained with DAPI (blue). PP, preplate; PC, progenitor population; IZ, intermediatezone; SP, subplate; CP, cortical plate; MZ, marginal zone; P, pial surface. Scale bar: 100m (A,B); 200m (D,E); 400m (F,G).

Fig. 5. Sall1 regulates the number ofcells committed to an early born fate.(A-C)Quantification of the proportion oftotal cells born (BrdU+) on E11.5 (A), E12.5(B) or E14.5 (C) in defined cortical layers atE18.5. (D,E)Quantification of cell numberin molecularly defined [Tbr1+: subplateand layer VI (LVI); Cux1+: layers II/III, IV(LII/III,IV)] cortical laminae at E18.5 (D) andthe proportion of cells in cortical laminaeas a percentage of total cortical plate cellnumber at E18.5 (E). *, P<0.05; **, P<0.01.PC, progenitor population; IZ,intermediate zone; SP, subplate; CP,cortical plate; MZ, marginal zone.

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

of the transcription factor Tbr2 can be used to identify IPCs(Englund et al., 2005). Quantifying Tbr2-positive IPC numberindicated that Tbr2+ IPCs were decreased by 48.1% by E14.5 (n3,P<0.01), unchanged at E17.5 (n3, P0.7) and increased by 23% atE18.5 (n3, P<0.05) in Sall1–/– mice compared with controls (Fig.6D; supplementary material Fig. S4). These findings identify a rolefor Sall1 in regulating the production of the Tbr2+ IPC populationfrom mid-neurogenesis.

The dramatic changes identified in PCs in Sall1-deficient animalsprompted us to examine the rate of neuronal differentiation. Theproportion of cells that re-enter versus exit the cell cycle in a 24-hour period was quantified using long-term BrdU incorporationstudies. Pregnant dams were injected with BrdU on E11.5, E14.5 orE17.5, and embryos were collected 24 hours later. The proportionof cells that differentiated into neurons was quantified(BrdU+Tuj1+/total BrdU+) and termed the quiescent-fraction (Q).Q gradually increases from 0 to 1 in control animals, whereas theproliferative fraction (P) decreases from 1 to 0 from E9.5 to E18.5(Takahashi et al., 1994). At E11, Q was 0.19±0.01 in control animalsand 0.24±0.02 in Sall1-deficient animals, which represents a 26.3%increase over controls (Fig. 6C; n4, P<0.05). This finding verifiesour previous results that showed increased numbers of neurons atE12.5 in Sall1-mutant animals. By E14.5, Q was 0.51±0.01 in controlanimals versus 0.44±0.01 in Sall1 mutants, which represents a 13.7%decrease compared with controls (Fig. 6C; n3, P<0.05). This suggeststhat more cells are re-entering the cell cycle in mutants than incontrols. Interestingly, the decrease in Q at E14.5 seemed to be inpart due to a decrease in the previously described rapidly exitingsubpopulation of cells born on E14.5 (supplementary material Fig.S3) (Takahashi et al., 1996a). By E17.5, Q increased to 0.76±0.01 incontrol animals and to 0.68±0.02 in Sall1-deficient animals (Fig. 6C),which represents a 10.5% decrease in Q in the absence of Sall1compared with controls (n4, P<0.05). Thus, in the absence of Sall1,early PCs differentiate rather than re-enter the cell cycle, whereas,later in neurogenesis, PCs preferentially re-enter the cell cycle ratherthan differentiate, compared with controls.

These findings indicate that E14.5 is a critical developmentalpoint, when Sall1 switches from promoting a proliferative fate toa differentiative fate. The early decrease (E14.5) and later increase(E18.5) in Tbr2-positive cells and the changes observed in neuronaldifferentiation in Sall1-mutant animals suggested that changes inthe type of division that a PC makes might contribute to theobserved phenotype. The proportion of E14.5 and E17.5proliferating cells that re-entered the cell cycle and gave rise toRGCs (Pax6+) or IPCs (Tbr2+) within a 24-hour period wasquantified using BrdU labeling. Although Pax6 expression does notexclusively identify RGC populations, because its expression ismaintained in ~25% of Tuj1+ newly differentiated neurons up to20 hours after cell division, (Kawaguchi et al., 2004) and becausecoexpression of Pax6 and Tbr2 has been observed in cells at theVZ-SVZ border (Englund et al., 2005), intense Pax6 staining(Pax6+++) can be used to identify RGCs and their immediateprogeny. We observed a 21.0% increase in the proportion of BrdU-labeled Pax6+++ RGCs (n3, P<0.001) with no change in theproportion of Tbr2+ IPCs (n4, P0.3) in Sall1–/– compared tocontrol animals at E14.5 (Fig. 6E). This suggests that, in the absenceof Sall1, proportionately more PCs are undergoing proliferativeRGC divisions at mid-neurogenesis. By E17.5, a 14.6% increase inthe proportion of cells that re-entered the cell cycle as Tbr2+ IPCswas observed in Sall1–/– animals compared with controls (P<0.05,n3; Fig. 6E). The fact that the proportion of cells re-entering thecell cycle as Pax6+++ RGCs was unchanged (P0.6, n3; Fig. 6E)suggests that, in the absence of Sall1, proportionately more IPCsare undergoing self-renewing divisions, rather than terminalneurogenic divisions, at this age. Together, these findings indicatethat Sall1 is a crucial regulator of PC maturation duringdevelopment.

Sall1 expression in dorsal progenitor cells regulates progenitorcell maturationOur results point to a role for Sall1 in regulating neuronaldifferentiation in the dorsal cortex. However, alterations in the ventral



Fig. 6. Sall1 regulates cell cycle exit.Quantification of VZ and SVZ cellnumber at E11.5, E12.5, E14.5, E17.5 andE18.5 in control and Sall1–/– mice (A).The labeling index was not altered inSall1-deficient animals at E11.5, E14.5 orE17.5 compared with controls (B). Therate of neuronal differentiation (Qfraction) was increased at E11.5 (E12.5:BrdU injected at E11.5), but decreased atE14.5 (E15.5: BrdU injected at E14.5) andE17.5 (E18.5: BrdU injected at E17.5) inSall1–/– animals compared with controls(C). Quantification of Tbr2 cell numberat E14.5, E17.5 and E18.5 (D). Alterationsin the rate of VZ (Pax6+) and SVZ(Tbr2+) cell cycle re-entry in Sall1–/–

animals compared with controls at E14.5and E17.5 (E). *, P<0.05; **, P<0.01; ***,P<0.001.

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

forebrain were histologically observed in Sall1-deficient mice.Interneurons derived from the ventral telencephalon tangentiallymigrate to their final laminar cortical position through the SVZ,intermediate zone and marginal zone (reviewed in Marin andRubenstein, 2001). Furthermore, dorsal progenitors have been shownto molecularly interact with ventrally derived migrating interneurons(Tiveron et al., 2006) and it is hypothesized that migratinginterneurons can influence dorsal PC differentiation (Haydar et al.,2000). To determine the contribution of ventrally derived cells to theobserved phenotype, we generated mice with a floxed Sall1 allele(Inoue et al., 2010; Yuri et al., 2009) and restricted deletion of Sall1to dorsal PCs using Emx1-Cre (mutant animals are termed Sall1-cKOEmx1). Emx1 expression initiates at E9.5, and Emx1-driven Crerecombination is evident from E10.5 (Gorski et al., 2002).Recombination is observed in the majority of excitatory neurons inthe cortex, but not in interneurons (Gorski et al., 2002). In order todetermine whether recombination of the floxed Sall1 allele hadoccurred, we examined Sall1 expression in the dorsal cortex at E12.5(Fig. 7A,B). In control animals, Sall1 expression extended into thedorsal cortex at E12.5 (Fig. 7A); however, in Sall1-cKOEmx1 embryos,strong Sall1 expression was limited to the ventral telencephalon anddid not extend beyond the cortico-striatial boundary (n3; arrow,Fig. 7B). Weak Sall1 expression was observed in the dorsal cortex atE12.5 in Sall1-cKOEmx1 animals, which might represent residual Sall1protein present in dorsal PCs produced prior to the recombinationevent. However, functional deletion of Sall1was confirmed by E12.5because the Sall1-cKOEmx1 cortical phenotype was identical to Sall1-null animals. An increase in the number of Tuj1-positive cells presentin the dorsal cerebral cortical wall was observed in Sall1-cKOEmx1

mice compared with controls (Fig. 7C,D). Also, similar to the Sall1-null animals, CSPG staining revealed an increase in the number ofcells in the subplate by E18.5 in Sall1-cKOEmx1 animals comparedwith controls (Fig. 7E,F). Deficits in the ventral telencephalon werenot observed, as expected.

Similar to null animals, the total length and dorsal surface areaof the brain was decreased by 3.6% (P<0.05, n3) and 5.8% (P<0.05,n3) at E18.5, respectively, in Sall1-cKOEmx1 animals compared withcontrols (supplementary material Fig. S5). Specifically, the cerebralhemispheres were decreased in length and dorsal surface area by12.8% (P<0.05, n3) and 12.8% (P<0.05, n3), respectively, in Sall1-cKOEmx1 animals compared with controls. As expected, noalteration in the length (P0.9, n3) or dorsal surface area (P0.9,n3) of the midbrain or other brain regions were observed(supplementary material Fig. S5). We quantified the number of cellsin the dorsal cortex in Sall1-cKOEmx1 animals and, similar to Sall1-deficient animals, observed an increase in the number of PCs(17.6%; n3, P<0.05) and a decrease in the number of cells in thecortical plate (23.3%; n3, P<0.01) at E18.5 in Sall1-cKOEmx1

animals compared with controls (supplementary material Fig. S5).To determine whether the increase in the number of PCs was dueto an increase in the number of IPCs, we examined Tbr2 stainingat E18.5. In Sall1-cKOEmx1 animals, an increase in the number ofTbr2-positive cells was observed in the dorsal cortex at E18.5compared with controls (n3; Fig. 7G,H). These findings suggesta requirement for Sall1 expression in dorsal PCs to regulate PCmaturation.

Cells born in the ventral telencephalon migrate through theintermediate zone and marginal zone during development enroute to the cortex. Although we observed an increase in subplatecell number (in the intermediate zone) and reelin-positive Cajal-Retzius cell number (in the marginal zone) in Sall1–/– animals, weobserved no alteration in total cell number in the intermediate zoneor marginal zone in Sall1-deficient animals compared with controls.In Sall1-cKOEmx1 animals, we also observed an increase in subplateand marginal zone populations; however, total cell number wasincreased by 16.2% in the intermediate zone (16.2%; n3, P<0.01)and by 25.5% in the marginal zone (28.5%; n3, P<0.001) comparedwith controls at E18.5 (supplementary material Fig. S5). These data



Fig. 7. Conditional deletion of Sall1 in dorsal PCs indicates that Sall1 regulates PC fate progression. Sall1 (red) expression is localized to the ventraltelencephalon and does not extend into the dorsal cortex in Sall1-cKOEmx1 animals (arrow, B), unlike controls (A) at E12.5. Tuj1 staining (green) of cortical neuronsat E12.5 identified an increase in preplate cell number in Sall1-cKOEmx1 (D) animals compared with controls (C). CSPG (red) immunostaining of the subplate andmarginal zone identified an increase in these structures in Sall1-cKOEmx1 (F) animals compared with controls (E) at E18.5. Tbr2 (red) immunostaining at E18.5identified an increase in IPCs in Sall1-cKOEmx1 (H) animals compared with controls (G). PC, progenitor population; PP, preplate; SP, subplate; CP, cortical plate; MZ,marginal zone. Scale bar: 100m (C,D,G,H); 350m (E,F); 400m (A,B).

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

suggest that a population of ventrally derived migrating cells isdecreased in Sall1–/– animals and that this deficit is rescued in Sall1-cKOEmx1 animals.

DISCUSSIONThese studies demonstrate that Sall1 regulates development of thecerebral cortex in mice and provide insight into neuralabnormalities that might be present in individuals with TBS. Sall1is expressed by CNS PCs throughout neurogenesis, as well as inperipheral organs. Using classical knockout studies, we identifieda unique requirement for Sall1 expression in dorsal cortical PCs.At E18.5, both the surface area and depth of the cerebral cortexwere decreased in size in Sall1–/– animals compared with controls.These alterations are a consequence of changes in the type ofdivision that cortical PCs undergo. Early in development, Sall1–/–PCs preferentially exit the cell cycle, whereas later in developmentIPCs preferentially re-enter the cell cycle. These changes areassociated with an increased commitment of neurons to an earlycortical fate and a decrease in the number of neurons committedto a later cortical fate, compared with controls. These findingssuggest that Sall1 temporally regulates cortical neurogenesis incortical PCs.

Sall1 regulates neuronal output in deep versus superficial corticallayersAlthough the inside-out pattern of laminar specification wasnormal in the absence of Sall1, the temporal order of deep tosuperficial laminar fate specification was altered. The first neuronallayer to be specified is the preplate, born from E11.5 to E12.5 incontrol animals, which splits to form the subplate and marginalzone at ~E13.5 (Gupta et al., 2002; Price et al., 1997; Sheppard andPearlman, 1997). Deep cortical layers (layers VI and V) aresubsequently specified from E12.5 to E13.5, with production ofsuperficial cortical layers (layers II, III, IV) predominating fromE14.5 (Angevine and Sidman, 1961; Caviness, 1982; Gillies andPrice, 1993; Levers et al., 2001; Price et al., 1997; Takahashi et al.,1999). From E11.5 to E12.5, Sall1-deficient PCs preferentially exitthe cell cycle at the expense of re-entering. Birth-dating studiesindicate that, early in development, proportionately more of thesedifferentiating neurons gave rise to subplate cells at the expense ofcommitting cells to a cortical layer fate in Sall1–/– animals. Thesefindings suggest that early in development Sall1 promotesproliferative symmetric divisions over asymmetric differentiativedivisions in RGCs and restricts neurogenesis of preplate neuronsin early cortical PCs.

Interestingly, increased early neurogenesis is often associatedwith depletion of the PC population. As a consequence, the corticalplate is decreased in size owing to the lack of available PCs laterin development (Bedford et al., 2005; Caviness et al., 2003; Handleret al., 2000; Roy et al., 2004; Wines-Samuelson et al., 2005). Ourfindings suggest that the decrease in cortical plate size in Sall1-mutant animals is not simply due to depletion of the PC population.Although Sall1 promotes cell cycle re-entry in early cortical PCsfrom E11.5 until E13.5, it facilitates cell cycle exit in late PCs. Theparadoxical role of Sall1 in early versus late PCs is mediated bychanges in the properties of RGCs versus IPCs and occurs as PCstransition from producing neurons with a deep layer fate to thosewith a superficial layer fate. We demonstrated that proportionately

more PCs re-entered the cell cycle as Pax6+++ RGCs and fewerexited the cell cycle and differentiated into neurons in Sall1-mutantanimals at E14.5, compared with controls. These findings indicatethat, from E14.5, differentiative divisions were decreased and thatsymmetric VZ proliferative divisions were increased in Sall1–/–animals compared with controls. Interestingly, these changes wereassociated with a decrease in the rapidly exiting subpopulation ofcells born on E14.5 (Takahashi et al., 1996a). Birth-dating of Sall1-deficient PCs at E14.5 indicated that proportionally more PCs gaverise to deep cortical layers (Tbr1+ layer VI neurons) andproportionately fewer PCs gave rise to superficial cortical layers(Cux1+ layer II/III, IV neurons) compared with controls.Coincident with these changes in laminar specification, RGCs wereincreased and IPCs were decreased in Sall1-deficient animals atE14.5, compared with controls. By E17.5, however, an increase inthe proportion of cells that re-entered the cell cycle as Tbr2+ IPCswas observed in Sall1–/– animals compared with controls,accompanied by a decrease in the proportion of cells thatdifferentiated into neurons, with no change in the proportion ofcells that re-entered the cell cycle as Pax6+++ RGCs. This suggeststhat Sall1–/– IPCs are undergoing symmetric self-renewing divisionsat the expense of neuronal differentiation. Because early (E12.5)IPC number was normal in Sall1-deficient animals, theseobservations support the hypothesis that Sall1 might influence thetransition from deep to superficial neuronal specification byregulating the terminal differentiation of IPCs.

A role for Sall1 in intermediate progenitor cell functionIt is interesting that Sall1 is highly expressed in RGCs anddownregulated in IPCs yet it has a major phenotype in regulatingIPC properties. What are the molecular mechanisms that couldmediate the actions of Sall1 in RGCs versus IPCs? Recently, severalmolecules have been implicated in regulating IPC proliferationand differentiation. Tbr2 is sufficient to suppress a radial glial cellphenotype and promote an IPC phenotype (Sessa et al., 2008).Disruption of Notch or Fragile-X mental retardation proteinsignaling promotes the transition of RGCs to IPCs (Mizutani etal., 2007; Saffary and Xie, 2011; Yoon et al., 2008). IPC numberis decreased in the absence of either Frs2, Foxg1 or insulinomaassociated 1, which indicates that these proteins regulate theexpansion of IPCs (Siegenthaler et al., 2008; Yamamoto et al.,2005). By contrast, Cux2 has been shown to limit the proliferationof IPCs. In the absence of Cux2, an increased number of IPCswas observed at E15.5, similar to the phenotype observed in Sall1-deficient mice (Cubelos et al., 2008). One of the most likelysignaling pathways that Sall1 could interact with is the Wntpathway. Similar to Sall1, analysis of Wnt signaling in cortical PCshas shown that the Wnt–b-catenin pathway has differentialeffects on RGCs versus IPCs (Wrobel et al., 2007; Munji et al.,2011). A downstream mediator of the Wnt pathway, b-catenin,is predominantly expressed in RGCs and its expression must bedownregulated to induce the production of IPCs (Mutch et al.,2010). Interestingly, numerous studies have shown that Sall1 isactivated in response to Wnt signaling and can interact with b-catenin (de Celis et al., 1996; de Celis et al., 1999; Farrell andMunsterberg, 2000; Sato et al., 2004). Sall1 might thereforemediate some of the effects of Wnt signaling in cortical PCs tocontrol the transition from an RGC to an IPC.


Sall1 regulates cortical neurogenesis RESEARCH ARTICLED

iseas

e M

odel

s & M

echa

nism

s

DM

M

Sall1- and Cux2-deficient animals both have an increasednumber of IPCs; however, in Cux2-deficient animals this increasewas associated with an increase in the number of cells in superficialcortical layers, whereas, in Sall1-deficient mice, superficial corticalneuron number is decreased. This decrease in Sall1-mutant animalsis a consequence of decreased neuronal differentiation from E14.5,as well as a decrease in the commitment of neurons to a superficialcortical layer fate. This does not exclude the possibility that theincreased number of IPCs present in Sall1–/– mice at E18.5 mightbe continuously committed to the production of superficial corticallayers postnatally, leading to normal neuronal number in superficiallayers in adults. Also, because Sall1 is predominantly expressed inradial glial cells, it is possible that Sall1 regulates migration ofneurons in a non-cell-autonomous manner. These potentials couldbe examined by analyzing the postnatal maturation of the cerebralcortex and the final organization of cortical laminae. Alternatively,gliogenesis might be altered in Sall1-deficient mice, because glialcells are born at late prenatal and early postnatal stages. However,we have not seen any differences in glial or oligodendrocytemarkers in Sall1-null or conditional knockout animals (ourunpublished observations). In summary, our findings support thehypothesis that, from mid-neurogenesis, Sall1 promotes terminalneurogenesis of PCs, initially by controlling the transition from anRGC to an IPC and later by regulating terminal neurogenesis ofIPCs.

The SALL family in human diseaseIn summary, we identified a previously unknown role for Sall1 inregulating PC fate progression in the developing cortex. In earlycortical PCs Sall1 promotes proliferative over neurogenic divisionsin RGCs, whereas later in development, Sall1 promotesdifferentiative over proliferative divisions in IPCs. Other membersof the Sall gene family have also been shown to restrict PCproliferation and must be eliminated in a cell to allow cellulardifferentiation in other systems. Sall1 is required for theproliferation or survival of embryonic stem cells (Karantzali et al.,2011) and in multipotent renal PCs in mice, but is not required fortheir generation or differentiation (Nishinakamura, 2008;Nishinakamura et al., 2011). In Drosophila, a Sall1 homolog, theSpalt gene, has been shown to restrict the number of sensory organprecursor cells and inhibit their neuronal differentiation (de Celiset al., 1999; Rusten et al., 2001). The related Sall family memberSall4 is required to maintain cells in a proliferative undifferentiatedstate (Barembaum and Bronner-Fraser, 2004; Elling et al., 2006;Sakaki-Yumoto et al., 2006). Of the four mammalian Sall genesidentified, expression studies indicate that they are expressed incortical PCs as well as in distinct and overlapping structures in thedeveloping nervous system and peripheral organs (Harrison et al.,2008a; Harrison et al., 2008b; Kohlhase et al., 2000; Kohlhase et al.,2002a; Ott et al., 2001) (and our unpublished observations).Mutations in three of these genes are associated with known humandisorders with neural abnormalities and overlapping phenotypesin peripheral organs, including TBS (SALL1), Okihirosyndrome/Duane radial ray syndrome, Holt-Oram syndrome, acro-renal-ocular syndrome (SALL4) and 18q deletion syndrome(SALL3) (Al-Baradie et al., 2002; Kohlhase et al., 1999a; Kohlhaseet al., 2002b; Kohlhase et al., 1998). Many of the mutationsidentified in individuals with TBS are located in exons 2 and 3 and

in intron 2 of Sall1, and are predicted to lead to prematuretermination of transcription (Blanck et al., 2000; Engels et al., 2000;Kohlhase et al., 1999b; Kohlhase et al., 2003; Kohlhase et al., 1999c;Kohlhase et al., 1998; Marlin et al., 1999; Salerno et al., 2000; Surkaet al., 2001; Walter et al., 2006). Although it is thought that manyof these transcripts will be degraded by nonsense-mediated RNAdecay, recent evidence has suggested that the truncated protein canfunction in a transdominant negative manner (Kiefer et al., 2003;Kiefer et al., 2008). These findings indicate that, in humans, TBScould be caused by a gain of function. However, deletions of SALL1have also been identified in some individuals with a milder formof TBS (Borozdin et al., 2006), indicating that haploinsufficiencycan also lead to the disorder. It is therefore likely that the differencesseen in the two phenotypes observed in humans and mice witheither a deletion or truncation of the Sall1 gene are due to the cell-type-specific protein interactions on target gene promoters.Previous studies demonstrated that Sall family members couldinteract with each other via their N-terminal region (Kiefer et al.,2003; Sakaki-Yumoto et al., 2006; Sweetman et al., 2003). Ourinvestigations and those of other researchers have found that therelated family members Sall2, Sall3 and Sall4 are also expressed incortical neural progenitor cells (Magdaleno et al., 2006; Ott et al.,2001; Parrish et al., 2004), suggesting that Sall family membersmight functionally compensate for each other and/or functionallyinteract to regulate neurogenesis in the developing cerebral cortex.The rarity of TBS in humans and the variable penetrance of thisdisorder make it difficult to assign a concise neural phenotype inindividuals with TBS. The findings presented here provideimportant insights into the role of Sall1 in neural precursors, withrelevance to human neural precursors, and will provide aframework for future studies aimed at examining neural functionin individuals with TBS.

METHODSAnimalsEmbryos were obtained from the mating of Sall1 heterozygote(Sall1+/–) animals, maintained on a C57BL/6J background, andgenotyped as previously described (Nishinakamura et al., 2001).No alterations in cortical development were observed in Sall1+/–

animals in our study (unpublished observations) and therefore theseembryos were also used as controls. Controls (Sall1+/+ or Sall1+/–mice) are jointly referred to as +/. Emx1-Cre animals (Gorski etal., 2002) were genotyped by PCR for the Cre gene, using previouslydescribed conditions (Furuta et al., 2000). For Sall1 expressionstudies, embryos were obtained from timed pregnant CD1 micefrom Charles Rivers Laboratories (Wilmington, MA). The day ofvaginal plug was designated as embryonic day 0.5 (E0.5). For birth-dating studies, pregnant dams were injected with BrdU (50 g/gof body weight) at the indicated times before embryo collection.For proliferation studies, BrdU was injected 60 minutes prior toembryo collection. Embryos were collected via cesarean sectionfrom E11.5 to E18.5. Brains from E18.5 embryos were dissectedfrom the skull prior to fixation. Embryos were either fixed in 4%paraformaldehyde (PFA; pH 7.4) and processed through increasingsucrose gradients for cryosectioning, or fixed in Carnoys solution(1:3:6 acetic acid: chloroform: 100% ethanol) and then processedthrough a butanol series for paraffin sectioning. PFA-fixed brainswere embedded in Tissue-Tek Optimal Cutting Temperature



iseas

e M

odel

s & M

echa

nism

s

DM

M

(O.C.T.) compound (EMS, Hatfield, PA) and sectioned at 20 m.Paraffin-embedded embryos were sectioned at 10 m. Animalprotocols and procedures were approved by the InstitutionalAnimal Care and Use Committee at the University of Pittsburghand adhered to the National Institutes of Health guidelines.

Measurement of cortical sizeBrains from E18.5 embryos were dissected from the skull and fixedovernight in 4% PFA. Brains were visualized on a Nikon (Melville,NY) dissecting microscope, photographed with a Photometrics(North Reading, MA) CoolSNAP digital camera and IP Labsoftware (Biovision Technologies, Exton, PA). Images weresubsequently imported into Photoshop 7.0 (Adobe Systems, SanJose, CA). Analyses were performed by an observer blind to thegenotype. Total brain length was measured as the distance fromthe rostral extent of the olfactory bulb to the caudal extent of theinferior colliculus. Cortical length was measured as the distancefrom the rostral extent of the cortex to the caudal extent of thecortex, at the lateral edge of the olfactory bulb. Midbrain lengthwas measured as the distance from the rostral extent of thesuperior colliculus to the caudal extent of the inferior colliculus.Surface area was analyzed in similarly defined regions. Statisticalanalysis was performed using an unpaired t-test with InStat 3software (GraphPad Software, San Diego, CA).

ImmunohistochemistryImmunohistochemistry and Nissl staining was performed aspreviously described (Harrison et al., 2008a; Roy et al., 2004), withthe following antibodies: mouse anti-BrdU (1:25, AmershamBiosciences, Piscataway, NJ); rabbit anti-Brn2 (1:500, Santa CruxBiotechnology, Santa Cruz, CA); rabbit anti-activated-caspase-3(1:200, Promega, Madison, WI); mouse anti-CSPG (1:5,Developmental Studies Hybridoma Bank, The University of Iowa,IA); rat anti-Ctip2 (1:500, Abcam, Cambridge, MA); rabbit anti-Cux1 (Santa Crux Biotechnology); chicken anti-nestin (1:500,Thermo Scientific); rabbit anti-Pax6 (1:500, Covance, Berkeley, CA);rabbit anti-PH3 (1:200, US Biological, Swampscott, MA); mouseanti-reelin (1:500, Abcam); mouse anti-Sall1 (1:500, PPMX PerseusProteomics, Tokyo, Japan); rabbit anti-Tbr1 (1:1000, Chemicon,Temecula, CA); rabbit anti-Tbr2 (1:1000, Chemicon); rabbit anti-Tuj1 (1:1000, Sigma, St Louis, MO). Sections were visualized on aNikon fluorescent microscope, photographed with a PhotometricsCoolSNAP digital camera and IP Lab software. Composite imageswere prepared using Photoshop 7.0. Contrast, color and brightnesswere adjusted in Photoshop 7.0.

Histological identification of cortical layersTo assign cellular subtypes and boundaries to cortical layers,sections were examined at 40� magnification. The VZ wasidentified as the cellular region that is adjacent to the ventricle andcontains organized parallel arrays of cells with elongated nucleiarranged perpendicular to the ventricular surface. The SVZ wascomposed of a compact layer of cells with round nuclei arrangedin a disorganized manner, adjacent to the VZ. In this study theprogenitor population was defined to include the VZ and SVZ. Theintermediate zone was cell sparse compared with the progenitorpopulation and contained a mixture of cells with round andelongated nuclei arranged both parallel and perpendicular to the

ventricular surface. The subplate was identifiable within theintermediate zone as a layer of large round nuclei, deep to thecortical plate. The cortical plate was evident from E14.5 andidentifiable owing to an increased packing density of round cellnuclei arranged in rows. The marginal zone was located superficialto the cortical plate, adjacent to the pial surface. The marginal zonecontained a mixture of cells with round nuclei and elongated nucleiarranged parallel to the ventricular surface.

Analysis and cell countsFor quantification of cell number in the dorsal cortex, stainedsections were imaged as described above. Sections werehistologically matched for rostral-caudal level between genotype.For cell counts, matched sections through the presumptivesomatosensory 1 hindlimb and forelimb region or through thepresumptive somatosensory 1 trunk region were quantified(Franklin and Paxinos, 1997). Cortical layers were histologicallydistinguished as described above. For all analyses a 150 m windowin the medial-lateral dimension spanning from the ventricular topial surface of the dorsal cortex from three non-adjacent sectionsper animal were counted, and the mean of the three sections peranimal was calculated. The means from three or four animals pergenotype were compared i.e. three or four mutant animals werecompared with three or four wild-type animals. The analyses wereperformed by an observer blind to the genotype. Cell counts wereperformed and quantified using a cell counting program, aspreviously described (Roy et al., 2004). The data were subsequentlydecoded and statistical analysis of the results was performed usingan unpaired t-test with InStat 3 software. Graphs were preparedusing Prism 4 software (GraphPad Software). Values arerepresented as mean ± s.e.m. Significance was set at P<0.05.

Cell death studiesFor studies of cell survival, the number of activated-caspase-3-positive cells spanning the ventricular to pial surface was quantified.

Proliferation studiesPregnant animals were injected with a single dose of BrdU (onE11.5, E14.5 or E17.5), and embryos were collected 60 minutes laterand processed for immunohistochemistry. Sections were stainedwith an anti-BrdU antibody and counterstained with DAPI. Tocalculate the labeling index, the total number of DAPI-positive cellswithin the VZ and SVZ was counted per window. The number ofBrdU-positive cells in the same window was counted and thepercent labeled calculated to determine the labeling index (numberBrdU positive cells/total number of DAPI-positive cells). E11.5sections were also counterstained with the neuronal marker Tuj1and the E11.5 VZ population was defined as the Tuj1-negativepopulation; E14.5 and E17.5 VZ and SVZ populations werehistologically distinguished using DAPI staining.

Laminar cell countsTo determine the number of cells in each cortical layer, individualmarkers were used to identify distinct laminae. Cells expressing ahigh level of Tbr1 identify layer VI, and Cux1 expression identifieslayers II/III and IV (Bulfone et al., 1995; Nieto et al., 2004). Tbr1-positive subplate cells, located in the intermediate zone, werehistologically distinguished from deep cortical plate cells by DAPI



iseas

e M

odel

s & M

echa

nism

s

DM

M

staining. The amount of Tbr1-positive or Cux1-positive cells wasexpressed as a percentage of total cortical plate cell number toidentify whether alterations in the proportion of cells committedto deep (Tbr1+/cortical plate cell number) versus superficial(Cux1+/cortical plate cell number) layers were present in Sall1–/–mice.

Birth-dating studiesFor birth-dating studies (E18.5 embryos injected with BrdU onE11.5, E12.5 or E14.5), BrdU-stained sections were examined andonly heavily labeled BrdU-positive cells were quantified. Heavilylabeled cells were assumed to be cells that incorporated BrdU andsubsequently exited the cell cycle. Those cells that re-entered thecell cycle will dilute the BrdU label in the nucleus and have a weakerBrdU staining. For laminar birth-dating studies, deep cortical layerVI cells were defined by high expression of Tbr1, and superficialcortical layers (layers II/III, IV) were defined by Cux1 expression.The proportion of BrdU+ cells within the cortical plate committedto a deep versus superficial layer fate was quantified: deep(Tbr1+BrdU+/BrdU+) and superficial (Cux1+Brdu+/BrdU+).

Cell cycle exit versus cell cycle re-entry studiesThe proportion of cells that exit the cell cycle in a defined periodis referred to as the quiescent fraction (Q). To identify the

proportion of cells that exit the cell cycle, embryos were injectedwith BrdU on either E11.5, E14.5 or E17.5 and collected 24 hourslater. Cells that exited the cell cycle to become neurons wereidentified as BrdU-positive and Tuj1-positive. Tuj1 is a pan neuralmarker that is expressed by neurons approximately 1 hour afterthey exit the cell cycle, and is maintained in migrating and matureneurons (Lee et al., 1990a; Lee et al., 1990b; Menezes and Luskin,1994; Hammerle and Tejedor, 2002). This marker is present in cellsin the VZ and SVZ and can be used to identify cells differentiatinginto neurons (Menezes and Luskin, 1994; Hammerle and Tejedor,2002). The quiescent fraction was determined as the proportionof cells that were double positive for BrdU and Tuj1 divided by thetotal BrdU-positive population (BrdU+Tuj1+/total BrdU).

The proportion of cells that re-enter the cell cycle in a definedperiod is referred to as the proliferative fraction. To identify theproportion of cells that re-entered the cell cycle, embryos wereinjected with BrdU on either E14.5 or E17.5 and collected 24 hourslater. PC cycle re-entry was determined as the proportion of cellsthat were double positive for BrdU and the RGC marker Pax6 orIPC marker Tbr2 divided by the total BrdU-positive population(BrdU+Pax6+/total BrdU or BrdU+Tbr2+/total BrdU). To quantifythe VZ and SVZ using molecular markers, the number of Pax6- orTbr2-positive cells within the PC population was quantified. Toquantify the IPC cell number at E12.5, the number of PH3-positivecells in abventricular regions in the progenitor domain wasquantified (Haubensak et al., 2004).ACKNOWLEDGEMENTSWe thank K. Mauro, E. Drill, W. Halfter, M. Parrish, C. Lagenaur, E. Thiels, L. Lillienand C. Lance-Jones for technical help and discussion. The chondroitin sulfateproteoglycan antibody developed by W. Halfter was obtained from theDevelopmental Studies Hybridoma Bank, developed under the auspices of theNICHD and maintained by The University of Iowa, Department of BiologicalSciences.

COMPETING INTERESTSThe authors declare that they do not have any competing or financial interests.

AUTHOR CONTRIBUTIONSS.J.H. and A.P.M. designed the research and interpreted the results; S.J.H.performed the research and analyses; R.N. generated the Sall1-null and Sall1-cKOanimals; K.R.J. provided the Emx1-Cre mice; S.J.H. and A.P.M. wrote the manuscript.

FUNDINGThis work was supported by the National Institutes of Health [NIMH MH60774 toA.P.M.]; the National Institute on Drug Abuse [NIDA grant AA13004 to A.P.M.]; theMinistry of Education, Culture, Sports, Science, and Technology of Japan [Grant-in-Aid for Scientific Research B23390228 to R.N.]; and the National Eye Institute (NEI)[NIH R01 EY014998 to K.R.J.].

SUPPLEMENTARY MATERIALSupplementary material for this article is available athttp://dmm.biologists.org/lookup/suppl/doi:10.1242/dmm.002873/-/DC1

REFERENCESAl-Baradie, R., Yamada, K., St Hilaire, C., Chan, W. M., Andrews, C., McIntosh, N.,

Nakano, M., Martonyi, E. J., Raymond, W. R., Okumura, S. et al. (2002). Duaneradial ray syndrome (Okihiro syndrome) maps to 20q13 and results from mutationsin SALL4, a new member of the SAL family. Am. J. Hum. Genet. 71, 1195-1199.

Angevine, J. B., Jr and Sidman, R. L. (1961). Autoradiographic study of cell migrationduring histogenesis of cerebral cortex in the mouse. Nature 192, 766-768.

Arlotta, P., Molyneaux, B. J., Chen, J., Inoue, J., Kominami, R. and Macklis, J. D.(2005). Neuronal subtype-specific genes that control corticospinal motor neurondevelopment in vivo. Neuron 45, 207-221.

Arnold, S. E. (1999). Neurodevelopmental abnormalities in schizophrenia: insightsfrom neuropathology. Dev. Psychopathol. 11, 439-456.

Attardo, A., Calegari, F., Haubensak, W., Wilsch-Brauninger, M. and Huttner, W. B.(2008). Live imaging at the onset of cortical neurogenesis reveals differential



TRANSLATIONAL IMPACT

Clinical issueMutations in the human SALL1 gene cause Townes Brocks syndrome (TBS).This condition is variably penetrant, and patients exhibit abnormalitiesprimarily in the limbs, kidneys, ears, heart, bone, esophagus and/or anus.Various neural deficits, including mental retardation, have been identified in asmall proportion of patients, although most patients are not formallyexamined for cognitive, emotional or behavioral alterations. Additionally, therarity of the disorder, coupled with variable penetrance and phenotype evenin individuals with similar mutations, has made it difficult to assess the impactof genetic lesions in SALL1 on cognitive or behavioral function in humans.

ResultsThis paper identifies and characterizes a distinct phenotype involving cerebralcortical neural precursor cells in a mouse model of Sall1 deficiency. Theauthors demonstrate that, although Sall1 is expressed in all central nervoussystem progenitor cells, the cerebral cortex is particularly sensitive to Sall1

deficiency in developing mice. In the absence of Sall1, early cortical progenitorcells exhibit enhanced neuronal differentiation, whereas later progenitor cellsproliferate rather than differentiate. In addition, deficiency of Sall1 leads to aprolonged production of neurons during development that are destined forearly cortical structures such as deep cortical layers.

Implications and future directionsAlthough neural deficits are not commonly reported in individuals with TBS,these data indicate a key role for Sall1 in regulating the rate of neural cellproduction in the cerebral hemispheres during mouse development. Becausethe cerebral cortex mediates higher-order thought processing, reasoning,memory and emotion, these findings suggest that individuals with TBS havepreviously unrecognized neurodevelopmental abnormalities, potentiallyaffecting neural processing, emotion and/or behavior. Future studies shouldexamine the cognitive and behavioral consequences of either Sall1 deficiencyor expression of a disease-associated human SALL1 allele in the mousecerebrum, and aim to identify the associated cellular alterations. Identifyingthe cellular processes affected by Sall1 deficiency or mutation might providepotential therapeutic targets in humans.

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

appearance of the neuronal phenotype in apical versus basal progenitor progeny.PLoS ONE 3, e2388.

Avram, D., Fields, A., Pretty On Top, K., Nevrivy, D. J., Ishmael, J. E. and Leid, M.(2000). Isolation of a novel family of C(2)H(2) zinc finger proteins implicated intranscriptional repression mediated by chicken ovalbumin upstream promotertranscription factor (COUP-TF) orphan nuclear receptors. J. Biol. Chem. 275, 10315-10322.

Barembaum, M. and Bronner-Fraser, M. (2004). A novel spalt gene expressed inbranchial arches affects the ability of cranial neural crest cells to populate sensoryganglia. Neuron Glia Biol. 1, 57-63.

Basson, M. and Horvitz, H. R. (1996). The Caenorhabditis elegans gene sem-4controls neuronal and mesodermal cell development and encodes a zinc fingerprotein. Genes Dev. 10, 1953-1965.

Bedford, L., Walker, R., Kondo, T., van Cruchten, I., King, E. R. and Sablitzky, F.(2005). Id4 is required for the correct timing of neural differentiation. Dev. Biol. 280,386-395.

Blanck, C., Kohlhase, J., Engels, S., Burfeind, P., Engel, W., Bottani, A., Patel, M. S.,Kroes, H. Y. and Cobben, J. M. (2000). Three novel SALL1 mutations extend themutational spectrum in Townes-Brocks syndrome. J. Med. Genet. 37, 303-307.

Borozdin, W., Steinmann, K., Albrecht, B., Bottani, A., Devriendt, K., Leipoldt, M.and Kohlhase, J. (2006). Detection of heterozygous SALL1 deletions by quantitativereal time PCR proves the contribution of a SALL1 dosage effect in the pathogenesisof Townes-Brocks syndrome. Hum. Mutat. 27, 211-212.

Botzenhart, E. M., Green, A., Ilyina, H., Konig, R., Lowry, R. B., Lo, I. F., Shohat, M.,Burke, L., McGaughran, J., Chafai, R. et al. (2005). SALL1 mutation analysis inTownes-Brocks syndrome: twelve novel mutations and expansion of the phenotype.Hum. Mutat. 26, 282.

Buck, A., Archangelo, L., Dixkens, C. and Kohlhase, J. (2000). Molecular cloning,chromosomal localization, and expression of the murine SALL1 ortholog Sall1.Cytogenet. Cell Genet. 89, 150-153.

Bulfone, A., Smiga, S. M., Shimamura, K., Peterson, A., Puelles, L. and Rubenstein,J. L. (1995). T-brain-1: a homolog of Brachyury whose expression defines molecularlydistinct domains within the cerebral cortex. Neuron 15, 63-78.

Cameron, T. H., Lachiewicz, A. M. and Aylsworth, A. S. (1991). Townes-Brockssyndrome in two mentally retarded youngsters. Am. J. Med. Genet. 41, 1-4.

Cantera, R., Luer, K., Rusten, T. E., Barrio, R., Kafatos, F. C. and Technau, G. M.(2002). Mutations in spalt cause a severe but reversible neurodegenerativephenotype in the embryonic central nervous system of Drosophila melanogaster.Development 129, 5577-5586.

Caviness, V. S., Jr (1982). Neocortical histogenesis in normal and reeler mice: adevelopmental study based upon [3H]thymidine autoradiography. Brain Res. 256,293-302.

Caviness, V. S., Jr, Goto, T., Tarui, T., Takahashi, T., Bhide, P. G. and Nowakowski, R.S. (2003). Cell output, cell cycle duration and neuronal specification: a model ofintegrated mechanisms of the neocortical proliferative process. Cereb. Cortex 13,592-598.

Cubelos, B., Sebastian-Serrano, A., Kim, S., Moreno-Ortiz, C., Redondo, J. M.,Walsh, C. A. and Nieto, M. (2008). Cux-2 controls the proliferation of neuronalintermediate precursors of the cortical subventricular zone. Cereb. Cortex 18, 1758-1770.

de Celis, J. F., Barrio, R. and Kafatos, F. C. (1996). A gene complex acting downstreamof dpp in Drosophila wing morphogenesis. Nature 381, 421-424.

de Celis, J. F., Barrio, R. and Kafatos, F. C. (1999). Regulation of the spalt/spalt-relatedgene complex and its function during sensory organ development in the Drosophilathorax. Development 126, 2653-2662.

Elling, U., Klasen, C., Eisenberger, T., Anlag, K. and Treier, M. (2006). Murine innercell mass-derived lineages depend on Sall4 function. Proc. Natl. Acad. Sci. USA 103,16319-16324.

Engels, S., Kohlhase, J. and McGaughran, J. (2000). A SALL1 mutation causes abranchio-oto-renal syndrome-like phenotype. J. Med. Genet. 37, 458-460.

Englund, C., Fink, A., Lau, C., Pham, D., Daza, R. A., Bulfone, A., Kowalczyk, T. andHevner, R. F. (2005). Pax6, Tbr2, and Tbr1 are expressed sequentially by radial glia,intermediate progenitor cells, and postmitotic neurons in developing neocortex. J.

Neurosci. 25, 247-251.Farrell, E. R. and Munsterberg, A. E. (2000). csal1 is controlled by a combination of

FGF and Wnt signals in developing limb buds. Dev. Biol. 225, 447-458.Franklin, K. B. J. and Paxinos, G. (1997). The Mouse Brain in Stereotaxic Coordinates.

New York: Academic Press.Furuta, Y., Lagutin, O., Hogan, B. L. and Oliver, G. C. (2000). Retina- and ventral

forebrain-specific Cre recombinase activity in transgenic mice. Genesis 26, 130-132.Gillies, K. and Price, D. J. (1993). The fates of cells in the developing cerebral cortex of

normal and methylazoxymethanol acetate-lesioned mice. Eur. J. Neurosci. 5, 73-84.

Gorski, J. A., Talley, T., Qiu, M., Puelles, L., Rubenstein, J. L. and Jones, K. R. (2002).Cortical excitatory neurons and glia, but not GABAergic neurons, are produced inthe Emx1-expressing lineage. J. Neurosci. 22, 6309-6314.

Gupta, A., Tsai, L. H. and Wynshaw-Boris, A. (2002). Life is a journey: a genetic lookat neocortical development. Nat. Rev. Genet. 3, 342-355.

Hammerle, B. and Tejedor, F. J. (2002). A method for pulse and chase BrdU-labelingof early chick embryos. J. Neurosci. Methods 122, 59-64.

Handler, M., Yang, X. and Shen, J. (2000). Presenilin-1 regulates neuronaldifferentiation during neurogenesis. Development 127, 2593-2606.

Harrison, S. J., Nishinakamura, R. and Monaghan, A. P. (2008a). Sall1 regulatesmitral cell development and olfactory nerve extension in the developing olfactorybulb. Cereb. Cortex 18, 1604-1617.

Harrison, S. J., Parrish, M. and Monaghan, A. P. (2008b). Sall3 is required for theterminal maturation of olfactory glomerular interneurons. J. Comp. Neurol. 507,1780-1794.

Haubensak, W., Attardo, A., Denk, W. and Huttner, W. B. (2004). Neurons arise in thebasal neuroepithelium of the early mammalian telencephalon: a major site ofneurogenesis. Proc. Natl. Acad. Sci. USA 101, 3196-3201.

Haydar, T. F., Wang, F., Schwartz, M. L. and Rakic, P. (2000). Differential modulationof proliferation in the neocortical ventricular and subventricular zones. J. Neurosci.20, 5764-5774.

He, X., Treacy, M. N., Simmons, D. M., Ingraham, H. A., Swanson, L. W. andRosenfeld, M. G. (1989). Expression of a large family of POU-domain regulatorygenes in mammalian brain development. Nature 340, 35-41.

Hevner, R. F. (2007). Layer-specific markers as probes for neuron type identity inhuman neocortex and malformations of cortical development. J. Neuropathol. Exp.Neurol. 66, 101-109.

Hevner, R. F., Shi, L., Justice, N., Hsueh, Y., Sheng, M., Smiga, S., Bulfone, A.,Goffinet, A. M., Campagnoni, A. T. and Rubenstein, J. L. (2001). Tbr1 regulatesdifferentiation of the preplate and layer 6. Neuron 29, 353-366.

Inoue, S., Inoue, M., Fujimura, S. and Nishinakamura, R. (2010). A mouse lineexpressing Sall1-driven inducible Cre recombinase in the kidney mesenchyme.Genesis 48, 207-212.

Ishikiriyama, S., Kudoh, F., Shimojo, N., Iwai, J. and Inoue, T. (1996). Townes-Brockssyndrome associated with mental retardation. Am. J. Med. Genet. 61, 191-192.

Karantzali, E., Lekakis, V., Ioannou, M., Hadjimichael, C., Papamatheakis, J. andKretsovali, A. (2011). Sall1 regulates embryonic stem cell differentiation inassociation with nanog. J. Biol. Chem. 286, 1037-1045.

Kawaguchi, A., Ogawa, M., Saito, K., Matsuzaki, F., Okano, H. and Miyata, T. (2004).Differential expression of Pax6 and Ngn2 between pair-generated cortical neurons. J.Neurosci. Res. 78, 784-795.

Kiefer, S. M., Ohlemiller, K. K., Yang, J., McDill, B. W., Kohlhase, J. and Rauchman,M. (2003). Expression of a truncated Sall1 transcriptional repressor is responsible forTownes-Brocks syndrome birth defects. Hum. Mol. Genet. 12, 2221-2227.

Kiefer, S. M., Robbins, L., Barina, A., Zhang, Z. and Rauchman, M. (2008). SALL1truncated protein expression in Townes-Brocks syndrome leads to ectopicexpression of downstream genes. Hum. Mutat. 29, 1133-1140.

Kohlhase, J., Wischermann, A., Reichenbach, H., Froster, U. and Engel, W. (1998).Mutations in the SALL1 putative transcription factor gene cause Townes-Brockssyndrome. Nat. Genet. 18, 81-83.

Kohlhase, J., Hausmann, S., Stojmenovic, G., Dixkens, C., Bink, K., Schulz-Schaeffer, W., Altmann, M. and Engel, W. (1999a). SALL3, a new member of thehuman spalt-like gene family, maps to 18q23. Genomics 62, 216-222.

Kohlhase, J., Kohler, A., Jackle, H., Engel, W. and Stick, R. (1999b). Molecular cloningof a SALL1-related pseudogene and mapping to chromosome Xp11.2. Cytogenet. CellGenet. 84, 31-34.

Kohlhase, J., Taschner, P. E., Burfeind, P., Pasche, B., Newman, B., Blanck, C.,Breuning, M. H., ten Kate, L. P., Maaswinkel-Mooy, P., Mitulla, B. et al. (1999c).Molecular analysis of SALL1 mutations in Townes-Brocks syndrome. Am. J. Hum.Genet. 64, 435-445.

Kohlhase, J., Altmann, M., Archangelo, L., Dixkens, C. and Engel, W. (2000).Genomic cloning, chromosomal mapping, and expression analysis of msal-2. Mamm.Genome 11, 64-68.

Kohlhase, J., Heinrich, M., Liebers, M., Frohlich Archangelo, L., Reardon, W. andKispert, A. (2002a). Cloning and expression analysis of SALL4, the murinehomologue of the gene mutated in Okihiro syndrome. Cytogenet. Genome Res. 98,274-277.

Kohlhase, J., Heinrich, M., Schubert, L., Liebers, M., Kispert, A., Laccone, F.,Turnpenny, P., Winter, R. M. and Reardon, W. (2002b). Okihiro syndrome is causedby SALL4 mutations. Hum. Mol. Genet. 11, 2979-2987.

Kohlhase, J., Liebers, M., Backe, J., Baumann-Muller, A., Bembea, M., Destree, A.,Gattas, M., Grussner, S., Muller, T., Mortier, G. et al. (2003). High incidence of theR276X SALL1 mutation in sporadic but not familial Townes-Brocks syndrome andreport of the first familial case. J. Med. Genet. 40, e127.



iseas

e M

odel

s & M

echa

nism

s

DM

M

Lee, M. K., Rebhun, L. I. and Frankfurter, A. (1990a). Posttranslational modification ofclass III beta-tubulin. Proc. Natl. Acad. Sci. USA 87, 7195-7199.

Lee, M. K., Tuttle, J. B., Rebhun, L. I., Cleveland, D. W. and Frankfurter, A. (1990b).The expression and posttranslational modification of a neuron-specific beta-tubulinisotype during chick embryogenesis. Cell Motil. Cytoskeleton 17, 118-132.

Levers, T. E., Edgar, J. M. and Price, D. J. (2001). The fates of cells generated at theend of neurogenesis in developing mouse cortex. J. Neurobiol. 48, 265-277.

Li, D., Dower, K., Ma, Y., Tian, Y. and Benjamin, T. L. (2001). A tumor host rangeselection procedure identifies p150(sal2) as a target of polyoma virus large Tantigen. Proc. Natl. Acad. Sci. USA 98, 14619-14624.

Lian, G. and Sheen, V. (2006). Cerebral developmental disorders. Curr. Opin. Pediatr.

18, 614-620.Magdaleno, S., Jensen, P., Brumwell, C. L., Seal, A., Lehman, K., Asbury, A.,

Cheung, T., Cornelius, T., Batten, D. M., Eden, C. et al. (2006). BGEM: an in situhybridization database of gene expression in the embryonic and adult mousenervous system. PLoS Biol. 4, e86.

Marin, O. and Rubenstein, J. L. (2001). A long, remarkable journey: tangentialmigration in the telencephalon. Nat. Rev. Neurosci. 2, 780-790.

Marlin, S., Blanchard, S., Slim, R., Lacombe, D., Denoyelle, F., Alessandri, J. L.,Calzolari, E., Drouin-Garraud, V., Ferraz, F. G., Fourmaintraux, A. et al. (1999).Townes-Brocks syndrome: detection of a SALL1 mutation hot spot and evidence fora position effect in one patient. Hum. Mutat. 14, 377-386.

Martinez-Cerdeno, V., Noctor, S. C. and Kriegstein, A. R. (2006). The role ofintermediate progenitor cells in the evolutionary expansion of the cerebral cortex.Cereb. Cortex 16 Suppl. 1, i152-i161.

McEvilly, R. J., de Diaz, M. O., Schonemann, M. D., Hooshmand, F. and Rosenfeld,M. G. (2002). Transcriptional regulation of cortical neuron migration by POU domainfactors. Science 295, 1528-1532.

Menezes, J. R. and Luskin, M. B. (1994). Expression of neuron-specific tubulin definesa novel population in the proliferative layers of the developing telencephalon. J.

Neurosci. 14, 5399-5416.Miyata, T., Kawaguchi, A., Saito, K., Kawano, M., Muto, T. and Ogawa, M. (2004).

Asymmetric production of surface-dividing and non-surface-dividing corticalprogenitor cells. Development 131, 3133-3145.

Mizutani, K., Yoon, K., Dang, L., Tokunaga, A. and Gaiano, N. (2007). DifferentialNotch signalling distinguishes neural stem cells from intermediate progenitors.Nature 449, 351-355.

Munji, R. N., Choe, Y., Li, G., Siegenthaler, J. A. and Pleasure, S. J. (2011). Wntsignaling regulates neuronal differentiation of cortical intermediate progenitors. J.

Neurosci. 31, 1676-1687.Mutch, C. A., Schulte, J. D., Olson, E. and Chenn, A. (2010). Beta-catenin signaling

negatively regulates intermediate progenitor population numbers in the developingcortex. PLoS ONE 5, e12376.

Nieto, M., Monuki, E. S., Tang, H., Imitola, J., Haubst, N., Khoury, S. J.,Cunningham, J., Gotz, M. and Walsh, C. A. (2004). Expression of Cux-1 and Cux-2in the subventricular zone and upper layers II-IV of the cerebral cortex. J. Comp.

Neurol. 479, 168-180.Nishinakamura, R. (2008). Stem cells in the embryonic kidney. Kidney Int. 73, 913-917.Nishinakamura, R., Matsumoto, Y., Nakao, K., Nakamura, K., Sato, A., Copeland, N.

G., Gilbert, D. J., Jenkins, N. A., Scully, S., Lacey, D. L. et al. (2001). Murinehomolog of SALL1 is essential for ureteric bud invasion in kidney development.Development 128, 3105-3115.

Nishinakamura, R., Uchiyama, Y., Sakaguchi, M. and Fujimura, S. (2011). Nephronprogenitors in the metanephric mesenchyme. Pediatr. Nephrol. 26, 1463-1467.

Noctor, S. C., Flint, A. C., Weissman, T. A., Dammerman, R. S. and Kriegstein, A. R.(2001). Neurons derived from radial glial cells establish radial units in neocortex.Nature 409, 714-720.

Noctor, S. C., Martinez-Cerdeno, V., Ivic, L. and Kriegstein, A. R. (2004). Corticalneurons arise in symmetric and asymmetric division zones and migrate throughspecific phases. Nat. Neurosci. 7, 136-144.

Noctor, S. C., Martinez-Cerdeno, V. and Kriegstein, A. R. (2008). Distinct behaviors ofneural stem and progenitor cells underlie cortical neurogenesis. J. Comp. Neurol. 508,28-44.

Ogawa, M., Miyata, T., Nakajima, K., Yagyu, K., Seike, M., Ikenaka, K., Yamamoto,H. and Mikoshiba, K. (1995). The reeler gene-associated antigen on Cajal-Retziusneurons is a crucial molecule for laminar organization of cortical neurons. Neuron 14,899-912.

Ott, T., Parrish, M., Bond, K., Schwaeger-Nickolenko, A. and Monaghan, A. (2001).A new member of the spalt-like zinc finger protein family, Msal3, is expressed in theCNS and sites of epithelial/mesenchymal interaction. Mech. Dev. 101, 203-207.

Parrish, M., Ott, T., Lance-Jones, C., Schuetz, G., Schwaeger-Nickolenko, A. andMonaghan, A. P. (2004). Loss of the Sall3 gene leads to palate deficiency,abnormalities in cranial nerves, and perinatal lethality. Mol. Cell. Biol. 24, 7102-7112.

Pilz, D., Stoodley, N. and Golden, J. A. (2002). Neuronal migration, cerebral corticaldevelopment, and cerebral cortical anomalies. J. Neuropathol. Exp. Neurol. 61, 1-11.

Polleux, F. and Lauder, J. M. (2004). Toward a developmental neurobiology of autism.Ment. Retard. Dev. Disabil. Res. Rev. 10, 303-317.

Pontious, A., Kowalczyk, T., Englund, C. and Hevner, R. F. (2008). Role ofintermediate progenitor cells in cerebral cortex development. Dev. Neurosci. 30, 24-32.

Price, D. J., Aslam, S., Tasker, L. and Gillies, K. (1997). Fates of the earliest generatedcells in the developing murine neocortex. J. Comp. Neurol. 377, 414-422.

Rosner, B. S. (1970). Brain functions. Annu. Rev. Psychol. 21, 555-594.Roy, K., Kuznicki, K., Wu, Q., Sun, Z., Bock, D., Schutz, G., Vranich, N. and

Monaghan, A. P. (2004). The Tlx gene regulates the timing of neurogenesis in thecortex. J. Neurosci. 24, 8333-8345.

Rusten, T. E., Cantera, R., Urban, J., Technau, G., Kafatos, F. C. and Barrio, R. (2001).Spalt modifies EGFR-mediated induction of chordotonal precursors in the embryonicPNS of Drosophila promoting the development of oenocytes. Development 128,711-722.

Saffary, R. and Xie, Z. (2011). FMRP regulates the transition from radial glial cells tointermediate progenitor cells during neocortical development. J. Neurosci. 31, 1427-1439.

Sakaki-Yumoto, M., Kobayashi, C., Sato, A., Fujimura, S., Matsumoto, Y., Takasato,M., Kodama, T., Aburatani, H., Asashima, M., Yoshida, N. et al. (2006). The murinehomolog of SALL4, a causative gene in Okihiro syndrome, is essential for embryonicstem cell proliferation, and cooperates with Sall1 in anorectal, heart, brain andkidney development. Development 133, 3005-3013.

Salerno, A., Kohlhase, J. and Kaplan, B. S. (2000). Townes-Brocks syndrome and renaldysplasia: a novel mutation in the SALL1 gene. Pediatr. Nephrol. 14, 25-28.

Sato, A., Kishida, S., Tanaka, T., Kikuchi, A., Kodama, T., Asashima, M. andNishinakamura, R. (2004). Sall1, a causative gene for Townes-Brocks syndrome,enhances the canonical Wnt signaling by localizing to heterochromatin. Biochem.Biophys. Res. Commun. 319, 103-113.

Schiffmann, S. N., Bernier, B. and Goffinet, A. M. (1997). Reelin mRNA expressionduring mouse brain development. Eur. J. Neurosci. 9, 1055-1071.

Schwartzkroin, P. A. and Walsh, C. A. (2000). Cortical malformations and epilepsy.Ment. Retard. Dev. Disabil. Res. Rev. 6, 268-280.

Sessa, A., Mao, C. A., Hadjantonakis, A. K., Klein, W. H. and Broccoli, V. (2008). Tbr2directs conversion of radial glia into basal precursors and guides neuronalamplification by indirect neurogenesis in the developing neocortex. Neuron 60, 56-69.

Sheppard, A. M. and Pearlman, A. L. (1997). Abnormal reorganization of preplateneurons and their associated extracellular matrix: an early manifestation of alteredneocortical development in the reeler mutant mouse. J. Comp. Neurol. 378, 173-179.

Sheppard, A. M., Hamilton, S. K. and Pearlman, A. L. (1991). Changes in thedistribution of extracellular matrix components accompany early morphogeneticevents of mammalian cortical development. J. Neurosci. 11, 3928-3942.

Siegenthaler, J. A., Tremper-Wells, B. A. and Miller, M. W. (2008). Foxg1haploinsufficiency reduces the population of cortical intermediate progenitor cells:effect of increased p21 expression. Cereb. Cortex. 18, 1865-1875.

Surka, W. S., Kohlhase, J., Neunert, C. E., Schneider, D. S. and Proud, V. K. (2001).Unique family with Townes-Brocks syndrome, SALL1 mutation, and cardiac defects.Am. J. Med. Genet. 102, 250-257.

Sweetman, D., Smith, T., Farrell, E. R., Chantry, A. and Munsterberg, A. (2003). Theconserved glutamine-rich region of chick csal1 and csal3 mediates proteininteractions with other spalt family members. Implications for Townes-Brockssyndrome. J. Biol. Chem. 278, 6560-6566.

Takahashi, T., Nowakowski, R. S. and Caviness, V. S., Jr (1994). Mode of cellproliferation in the developing mouse neocortex. Proc. Natl. Acad. Sci. USA 91, 375-379.

Takahashi, T., Nowakowski, R. S. and Caviness, V. S., Jr (1996a). Interkinetic andmigratory behavior of a cohort of neocortical neurons arising in the early embryonicmurine cerebral wall. J. Neurosci. 16, 5762-5776.

Takahashi, T., Nowakowski, R. S. and Caviness, V. S., Jr (1996b). The leaving or Qfraction of the murine cerebral proliferative epithelium: a general model ofneocortical neuronogenesis. J. Neurosci. 16, 6183-6196.

Takahashi, T., Goto, T., Miyama, S., Nowakowski, R. S. and Caviness, V. S., Jr (1999).Sequence of neuron origin and neocortical laminar fate: relation to cell cycle oforigin in the developing murine cerebral wall. J. Neurosci. 19, 10357-10371.

Tiveron, M. C., Rossel, M., Moepps, B., Zhang, Y. L., Seidenfaden, R., Favor, J.,Konig, N. and Cremer, H. (2006). Molecular interaction between projection neuronprecursors and invading interneurons via stromal-derived factor 1 (CXCL12)/CXCR4signaling in the cortical subventricular zone/intermediate zone. J. Neurosci. 26,13273-13278.

Toker, A. S., Teng, Y., Ferreira, H. B., Emmons, S. W. and Chalfie, M. (2003). TheCaenorhabditis elegans spalt-like gene sem-4 restricts touch cell fate by repressing



iseas

e M

odel

s & M

echa

nism

s

DM

M

the selector Hox gene egl-5 and the effector gene mec-3. Development 130, 3831-3840.

Townes, P. L. and Brocks, E. R. (1972). Hereditary syndrome of imperforate anus withhand, foot, and ear anomalies. J. Pediatr. 81, 321-326.

Walter, K. N., Greenhalgh, K. L., Newbury-Ecob, R. A. and Kohlhase, J. (2006).Mosaic trisomy 8 and Townes-Brocks syndrome due to a novel SALL1 mutation inthe same patient. Am. J. Med. Genet. A 140, 649-651.

Wines-Samuelson, M., Handler, M. and Shen, J. (2005). Role of presenilin-1 in corticallamination and survival of Cajal-Retzius neurons. Dev. Biol. 277, 332-346.

Wrobel, C. N., Mutch, C. A., Swaminathan, S., Taketo, M. M. and Chenn, A. (2007).Persistent expression of stabilized beta-catenin delays maturation of radial glial cellsinto intermediate progenitors. Dev. Biol. 309, 285-297.

Yamamoto, S., Yoshino, I., Shimazaki, T., Murohashi, M., Hevner, R. F., Lax, I.,Okano, H., Shibuya, M., Schlessinger, J. and Gotoh, N. (2005). Essential role ofShp2-binding sites on FRS2alpha for corticogenesis and for FGF2-dependentproliferation of neural progenitor cells. Proc. Natl. Acad. Sci. USA 102, 15983-15988.

Yoon, K. J., Koo, B. K., Im, S. K., Jeong, H. W., Ghim, J., Kwon, M. C., Moon, J. S.,Miyata, T. and Kong, Y. Y. (2008). Mind bomb 1-expressing intermediateprogenitors generate notch signaling to maintain radial glial cells. Neuron 58, 519-531.

Yuri, S., Fujimura, S., Nimura, K., Takeda, N., Toyooka, Y., Fujimura, Y., Aburatani,H., Ura, K., Koseki, H., Niwa, H. and Nishinakamura, R. (2009). Sall4 is essential forstabilization, but not for pluripotency, of embryonic stem cells by repressingaberrant trophectoderm gene expression. Stem Cells 27, 796-805.



iseas

e M

odel

s & M

echa

nism

s

DM

M

sall1 regulates cortical neurogenesis and laminar fate specification … · sall1 in neural pcs and...

Documents