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Spatial transcriptomics/genomics with subcellular resolution

Bintu Lab

bbintu@health.ucsd.edu

Cellular and Molecular Medicine

Bioengineering

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Microfluidics

Microscope lasers

Spatial transcriptomics (Method of the year in 2020 - Nature methods)

sample

5 fluorescent colors > 1,000-10,000 effective colors

Multiplex Error Robust In Situ Hybridization (MERFISH)

Chen et al Science 2015

X. Zhuang, Nature Methods, 2021

Advances in DNA synthesis (labelling thousands of molecular types)

Combinatorial encoding and decoding scheme

+

=

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Microfluidics

Microscope

Flavors of spatial transcriptomics/genomics

Next gen. sequencing

In situ single molecule imaging

In vitro single-molecule imaging

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Microfluidics

Microscope

Flavors of spatial transcriptomics/genomics

MERFISH (Zhuang lab)

seqFISH (Cai lab)

Spatial transcriptomics (Frisén lab)

SlideSeq (Chen&Makasco labs)

FISSEQ (Church lab)

STAR-map (Deisseroth lab)

High sensitivity (>80% efficiency) and spatial resolution (subcellular)
Low genomic throughput

(~500 colors)

Low sensitivity and spatial resolution

High genomic throughput (>30,000 colors)

Ståhl et al. Science 2016

Rodriques et al Science 2019

Chen et al Science 2015

Lubeck et al., 2014

Wang et al Science 2019

Lee et al Science 2014

Next gen. sequencing

Chen et al Science 2015 X. Zhuang, Nature Methods, 2021

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MERFISH - applications

Spatial genomics

Su,...,Bintu*,Zhuang* Cell 2020

Inducing neurogenesis in aged mammalian brains

Collaboration with Don Cleveland

Imaging ecDNA

Collaboration with Frank Furnari Don Cleveland and Bing Ren

Olfactory epithelium(nose)

Olfactory bulb (brain)

Receptor

RNA

Epithelium

Glomeruli

RNA MERFISH

DNA MERFISH

EGFR

ecDNA

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Inducing neurogenesis

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dying

convert

Inducing neurogenesis

In the adult centers, the neural paths are something fixed and immutable:

everything may die, nothing may be regenerated

Ramon y Cajal, 1928

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Approaches to glia-to-neuron transdifferentiation

A therapeutically viable approach

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Maimon et al, Nat Neurosci, 2021

+Tamoxifen

Glia promoter

Genetically label glial cells

New neurons after 2 months

+PTB-ASO

PTB mRNA

Glial cells

Improved memory

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What cell types convert into neurons?

Which types of new neuron and in what brain regions?

Can we capture intermediate states?

Maimon et al, Nat Neurosci, 2021

+Tamoxifen

Glia promoter

Genetically label glial cells

New neurons after 2 months

+PTB-ASO

PTB mRNA

Glial cells

Improved memory

1. Genetically/AAV mediated labelling is imprecise

2. Prior works did not characterize the conversion process

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Identifying the cells that convert into new neurons: spatial transcriptomics (MERFISH Multiplexed Error-Robust Fluorescence In-Situ Hybridization)

�

Thalamic

Neurons

�

Oligos

�

Cortical

Neurons

�

Microglia

�

Astrocytes

�

Choroid

Plexus

�

Striatal

Neurons

�

Ependymal

�

Intermediates

�

Hippocampal

Neurons

�

DG

�

Maimon, Marinas, Vazquez et al. Unpublished

Huntington's disease (HD) is an inherited disorder that results in the death of brain cells

transcription, cell signaling, and intracellular transporting

Histone H3 is a nuclear core histone protein of DNA chromatin, with an important role in chromosome condensation and cell-cycle progression during mitosis and meiosis after phosphorylation of serine-10 and serine-28 residues. Phosphorylation occurs during late G2 to early prophase, while dephosphorylation occurs slowly from late anaphase to early telophase. Therefore in metaphase, histone H3 is always heavily phosphorylated and positive for PHH3,

Several targets have been identified for potential exploitation in therapy, with strategies that include HTT lowering and immunomodulation²⁵⁵. Ac, acetyl group; ASO, antisense oligonucleotide; BDNF, brain-derived neurotrophic factor; CB2, cannabinoid receptor 2; EAAT2, excitatory amino acid transporter 2; GM1, monosialotetrahexosylganglioside; HDAC4, histone deacetylase 4; JNK, c-Jun N-terminal kinase (MAPK8, MAPK9 and MAPK10); KMO, kynurenine 3-monooxygenase; MAPK, mitogen-activated protein kinase; NMDA, N-methyl-D-aspartate; P, phosphate group; p38, mitogen-activated protein kinase (MAPK11, MAPK12, MAPK13 and MAPK14); PDE, phosphodiesterase; PPAR-γ, peroxisome proliferator-activated receptor-γ; RNAi, RNA interference; Su, sumoyl group; TrkB, tyrosine receptor kinase B. Figure adapted from Ref. 6, Nature Publishing Group

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MERFISH in the mouse brain

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Astrocytes

Cortical excitatory neurons

Cortical inhibitory neurons

CA2

CA1

Oligodendrocytes

DG

Pericytes

CA3

Endothelial

Thalamic neurons

MERFISH (230 genes)

Single-cell UMAP (~57,000 cells)

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Ependymal cells

Immature SVZ neurons

Mature Dentate gyrus neurons

Astrocytes

Immature dentate gyrus neurons

Zhao, Deng, Gage, Cell, 2008

SGZ

Neurogenesis within the young mouse brain

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Ependymal (Progenitors)

Immature neurons

Dentate gyrus neurons

Astrocytes

8 week (young) mouse

Immature neurons

Immature

neurons

1 year (old) mouse

1 year mouse - PTBP1 ASO

Ependymal (Progenitors)

Astrocytes

Dentate gyrus neurons

Neurogenesis stops in the aged brain

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Ependymal (Progenitors)

Immature neurons

Dentate gyrus neurons

Astrocytes

8 week (young) mouse

Immature neurons

Immature

neurons

1 year (old) mouse

1 year mouse - PTBP1 ASO

Ependymal (Progenitors)

Astrocytes

Dentate gyrus neurons

Neurogenesis stops in the aged brain

Number of cells/section

4 weeks

1 year

Immature

DG

Immature

SVZ

8 weeks

4 weeks

1 year

8 weeks

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20

0

Number of PTBP1

transcripts/cell

Ventricular zone

Maimon, Marinas, Vazquez et al. Unpublished

Determining which cell types express PTBP1 in the aged mouse brain

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Comparing cell type definition and gene expression between

single-nucleus RNA sequencing with MERFISH data

Single nuclear

mRNA sequencing

MERFISH

Huntington's disease (HD) is an inherited disorder that results in the death of brain cells

transcription, cell signaling, and intracellular transporting

Histone H3 is a nuclear core histone protein of DNA chromatin, with an important role in chromosome condensation and cell-cycle progression during mitosis and meiosis after phosphorylation of serine-10 and serine-28 residues. Phosphorylation occurs during late G2 to early prophase, while dephosphorylation occurs slowly from late anaphase to early telophase. Therefore in metaphase, histone H3 is always heavily phosphorylated and positive for PHH3,

Several targets have been identified for potential exploitation in therapy, with strategies that include HTT lowering and immunomodulation²⁵⁵. Ac, acetyl group; ASO, antisense oligonucleotide; BDNF, brain-derived neurotrophic factor; CB2, cannabinoid receptor 2; EAAT2, excitatory amino acid transporter 2; GM1, monosialotetrahexosylganglioside; HDAC4, histone deacetylase 4; JNK, c-Jun N-terminal kinase (MAPK8, MAPK9 and MAPK10); KMO, kynurenine 3-monooxygenase; MAPK, mitogen-activated protein kinase; NMDA, N-methyl-D-aspartate; P, phosphate group; p38, mitogen-activated protein kinase (MAPK11, MAPK12, MAPK13 and MAPK14); PDE, phosphodiesterase; PPAR-γ, peroxisome proliferator-activated receptor-γ; RNAi, RNA interference; Su, sumoyl group; TrkB, tyrosine receptor kinase B. Figure adapted from Ref. 6, Nature Publishing Group

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Maimonet al. Unpublished

Reduction in PTBP1 activates expression of genes characteristic of immature neurons

Control

ASOs

Control

Igfbpl1 (marker of immature neurons)

DG

SVZ

DG

SVZ

MERFISH

Ependymal

Hippocampal Astro

PTBP1

REST

RTN4

DCX

PTBP1

REST

RTN4

DCX

0

10

20

0

6

12

mRNA per cell

Control

PTBP1-ASO

mRNA per cell

Huntington's disease (HD) is an inherited disorder that results in the death of brain cells

transcription, cell signaling, and intracellular transporting

Histone H3 is a nuclear core histone protein of DNA chromatin, with an important role in chromosome condensation and cell-cycle progression during mitosis and meiosis after phosphorylation of serine-10 and serine-28 residues. Phosphorylation occurs during late G2 to early prophase, while dephosphorylation occurs slowly from late anaphase to early telophase. Therefore in metaphase, histone H3 is always heavily phosphorylated and positive for PHH3,

Several targets have been identified for potential exploitation in therapy, with strategies that include HTT lowering and immunomodulation²⁵⁵. Ac, acetyl group; ASO, antisense oligonucleotide; BDNF, brain-derived neurotrophic factor; CB2, cannabinoid receptor 2; EAAT2, excitatory amino acid transporter 2; GM1, monosialotetrahexosylganglioside; HDAC4, histone deacetylase 4; JNK, c-Jun N-terminal kinase (MAPK8, MAPK9 and MAPK10); KMO, kynurenine 3-monooxygenase; MAPK, mitogen-activated protein kinase; NMDA, N-methyl-D-aspartate; P, phosphate group; p38, mitogen-activated protein kinase (MAPK11, MAPK12, MAPK13 and MAPK14); PDE, phosphodiesterase; PPAR-γ, peroxisome proliferator-activated receptor-γ; RNAi, RNA interference; Su, sumoyl group; TrkB, tyrosine receptor kinase B. Figure adapted from Ref. 6, Nature Publishing Group

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Progenitors

Immature neurons

Dentate gyrus neurons

Astrocytes

8 week mouse

Immature

neurons

1 year mouse - PTBP1 ASO (2 weeks)

Immature

neurons

1 year mouse - no ASO

Immature neuronal states emerging upon PTBP suppression in SVZ

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L6 cortical

Neurons

OB

Neurons

Striatal

Neurons

Immature

Ependymal

Medial

Ependymal

lateral

Stage 1

Stage 2

Stage 3

Oligo

Posterior

Striatal

neurons

Granular

neurons

cells

Choroid

plexus

200µm

Neurogenesis cell atlas – SVZ 4week mouse

Olfactory Bulb Path

Sox11

Ki67

Prox1

Stage 3

Pearson Correlation heatmap

A cells

Mitosis

Ependymal

B cells

Lateral

Ependymal

Medial

Ependymal

Stage 1

Stage 2

Buylle 2021 (SNuc-Seq)

Maimon et al, 2024

(MERFISH)

Maimon et al (MERFISH)

Ependymal

B cells

Mitosis

A cells

Medial Ependymal

Stage 1

Lateral Ependymal

Stage 2

Stage 3

Ascl1

Dcx

Sox11

Vim

Ki67

Prox1

Ascl1

Vim

Dcx

Buylea, 2021, eLife

Ki67

Sox11

Prox1

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L6 cortical

Neurons

OB

Neurons

Striatal

Neurons

Immature

Ependymal

Medial

Ependymal

lateral

Stage 1

Stage 2

Stage 3

Oligo

Posterior

Striatal

neurons

Granular

neurons

cells

Choroid

plexus

200µm

Neurogenesis cell atlas – SVZ 4week mouse

Olfactory Bulb Path

Vim

Slc2a1

Zfp36l1

Sox9

Sox2

Nnat

Slc1a3

Fam107a

Mfge8

Ascl1

Hes5

Sox4

Sox11

Dlx1

Dlx2

Igfbpl1

Dcx

Ngef

Pde10a

Nrgn

Ptbp1

Rest

Brn2

Pseudo

Slc2a1

Zfp36l1

Sox9

Sox2

Nnat

Slc1a3

Fam107a

Mfge8

Ascl1

Hes5

Sox4

Sox11

Dlx1

Dlx2

Igfbpl1

Dcx

Gad1

Rbfox3

Ptbp1

Rest

Vim

Pseudo

Ependymal

cells

Immature cells

Mature Neurons

Brn2

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PC1

PC2

Stage 1

Saline

PTBP1-ASO

Sox11

Dpysl3

Ascl1

Ki67

Dcx

Mfge8

Mcm2

Top2a

Vim

Aqp4

Gja1

Sox9

S100b

Zfp361l

Unc13c

Slc2a1

Saline

PTBP1-ASO

Vim

S100b

Ascl1

Dcx

Sox11

0

120

18

12

10

20

0

Ki67

14

0

Saline injection

PTBP1-ASOs

Stage 1

Stage 2

Stage 3

Stage 1

Stage 2

Stage 3

#cells/section

Non

ASO

Saline

# cells/section

1 year old mice

Non

ASO

Saline

Non

ASO

Saline

Dcx

Sox11

Ccnd1

Mcm5

Ki67

Ccnd2

Ependymal

Choroid Plexus

5µm

Stage 3

PC1

PC2

4week

PTBP1-ASO

Dcx

Dlx2

Sox11

Sgk1

Ple1

Ser1

Grp

Serf1

Rasl

Crmp4

Reln

Vim

Gabr2

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EdU staining of new neurons upon cell division

4 week

EdU + 3 days

DG

SVZ

DG

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PTBP1 and its targets are express in the human SVZ

Maimon et al. Unpublished

Ependymal 1

Ependymal 2

PTBP1

Rest

PTBP2

Brn2

Huntington's disease (HD) is an inherited disorder that results in the death of brain cells

transcription, cell signaling, and intracellular transporting

Histone H3 is a nuclear core histone protein of DNA chromatin, with an important role in chromosome condensation and cell-cycle progression during mitosis and meiosis after phosphorylation of serine-10 and serine-28 residues. Phosphorylation occurs during late G2 to early prophase, while dephosphorylation occurs slowly from late anaphase to early telophase. Therefore in metaphase, histone H3 is always heavily phosphorylated and positive for PHH3,

Several targets have been identified for potential exploitation in therapy, with strategies that include HTT lowering and immunomodulation²⁵⁵. Ac, acetyl group; ASO, antisense oligonucleotide; BDNF, brain-derived neurotrophic factor; CB2, cannabinoid receptor 2; EAAT2, excitatory amino acid transporter 2; GM1, monosialotetrahexosylganglioside; HDAC4, histone deacetylase 4; JNK, c-Jun N-terminal kinase (MAPK8, MAPK9 and MAPK10); KMO, kynurenine 3-monooxygenase; MAPK, mitogen-activated protein kinase; NMDA, N-methyl-D-aspartate; P, phosphate group; p38, mitogen-activated protein kinase (MAPK11, MAPK12, MAPK13 and MAPK14); PDE, phosphodiesterase; PPAR-γ, peroxisome proliferator-activated receptor-γ; RNAi, RNA interference; Su, sumoyl group; TrkB, tyrosine receptor kinase B. Figure adapted from Ref. 6, Nature Publishing Group

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Conclusions

PTBP1 suppression via ASO reactivates the neurogenesis pathway in an otherwise dormant subependymal (Stage 1)

Some of these cells upon cell division are likely incorporated into the striatum as mature neurons close to the ventricle

Human SVZ contains these dormant PTBP1 positive progenitor cells

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DNA MERFISH - Applications

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Model relating DNA organization and transcriptional activity

Microglia (brain)

Sall1 -mRNA

Csf1r

Cx3cr1

Sall1 - nascent RNA

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Model relating DNA organization and transcriptional activity

Microglia (brain)

Genes

Enhancers

Repressors

Transcription

factors

Pol II

Sall1

Nucleus

Sall1 - nascent RNA

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Sequencing methods point towards regulation model

Model

Sequencing and genetic tools

Fixsen et al, 2022 - Chris Glass’ lab

Microglia (brain)

Sall1 - nascent RNA

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Super-resolution chromatin tracing

2

STORM

300nm

(Bintu et al. Science 2018)

chr21:28.03-28.06Mb

2^nd 30kb segment

...

chr21:29.20-29.23Mb

41^st 30 kb segment

...

chr21:28-28.03Mb

1^st 30kb segment

1μm

Diffraction

limited

Readout sequence

Labelling scheme

chr21:28-29.23Mb

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Super-resolution chromatin tracing

28Mb chr 21 29.12Mb

Labelling scheme

chr21:28-29.23Mb

The structure of 1.2 Mb region at ~20nm x,y,z resolution and 30kb genomic resolution

300nm

28Mb chr 21 29.12Mb

300nm

(Bintu et al. Science 2018)

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TADs emerge from single-cell TAD-like structures

Bintu et al. Science 2018

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TADs emerge from single-cell TAD-like structures

Rao et al., Cell, 2014

Pearson correlation of 0.96

Median distance

(1,200 chromosomes)

Bintu et al. Science 2018

Distance matrix

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Extrachromosmal DNA (ecDNA)

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antibody EGFRvIII

DAPI

GBM39 cells

EGFR ecDNA

EGFRvIII

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Recent works suggest an epigenetic structure-function relationship between ecDNA

Wu et al Nature 2020

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1st 5kb of ecDNA

chr7:54.9MB

chr7:56.1Mb

EGFR ecDNA

Imaging ecDNA in GBM39 cells

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loci 4,5,6

loci 1,2,3

…

1st 5 kb

2nd 5 kb

3rd 5 kb

252nd 5 kb

4th 5 kb

… ~80

hybridizations

DNA MERFISH imaging of ecDNA

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1st 5kb

1nd 5kb reimaged

1nd 50kb reimaged

1st 50kb

Preserving structure after hundreds of hybridizations

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1st 5kb

Identify the endogenous chr7 locus (part of chr7)

chr7:54.9MB

chr7:56.1Mb

EGFR ecDNA

chr7

54.9Mb

56.1Mb

50kb outside the ecDNA on chr7

chr7

54.9Mb

56.1Mb

chr7:54.9MB

chr7:56.1Mb

EGFR ecDNA

EGFR deletion

chr7

54.9Mb

56.1Mb

chr7:54.9MB

chr7:56.1Mb

EGFR ecDNA

ΔEGFR

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ecDNA structure and composition in single-cells

chr7:54.9MB

chr7:56.1Mb

EGFR

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EGFR Intron

EGFR Exons

LaminA

The expression profile from the ecDNA is heterogeneous

Nucleoli

Spliceosomes

Pol2(Phos)

LaminA

WGA

Antibodies

1. Is there a special structure-function relation between the ecDNA 3d structure and transcription?

2. Upon drug treatment, why do dormant echidna become reactivated?

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Non-injected mouse

GBM

Human glioblastoma cells injected in the mouse brain

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GBM

Human glioblastoma cells injected in the mouse brain

1st 5kb dapi

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GBM

Human glioblastoma cells injected in the mouse brain

1st 5kb dapi

EGFRvIII dapi

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GBM

Human glioblastoma cells injected in the mouse brain

1st 5kb dapi

Aqp4 dapi

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ecDNA formation - chromosome spreads

chr6: 18Mb-18.03Mb

chr6: 81Mb-81.03Mb

Stained genome wide probes

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Chr6 (dapi)

chr6: 18Mb-18.03Mb

chr6: 81Mb-81.03Mb

Chromatin tracing of chromosome spreads

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chr6: 18Mb-18.03Mb

chr6: 33Mb-33.05Mb

chr6: 81Mb-81.03Mb

Chr6 (dapi)

chr6: 18Mb-18.03Mb

chr6: 33Mb-33.05Mb

chr6: 48Mb-48.05Mb

chr6: 81Mb-81.03Mb

Chr6 (dapi)

Chromatin tracing of chromosome spreads

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chr6: 18Mb-18.03Mb

chr6:33Mb-33.05Mb

chr6:48Mb-48.05Mb

chr6:66Mb-66.05Mb

chr6: 81Mb-81.03Mb

Chr6 (dapi)

chr6: 18Mb-18.03Mb

chr6:33Mb-33.05Mb

chr6:48Mb-48.05Mb

chr6:66Mb-66.05Mb

chr6: 81Mb-81.03Mb

…

Chromatin tracing of chromosome spreads

Use this methodology to look at chromothripsis events/ecDNA formation

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Collaborators

Don Cleveland

Roy Maimon

Carlos Marinas

Quan Zhu

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Acknowledgments

Frank Furnari

Brett Taylor

Prasad Trivedi

Ideally we would connect DNA organization with the activity status of the genes within these structures and with some the key landmarks inside the nucleus that help organize the DNA such as nuclear speckles concentrate active chromatin for performing RNA splicing or the nuceloli and nuclear lamina which interact with inactive chromatin.

For this purpose I combined multiplexed imaging of DNA (previously described) with an analogous method for measuring multiplexed imaging of nascent RNA and with protein imaging of components of nuclear landmarks.

Thus within the same single cells we can measure both the chromatin organization for >1,000 regions together with the activity of the >1,000 genes that reside in these regions (here each colored sphere is a gene that is bursting) and the key nuclear landmarks.

This has a lot of potential for basic cell biology asking questions like

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Questions

Spatial genomics

Su,...,Bintu*,Zhuang* Cell 2020

Inducing neurogenesis in aged mammalian brains

Collaboration with Don Cleveland

Imaging ecDNA

Collaboration with Frank Furnari Don Cleveland and Bing Ren

Olfactory epithelium(nose)

Olfactory bulb (brain)

Receptor

RNA

Epithelium

Glomeruli

RNA MERFISH

DNA MERFISH

EGFR

ecDNA

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