(2024) 22:158
Goodkey et al. BMC Medicine
https://doi.org/10.1186/s12916-024-03363-6

BMC Medicine

RESEARCH ARTICLE

Open Access

Olfactory bulb anomalies in KBG syndrome
mouse model and patients
Kara Goodkey1,2, Anita Wischmeijer3, Laurence Perrin4, Adrianne E. S. Watson1,2, Leenah Qureshi1,
Duccio Maria Cordelli5, Francesco Toni6, Maria Gnazzo7, Francesco Benedicenti3, Monique Elmaleh‑Bergès8,
Karen J. Low10,9 and Anastassia Voronova1,11,12,2*   

Abstract
ANKRD11 (ankyrin repeat domain 11) is a chromatin regulator and the only gene associated with KBG syndrome,a rare neurodevelopmental disorder. We have previously shown that Ankrd11 regulates murine embryonic corticalneurogenesis. Here, we show a novel olfactory bulb phenotype in a KBG syndrome mouse model and two diagnosed
patients. Conditional knockout of Ankrd11 in murine embryonic neural stem cells leads to aberrant postnatal olfac‑
tory bulb development and reduced size due to reduction of the olfactory bulb granule cell layer. We further showthat the rostral migratory stream has incomplete migration of neuroblasts, reduced cell proliferation as well as aber‑rant differentiation of neurons. This leads to reduced neuroblasts and neurons in the olfactory bulb granule cell
layer. In vitro, Ankrd11-deficient neural stem cells from the postnatal subventricular zone display reduced migration,
proliferation, and neurogenesis. Finally, we describe two clinically and molecularly confirmed KBG syndrome patientswith anosmia and olfactory bulb and groove hypo-dysgenesis/agenesis. Our report provides evidence that Ankrd11
is a novel regulator of olfactory bulb development and neuroblast migration. Moreover, our study highlights a novel
clinical sign of KBG syndrome linked to ANKRD11 perturbations in mice and humans.
Keywords Anosmia, Olfactory nerve, Olfactory bulb, Rostral migratory stream, RMS, Neural stem cell, Epigenetics,
Chromatin, Neuroblast, Migration, ANKRD11*Correspondence:
Anastassia Voronova
voronova@ualberta.ca
1
Department of Medical Genetics, Faculty of Medicine & Dentistry,
University of Alberta, Edmonton, AB T6G 2H7, Canada
2
Women and Children’s Health Research Institute, University of Alberta,
5‑083 Edmonton Clinic Health Academy, Edmonton, AB T6G 1C9, Canada
3
Clinical Genetics Service and Coordination Center for Rare Diseases,
Department of Pediatrics, Regional Hospital of Bolzano, Bolzano, Italy
4
Clinical Genetics Unit, Hôpital Robert-Debré, Paris, France
5
IRCCS Istituto Delle Scienze Neurologiche Di Bologna, UOC
Neuropsichiatria Dell’età Pediatrica, Bologna, Italy
6
IRCCS Istituto Delle Scienze Neurologiche Di Bologna, Programma Di
Neuroradiologia Con Tecniche Ad Elevata Complessità (PNTEC), Bologna,
Italy
7
Laboratory of Medical Genetics, Translational Cytogenomics Research
Unit, Bambino Gesù Children’s Hospital, IRCCS, 00165 Rome, Italy
8
Service d’Imagerie Pédiatrique, Hôpital Universitaire Robert Debré, Paris,
France9Department of Academic Child Health, Bristol Medical School,
Population Health Sciences, University of Bristol, Bristol, UK

10

Clinical Genetics Service, St. Michaels Hospital, Bristol, UK
Department of Cell Biology, Faculty of Medicine & Dentistry, Universityof Alberta, Edmonton, AB T6G 2H7, Canada
12
Faculty of Medicine & Dentistry, Neuroscience and Mental HealthInstitute, Edmonton, AB T6G 2E1, Canada
11© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom‑mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Goodkey et al. BMC Medicine(2024) 22:158

Introduction
The mammalian olfactory bulb (OB) is a twin ovoid
structure located directly behind the nasal cavity andis part of the central nervous system (CNS). The OB is
one of the few CNS structures known to undergo postnatal and adult neurogenesis, which plays an importantrole in OB growth and neuronal plasticity [1–3]. While
the main function of the OB is olfaction [4], it is also
involved in mood regulation [5]. Olfactory bulbectomy or
OB neuronal ablation induces depressive-like behaviors
in rodents [6–8]. Moreover, reduction or loss of olfaction
is linked to behavioral changes in patients with major
depressive disorder, bipolar disorder, and schizophrenia[9, 10].
The OB hosts a large diversity of cell types, including neuronal and glial cells of various origins and timepoints. Glutamatergic excitatory neurons are formed
embryonically from the local neural stem and progenitor
cells (NPCs) located in the OB ventricular zone [11–14].
GABAergic inhibitory neurons (interneurons), which
include parvalbumin, calretinin, calbindin (CALB +),and neurogranin (NG +) cells, are generated during
embryonic and/or postnatal development from the NPCs
located in the subventricular zone (SVZ), which lines
the lateral ventricles [2, 13, 15, 16]. Glial cells, including
astrocytes (GFAP +) and oligodendroglia (PDGFRα +),have supporting functions in the OB. While some glial
cells, such as oligodendrocyte progenitor cells (OPCs),
arise embryonically from the medial ganglionic eminence
and anterior entopeduncular area NPCs, the majority of
proliferation and migration of glial cells occurs postnatally from the SVZ [2, 13].
The OB hosts several discrete neuronal layers, known
as the granule cell layer (GCL), the mitral cell layer(MCL), the external plexiform layer (EPL), and the glomerular layer (GL) [17]. Starting with the deepest layer,
the GCL primarily contains inhibitory interneurons [18].
Mitral/tufted (M/T) excitatory neuronal cell bodies are in
the next layer above termed MCL, whereas their lateral
dendrites are in the EPL, located above the MCL [19].
The most superficial neuronal layer is the GL, which iscomposed of spherical glomeruli, containing periglomerular cells, dendrites from excitatory M/T cells, and axons
from olfactory sensory neurons (OSN) whose cell bodies
are located in the olfactory nerve layer (ONL) of the nasal
cavity [20, 21]. Progenitors and neuroblasts that arrivein the OB do so via tangential migration from the SVZ
along the rostral migratory stream (RMS). Once neuroblasts reach the GCL, they migrate radially to populate
the remaining layers [13]. These neuroblasts contributeto postnatal and adult OB neurogenesis [13].
While adult OB neurogenesis is well characterizedthroughout rodent life, adult OB neurogenesis in humans

Page 2 of 24

is debated. Several studies have provided evidence ofadult human OB neurogenesis persisting throughout
adulthood, while others have found a sharp decrease in
human OB neurogenesis after childhood (reviewed in [3,16]). Nevertheless, OB neurogenesis does occur postnatally in both the rodent and human brain development[22–24]. Migrating neuroblasts are ensheathed by astrocytes and guided by a variety of molecular and structuralcues including chemotactic factors, blood vessels, extracellular matrix (ECM) components, and adhesion molecules [25–27]. This process contributes to OB growth andneuronal plasticity [2, 28].
OB development and maintenance are regulatedtranscriptionally and by sensory experiences. Odor
deprivation decreases the number and maturation of
postnatal newborn granule cells [29, 30], while odor
training increases OB adult neurogenesis [31]. With
regard to gene expression regulation, most research
has focused on the role of transcription factors, such as
homeobox and T-box genes, during OB development[17, 32, 33], while chromatin and epigenetic regulation
has been understudied. What is known is that chromatin regulators, such as HDAC2 (histone deacetylase 2),
regulate adult OB neurogenesis via adult-born OB neuron survival and differentiation [34]. With regard to OB
development, chromatin remodelers, such as MeCP2(methyl-CpG binding protein 2; Rett syndrome diseasegene), regulate OSN development in the olfactory epithelium [35, 36]. Moreover, HDACs regulate neurotransmitter expression in the OB interneurons [37].
ANKRD11 is a chromatin regulator that interacts with
HDACs and HATs (histone acetylases) to regulate global
gene expression [38–40]. ANKRD11 is one of the mostfrequently de novo mutated genes in monogenic neurodevelopmental disorders (NDDs) and is moderately
de novo mutated in autism spectrum disorder (ASD)[41–44]. Inherited or de novo mutations in or deletionsof ANKRD11 are associated with KBG syndrome (OMIM#148050; named after the initials of the first threepatients) [41]. Patients with KBG syndrome can present
with short stature, distinctive facial features, macrodontia, skeletal anomalies, congenital heart defects, intellectual disability, and seizures [45–49]. Many patients will
also have one or a combination of neurobehavioral issues,
such as attention-deficit/hyperactivity disorder (ADHD),
hyperactive/restless behaviors, performance anxiety, lowfrustration tolerance, obsessive–compulsive, and impulsive-aggressive behaviors, ASD, and mood disorders suchas depression [50–52]. CNS anomalies include dilated
brain ventricles, heterotopias, callosal abnormalities,
and seizures [45, 50, 53–56]. We have previously shownthat Ankrd11 regulates murine NPC proliferation, differentiation, and neuron localization in the developing

Goodkey et al. BMC Medicine(2024) 22:158

cortex and during adult OB neurogenesis [38]. Whether
Ankrd11 regulates embryonic and/or postnatal OB
development has not been previously studied.
Here, we show that the loss of Ankrd11 in the embryonic murine NPCs in vivo leads to hypoplastic postnatal
OB with reduced GCL layer. We further show that loss of
Ankrd11 leads to reduced NPC proliferation, migration,
and aberrant neurogenesis in vitro and in vivo. Clinicalobservations and magnetic resonance imaging (MRI)
from two patients with molecularly confirmed KBG syndrome show absence or hypoplasia of olfactory nerves
and bulbs and/or clinical anosmia. Together, our results
identify a new role for Ankrd11 in OB development anda novel OB-related clinical phenotype linked to KBG
syndrome.

Materials and methods
Ethical complianceThis study is in accordance with the Helsinki declarationand its following modifications. The reported KBG syndrome patients’ data were obtained through routine clinical care and are not considered research at the involved
institutions; formal review board approval was therefore not requested. Patient 1 was initially evaluated andtreated at the Rare Disease Unit and the Pediatric Neurology Unit of University Hospital S.Orsola-Malpighi—
Bologna, and subsequently at the Clinical Genetics Unit,
Regional Hospital Bolzano, Italy. Patient 2 was diagnosed
and in follow-up at the Clinical Genetics Unit, Hôpital
Robert-Debré, Paris, France. Both parents of patients 1
and 2, and patient 1 himself, provided written informed
consent for genetic testing, and publication of clinicaldata and/or pictures, and they authorized the processingof personal data according to the Italian and French bioethics laws.
All animal use was approved by the Animal Care Committee of the University of Alberta in accordance with theCanadian Council of Animal Care policies.
Patients

Patient 1 (see Fig. 7A–D) is a 20-year-old male, the only
child of healthy non-consanguineous Caucasian parents. The boy was born at term (40 WG + 1 day) withoutcomplications. He did not present with feeding difficulties, but he consistently refuses to eat any kind of fruit or
raw vegetables. At 10 years, his linear and weight growthparameters were mostly 40th–50th centile (clearly below
parental target of > 90th centile), with a slightly smaller
but normal head circumference. At the age of 11.5 years,
he was first investigated because of excess weight gain
without linear growth. Initially, a constitutional delay ofpuberty was suspected since his bone age was delayed. He
was already known to be anosmic and at age 13, a deficit

Page 3 of 24

of growth hormone (GH) was diagnosed, in the absenceof other endocrinological anomalies. He started biosynthetic GH therapy at the age of 13 years and 10 months
when his height was 148.8 cm (18th centile, target height
188 cm), and shortly thereafter, normal pubertal development followed. GH therapy was stopped after 3 years,
due to a lack of gain in length, and other health problems.
His final height aged 20 is 168.5 cm (13th centile), hisweight is 64 kg (27th centile), and his head circumference is 54.5 cm (34th centile). At the age of 16 years and8 months, he presented with focal, secondary generalized
epileptic seizures. Seizures were resistant despite treatment with valproic acid, levetiracetam, carbamazepine,
perampanel, and clobazam. He developed a possible
drug-induced liver injury 2 weeks after the introduction of low dose carbamazepine. Ongoing therapy withlamotrigine has been successful in seizure remission. The
clinical diagnosis of KBG syndrome was molecularly confirmed by the identification of the de novo heterozygous
variant c. 3221_3222delAA, p. Lys1074ArgfsTer27 in the
ANKRD11 gene. This loss-of-function mutation was previously reported in another patient with KBG syndrome[57]. Patient 1 developed typical KBG craniofacial dysmorphic features from puberty, such as a triangular face,
heavy eyebrows, bulbous nose, full lips, large central incisors, and low anterior hairline. He also presents with thorax asymmetry, thoracal dextro-convex scoliosis, thoracal
kyphosis, anteverted shoulders, hyperextensible knees
and interphalangeal articulations of fingers, myopia, andcorneal dystrophy of the right eye. Cardiac and renal
ultrasound were normal. Audiometric testing remains
within normal limits.
Psychomotor development of this patient was
unremarkable; he was able to walk independently at
13 months and pronounced his first words at 10 months,
followed by a fast progression of language development.
His school performance was normal, with above-average results; however, he showed poor adaptive behavior
and occasional tantrums. At the age of almost 13 years,
because of suspicion of dysgraphia, a neuropsychological evaluation was performed, which concluded: normal cognitive profile in average-high range (WISC-IV
total QI 119), motor coordination disorder particularly
regarding visual-motor integration (ICD-10 F82 specificdevelopmental disorder of motor function), and leading
to dysgraphia (ICD-10 F81.8) at school, fragile emotional
control, and poor adaptation to changes. His behavioral issues worsened over time: currently, he displays
impulsive-aggressive and obsessive–compulsive behaviors, a reduced ability to relate to others and low selfconfidence. He tends to be anxious and depressive andis currently unable to live or work independently. Moodand behavior might be negatively influenced by his at

Goodkey et al. BMC Medicine(2024) 22:158

least partial awareness of his limitations. He is receiving
psychotherapy. A recent neuropsychologic re-evaluation
confirmed normal intellectual functioning profile, butwith disharmony in the functions of motor integration,social, and relational correspondence, which still require
specialist investigations.
Patient 2 (see Fig. 7I) is an 8-year-old girl born to
French non-consanguineous parents. She came to genetic
counseling for psychomotor retardation and olfactory
bulb agenesis. An array and testing for fragile X syndrome were performed but they were both negative. The
sequencing of the EHTM1 gene was normal. An ID genepanel found a variant c.6152dupC de novo in ANKRD11.
She has hypertelorism, plagiocephaly, upwardly sloping labial commissures, large incisors, and short fingers
especially the fifth. The proband was described as a very
calm baby. The neonatal period was marked by failure to
thrive and gastric reflux which needed treatment. She sataround 14 months and walked alone at the age of 2 years
and 6 months. Her first words appeared around 1 year ofage. At the age of 8 years, she still has a severe language
delay. She has behavioral disorders with frustration intolerance, emotional immaturity, and crises of anger. She
has poor safety awareness and has concentration difficulties. She had recurrent ear nose and throat infections andrecurrent left lumbar tumefaction of unknown etiology
which can evoke a vascular malformation. Her height has
followed − 1SD trajectory and her head circumferenceplots on 2SD.
MRI

MRI exams were performed at the age of 14 years forpatient #1 and 3 years for patient #2 with a 3 T (Tesla)scanner (MAGNETOM Skyra, Siemens Healthcare,
Erlangen, Germany) equipped with a 64-channel headcoil. High-resolution heavily T2-weighted sequences sagittal sequences were acquired, completed with coronalmultiplanar reconstruction (MPR). Axial brain images
were made in FLAIR T2-weighted, coronal brain images
in FSE T2-weighted. For the nasoethomodial region sagittal and coronal multiplanar reformatting three-dimensional high resolution heavy T2-weighted images were
made. Controls with normal appearing OB anatomy
underwent the same MRI exam and the resulting images
were generated as described above.
Prevalence patient survey

We collaborated with the KBG Foundation to obtain asnapshot of parent or self-reported anosmia or hyposmia.
Families who are members of the KBG Foundation Facebook group were asked to answer a Facebook poll andselect from the options (1) no sense of smell; (2) reduced
sense of smell; (3) normal sense of smell/never noticed aPage 4 of 24

problem; or (4) not possible to know. We specified thatchange of sense of smell following COVID infection
should not be reported in this poll.
Animals

All mice were housed in a University of Alberta Animal Facility and serviced by Health Sciences Laboratory Animal Services (HSLAS). Mice were maintainedon a 12-h light or dark cycle, and food and water were
provided ad libitum. Embryos at developmental ages
embryonic day (E)18.5 and postnatal mice at age postnatal day (P)5, 10, 15, and P30 of either sex were used for all
experiments.
Ankrd11fl/fl mice in which exon 7 of the Ankrd11
gene is flanked by LoxP sites are described in [58, 59].
­RosaYFPSTOP/STOP (B6.129X1-Gt(ROSA)26Sortm1(EYFP)
Cos/J, stock # 006148) mice were obtained from JacksonLaboratories [60]. ­NestinCreERT2 mice were obtained
from [61] and used to induce recombination in Nestin + NPCs [62, 63]. To generate Ankrd11 NPC-specificconditional knockout mice, ­Ankrd11fl/fl;RosaYFPSTOP/
STOP
females were mated with ­Ankrd11fl/fl;RosaYFPSTOP/
STOP;NestinCreERT2 male mice to generate ­Ankrd11fl/
fl
STOP/STOP
Contol;RosaYFP­(Ankrd11) and A­nkrd11fl/
fl
STOP/STOP
ERT2
nscKO
;RosaYFP;NestinCre­(Ankrd11) progeny. Mice were mated in the evenings and a positive plug,
determined in the morning, was considered as E0.5.
CD1 timed pregnant females were purchased fromCharles River and used to foster ­Ankrd11Contol andnscKO­Ankrd11
pups as per previously published cesarean
section and cross-fostering protocols [64, 65].
Tamoxifen and BrdU injections

Pregnant murine mothers were injected once intraperitoneally (IP) with 90 mg/kg of tamoxifen (TMX; Sigma)
at E14.5. TMX dosage was optimized using ­Ankrd11fl/
fl
dams between P70 and P90 to maximize survival ofpups while maintaining efficient recombination (datanot shown). TMX was dissolved in a 9.5% ethanol insunflower seed oil (Sigma) as described in [62, 63] toinduce recombination in Nestin + NPCs. P14 pups wereIP injected with 100 mg/kg dose of BrdU (Bromodeoxyuridine) dissolved in sterile 1 × PBS (phosphate bufferedsaline) 24 h prior to euthanasia [66].
Genotyping

All mice were bred and genotyped as recommended
by Jackson Laboratories and European Mouse Mutant
Archive (EMMA) using the following primers: Ankrd11:
forward,5’-CTG​TCT​CAG​AGA​GGA​GAG​TGA​GGA​
GGAC-3’; reverse, 3’-TAC​CTT​ACA​CCC​TGA​GAC​GGC​
GTC​-5’, 34 cycles of: 94 °C-30 s, 62 °C-45 s, 72 °C-60 s.
Pan-Cre: forward, 5’-TTC​CCG​CAG​AAC​CTG​AAG​

Goodkey et al. BMC Medicine(2024) 22:158

ATG-3’; reverse, 3’-11 CCC​CAG​AAA​TGC​CAG​ATT​
ACG-5’; control forward primer 5’-AAC​AAC​AAT​GGC​
ACA​ACC​TAAT-3’; control reverse 3’-ACT​TTC​TCC​
CCA​CCC​GTC​TA-5’; 35 cycles of: 94 °C-15 s, 60 °C-30 s,
72 °C-90 s. ­YFPSTOP: wild-type, 5’-GGA​GCG​GGA​GAA​
ATG​GAT​ATG-3’; common, 5’-AAA​GTC​GCT​CTG​AGT​
TGT​TAT-3’; mutant, 5’-AAG​ACC​GCG​AAG​AGT​TTG​
TC-3’, 35 cycles of 94 °C-15 s, 65 °C-15 s, 72 °C-60 s.
Primary cultures
Primary neurospheres

Cells from embryonic cortex or postnatal SVZ weremechanically triturated and cultured at clonal density (10 cells/μl) as described in [67]. Briefly, cells were
cultured in a 6-well polystyrene microplate (08–7721B; Fisher) for 6 days in vitro (DIV) in 2 ml/well ofserum-free media (SFM: equal parts Dulbecco’s modified Eagle medium low glucose (DMEM, 10567–014,(Sigma), 5 mM HEPES (Gibco), 0.1125% sodium bicarbonate (NaHCO3, Gibco), 1% penicillin–streptomycin(Gibco), and 100 μg/mL L-glutamine (Gibco), supplemented with 10 ng/mL FGF (fibroblast growth factor;
Peprotech), 2% B27 (Gibco), 20 ng/mL EGF (epidermal
growth factor; Peprotech), and 2 μg/mL heparin sodium
salt (H3149-50KU; Sigma) [38, 67, 68].
For E15.5 cultures, TMX was injected into pregnant murine mothers at E14.5 as described above, andthe cerebral cortex was microdissected from E15.5­Ankrd11control and ­Ankrd11nscKO embryos to generate
primary neurospheres. Primary neurospheres were thencentrifuged at 465 g for 7 min at room temperature andthe cell pellet was collected for total RNA purificationaccording to manufacturer’s instructions (Omega BioTek E.Z.N.A kit).
For P7 cultures, the SVZ was dissected from naïve
(non-injected)­Ankrd11fl/fl;RosaYFPSTOP/STOP
and
fl/fl
­Ankrd11 ;RosaYFPSTOP/STOP;NestinCreERT2 pups. Cells
were cultured in a 6-well polystyrene microplate (08–772-1B; Fisher) with 2 ml of supplemented SFM per wellfor 6 DIV as described in [67]. To induce recombinationin vitro, primary neurospheres on 5 DIV were incubatedwith 1 μM 4-hydroxytamoxifen (4OH-TMX) for 24 h.
Primary neurospheres on 6 DIV were used in downstream assays described below.
Secondary neurospheres

P7 4-OHT treated primary neurospheres were pooled
and dissociated as in [67]. Resulting neurosphere cells
were cultured at clonal density (2 cells/μl) as secondary
neurospheres as described in [67] for 7 DIV in a 6-wellpolystyrene microplate with 2 ml of SFM supplemented
with FGF, EGF, B27, and heparin. Secondary neurospheres were counted and then centrifuged at 465 g forPage 5 of 24

7 min at room temperature, and the cell pellet was collected for total RNA purification according to manufacturer’s instructions (Omega Bio-Tek E.Z.N.A kit).
Primary neurosphere migration assay

Ten to 15 of P7 4-OHT treated primary neurospheres
were randomly selected and plated on a 40 μg/mL polyD-lysine (PDL) (Sigma, P6407) and 4 μg/mL laminin(VWR, CACB354232) coated coverslips [69, 70]. Spheres
were allowed to adhere to the plate for 5–10 min in the
incubator prior to the addition of SFM supplemented
with FGF, EGF, and B27 [67]. Individual spheres wereimaged at plating and 24 h later (1 DIV).
NPC scratch assay

P7 4-OHT treated primary neurospheres were pooled
and dissociated as in [67]. Resulting neurosphere cells
were plated at high density (260,000 cells/cm2) on
PDL- and laminin-coated 24-well plates in SFM supplemented with FGF, EGF, and B27. After 3–4 h (after NPCs
adhered), a single scratch was made in each well using
a 10-μl pipette tip. Images of each scratch were taken
directly post scratch (0 DIV), 1, and 2 DIV.
NPC monolayer cultures

P7 4-OHT treated primary neurospheres were pooled
and dissociated as in [67]. Resulting neurosphere cells
were plated at 40,000 cells/cm2 density, optimal for differentiation into neurons, astrocytes, and oligodendroglial cells [67], on PDL- and laminin-coated coverslips.
Cells were cultured in SFM supplemented with FGF, EGF,
and B27 for 4 DIV [67].
qRT‑PCR (quantitative reverse transcriptase‑polymerase
chain reaction)

cDNA was generated from 20 to 70 ng of total RNA isolated from primary or secondary neurospheres using
QuantiTect Reverse Transcription Kit (Qiagen) as per
the manufacturer’s instructions. A no-RT control was
included to verify genomic DNA elimination. 1/100thof the RT reaction was used as a template for the QPCR
amplification. QPCR was performed according to MIQE
guidelines [71] using the Ssoadvanced SYBR Green kit(BioRad) on the Roche LightCycler 96 machine with thefollowing primers: Ankrd11 exon 7: Forward 5’-GCG​TGT​
AAC​CGG​GGC​TAT​TAC-3’; Reverse 5’-CGT​TGT​TGG​
CGG​CAT​CAT​G-3’, 40 cycles of; 95 °C 15 s, 60 °C 15 s,
72 °C 20 s. Gapdh; Forward 5’-AAA​TAC​GGA​CTG​CAG​
CCC​TC-3’; Reverse 5’-AAA​TCC​GTT​CAC​ACC​GAC​
CTT-3’, 40 cycles of; 95 °C 15 s, 60 °C 15 s, 72 °C 20 s.

Goodkey et al. BMC Medicine(2024) 22:158

Page 6 of 24

Fig. 1 Gross morphological changes in postnatal ­Ankrd11nscKO OB. A Schematic of breeding scheme. ­Ankrd11fl/fl;RosaYFPSTOP/STOP females weremated with ­Ankrd11fl/fl;RosaYFPSTOP/STOP;NestinCreERT2 males. Pregnant dams were injected with tamoxifen (TMX) at E14.5. Resulting embryos­(Ankrd11control and ­Ankrd11nscKO) were collected at E18.5 or allowed to be born with subsequent collection at P5-P30. Loss of Ankrd11 only occurred
after TMX injection in embryos carrying ­NestinCreERT2 allele. B–I Representative images of ­Ankrd11control and ­Ankrd11nscKO OB at P5 (B), P10 (D),P15 (F), and P30 (H) that were injected with TMX at E14.5. Uninjected ­Ankrd11fl/fl and ­Ankrd11fl/fl;NestinCreERT2 control images have gray and pinkborders in (I). Quantification of OB area of ­Ankrd11control and ­Ankrd11nscKO at P5 (C), P10 (E), P15 (G), and P30 (J). Gray and pink bars in J show P30
OB area from uninjected ­Ankrd11fl/fl and ­Ankrd11fl/fl;NestinCreERT2. Scale bar = 250 mm. K Quantitative RT-PCR for Ankrd11 mRNA in E15.5 corticalprimary neurosphere cultures dissected from ­Ankrd11control and ­Ankrd11nscKO embryos that received TMX at E14.5. Data were normalized to GapdhmRNA and calibrated against ­Ankrd11Control samples. Error bars represent SEM. n = 5–13 mice per genotype from at least 2 independent litters. Data
were analyzed using unpaired t-test (C,E,G,K) or two-way ANOVA followed by Tukey’s multiple comparisons post hoc test (J), ns not significant,**p < 0.01, ***p < 0.001. Ctrl control, E embryonic day, P postnatal day, TMX tamoxifen, YFP yellow fluorescent protein, OB olfactory bulb

Immunocytochemistry and Immunohistochemistry
Tissue preparation

Pregnant murine dams were euthanized with CO2 andbrains from E18.5 embryos were dissected and incubated
in 4% paraformaldehyde (PFA, Fisher, 50980487) for
24 h at 4 °C. P15 pups were euthanized via IP injectionof Euthanyl (Bimeda-MTC) at 192 mg/kg of body weight,
followed by transcardiac perfusion with HBSS (Hanks’

Balanced Salt Solution, Gibco) and 4% PFA. At this point,
postnatal brain areas encompassing OB were photographed using an iPhone 11 pro (Apple) at 2 × zoom togenerate top-down images in Fig. 1B–I. Dissected brains
were further fixed in 4% paraformaldehyde for 24 h at
4 °C, after which tissues were incubated in 30% sucrose in
1 × PBS for 72 h. Samples were embedded and flash frozen in an optimal cutting temperature (OCT) compound

Goodkey et al. BMC Medicine(2024) 22:158(Epredia) and sectioned sagitally with 16 µm thickness ona cryotome (Leica Biosystems, Germany). The sections
were transferred onto glass microscope slides and storedat − 80 °C.
Immunofluorescence

Sections were processed for immunohistochemistry
as described in [62, 63, 66]. Briefly, sections were rehydrated with PBS and blocked for 1 h with 5% bovine
serum albumin (BSA, Jackson ImmunoResearch) and
0.3% Triton-X100 in PBS, then incubated with primary
antibodies listed in the “Antibodies” section diluted in
5% BSA overnight at 4 °C. Sections were then washedin PBS and incubated with appropriate secondary antibodies listed in the “Antibodies” section for 1 h at room
temperature. Some sections also underwent BrdU (Bromodeoxyuridine) staining, which was performed as
described in [63]. Briefly, immunostained sections were
post-fixed with 4% PFA and underwent bathing in 1 M
and 2 M HCL, followed by incubation with anti-BrdU
primary antibody and appropriate secondary antibody(see “Antibodies” section below) incubation thereafter.
Samples were then counterstained with the nuclear stain
Hoechst (33217; Riodel-De Haen Ag) at 1:1000, washedextensively with PBS, and mounted with FluoromountG mounting media (ThermoFisher). Whenever possible,
immunostaining was performed on all slides at once tolimit batch-to-batch variability.
Four DIV NPC monolayer cultures were immunostained as described in [62]. Briefly, cultures were fixedwith 4% PFA, washed in PBS, permeabilized with 0.2%
IGEPAL CA-630 (Sigma) solution, and blocked with 6%
donkey serum and 0.5% BSA in PBS for 1 h. Fixed cultures were then incubated with primary antibodies overnight at 4 °C and appropriate secondary antibodies for
1 h at room temperature (see “Antibodies” section below
for more details). Samples were counterstained with
Hoechst as above and mounted onto glass coverslips with
Fluoromount-G mounting media (ThermoFisher).
Antibodies

Primary antibodies: anti-BrdU (Abcam, ab1893,
RRID:AB_302659, 1:1000), anti-βIII-tubulin (Biolegend, 802001, RRID:AB_2564645, 1:2000), anti-CALB(Swant, cb38, RRID:AB_10000340, 1:1000), anti-CC3(Millipore, AB3623, RRID:AB_91556, 1:250), anti-DCX(Abcam, ab18723, RRID:AB_732011, 1:300), antiGFAP (Thermo Fisher, 13–0300, RRID:AB_2532994,
1:2000), anti-NEUN-Alexa555 (Millipore, MAB377A5,
RRID:AB_2814948, 1:500), anti-NG (Abcam, AB5620,
RRID:AB_91937, 1:600), anti-PSA-NCAM (Sigma,
MAB5324, RRID:AB_95211, 1:400), anti-PDGFRα(R&D Systems, AF1062, RRID:AB_2236897, 1:400),

Page 7 of 24

and anti-SOX2 (Novus Bio, AF2018, RRID:AB_355110,
1:1000 or Cell Signaling, 3728S, RRID:AB_2194037,
1:1500).
Secondaryantibodies:anti-goat-555(Jackson,705–165-147,
RRID:AB_2307351,
1:1000),antigoat-647 (Jackson, 705–605-147, RRID:AB_2340437,
1:1000), anti-mouse IgM-555 (Jackson, 715–165-140,
RRID:AB_2340812, 1:1000), anti-rabbit-488 (Jackson, 711–545-152, RRID:AB_2313584, 1:1000), antirat-555 (Jackson, 712–165-153, RRID:AB_2340667,
1:1000), and anti-sheep-647 (Jackson, 713–175-147,
RRID:AB_2340730, 1:1000).
Microscopy

Live images of neurospheres as well as NPC scratch assay
were captured with a Zeiss Primovert inverted cell culture microscope, Zeiss Axiocam ERc5s camera, andZen software (Zeiss). Primary neurospheres or NPCs
that underwent scratch assays (migration assays) wereimaged at 100 × magnification. Secondary neurospheres
were swirled into the center of the well and imaged at
40 × magnification.
Immunostained brain sections and cultures wereimaged with a Zeiss Axio Imager M2 fluorescencemicroscope, ORCA-Flash LT sCMOS Camera, and Zen
software (Zeiss). Four to six sections in every sample
encompassing medial and lateral planes were imaged
at 100 × magnification in a single plane. Representative
images show tiled images encompassing OB in a sagittal plane or cropped images encompassing OB neuronallayers. Images from each set of immunostaining wereacquired using identical settings.
Data analysis

All data were analyzed and presented from at least three
animals per genotype. E18.5 samples were collected from
two independent litters and P15 samples were collectedfrom three independent litters. Cell culture data was
obtained from at least two independent litters. Sample
sizes (n) are indicated in the figure legends and indicate
the number of animals analyzed. All data were graphedusing GraphPad Prism (version 8.0.2) and presented asmean ± SEM. Representative images were acquired with
Zen software and processed in Adobe Photoshop CC
2015. Figures were generated in Adobe Illustrator CC
2015. Schematics were generated using Biorender and
Adobe Illustrator.
qRT‑PCR analysis

Quantitative RT-PCR data were collected from 13 E15.5
embryos (5 A
­ nkrd11nscKO and 8 A­ nkrd11Control) and 6 P7
nscKO
pups (3 ­Ankrd11
and 3 ­Ankrd11Control) performedin technical duplicates. Average cycle threshold (Ct)Goodkey et al. BMC Medicine(2024) 22:158

values were normalized to Gapdh and calibrated against
a ­Ankrd11control sample from each litter using the 2­ −ΔΔCt
method [72].
Image analysis

Data analysis from E18.5 brains was blinded to the genotype until analysis completion. Since ­Ankrd11nscKO postnatal mice had visibly smaller OBs, blinded analysis at
this age was not possible. Data at least from eight animals
from two independent litters was analyzed for all ages.
In Fig. 1, area of whole OBs was outlined in ImageJ andmeasured. In Figs. 2 and S1, area of one OB (excludingONL) in anatomically matched sagittal sections was outlined in ImageJ and measured. GL and ONL were identified using Hoechst signal.
For analysis in Figs. 2, 3 and 4, S1-3, anatomicallymatched sections from each biological replicate were analyzed. Image contrast was enhanced to the same degreeand only using linear enhancement. Due to the varying size of the sagittal OB area between ­Ankrd11control
and ­Ankrd11nscKO animals, samples were anatomicallymatched by selecting the two largest OB areas from eachanimal that contained both visible RMS and well-defined
OB layers. To identify the center of the OB in an unbiased manner, we overlayed a crosshair pattern over eachanatomically matched OB section. We then applied acircle that would encompass OB borders. Finally, weapplied the same size column (rectangle), which originated from the OB center and emanated outwards in a
rostral direction (towards circle edge) (Figs. 2E, S1E). Theedge of the column was aligned so it was perpendicular(green square indicating 90°) to the edge of the OB andyielded horizontally aligned OB layers for downstream
analysis (Figs. 2F-G, S1F). Further identification of the

Page 8 of 24

OB neuronal layers was performed using Hoechst staining (Figs. 2, S1).
E18.5 and P15 OB sections were subjected tomarker + cell counting as described in [38, 73]. Briefly,
the entire OB was imaged and a defined size column spanning OB center to ONL was counted formarker + cells (Figs. 2E, S1E). Results are presented
as mean marker + cells per entire column or per layer.
For BrdU quantification, results are presented as mean
number or density of BrdU + cells and as a proportionof BrdU + marker + cells relative to total marker + cells.
Images in Figs. 3C, D, S2B and S3A were analyzed using
integrated density, which integrates the area size with themean gray value of the fluorescent signal, using Fiji software [74].
For RMS analysis, RMS was identified with PSANCAM or DCX immunostaining and divided into three
regions; region 1 is a caudodorsal (CD) zone, region 2 isa ventral intermediate (VI) zone according to [75, 76],
and region 3 is OB as depicted in Fig. 3B. Integrated density and area size for the three regions were analyzed as
described above.
In all immunostained culture experiments, three to fiverandom fields of view were captured with 20 × objective.
For cell identity analysis, at least 300 cells from different
fields were counted per condition and results are from atleast two technical replicates from each biological replicate. Images were enhanced linearly and to the same
extent to facilitate marker + cell analysis. Marker + cells
are expressed as proportion of healthy (non-condensed)
Hoechst + nuclei, except CC3 + cells are expressed as
proportion of total (sum of cells with condensed andhealthy) Hoechst + nuclei.
For secondary neurosphere analysis, each biologicalreplicate contained 2–3 technical replicates and every
sphere containing 50 or more cells was counted [63, 77].

(See figure on next page.)
Fig. 2 Loss of Ankrd11 in E14.5 NPCs results in reduced P15 OB with aberrant cellular composition. Also see Supplemental Fig. 1. A ­Ankrd11fl/
fl;RosaYFPSTOP/STOP dams mated with ­Ankrd11fl/fl;RosaYFPSTOP/STOP;NestinCreERT2 males were injected with TMX at E14.5. Resulting pups ­(Ankrd11control
and ­Ankrd11nscKO) were collected at P15. Images were collected from anatomically matched medial (M) and lateral (L) sagittal sections
encompassing OB. B Representative images of ­Ankrd11control and ­Ankrd11nscKO sagittal sections stained with Hoechst (white). Solid outline shows
OB area (excluding ONL) of ­Ankrd11control (yellow) and ­Ankrd11nscKO (red). C Representative overlap of ­Ankrd11control and ­Ankrd11nscKO OB areafrom (B). D Quantification of B. E Representative P15 OB schematic that displays identification of OB center by circle and crosshairs method followed
by demarcation of the OB column and layers. Please see “Materials and methods” for more details. RMS rostral migratory stream, GL glomerular
layer, MCL mitral cell layer, EPL external plexiform layer, GCL granule cell layer. F, G Representative images of ­Ankrd11control (F) and ­Ankrd11nscKO(G) OB stained with Hoechst (white), blue crosshair shows center of OB used to demarcate the OB column (green rectangle). OB column cropis on the right side with demarcated layers. H Quantification of OB layer depth shown in F and G. I–T Representative images and quantificationof marker + cells in ­Ankrd11control and ­Ankrd11nscKO OB column layers immunostained for DCX (green) (I, J), NEUN (red) (K, L), CALB (green) (M, N),NG (pink) (O, P), GFAP (red) (Q, R), and PDGFRα (yellow) (S, T). Hoechst is counterstained in blue. The Hoechst panels in I, Q, and S were obtained
from the same image immunostained for DCX, GFAP, and PDGFRα; and in K and M from the same image immunostained for NEUN and CALB. Error
bars represent SEM. Data was analyzed using two-way ANOVA followed by Tukey’s multiple comparisons post hoc test. n = 5 mice per genotype
from at least two independent litters. Scale bars represent 1000 μm (B), 500 μm (F and G), 100 μm (I, K, M, O, Q and S). CB cerebellum, Ctx cortex, Eembryonic day, L lateral, M medial, OB olfactory bulb, P postnatal day

Goodkey et al. BMC Medicine(2024) 22:158

Page 9 of 24

Fig. 2 (See legend on previous page.)

Each data point in Fig. 6C represents an average numberof spheres per technical replicate. After counting, spheres
were swirled to the center and imaged as describedabove. Sphere diameter analysis was performed using

ImageJ on spheres that fit within the field of view. In
cases of uneven sphere shape, the longest diameter wasrecorded. A minimum of ~ 100 spheres per field of view
was measured.

Goodkey et al. BMC Medicine(2024) 22:158

Page 10 of 24

Fig. 3 Loss of Ankrd11 in E14.5 NPCs leads to abrogated P15 RMS migration. Also see Supplemental Fig. 2. A ­Ankrd11fl/fl;RosaYFPSTOP/STOP dams
mated with ­Ankrd11fl/fl;RosaYFPSTOP/STOP;NestinCreERT2 males were injected with TMX at E14.5. Resulting mice ­(Ankrd11control and ­Ankrd11nscKO)
were collected at P15. Images were collected from anatomically matched medial (M) and lateral (L) sagittal sections encompassing OB. RMS
indicated in green. B Schematic of areas measured in P15 RMS analysis. Region 1 encompassed the caudodorsal (CD) zone in the RMS (indicatedby a blue arrowhead), region 2 the ventral intermediate zone in the RMS (indicated by a red arrow), and region 3 where the RMS meets the OB(indicated by a yellow star). C, D Representative images from ­Ankrd11control and ­Ankrd11nscKO P15 medial (C) and lateral (D) OB encompassing RMS
immunostained for DCX (green) and Hoechst (white). Blue arrowhead indicates CD RMS (1), red arrow indicates VI RMS (2), yellow star indicatesOB (3). White dashed boxes in lateral Hoechst panel in D are shown in the right column at higher magnification and labeled with regions #1–3
corresponding the regions identified in B. E Quantification of integrated density of Hoechst signal in CD RMS (1), VI RMS (2) and OB (3) regions
outlined in D. F Quantification of region #1 (CD RMS) area. Error bars represent SEM. Data was analyzed using unpaired t-test. ns not significant,*p < 0.05, **p < 0.01, ***p < 0.001. n = 4–5 mice per genotype from at least two independent litters. Scale bars represent 1000 μm (C, D), 200 μm(insets of D). E embryonic day, L lateral, LV lateral ventricle, M medial, OB olfactory bulb, RMS rostral migratory stream

For primary neurosphere migration analysis, 5–10
spheres were plated in each PDL- and laminin-coatedwell in a 24-well dish in triplicate. Adhered spheres

were imaged as described above at 3 h post plating (0DIV, prior to migration) and again after 24 h (1 DIV,
after migration). Area of each sphere was outlined andGoodkey et al. BMC Medicine(2024) 22:158

quantified using ImageJ. Percent sphere area change from0 to 1 DIV was calculated using the biological averageand normalized to its littermate control. Each data point
in Fig. 5L, M represents an average of a 12–48 individual primary neurospheres measured per each biological replicate from 3 independent litters. Representative­Ankrd11control and A­ nkrd11nscKO 0 DIV and 1 DIV primary neurosphere area overlays are shown in Fig. 5K.
For scratch analysis, cells were plated on PDL- andlaminin-coated well in a 24-well dish in triplicate andimaged as above. 0 DIV scratch boundary was overlayed onto 1 DIV and 2 DIV of the same well and region,
cells within the scratch boundary were quantified using
ImageJ. Number of migrated cells were quantified andnormalized to a littermate control in each experiment.
All representative immunohistochemisty (IHC) andimmunocytochemistry (ICC) images were enhanced
post-acquisition linearly except representative images
for DCX, PDGFRα, βIII, and GFAP stains (Figs. 2I, Q, S
and 6E, G, I) were enhanced non-linearly to visualize cellprocesses.
Statistical analysis

All data passed normality test using the Kolmogorov–
Smirnov or Shapiro–Wilk tests, except for the data from
migration assays (Fig. 5), for which we performed a nonparametric Mann–Whitney test. For normal data, parametric tests were performed as following. For two-groupcomparisons, two-tailed unpaired Student’s or multiple
t-tests were used to assess statistical significance between
means. For three or more group comparisons, one- or
two-way unpaired ANOVA was followed by Tukey’s multiple comparisons post hoc tests. In all cases, GraphPad
Prism (version 8.0.2) was used for statistical tests. In the

Page 11 of 24

figures, an asterisk marks statistical significance indicated by *p-value of < 0.05, **p < 0.01, ***p < 0.001.

Results
Loss of Ankrd11 in NPCs results in aberrant postnatal OB
development

Ankrd11 is expressed in both NPCs and neurons during embryonic brain development [38]. To determine
the role of Ankrd11 in OB development, we induced
knockout of Ankrd11 in embyronic day (E) 14.5 NPCs
and their subsequent progeny by breeding ­Ankrd11fl/
fl
mice, where exon 7 of the Ankrd11 gene is flanked byloxP sites, with TMX-inducible N­ estinCreERT2 recombinase that is expressed specifically in Nestin + NPCs
(Fig. 1A) [61–63]. Upon TMX administration, Crecauses recombination at the floxed exon 7 of Ankrd11 in
NPCs, creating a frameshift mutation that encompasses
all known functional domains of the protein [58, 59].
The progeny from this TMX-injected cross that contain­NestinCreERT2 will be referred to as ­Ankrd11nscKO, andlitter mates that do not contain a ­NestinCreERT2 will bereferred to as A
­ nkrd11Contol (Fig. 1A). To confirm lossof Ankrd11 in NPCs, we dissected E15.5 ­Ankrd11nscKO
and ­Ankrd11Contol neocortices 24 h post TMX injection.
mRNA from cortical NPCs, which were enriched via
neurosphere protocol [67], was subjected to qRT-PCR
analysis. Figure 1K shows a ~ 96% decrease (p < 0.0001) in
Ankrd11 expression in A
­ nkrd11nscKO NPCs compared tocontrolthe ­Ankrd11
NPCs.
In mice, E14.5 is a timepoint that precedes the formation or migration of the majority of interneurons, glial
cells, and glutamatergic cells in the developing OB [13].
Thus, for all in vivo experiments, ­Ankrd11nscKO was
induced at E14.5. To identify if the loss of Ankrd11 in(See figure on next page.)
Fig. 4 Loss of Ankrd11 in E14.5 NPCs leads to decreased proliferation and aberrant neurogenesis in P15 RMS. Also see Supplemental Fig. 3. A
­Ankrd11fl/fl;RosaYFPSTOP/STOP dams mated with ­Ankrd11fl/fl;RosaYFPSTOP/STOP;NestinCreERT2 males were injected with TMX at E14.5 and resulting pups
were injected with BrdU at P14. Resulting mice ­(Ankrd11control and ­Ankrd11nscKO) were collected at P15. Images were collected from anatomicallymatched medial (M) and lateral (L) sagittal sections encompassing OB. RMS indicated in green. B Schematic of areas measured in P15 RMS analysis.
Region 1 encompassed the caudodorsal (CD) zone in the RMS, region 2 the ventral intermediate zone in the RMS, and region 3 where the RMS
meets the OB. Black box shows analysis area of region 1 in C. C Representative images of ­Ankrd11control and ­Ankrd11nscKO P15 RMS region 1
immunostained for BrdU (pink) and SOX2 (green). Open white arrow indicates BrdU + SOX2- cells, closed white arrow indicates BrdU-SOX2 + cells,
and yellow open arrow indicates BrdU + SOX2 + cells. Please see Fig. S3A for approximate image locations. D–J Quantification of C for BrdU + cellnumber (D) and density (E), SOX2 + cell number (F) and density (G), BrdU + SOX2 + cell number (H) and density (I) and proliferative index
of Sox2 + cells (% BrdU + SOX2 + /SOX2 + cells) (J). K Representative images of ­Ankrd11control and ­Ankrd11nscKO P15 RMS region 1 immunostainedfor PSA-NCAM (light blue) and CC3 (green). RMS outline in yellow dashed line is defined by the PSA-NCAM boundaries. White arrows indicate
CC3 + cells. Counterstained with Hoechst in white. Please see Fig. S3C for approximate image locations. L–M Quantification of (K) for CC3 + cellnumber (L) and density (M). N Representative images of ­Ankrd11control and ­Ankrd11nscKO P15 RMS region 1 immunostained for PSA-NCAM (lightblue) and NEUN (red). RMS outline in yellow dashed line is defined by the PSA-NCAM boundaries. Counterstained with Hoechst in white. PSA-NCAM
image is also presented in Fig. S3C inset, along with a precise image location in the OB sections. O–P Quantification of (N) for NEUN + cell number(O) and density (P). Error bars represent SEM. Data was analyzed using unpaired t-test. ns not significant, *p < 0.05 **p < 0.01, ***p < 0.001. n = 4–5
mice per genotype from at least two independent litters. Scale bars represent 100 μm (C, K), 200 μm (N). E embryonic day, L lateral, LV lateral
ventricle, M medial, OB olfactory bulb, P postnatal day, RMS rostral migratory stream

Goodkey et al. BMC Medicine(2024) 22:158

Fig. 4 (See legend on previous page.)

Page 12 of 24

Goodkey et al. BMC Medicine(2024) 22:158

E14.5 NPCs influenced OB development, we first determined any gross changes in the OB at various endpoints
using low-resolution photographs. At E18.5 (~ 1 day
before birth) OBs in the ­Ankrd11nscKO mice were visuallyindistinguishable from A­ nkrd11control (Fig. S1B-D). Analysis of postnatal animals revealed that A­ nkrd11Control
OB continued to increase in size with age, as expected(Fig. 1B–J). While at P5, the gross OB size was comparable between A­ nkrd11nscKO and A­ nkrd11Control (Fig. 1B–C), starting from P10 and until P30 (latest timepoint
analyzed), ­Ankrd11nscKO OB was ~ 37–56% smaller compared to A
­ nkrd11Control OB (Fig. 1D–J). Notably, thesechanges were not due to non-specific effects of Crerecombinase or TMX as OB area between uninjected­Ankrd11fl/fl and A­ nkrd11fl/fl;NestinCreERT2 mice was notdifferent and comparable to TMX-injected A­ nkrd11Control(Fig. 1I–J). Importantly, OB area from P30 TMX-injected­Ankrd11nscKO animals was still decreased when compared to uninjected controls (Fig. 1J).
Loss of Ankrd11 in NPCs does not affect OB development
at E18.5 in vivo

To determine the effect of Ankrd11 loss on OB development in more detail, we first analyzed the resultant E18.5
OB tissue following Ankrd11 nscKO at E14.5. Analysisof the whole OB area (excluding ONL) showed no difference between E18.5 ­Ankrd11nscKO and ­Ankrd11control
anatomically matched sagittal sections (Fig. S1A-D).
To identify the center of the OB in an unbiased manner, we overlayed a crosshair pattern over each anatomically matched OB section. We then applied a circle thatwould encompass OB borders. Finally, we applied thesame size column (rectangle), which originated fromthe OB center and emanated outwards in a rostral direction (towards circle edge), so all OB layers were captured(Fig. S1E). Resultant OB column boxes allowed us to analyze layer depth and cellular composition. Subsequent

Page 13 of 24

analysis of the OB neuronal layers, which were identified by Hoechst staining and as indicated in Fig. S1E-F,
showed no changes in layer depth (Fig. S1G). Next, weanalyzed the presence of NPCs and OB neurons. During
the late embryonic stage, the majority of the M/T neurons and some interneurons have formed and migrated
to their specific OB layers [13, 18]. Serial sagittal sectionsof E18.5 A­ nkrd11nscKO and A­ nkrd11control brains were
immunostained for markers expressed in precursors andpostmitotic neuronal cell types prevalent to OB development, including SOX2 (SRY-box transcription factor
2, a marker of NPCs [78]), DCX (doublecortin, markerof neuroblasts and immature neurons [79]) as well as
NEUN (neuronal nuclear protein, a marker of excitatory
and inhibitory neurons), CALB (calbindin, marker of periglomerular interneurons), and NG (neurogranin, marker
of granule cell inhibitory interneurons) [15, 80, 81]. The
identification of individual OB layers was determined by
Hoechst nuclear staining and is indicated in Fig. S1F. We
found no significant changes in the number of SOX2 + ,DCX + , NEUN + , CALB + , and NG + cells between­Ankrd11control and ­Ankrd11nscKO OB layers (Fig. S1H-Q).
Finally, there were no changes between number of OPCs,
detected using PDGFRα (platelet-derived growth factor
receptor alpha) specific antibody [82], in any layer (Fig.
S1R-S). Together, these data suggest that the knockoutof Ankrd11 in embryonic NPCs does not affect OB cell
development at E18.5.
Loss of Ankrd11 in NPCs causes aberrant OB development
at P15 in vivo

Figure 1 demonstrated that OB in the A­ nkrd11nscKO
mice was significantly reduced after P10 and at least
until P30. Therefore, we chose to perform an in-depth
analysis of OB cell development at P15, a timepoint
when postnatal interneuron genesis and glial cell proliferation is ongoing [2, 13]. Comparison of A
­ nkrd11control(See figure on next page.)
Fig. 5 Loss of Ankrd11 results in reduced SVZ NPC migration in vitro. A Schematic: P7 SVZ primary neurospheres were cultured from naive
uninjected ­Ankrd11fl/fl and ­Ankrd11fl/fl;NestinCreERT2 pups for 6 DIV. Neurospheres were treated with 4OH-TMX for 24 h prior to dissociationon 6 DIV resulting in ­Ankrd11Control and ­Ankrd11nscKO NPCs. Treated primary neurospheres were directly plated onto adherent wells for primaryneurosphere migration assay (H–M). Treated primary neurospheres were also dissociated and cultured as adherent NPC monolayer for scratch
assay (C–G) or secondary neurospheres (B). B Quantitative RT-PCR for Ankrd11 mRNA in P7 SVZ secondary neurosphere cultures from ­Ankrd11control
and ­Ankrd11nscKO NPCs. Data were normalized to Gapdh mRNA and calibrated against ­Ankrd11Control samples. C Schematic: 4OH-TMX treated
P7 SVZ primary neurosphere cells were cultured as adherent NPCs and subjected to scratch assay. D, E Representative images of ­Ankrd11control
and ­Ankrd11nscKO NPC scratch assay at 0, 1, and 2 DIV. 0 DIV indicates images taken immediately after scratch, and 1 and 2 DIV indicate images
taken 1–2 days after scratch. Scratch region at 0 DIV is outlined in black. F, G Quantification of cell migration into scratch region at 1 DIV (F) and 2
DIV (G) normalized to control. H Schematic: 4 OH-TMX treated selected P7 SVZ primary neurosphere cells were allowed to adhere and neurospherecell migration was assessed at 1 DIV. I, J Representative images of ­Ankrd11control and ­Ankrd11nscKO primary neurospheres assay at 0 and 1 DIV. Blackoutline shows the area of the neurosphere boundary (K). L Quantification of primary neurosphere area at 0 DIV. M Quantification of percent areachange on 1 DIV relative to 0 DIV and normalized to control. Error bars represent SEM. Data was analyzed the non-parametric Mann–Whitney test.
ns not significant, *p < 0.05, **p < 0.01 n = 4–6 mice per genotype from at least two independent litters. Scale bars represent 100 μm (D, E), 200 μm (I,J). DIV day in vitro, NPC neural precursor cells, NS neurospheres, 4OH-TMX 4-hydroxytamoxifen, P postnatal day, SVZ subventricular zone

Goodkey et al. BMC Medicine(2024) 22:158

Fig. 5 (See legend on previous page.)

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Goodkey et al. BMC Medicine(2024) 22:158

and ­Ankrd11nscKO P15 OBs showed a ~ 38% reduction (p < 0.001) in the whole OB area (excluding ONL)
(Fig. 2A–D). Next, we immunostained tissue sections
for markers of neurons and glia. As in Fig. S1, weapplied a circle and crosshairs method to determine
OB center in an unbiased manner (Fig. 2E). Resultant
OB column permitted analysis of layer depth and cellular composition. The identification of individual OB
layers was determined by Hoechst nuclear staining andis indicated in Fig. 2F, G. Overall, we found a ~ 33%
decrease (p < 0.0001) in the depth of the ­Ankrd11nscKO
GCL layer when compared to A
­ nkrd11Control, resulting
in a ~ 16% overall decrease of OB column (total layers) (p < 0.0001) in the ­Ankrd11nscKO samples (Fig. 2H).
We next analyzed cellular composition of each layer.
­Ankrd11nscKO OB showed a ~ 42% (p < 0.001) reduction in total DCX + cells (Fig. 2I, J). This was due toa ~ 51% (p < 0.0001) decrease of DCX + cells in the
GCL (Fig. 2I, J). This was accompanied by ~ 26%
decrease (p < 0.0001) in total NEUN + cells due to a
specific ~ 24% decrease (p < 0.0003) in NEUN + cells
within the GCL (Fig. 2K, L). Notably, NEUN + cells inthe ­Ankrd11nscKO MCL and GCL exhibited a diffuse
pattern of organization as compared to a well-definedpattern in A
­ nkrd11Control (Fig. 2K). This may indicate
abnormal localization of neurons in the A­ nkrd11nscKO
mice, which supports and extends prior reports that
demonstrated aberrant cortical neuronal localization in the Ankrd11-deficient mice [38, 83]. Finally,­Ankrd11Control and ­Ankrd11nscKO samples showed comparable CALB + , NG + , GFAP + (glial fibrillary acidic
protein), an astrocyte marker [84], and PDGFRα + cells
in all OB layers (Fig. 2M–T). In summary, our results
demonstrate a smaller OB size in P15 ­Ankrd11nscKO
mice accompanied by a specific decrease in DCX + and
NEUN + cells in the GCL and reduced GCL size.
Therefore, the OB size in the A­ nkrd11nscKO is mostprobably reduced due to aberrant formation and/or
maintenance of the GCL.

Page 15 of 24

Loss of Ankrd11 in NPCs results in aberrant postnatal RMS
development in vivo

Postnatal OB development and maintenance are dependent on the rostral migratory stream (RMS) [13, 18, 76].
DCX + neuroblasts originating in the SVZ travel along
the RMS during postnatal development and in adulthood via a GFAP + astrocyte network into the OB, where
they differentiate and integrate into OB outer neuronallayers, thus contributing to OB growth [2, 16, 22]. To
ask whether the RMS in the A­ nkrd11nscKO brains is perturbed, we immunostained serial E18.5 and P15 sagittal
sections of ­Ankrd11control and ­Ankrd11nscKO brains for
DCX, GFAP, and SOX2 (Figs. S2 and 3).
In the E18.5 sections, DCX stained all neurons in thebrain as they represent immature neurons (data notshown). To identify RMS, we instead used SOX2 staining, which at this timepoint is restricted to select areas
including a thin layer of cortical SVZ and RMS (Fig.
S2B). SOX2 + cells formed a continuous stream ofcells that connect to the OB in both ­Ankrd11nscKO and­Ankrd11control E18.5 brains (Fig. S2B). Integrated density analysis showed no significant changes in the SOX2,
DCX, or GFAP fluorescence levels in the RMS (Fig. S2C–E). There were also no gross apparent changes in RMS
integrity at this timepoint (Fig. S2B).
In contrast to E18.5, RMS in the P15 ­Ankrd11nscKO
brain sections appeared to be interrupted between the
SVZ and OB (indicated by red arrows in Fig. 3A-D). This
was apparent in images with DCX immunostaining andnuclear (Hoechst) staining (Fig. 3C, D). To quantify thisand explore the potential consequences on the downstream migration and population within the OB, wemeasured the nuclei density from three locations along
the RMS (Fig. 3E). Region 1 encompassed the caudodorsal (CD) RMS (indicated by blue arrowheads in Fig. 3C,D) and region 2 measured the ventral intermediate (VI)zones (indicated by red arrows in Fig. 3C, D), which wereidentified and demarcated according to [75, 76]. Region
3 sampled the area where the RMS connects to the OB(See figure on next page.)
Fig. 6 Loss of Ankrd11 decreases SVZ NPC proliferation and causes aberrant neurogenesis in vitro. A Schematic: P7 SVZ primary neurospheres were
cultured from ­Ankrd11fl/fl and ­Ankrd11fl/fl; ­NestinCreERT2 pups for 6 DIV. Neurospheres were treated with 4OH-TMX for 24 h prior to dissociationon 6 DIV resulting in Control and nscKO NPCs. Primary neurospheres were dissociated and further cultured as secondary neurospheres for 7
DIV or adherent NPC monolayer for 4 DIV. B Representative images of ­Ankrd11control and ­Ankrd11nscKO secondary neurospheres at 7 DIV. Inset
is shown at the bottom left corner in higher magnification. C, D Quantification of B for secondary neurosphere number (C) and diameter (D).
E–L Representative images and quantification of ­Ankrd11control and ­Ankrd11nscKO 4 DIV NPC monolayers immunostained for βIII (yellow) (E, F),
GFAP (green) (G, H), PDGFRα (white) (I, J), CC3 (red) (K, L). Arrowheads indicate marker + cells. Yellow insets in K are shown at higher magnificationon the right side. Cells were counterstained with Hoechst (blue). Data are presented as % marker + cells from healthy (non-condensed nuclei)
except % CC3 + cells is presented as % from all nuclei (healthy and condensed). Examples of condensed nuclei positive for CC3 are shown in K
and insets (yellow boxes) on right side. Error bars represent SEM. Data was analyzed using unpaired t-test. ns not significant, *p < 0.05, **p < 0.01,***p < 0.001. n = 3–4 mice per genotype from at least two independent litters. Scale bars represent 500 μm (B), 100 μm (inset of B), 50 μm (E, G, I, K).
DIV day in vitro, NPC neural precursor cells, NS neurospheres, 4OH-TMX 4-hydroxytamoxifen, P postnatal day, SVZ subventricular zone

Goodkey et al. BMC Medicine(2024) 22:158

Page 16 of 24

Fig. 6 (See legend on previous page.)(indicated by yellow star in Fig. 3C, D). Integrated density analysis of the nuclear stain Hoechst revealed a ~ 45%
and ~ 25% significant decreases (p < 0.05) in cell density

in ­Ankrd11nscKO regions 2 and 3, respectively (Fig. 3E).
While the density of nuclei was not changed in a defined
region of interest inside region 1 of RMS, the size of theGoodkey et al. BMC Medicine(2024) 22:158

whole region 1 of the A­ nkrd11nscKO RMS area was ~ 70%(p = 0.008) increased when compared to ­Ankrd11control,
further highlighting the aberrant RMS (Figs. 3F, S3A,C).
Together, this indicates aberrant RMS development inthe ­Ankrd11nscKO brain.
Next, we asked if A
­ nkrd11nscKO RMS displayed aberrant cell proliferation, differentiation, and/or apoptosis. First, mice were injected with BrdU 24 h prior to
perfusion to label actively proliferating cells (Fig. 4A).
RMS was identified with neuroblast-specific anti-PSANCAM (polysialylated neuronal cell adhesion molecule),
which completely overlaps with DCX (data not shown)[85]. Analysis of region 1 (caudodorsal RMS adjacent toLV) (Figs. 4B, S3A) revealed that the number and density of BrdU + cells was ~ 76% (p < 0.0001) decreasedin ­Ankrd11nscKO when compared to ­Ankrd11Control(Fig. 4C–E). This was not due to a decrease in progenitors as the number and density of SOX2 + cells and integrated density of PSA-NCAM was similar between­Ankrd11nscKO and A­ nkrd11Control (Figs. 4F, G, S3B). However, the number, density, and proportion of proliferating BrdU + SOX2 + cells were decreased by ~ 50% (p <0.01) in ­Ankrd11nscKO when compared to A
­ nkrd11Control(Fig. 4H–J). Next, we asked whether ­Ankrd11nscKO RMS
displayed aberrant cell apoptosis. Analysis of region 1
using anti-CC3 (cleaved caspase 3), an apoptosis marker[63], revealed no differences in the number or density ofCC3 + cells between A­ nkrd11nscKO and A­ nkrd11Control
nscKO(Fig. 4K-M). Thus, ­Ankrd11progenitors in the
RMS display reduced proliferation, which supports andextends previous reports showing reduced proliferationof Ankrd11-deficient cortical progenitors [38].
The RMS is populated by migrating progenitors thatterminally differentiate into postmitotic interneurons
once they reach the OB [86]. However, overexpression
or knockout of NeuroD1 or NCAM, respectively, haveresulted in RMS with prematurely differentiated neurons[87, 88]. We thus asked whether A­ nkrd11nscKO RMS contained any prematurely differentiated neurons. We analyzed RMS region 1, which was identified by PSA-NCAM
staining (Fig. S3C). We observed a > 2.8-fold increase in
the number and density (p = 0.001) of NEUN + cells inthe ­Ankrd11nscKO RMS when compared to ­Ankrd11Control(Fig. 4N–P). These results suggest a precocious differentiation of RMS progenitors into mature neurons before
they can reach the OB and integrate into the GCL. Theseobservations support and extend previous reports that
demonstrate precocious neuronal differentiation in progenitor-rich and usually neuron-devoid cortical SVZ inmice with Ankrd11 knockdown or mutations [38].

Page 17 of 24

Together, these results indicate an apparently normal
RMS in embryonic ­Ankrd11nscKO mice and a discontinuous RMS in postnatal ­Ankrd11nscKO mice, which may
suggest aberrant progenitor migration. Moreover, ourresults indicate a decrease in RMS progenitor proliferation and aberrant differentiation. To corroborate andtease apart the roles of Ankrd11 in migration, proliferation, and differentiation, we pursued in vitro cultures.
Loss of Ankrd11 results in decreased migration of SVZ
NPCs in vitro

To confirm the effect on migration of SVZ-derived­Ankrd11nscKO NPCs, we cultured P7 SVZ NPCs from­Ankrd11fl/fl and ­Ankrd11fl/fl;NestinCreERT2 pups that were
not injected with TMX, using a neurosphere protocol for 6
DIV (days in vitro) [62, 67]. We have previously shown thatthese neurospheres are highly enriched in Nestin + NPCs[67]. Recombination in primary neurospheres was induced byadding 4OH-TMX (active component of TMX) to the media
for 24 h prior to downstream migration assays (Fig. 5A). RTqPCR analysis of 4-OHT treated neurospheres revealed ~ 80%
Ankrd11 mRNA reduction (p = 0.012) in A
­ nkrd11nscKO culControl
tures when compared to ­Ankrd11(Fig. 5B).
First, we dissociated 4-OHT treated primary neurospheres from ­Ankrd11nscKO and ­Ankrd11Control culturesand plated them in a high-density monolayer. We thenperformed a scratch assay and assessed cell migration
immediately after scratch formation and 1 and 2 DIV
thereafter (Fig. 5C). ­Ankrd11control cells gradually filledin the scratch area over 2 DIV (Fig. 5D). In contrast,­Ankrd11nscKO cells showed ~ 51–64% decrease in thenumber of migrated cells on 1 and 2 DIV, respectively(p < 0.01), when compared to ­Ankrd11Control (Fig. 5E-G).
We have corroborated these results with a neurospheremigration assay [69, 89]. Here, individual 4OH-TMX
treated primary neurospheres were plated without dissociation onto PDL- and laminin-coated wells; the migration of cells out of the adhered spheres was assessed at
the time of plating and after 1 DIV (Fig. 5H). Migration
was determined by analyzing the change in total neurosphere area from 0 to 1 DIV (Fig. 5I–K). At the time ofplating (0 DIV), neurosphere area was similar between­Ankrd11control and ­Ankrd11nscKO (Fig. 5I–L). On 1 DIV,
­Ankrd11control neurospheres were visually more uniform creating a flattened surface of migrated NPCs
(Fig. 5I), while A­ nkrd11nscKO neurospheres remained asa dense sphere with decreased individual cells migrating out (Fig. 5J). Quantification showed a ~ 34% decreasein ­Ankrd11nscKO neurosphere area change relative to­Ankrd11control (p = 0.0095) on 1 DIV (Fig. 5K, M).

Goodkey et al. BMC Medicine(2024) 22:158

Together, these results show that loss of Ankrd11 leads
to decreased NPC migration in vitro.
Loss of Ankrd11 leads to reduced NPC proliferationand neurogenesis in vitro

To corroborate the effect of Ankrd11 deficiency on NPC
proliferation, we first generated primary neurospheresfrom P7 ­Ankrd11fl/fl and ­Ankrd11fl/fl;NestinCreERT2SVZ NPCs and treated them with 4OH-TMX to induce
recombination (Fig. 6A and as described above). Twentyfour hours after 4OH-TMX treatment, these spheres
were dissociated and plated at clonal density for 7 DIV toform secondary neurospheres, which were used as a readout of NPC proliferation [77]. Analysis of secondary neurospheres revealed a ~57% decrease (p = 0.0078) in thenumber and ~24% decrease in the diameter (p = 0.0002)
of ­Ankrd11nscKO spheres compared to ­Ankrd11control(Fig. 6B–D). These results show that ­Ankrd11nscKO NPCs
have reduced proliferation in vitro (Fig. 6B–D) and corroborate our in vivo results (Fig. 4C–J).
To determine whether Ankrd11 regulates NPC differentiation, we dissociated 4OH-TMX treated primaryneurospheres and plated them as adherent monolayers at a density that we have previously showed was
optimal for differentiation into neurons and glia by 4–5
DIV [67] (Fig. 6A). Immunostaining on 4 DIV revealeda ~ 22% decrease in the formation of βIII + neurons(p = 0.0095) in the ­Ankrd11nscKO cultures when comparedto ­Ankrd11Control (Fig. 6E, F) without significant effect on
GFAP + astrocytes, PDGFRα + oligodendrocyte precursor
cells, or apoptotic CC3 + cells (Fig. 6G–L). Therefore, lossof Ankrd11 results in decreased neurogenesis in vitro.
OB anomalies are detected in KBG syndrome patients

An MRI scan of patient #1 (male; de novo heterozygous
variant c. 3221_3222delAA, p. Lys1074ArgfsTer27 in the
ANKRD11 gene) showed normal brain structures; particularly, the sella turcica and hypophysis were morphologically normal, but the olfactory bulbs were not clearly
depicted compared to normal control images. The olfactory grooves looked severely hypoplastic and the straightgyri had a dysmorphic aspect with less prominent andorganized adjacent sulci as in normal anatomy (Fig. 7A–
H). These observations were interpreted as dysgenesis

Page 18 of 24

or hypoplasia of the olfactory bulbs and nerves, possibly
associated with the clinically confirmed anosmia. Patient#2 (female; c.6152dupC de novo in the ANKRD11 gene)came to genetic counseling for psychomotor retardationand olfactory bulb agenesia. Her olfactory bulbs were notseen on cerebral MRI confirming OB hypoplasia, dysplasia, or agenesis (Fig. 7I-J). Other relevant clinical observations included failure to thrive for patient #2 and relatively
poor response to growth hormone therapy in patient #1.
While patient #2 has psychomotor retardation and severe
speech delay, patient #1 is cognitively average and wasable to complete school education. Finally, both patients
present with mood disorders, including depressive behavior, anxiety, frustration intolerance, and/or anger.
We have also performed a Facebook KBG foundationpoll, which was answered by 103 families; 73 reported
normal sense of smell with 21 stating that it was not possible to know if the sense of smell was normal. Notably, 9(8.7%) reported alterations to sense of smell, with 5 (4%)
reporting absent sense of smell, and 4 (3.8%) reporting a
reduced sense of smell.
Together, these results identify anosmia, hyposmia,
olfactory nerve and/or OB dysgenesis or hypoplasia asnovel clinical symptoms of KBG syndrome.

Discussion
In this study, we show that conditional ablation of
Ankrd11 in murine embryonic NPCs results in abnormal
postnatal OB development, including abrogated RMS,
reduced GCL, and overall smaller OB size. We further
show Ankrd11-deficient NPCs display reduced proliferation, migration, and neurogenesis. Finally, we also show
two KBG syndrome patients with clinical anosmia and/or
OB hypoplasia. Thus, ANKRD11 is required for proper
olfactory bulb development in mice and humans.
Several transcription regulators and signaling receptors are critical for tangential neuroblast migration in
the RMS and subsequent OB development. For example,
NFIX (Nuclear Factor IX) constitutive knockout micedisplay enlarged SVZ, reduced RMS, and postnatal OB
hypoplasia [90]. Conditional knockout of Tbr2 (T-box
transcription factor 2) in E11 NPCs results in stunted
embryonic and postnatal OB development at least inpart due to enlarged cellular density in and size of the(See figure on next page.)
Fig. 7 OB hypoplasia in KBG syndrome patients. Coronal multiplanar reformatting three-dimensional high resolution heavy T2-weighted imagesof the naso-ethmoidal region. A–D Patient #1; MRI at 14 years of age. E–H Normal control anatomy. The olfactory bulb is not clearly visible in KBG
syndrome patient as compared to the normal subject (arrowheads). In addition, the olfactory grooves are severely hypoplastic and the straightgyri (dotted arrows in A–D) are dysmorphic with less prominent and organized adjacent sulci with respect to the normal anatomy (arrow in E–H).
I Patient #2; MRI at 3 years of age. J Normal control anatomy. No olfactory bulbs are seen in the KBG syndrome patient and consequently there
are no olfactory grooves. The olfactory sulci are visible (arrow). Olfactory bulbs (arrowhead) are visible within well-formed olfactory groovesof the control. The olfactory sulci (arrow) can also be seen

Goodkey et al. BMC Medicine(2024) 22:158

Fig. 7 (See legend on previous page.)

Page 19 of 24

Goodkey et al. BMC Medicine(2024) 22:158

RMS as early as E16.5 [91]. Furthermore, conditional
knockout of Mll1 (mixed lineage leukemia protein-1) inE13.5 NPCs leads to postnatal OB hypoplasia in vivo,
reduced neuroblast density in the RMS ex vivo as well as
diminished neuroblast chain migration from SVZ neurospheres in vitro [92]. Finally, constitutive knockout of
Prokr2 (prokineticin receptor 2), which binds PROK2, achemoattractant for neuroblasts in the RMS [93], resultsin fewer neuroblasts in the OB in vivo and an impaired
migration of SVZ NPCs towards Prok2 in vitro [94]. In
agreement, Sp8/Sp9 knockout mice show aberrant RMS
migration and OB development at least in part via downregulation of the Prokr2 expression [95]. Our results
showing that ablation of Ankrd11 in NPCs leads toreduced NPC migration in vitro and aberrant RMS development in vivo support and extend these reports.
Notably, we show a distinct OB phenotype compared tostudies cited above. Constitutive or NPC-specific knockouts of NFIX, Tbr2, Mll1, or Prokr2 lead to gross changes
in all OB layers, including GCL [90–95]. However, weshow that Ankrd11 conditional knockout in embryonic
NPCs leads to reduced neuroblasts and postmitotic neurons only in the GCL. The inhibitory granule neurons in
the GCL are one of the most abundant neuron types in
the OB and play a critical role in the discrimination ofodors through their synaptic interactions with the excitatory M/T neurons located in the MCL [96, 97]. Notably,
our results demonstrate abnormal neuronal localizationin the GCL and MCL of the A­ nkrd11nscKO OB. We suspect this could be due, at least in part, to the aberrant neuronal migration. This is supported by our in vitro resultsthat show decreased progenitor migration. It is tempting
to speculate that these changes may have a direct impact
on olfaction. While we did not perform behavioral olfaction experiments, our clinical data align well with murine
results and demonstrate absence or hypoplasia of OB
and OB-associated structures co-present with anosmia/
hyposmia in two KBG syndrome patients.
Contribution of migrating postnatal neuroblasts in the
RMS to postnatal and adult OB development and function is supported by prior literature [2]. For example, sensory deprivation via naris occlusion (nasal cavity blockage)leads to reduced neuroblast migration in the RMS andstunted OB growth, while removal of the naris occlusion
restores neuroblast migration and OB development [98].
Notably, the restoration of OB growth in olfaction deprived
mice requires neuroblast formation from SVZ NPCs [98].
Mouse genetic models demonstrate that proper RMS and
OB development is also regulated by several neurogenesisrelated genes. For example, DCX deficiency leads to abnormal SVZ NPC migration, precocious differentiation in the
RMS, and a smaller OB due to loss of Calretinin + granulecells [88, 99]. Overexpression of NeuroD1, a neurogenic

Page 20 of 24

transcription factor, induces premature differentiation ofneurons in RMS [87]. Our data suggest that the reduction
in OB size in the A­ nkrd11nscKO mice is due to reduced NPC
proliferation, migration, and aberrant neurogenesis. Notably, while the ­Ankrd11nscKO OB GCL contained reduced
number of neurons, the RMS contained more mature neurons when compared to control. It is likely that theAnkrd11-deficient progenitors that reached the OB have
reduced neurogenesis, while progenitors that are present in
the abrogated RMS differentiate locally before reaching theOB, similarly to DCX-deficient mice.
We and others have previously shown aberrant neuronal positioning in the cortex of mice that carry a splicemutation in the Ankrd11 gene (Yoda mice) or in micewith Ankrd11 knockdown in murine embryonic cortical
NPCs, which could suggest aberrant neuronal migration[38, 83]. Our results extend and support these conclusions by showing neuronal mislocalization (heterotopia)
in the A­ nkrd11nscKO RMS. Together, this suggests thatsome Ankrd11 mechanisms may be shared between OB
and cortical development. Furthermore, our report isthe first to show that Ankrd11 deficiency in NPCs causes
severe migration defects via the abrogated postnatal RMS
in vivo and reduced migration in vitro.
From a clinical perspective, this is the first case of aclear link between ANKRD11, KBG syndrome, and OB
deficits. Previously, one patient with 16q24.3 microdeletion, which encompasses ANKRD11, was reported tohave anosmia [100]. Since clinical phenotypes between
patients with ANKRD11 variants vs 16q24.3 microdeletions may vary in nature, numbers, penetrance, andseverity [100], it was not clear whether the anosmia in
the 16q24.3 microdeletion patient was due to the deletionof ANKRD11 and/or any other genes. Our report showing OB deficiencies in two patients with ANKRD11 variants as well as clear OB developmental reduction in sizein mice lacking Ankrd11 in embryonic NPCs and theirderivatives establishes a strong causative link between
ANKRD11 perturbations and OB deficiencies. Hypo/
anosmia is not a phenotype that would routinely beenquired about or reported. Indeed, if one has congenitally reduced sense of smell one may never know that itis reduced. To this end, the Facebook survey on senseof smell was responded to by 103 families with KBG
syndrome patients, which represents approximately aquarter of known families registered with the KBG foundation. Of these 8.7% reported reduced or absent senseof smell. Notably, ~ 29% of respondents stated that it was
not possible to know if the sense of smell was normal.
Our report demonstrates OB defects in two KBG syndrome patients, and this along with the results of the pollsuggest this may be a relatively rare feature in KBG syndrome or an under recognized/reported one.

Goodkey et al. BMC Medicine(2024) 22:158

OB malformations, including uni- and bilateral OB
hypoplasia or aplasia, are reported in ~ 30% of patientswith Mendelian Disorders of the Epigenetic Machinery[101]. Notably, chromatin or epigenetic regulation represents the largest group of NDD risk genes [102–104]. Not
surprisingly, OB malfunctions, which include anosmia(loss of smell), hyposmia (reduced scent detection), andhyperosmia (enhanced scent detection) or OB dysplasia have been detected in patients with ASD [105–108],
including patients with variants in epigenetic regulators
like CBP (cyclic AMP response element binding protein binding protein; Rubinstein–Taybi syndrome riskgene) and FMR1 (fragile X messenger ribonucleoprotein
1; fragile X Syndrome risk gene) [109–111]. Our results
showing absent or altered OB with/without anosmia in
KBG syndrome patients and reduced postnatal OB size
and altered RMS development in the A­ nkrd11nscKO mice
support and extend these reports suggesting that epigenetic risk genes of developmental disorders may be key
regulators of OB development and function.
The sense of smell is strongly connected to the emotional processing via the connection of the olfactory system with reward pathways and the hippocampus [8, 112].
Reductions in the ability to smell or OB ablation candrastically change the food intake [113, 114]. For example, OB ablation results in altered feeding patterns in rats
and sheep [115, 116]. Human patients with traumaticbrain injury that show changes in olfaction and the OB
have difficulties in feeding [117]. With regard to behavior,
reduction or loss of olfaction has close ties to behavioral changes in mood-altering disorders, and OB changes
themselves can be used as a marker for depression [9,10, 118]. Furthermore, OB bulbectomy or OB neuronalablation in rodents induces depressive-like behaviors[6–8]. Notably, some patients with NDDs, including KBG
syndrome, experience feeding difficulties and failure to
thrive as well as frustration and depressive behavior [45,51, 52, 119–121]. While the link between OB defects ormalfunction and NDD-related behaviors is not currently
known, our work and previous reports suggest a more
systematic recording of sense of smell in newly diagnosed patients along with continued efforts to record thedetailed feeding and neurobehavioral phenotypes may
be warranted. While it is unlikely that hyposmia or anosmia would be amenable to clinical treatment, it may be
important for appropriate genetic counseling.

Conclusions
Taken together, our results demonstrate a novel and critical role for ANKRD11 in OB development and suggest
OB size or olfaction evaluations should be considered
upon KBG syndrome diagnosis for appropriate genetic
counseling and to improve clinical care.

Page 21 of 24

Abbreviations
4OH-TMX	4-Hydroxytamoxifen
ADHD	Attention-deficit/hyperactive disorder
Ankrd11	Ankyrin repeat domain 11
ASD	Autism spectrum disorderβIII	Beta-III tubulin
BrdU	Bromodeoxyuridine
BSA	Bovine serum albumin
CALB	Calbindin
CBP	CREB binding protein
CC3	Cleaved caspase 3
CD	Caudodorsal
CNS	Central nervous system
DCX	Doublecortin
DIV	Day in vitro
E	Embryonic day
ECM	Extracellular matrix
EGF	Epidermal growth factor
EPL	External plexiform layer
FGF	Fibroblast growth factor
FMR1	Fragile X messenger ribonucleoprotein 1
GCL	Granule cell layer
GFAP	Glial fibrillary acidic protein
GL	Glomerular layer
HAT	Histone acetyltransferase
HBSS	Hanks’ Balanced Salt Solution
HDACs	Histone deacetylase
HEP	Heparin
IP	Intraperitoneal
LV	Lateral ventricle
M/T	Mitral tufted cells
MCL	Mitral cell layer
MeCP2	Methyl CpG binding protein 2
MRI	Magnetic resonance imaging
NG	Neurogranin
NDD	Neurodevelopmental disorders
NPC	Neural precursor cells
NEUN	Neuronal nuclei
OB	Olfactory bulb
ONL	Olfactory nerve layer
OPC	Oligodendrocyte precursor cells
OSN	Olfactory sensory nerve
P	Postnatal day
PSA-NCAM	Polysialylated-neural cell adhesion molecule
PDGFRα	Platelet-derived growth factor receptor alpha
PDL	Poly-d-lysine
Prok2	Prokineticin 2
Prokr2	Prokineticin receptor 2
RMS	Rostral migratory stream
SOX2	
Sex-determining region Y (SRY)-related high-mobility-group(HMG)-box 2
SVZ	Subventricular zone
TMX	Tamoxifen
VI	Ventral intermediate

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12916-​024-​03363-6.
Supplementary Material 1.
Acknowledgements
We thank the patients and families for their participation in this work. We
thank Dr. Emanuela Scarano (Rare Diseases Unit, Department of Pediatrics,
IRCCS Azienda Ospedaliero-Universitaria di Bologna, Italy) for previous clinicalcare for patient 1, as well as Paige Fumo Fox and Annette Maughan for help
with the KBG Foundation Facebook poll on smell. We would also like to thankNicole Dittmann and Yana Kibalnyk for feedback on the manuscript.

Goodkey et al. BMC Medicine(2024) 22:158

Authors’ contributions
Conceptualization, KG, AW, LP, KL, and AV. Methodology, KG, AW, AESW, LP,
DMC, FT, FB, MEB, and AV. Formal analysis, DMC, MEB, KG, and LQ. Investiga‑
tion, KG, AW, AESW, LQ, LP, DMC, FT, FB, MEB, and AV. Resources, AV. Writ‑
ing—original draft, KG. Writing—review and editing, AW, LP, KL, KG, and AV.
Supervision, AV. Funding acquisition, AV.
Funding
KG was supported by the Women and Children’s Health Research Institute(WCHRI) Graduate studentship and Natural Sciences and Engineering
Research Council (NSERC) Canada Graduate Scholarship—Master’s level. This
work was supported by private donations from families affected by the KBG
syndrome, KBG Foundation seed funding and an award from The Scottish Rite
Charitable Foundation of Canada awarded to AV. AV was also supported by
the Canada Research Chair in Neural Stem Cell Biology and Sloan Research
Fellowship in Neuroscience awards. KL is supported by a National Institutefor Health and Care Research (NIHR) Doctoral Research Fellowship (302303).
AESW was supported by Canadian Institutes for Health Research (CIHR)
Canada Graduate Scholarship—Doctoral level.
Availability of data and materials
All data generated or analyzed during this study are included in this publishedarticle and its supplementary information files.

Declarations
Ethics approval and consent to participate
All relevant information is described in “Materials and methods”.
Consent for publication
All authors consent to publication.
Competing interests
The authors declare no competing interests.
Received: 2 August 2023 Accepted: 18 March 2024

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