Understanding embryonic head development. ANAT2341 Tennille Sibbritt Embryology Unit Children s Medical Research Institute

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1 Understanding embryonic head development ANAT2341 Tennille Sibbritt Embryology Unit Children s Medical Research Institute

2 Head malformations among the most common category of congenital malformations in the neonate population Thought to originate from errors of early development caused by mutations that impact on gene function Genetic causes of many cases are still unknown Lack of knowledge has hampered the ability to provide a clinical diagnosis of the affected individuals. Need to identify potential malformation-causing genes.

3 Mouse embryonic development

4 Stages of mouse embryonic development Fertilisation Gestation period of a mouse is ~20 days 20 days: Birth 6 days: Gastrulation

5 Gastrulation Early phase in embryonic development Formation of the primary germ layers from which all tissues of the embryos will be generated Necessary for correct placement of precursor tissues for subsequent morphogenesis Following implantation, mouse embryos become structurally distinct from non-mammalian and even other mammalian embryos (e.g. humans) Tang et al., Nat. Rev. Gen., 2016

6 Mouse gastrulation After implantation, mouse embryo develops into a highly polarised cylindrical structure: egg cylinder (proximal-distal axis) proximal Column of 3 tissues: Arkell & Tam, Open Biol., Extraembryonic ectoderm (proximal): derived from trophectoderm - Epiblast (distal): gives rise to the entire embryo distal - Visceral endoderm: Envelopes the above two structures and derived from the primitive endoderm

7 Formation of the primitive streak proximal Transient embryonic structure Formation occurs at E6.25-E6.5 Arkell & Tam, Open Biol., 2012 distal Involves movement and rearrangement of epiblast cells (as well as high signalling activity à more later) Epiblast cells undergo epithelial-to-mesenchymal transition (EMT) à lose cell-cell adhesion

8 Canonical WNT signalling Primitive streak and WNT signalling Primitive streak determines the anterior-posterior axis of embryo à crucial for development of the body plan! WNT signalling plays a major role in this process Causes accumulation of β-catenin in the cytoplasm that translocates to the nucleus à acts as a transcriptional coactivator of transcription factors of the TCF/LEF family These transcription factors drive lineage specification, germ layer formation and cell/tissue movement Fossat et al., Development, 2011 High WNT in the primitive streak and surrounding tissues à concentration gradient of high WNT in posterior and low WNT in anterior

9 Mouse craniofacial development The head is comprised of all three primary germ layers: - Ectoderm: brain, neural crest, surface ectoderm (skin) - Mesoderm: cranial mesochyme and heart - Endoderm: foregut Head progenitors expressed in anterior region of embryo Arkell & Tam, Open Biol., 2012

10 Mouse craniofacial development Arkell & Tam, Open Biol., 2012 Determined by a collection of molecular regulators (including transcription factors) that interact with one another to govern gene expression (gene regulatory networks) Suppression of WNT signalling activity in the anterior region required for normal head development: - WNT signalling agonists expressed in the posterior while antagonists expressed in the anterior - Head progenitor tissues spatially coincide with the anterior region

11 Using knockouts to determine gene function

12 Global knockout of a gene To knockout a specific gene, a gene targeting approach can be used Use a targeting vector containing an antibiotic resistance gene to target gene of interest on one allele Genomic DNA Gene A Introduce vector into embryonic stem cells (ESCs) à derived from ICM of a blastocyst and can generate any type of cell

13 Global knockout of a gene Through a process known as homologous recombination, the vector will target the gene and the DNA swapped between the vector and one allele of the gene Introduces the antibiotic resistance gene- allows selection of cells that have been targeted by growing them in antibiotics Genomic DNA Gene A Homologous recombination Foreign DNA

14 Global knockout of a gene This generates cells that are heterozygous mutants Genomic DNA - Genomic DNA + Foreign DNA

15 Antibiotic selection and confirmation of genotype of ESCs Global knockout of a gene +/+ +/+ Inject cells into the blastocyst of a mouse embryo Blastocyst +/- +/- Chimeric Blastocyst

16 Global knockout of a gene +/+ Chimera +/- +/+ +/- -/- +/- +/+ Gene is completely knocked out

17 Conditional knockout using Cre-LoxP This system can be used to knock out genes in a specific cell type or tissue, or specific time Useful if a global knockout of the gene is lethal Cre recombinase: derived from P1 bacteriophage that s responsible for site-specific recombination LoxP site: 34bp, containing 8bp asymmetric sequence and two 13bp palindromic sequences that are recognised by Cre 13bp 8bp 13bp ATAACTTCGTATA - NNNTANNN -TATACGAAGTTAT

18 Conditional knockout using Cre-LoxP Cre binds to each palindromic sequence of LoxP, forming a dimer, and this dimer binds to another dimer on another LoxP site, forming a tetramer Double stranded DNA is cut at both LoxP sites and the strand rejoined by DNA ligase Cre Otx2 Otx2 Otx2 Cre

19 Tissue specification of knockout How can we specify Cre recombinase expression in a particular tissue type? Can place the Cre recombinase gene downstream of the promoter of a gene expressed in a specific tissue Promoters are located near the transcription start site and are bound by RNA polymerase to drive transcription Promoter Cre LoxP Cre Gene Cre

20 Specifying timing of a knockout Tamoxifen-induced Cre recombinase (CreER T2 ) Fusion between Cre recombinase and the estrogen receptor Estrogen Receptor CreER T2 Cre

21 Time specification of a knockout This fusion in the absence of tamoxifen renders the Cre recombinase inactive Inactivity is a result of it being in complex with a protein called HSP90 Keeps Cre in the cytoplasm where it is unable to gain access to the gene of interest CreER T2 HSP90 Cytoplasm Nucleus

22 Time specification of a knockout The complex between CreER T2 and Hsp90 is disrupted in the presence of tamoxifen Allows CreER T2 to translocate to the nucleus where it can catalyse LoxP-specific recombination events like I described before. Tamoxifen CreER T2 Hsp90 Cytoplasm Nucleus

23 Understanding head development

24 LHX1: a regulator of head development LIM domain homeobox transcription factor that regulates the expression of its target genes à generally an activator Interacts with LDB1 (via LIM domains) and forms a complex with SSBP3 to regulate transcription of genes (e.g. WNT signalling antagonists) Globally knocking out Lhx1 in mice results in a head truncation phenotype Lhx1 -/- Lhx1 +/+ Shawlot et al., Nature 1995

25 LHX1: a regulator of head development LIM domain homeobox transcription factor that regulates the expression of its target genes à generally an activator Interacts with LDB1 (via LIM domains) and forms a complex with SSBP3 to regulate transcription of genes (e.g. WNT signalling antagonists) Globally knocking out Lhx1 in mice results in a head truncation phenotype Lhx1 -/- Lhx1 +/+ LHX1 is involved in head development. How? Shawlot et al., Nature 1995

26 Where is Lhx1 expressed? In situ hybridisation (ISH): Uses labelled complementary RNA to determine position of RNA in tissue/embryo At E7.25, Lhx1 expression is in the primitive streak/epiblast and anterior visceral endoderm (AVE) Fossat et al., Development, 2015

27 Where is Lhx1 expressed? In situ hybridisation (ISH): Uses labelled complementary RNA to determine position of RNA in tissue/embryo At E7.25, Lhx1 expression is in the primitive streak/epiblast and anterior visceral endoderm (AVE) By E7.75, expression is in the anterior mesendoderm (AME) Fossat et al., Development, 2015

28 Knocking out Lhx1 specifically in the epiblast Conditional knockout of Lhx1 in the epiblast (remember, epiblast gives rise to 3 primary germ layers) By E7.75, complete ablation of Lhx1 By E9.5, no cranium Fossat et al., Development, 2015

29 Knocking out Lhx1 specifically in the epiblast Conditional knockout of Lhx1 in the epiblast (remember, epiblast gives rise to 3 primary germ layers) By E7.75, complete ablation of Lhx1 By E9.5, no cranium How does knocking out Lhx1 in the epiblast affect head development? Fossat et al., Development, 2015

30 Loss of head tissue precursors in Lhx1epiCKO ISH with markers of different regions of the head (E8.5 embryos) Dorsal and ventral forebrain Midbrain Hindbrain Fossat et al., Development, 2015 Mid-hindbrain junction

31 Loss of head tissue precursors in Lhx1epiCKO ISH with markers of different regions of the head (E8.5 embryos) Dorsal and ventral forebrain Midbrain Hindbrain Fossat et al., Development, 2015 Mid-hindbrain junction

32 Loss of head tissue precursors in Lhx1epiCKO ISH with markers of different regions of the head (E8.5 embryos) Dorsal and ventral forebrain Midbrain Hindbrain Fossat et al., Development, 2015 Mid-hindbrain junction Loss of precursors of dorsal and ventral forebrain, midbrain and the mid-hindbrain junction

33 Loss of head tissue precursors in Lhx1epiCKO ISH with markers of different regions of the head (E8.5 embryos) Dorsal and ventral forebrain Midbrain Hindbrain Fossat et al., Development, 2015 Mid-hindbrain junction Loss of precursors Lhx1 is also of expressed dorsal and ventral in the forebrain, AME at E7.75. midbrain and the mid-hindbrain junction How does knocking it out here affect head development?

34 Loss of forebrain tissue in Lhx1ameCKO Conditional KO of Lhx1 in the anterior mesendoderm (AME) Tamoxifen-induced Cre recombinase ISH Fossat et al., Development, 2015

35 Loss of forebrain tissue in Lhx1ameCKO Conditional KO of Lhx1 in the anterior mesendoderm (AME) Tamoxifen-induced Cre recombinase ISH Head truncation not as strong as Lhx1epiCKO. Why? Fossat et al., Development, 2015

36 Loss of forebrain tissue in Lhx1ameCKO Conditional KO of Lhx1 in the anterior mesendoderm (AME) Tamoxifen-induced Cre recombinase Loss of forebrain markers and tissue à essential role for LHX1 in head formation ISH ISH Head truncation not as strong as Lhx1epiCKO. Why? Fossat et al., Development, 2015

37 Loss of forebrain tissue in Lhx1ameCKO Conditional KO of Lhx1 in the anterior mesendoderm (AME) Tamoxifen-induced Cre recombinase Loss of forebrain markers and tissue à essential role for LHX1 in head formation ISH ISH What is causing the loss of head tissue precursors in epiblast and AME knockout embryos? Is it WNT signalling? Head truncation not as strong as Lhx1epiCKO. Why? Fossat et al., Development, 2015

38 Is WNT signalling affected in Lhx1CKO embryos? Similar head phenotype to those embryos with ectopic expression of WNT signalling activity Dkk1 is a WNT antagonist Lewis et al., Development, 2008

39 Decreased expression of WNT antagonists in Lhx1epiCKO ISH on WNT antagonists (remember Lhx1 is expressed anteriorly and the anterior region expresses WNT antagonists) Decreased expression of WNT antagonists in Lhx1epiCKO Fossat et al., Development, 2015

40 Decreased expression of WNT antagonists in Lhx1ameCKO ISH on WNT antagonists (remember Lhx1 is expressed anteriorly and the anterior region expresses WNT antagonists) Quantitative reverse transcription polymerase chain reaction (RT-qPCR) on WNT antagonists RT-qPCR: RNA is transcribed into complementary DNA (cdna). The cdna is amplified and a fluorescent dye is used to intercalate the double stranded cdna, giving a readout of the amount of RNA à gene expression Decreased expression of WNT antagonists in Lhx1ameCKO Fossat et al., Development, 2015

41 Elevated WNT signalling in Lhx1CKO embryos contributes to head phenotype RT-qPCR results on anterior tissue of Lhx1epiCKO and Lhx1ameCKO show increased expression of WNT targets (although not significant for Lhx1ameCKO) What do you make of the Lhx1ameCKO results compared to Lhx1epiCKO results? Axin2 and Lef1 are direct WNT targets Fossat et al., Development, 2015

42 Elevated WNT signalling in Lhx1CKO embryos contributes to head phenotype Examined the phenotypic effect of an increase in WNT signalling in conjunction with reduced Lhx1 activity in compound mutant embryos Compound mutant: two different heterozygous mutant alleles Lhx1 +/-, Dkk1 +/- (WNT antagonist) and β-catenin (Ctnnb1 +/- ) mutants have no phenotype If LHX1 is involved in WNT signalling, then Lhx1 +/- /Dkk1 +/- and Lhx1 +/- /Ctnnb1 +/- compound mutants will have a head phenotype Fossat et al., Development, 2015

43 Elevated WNT signalling in Lhx1CKO embryos contributes to head phenotype Examined the phenotypic effect of an increase in WNT signalling in conjunction with reduced Lhx1 activity in compound mutant embryos Compound mutant: two different heterozygous mutant alleles Lhx1 +/-, Dkk1 +/- (WNT antagonist) and β-catenin (Ctnnb1 +/- ) mutants have no phenotype If LHX1 is involved in WNT signalling, then Lhx1 +/- /Dkk1 +/- and Lhx1 +/- /Ctnnb1 +/- compound mutants will have a head phenotype Fossat et al., Development, 2015

44 Conclusions LHX1 is imperative for head development, resulting in loss of head tissue precursors for forebrain, midbrain and hindbrain formation Loss of Lhx1 results in reduction of WNT antagonists and an increase in WNT signalling activity à regulates the expression of these factors

45 What next? LHX1 is a transcription factor, so it regulates the expression of many genes. What genes does it target? Are these genes involved in head development and/or WNT signalling?

46 Embryology Unit, CMRI (past and present) Patrick Tam Nicolas Fossat Vanessa Jones Jane Sun Joanne Shen Kenny Ip Emilie Wilkie Joshua Studdert Renée Rawson Acknowledgements