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Gene Targeting in Neuroendocrinology

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ABSTRACT

Research in neuroendocrinology faces particular challenges due to the complex interactions between cells in the hypothalamus, in the pituitary gland and in peripheral tissues. Within the hypothalamus alone, attempting to target a specific neuronal cell type can be problematic due to the heterogeneous nature and level of cellular diversity of hypothalamic nuclei. Because of the inherent complexity of the reproductive axis, the use of animal models and in vivo experiments are often a prerequisite in reproductive neuroendocrinology. The advent of targeted genetic modifications, particularly in mice, has opened new avenues of neuroendocrine research. Within this review, we evaluate various mouse models used in reproductive neuroendocrinology and discuss the different approaches to generate genetically modified mice, along with their inherent advantages and disadvantages. We also discuss a variety of versatile genetic tools with a focus on their potential use in reproductive neuroendocrinology. © 2015 American Physiological Society. Compr Physiol 5:1645‐1676, 2015.

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Figure 1. Figure 1. Schematic model of the hypothalamic pituitary gonadal (hpg) axis and various factors that affect its activation and function. Reprinted, with permission, from (242).
Figure 2. Figure 2. Approaches to generate mouse models. (A) For conventional and BAC transgenic mice, fertilized eggs are collected and matured, the vector is microinjected into the male pronucleus where random integration occurs and the embryos are then transferred into a pseudopregnant female mouse. (B) Gene targeting involves the electroporation of the construct into mouse embryonic stem (ES) cells, with correct insertion (homologous recombination) ensured by positive and negative selection. Gene‐targeted ES cells are then injected into the blastocyst and transferred into a pseudopregnant female. Chimeric mice with the insert present in the germline are backcrossed to generate heterozygous mutant mice. Adapted, with permission, from (69).
Figure 3. Figure 3. Genome editing works on the principle of creating a targeted break in the DNA. With zinc finger nucleases (ZFNs) and transcription activator‐like effector nucleases (TALENs) DNA binding is mediated by protein‐DNA interactions. In the clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas9 system, DNA binding is mediated by RNA/DNA hybridization. Adapted, with permission, from (158).
Figure 4. Figure 4. The effect of Cre‐mediated recombination differs depending on the position and orientation of the loxP sites. (A) When loxP sites are in the same orientation, the loxP‐flanked sequence is excised (with the forward reaction favored). (B) When the loxP sites are in opposing directions the loxP‐flanked sequence is reversibly inversed. (C) When individual loxP sites are located on nonhomologous chromosomes in the same orientation, chromosomal rearrangements occur. Adapted, with permission, from (28).
Figure 5. Figure 5. Schematic of the double‐floxed inverted open reading frame (DIO) strategy to attain expression of a transgene exclusively in Cre‐expressing cells. Two pairs of incompatible loxP sites flank the inverted ORF. Upon completion of Cre‐mediated recombinated, two incompatible loxP sites remain resulting in a one‐way reaction and the ORF being in the sense orientation. Adapted, with permission, from (40).
Figure 6. Figure 6. Comparison of IRES and 2A. (A) Two knockin mouse lines were generated, one with an IRES‐EGFP cassette downstream of the Sox9 stop codon (Sox9IE), the other with a self‐cleavable 2A peptide‐EGFP cassette downstream of the Sox9 stop codon (Sox9FE). (B) As can be seen, EGFP expression is appreciably weaker when downstream of IRES compared to 2A. (C) Using 2A instead of IRES resulted in the production of significant amounts of Sox9‐EGFP fusion protein. Adapted, with permission, from (44).
Figure 7. Figure 7. Control of gene expression using the Tet‐On and Tet‐Off systems. (A) A tissue‐specific promoter (TSP) drives expression of tTA (Tet‐Off system) that, in the absence of doxycycline, facilitates transcription. Upon binding doxycycline, tTA dissociates from the Tet‐O sequences resulting in transcriptional repression. (B) By expressing rtTA under the control of a tissue specific promoter (Tet‐ON), the presence of doxycycline facilitates binding to the Tet‐O sequences, thus activating gene expression. Adapted, with permission, from (157).
Figure 8. Figure 8. (A) The rtTA system can be used in combination with the Cre recombinase system to attain temporal control over gene expression. (B) Inducible Cre systems consist of fusion proteins of Cre and the ligand‐binding domain of a steroid hormone receptor. In the absence of the inducer, Cre is sequestered to the cytoplasm by association with heat shock proteins (HSP). Upon binding the inducer, Cre dissociates from the HSPs, facilitating nuclear translocation and thus recombination. Adapted, with permission, from (157).
Figure 9. Figure 9. Schematic of the split‐Cre system. Two distinct promoter elements drive the expression of either NCre or CCre. In cells where both promoter elements are sufficiently active, NCre and CCre will be coexpressed and form a dimer, facilitating Cre‐mediated recombination. Adapted, with permission, from (118).
Figure 10. Figure 10. (A) Restriction map and targeting strategy of the R26 locus. The reporter construct consists of a splice acceptor (SA) sequence, a neomycin (neo) cassette followed by the lacZ gene and a bovine polyadenylation signal (bpA). The neo cassette is flanked by loxP sites, with multiple polyadenylation signal at the 3' end, to facilitate Cre‐dependent neo cassette removal and lacZ expression. The resulting construct was inserted in the unique XbaI site of a plasmid vector (pROSA‐1) containing a 5 kb subcloned genomic fragment of the original locus together with a diphtheria toxin A expression cassette for negative selection (pROSA26‐R). The unique XbaI site is 300 bp 5 distant from the original ROSA26 β‐geo strain insertion site. (B) Generalized LacZ expression after Cre‐mediated recombination in mouse embryos. R26‐R heterozygous (left) or R26‐Cre/R26‐R embryos stained with X‐gal. Adapted, with permission, from (246).
Figure 11. Figure 11. Endogenous τGFP expression in gonadotropes. (A) Three‐dimensional reconstruction (20 μm z‐stack) of the caudal area of the postpubertal female pituitary gland prepared from a 14‐week‐old GRIC/eR26‐τGFP mouse at diestrus after perfusion with rhodamine‐coupled gelatin (to paint blood vessels). The arrows mark gonadotrope protrusions extending in the direction of blood vessels (red). (B) A three‐dimensional surface rendering (Imaris) of (A) for better visualization of blood vessels. Scale bars, 15 μm. Adapted, with permission, from (7).
Figure 12. Figure 12. Genetically encoded transneuronal tracing. (A) Genetic strategy to express a retrograde tracer (GFP‐TTC), bidirectional tracer (BL) and a stationary marker (τlacZ) exclusively in kisspeptin neurons. (B) Some, but not all, GnRH neurons in the medial septum (MS), preoptic area (POA), horizontal diagonal band of Broca (HDB) and organum vasculosum of lamina terminalis (OVLT) contain the tracer. Adapted, with permission, from (145).
Figure 13. Figure 13. GCaMP3 imaging in gonadotropes. GCaMP3 expressing cells (green) before (A) and after (B) 10 nmol/L GnRH application. (C) Traces of fluorescence change in two individual cells (red, yellow) marked in (A) and (B) are shown. The time point and duration of 10 nmol/L GnRH application is indicated. (Götz and Boehm, unpublished data.)
Figure 14. Figure 14. (A) Kiss‐IRES‐Cre/ERαlox/lox (KERKO) female mice exhibit premature vaginal opening (VO) and (B) higher serum luteinizing hormone (LH) levels in juvenile (P15) and peripubertal (P25) periods compared to wild type. (C) Cyclicity is impaired in KERKO female mice with long persistent cornified or leucocytic stages. C, cornified (estrus); N, nucleated (proestrus), L, leucocytic (metestrus and diestrus). (D) Proposed model for the control of puberty by ERα signaling in kisspeptin neurons. In the prepubertal period, ERα signaling in arcuate nucleus kisspeptin neurons mediates a brake on GnRH release, while a successive increase of ERα signaling in the anteroventral periventricular nucleus kisspeptin neurons mediates puberty progression, by increasing GnRH release. Adapted, with permission, from (173).
Figure 15. Figure 15. Selective chronic ablation of gonadotropin‐releasing hormone receptor (GnRHR)+ cells in the double knockin GRIC/R26‐DTA mouse line. (A) GRIC/R26‐DTA mice are obtained by crossing the GnRHR‐IRES‐Cre (GRIC) mice with R26‐DTA mice. Coexpression of Cre in the presence of the R26‐STOP‐DTA background mediates the Cre‐dependent removal of the loxP‐flanked STOP cassette allowing DTA expression and subsequent death of GnRHR‐expressing cells. (B) GRIC/R26‐DTA 12‐week‐old mice show hypogonadism compared to control R26‐DTA littermates. Male (top) and female (bottom) gonads are drastically smaller in GRIC/R26‐DTA (left) compared to control R26‐DTA littermates (right). (C) Proposed model of embryonic GnRH signaling‐dependent gonadotrope development in male mice. Increased GnRH release from axon terminals at the median eminence triggers an increase in LH secretion around E16 (I). At this stage, only LH+ cells express GnRHR (green) and are GnRH‐responsive. The embryonic increase in LH secretion promotes proper development of FSH+ gonadotropes in the anterior pituitary. FSH+ gonadotropes coexpress TSH+ and LHR, suggesting a direct LH effect on FSH+ gonadotrope development (II). Between E17 and P7 (III), FSH+ gonadotropes upregulate FSHβ and lose TSHβ expression and start to express GnRHR (green). Adapted, with permission, from (284).
Figure 16. Figure 16. Ablation efficiency comparison between systemic and intracerebroventricular DT injections in POMC‐IRES‐Cre/R26‐DTR/R26‐LacZ mice. (A) Hypothalamic POMC+ neurons visualized by β‐gal staining in POMC/R26‐LacZ intracerebroventricular‐treated controls (left), and both IP‐treated (central) and ICV‐treated (right) POMC/R26‐DTR/R26‐LacZ, 15 days after DT treatment. (B) Bilateral hypothalamic POMC+ cell counting. (C) Relative pituitary POMC mRNA levels (%). Notably, pituitary POMC mRNA expression is unaffected following icv injection but reduced by 72% in systemic treatment. POMC: pro‐opiomelanocortin. Adapted, with permission, from (222).
Figure 17. Figure 17. Overview of the main classes of optogenetic tools. (A) Light‐gated opsins are naturally found in microbial organisms. ChR2, an excitatory cation‐conducting light‐gated channel was identified in the unicellular algae Chlamydomonas reinhardtii (left) and responds to blue light. VChR1, a cation‐conducting channelrhodopsin from the algae Volvox carteri (center), elicits large depolarizing photocurrents at red‐shifted wavelengths. Halorhodopsin (right) identified in haloarchea Natronomonas pharaonis (NpHR) elicit inward hyperpolarizing Cl photocurrents in response to yellow light. (B) Chimeric opsin‐GPCRs, called “optoXRs” were developed to achieve optical control of G‐coupled intracellular signaling. Adapted, with permission, from (299).
Figure 18. Figure 18. (A) Structural alignment between bacteriorhodopsin (BR) and channelrhodopsin‐2 (ChR2) based on homology modeling. (B) ChR2 photocycle model [details in (22,286)]. (C) Photocurrents elicited by single pulses of blue light, showing kinetics over time. tinact: inactivation time, intrinsic switching to the closed state during stimulation. tdeact: deactivation time. trec: recovery time in the dark pulses. (D) Retinal‐binding pocket of BR showing the most relevant residues in proton transfer and the ChR2 residues critical for gating and cation transfer, based on sequence homology with BR. The opsins light‐sensing ability is due to the formation of a covalent complex with the all‐trans‐retinal rhodopsin. A noncovalent interaction between a conserved lysine within the 7TM structure and the all trans‐retinal constitutes a retinal Schiff base (RSB) critical for signal transduction. In type I opsins, photon absorption by the retinal molecule causes its isomerization from all trans‐retinal to 13 cis‐retinal. Following isomerization, the RSB undergoes dipole shift triggering conformational changes overall the 7TM structure, allowing gating and ions conductance. The residues lining the retinal‐binding pocket strongly influence the ionic environment and the biochemical properties of the RSB, defining the identity of any single channel (in terms of ions conductance, gating kinetics, light sensitivity, and wavelength tuning). Adapted, with permission, from (300).


Figure 1. Schematic model of the hypothalamic pituitary gonadal (hpg) axis and various factors that affect its activation and function. Reprinted, with permission, from (242).


Figure 2. Approaches to generate mouse models. (A) For conventional and BAC transgenic mice, fertilized eggs are collected and matured, the vector is microinjected into the male pronucleus where random integration occurs and the embryos are then transferred into a pseudopregnant female mouse. (B) Gene targeting involves the electroporation of the construct into mouse embryonic stem (ES) cells, with correct insertion (homologous recombination) ensured by positive and negative selection. Gene‐targeted ES cells are then injected into the blastocyst and transferred into a pseudopregnant female. Chimeric mice with the insert present in the germline are backcrossed to generate heterozygous mutant mice. Adapted, with permission, from (69).


Figure 3. Genome editing works on the principle of creating a targeted break in the DNA. With zinc finger nucleases (ZFNs) and transcription activator‐like effector nucleases (TALENs) DNA binding is mediated by protein‐DNA interactions. In the clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas9 system, DNA binding is mediated by RNA/DNA hybridization. Adapted, with permission, from (158).


Figure 4. The effect of Cre‐mediated recombination differs depending on the position and orientation of the loxP sites. (A) When loxP sites are in the same orientation, the loxP‐flanked sequence is excised (with the forward reaction favored). (B) When the loxP sites are in opposing directions the loxP‐flanked sequence is reversibly inversed. (C) When individual loxP sites are located on nonhomologous chromosomes in the same orientation, chromosomal rearrangements occur. Adapted, with permission, from (28).


Figure 5. Schematic of the double‐floxed inverted open reading frame (DIO) strategy to attain expression of a transgene exclusively in Cre‐expressing cells. Two pairs of incompatible loxP sites flank the inverted ORF. Upon completion of Cre‐mediated recombinated, two incompatible loxP sites remain resulting in a one‐way reaction and the ORF being in the sense orientation. Adapted, with permission, from (40).


Figure 6. Comparison of IRES and 2A. (A) Two knockin mouse lines were generated, one with an IRES‐EGFP cassette downstream of the Sox9 stop codon (Sox9IE), the other with a self‐cleavable 2A peptide‐EGFP cassette downstream of the Sox9 stop codon (Sox9FE). (B) As can be seen, EGFP expression is appreciably weaker when downstream of IRES compared to 2A. (C) Using 2A instead of IRES resulted in the production of significant amounts of Sox9‐EGFP fusion protein. Adapted, with permission, from (44).


Figure 7. Control of gene expression using the Tet‐On and Tet‐Off systems. (A) A tissue‐specific promoter (TSP) drives expression of tTA (Tet‐Off system) that, in the absence of doxycycline, facilitates transcription. Upon binding doxycycline, tTA dissociates from the Tet‐O sequences resulting in transcriptional repression. (B) By expressing rtTA under the control of a tissue specific promoter (Tet‐ON), the presence of doxycycline facilitates binding to the Tet‐O sequences, thus activating gene expression. Adapted, with permission, from (157).


Figure 8. (A) The rtTA system can be used in combination with the Cre recombinase system to attain temporal control over gene expression. (B) Inducible Cre systems consist of fusion proteins of Cre and the ligand‐binding domain of a steroid hormone receptor. In the absence of the inducer, Cre is sequestered to the cytoplasm by association with heat shock proteins (HSP). Upon binding the inducer, Cre dissociates from the HSPs, facilitating nuclear translocation and thus recombination. Adapted, with permission, from (157).


Figure 9. Schematic of the split‐Cre system. Two distinct promoter elements drive the expression of either NCre or CCre. In cells where both promoter elements are sufficiently active, NCre and CCre will be coexpressed and form a dimer, facilitating Cre‐mediated recombination. Adapted, with permission, from (118).


Figure 10. (A) Restriction map and targeting strategy of the R26 locus. The reporter construct consists of a splice acceptor (SA) sequence, a neomycin (neo) cassette followed by the lacZ gene and a bovine polyadenylation signal (bpA). The neo cassette is flanked by loxP sites, with multiple polyadenylation signal at the 3' end, to facilitate Cre‐dependent neo cassette removal and lacZ expression. The resulting construct was inserted in the unique XbaI site of a plasmid vector (pROSA‐1) containing a 5 kb subcloned genomic fragment of the original locus together with a diphtheria toxin A expression cassette for negative selection (pROSA26‐R). The unique XbaI site is 300 bp 5 distant from the original ROSA26 β‐geo strain insertion site. (B) Generalized LacZ expression after Cre‐mediated recombination in mouse embryos. R26‐R heterozygous (left) or R26‐Cre/R26‐R embryos stained with X‐gal. Adapted, with permission, from (246).


Figure 11. Endogenous τGFP expression in gonadotropes. (A) Three‐dimensional reconstruction (20 μm z‐stack) of the caudal area of the postpubertal female pituitary gland prepared from a 14‐week‐old GRIC/eR26‐τGFP mouse at diestrus after perfusion with rhodamine‐coupled gelatin (to paint blood vessels). The arrows mark gonadotrope protrusions extending in the direction of blood vessels (red). (B) A three‐dimensional surface rendering (Imaris) of (A) for better visualization of blood vessels. Scale bars, 15 μm. Adapted, with permission, from (7).


Figure 12. Genetically encoded transneuronal tracing. (A) Genetic strategy to express a retrograde tracer (GFP‐TTC), bidirectional tracer (BL) and a stationary marker (τlacZ) exclusively in kisspeptin neurons. (B) Some, but not all, GnRH neurons in the medial septum (MS), preoptic area (POA), horizontal diagonal band of Broca (HDB) and organum vasculosum of lamina terminalis (OVLT) contain the tracer. Adapted, with permission, from (145).


Figure 13. GCaMP3 imaging in gonadotropes. GCaMP3 expressing cells (green) before (A) and after (B) 10 nmol/L GnRH application. (C) Traces of fluorescence change in two individual cells (red, yellow) marked in (A) and (B) are shown. The time point and duration of 10 nmol/L GnRH application is indicated. (Götz and Boehm, unpublished data.)


Figure 14. (A) Kiss‐IRES‐Cre/ERαlox/lox (KERKO) female mice exhibit premature vaginal opening (VO) and (B) higher serum luteinizing hormone (LH) levels in juvenile (P15) and peripubertal (P25) periods compared to wild type. (C) Cyclicity is impaired in KERKO female mice with long persistent cornified or leucocytic stages. C, cornified (estrus); N, nucleated (proestrus), L, leucocytic (metestrus and diestrus). (D) Proposed model for the control of puberty by ERα signaling in kisspeptin neurons. In the prepubertal period, ERα signaling in arcuate nucleus kisspeptin neurons mediates a brake on GnRH release, while a successive increase of ERα signaling in the anteroventral periventricular nucleus kisspeptin neurons mediates puberty progression, by increasing GnRH release. Adapted, with permission, from (173).


Figure 15. Selective chronic ablation of gonadotropin‐releasing hormone receptor (GnRHR)+ cells in the double knockin GRIC/R26‐DTA mouse line. (A) GRIC/R26‐DTA mice are obtained by crossing the GnRHR‐IRES‐Cre (GRIC) mice with R26‐DTA mice. Coexpression of Cre in the presence of the R26‐STOP‐DTA background mediates the Cre‐dependent removal of the loxP‐flanked STOP cassette allowing DTA expression and subsequent death of GnRHR‐expressing cells. (B) GRIC/R26‐DTA 12‐week‐old mice show hypogonadism compared to control R26‐DTA littermates. Male (top) and female (bottom) gonads are drastically smaller in GRIC/R26‐DTA (left) compared to control R26‐DTA littermates (right). (C) Proposed model of embryonic GnRH signaling‐dependent gonadotrope development in male mice. Increased GnRH release from axon terminals at the median eminence triggers an increase in LH secretion around E16 (I). At this stage, only LH+ cells express GnRHR (green) and are GnRH‐responsive. The embryonic increase in LH secretion promotes proper development of FSH+ gonadotropes in the anterior pituitary. FSH+ gonadotropes coexpress TSH+ and LHR, suggesting a direct LH effect on FSH+ gonadotrope development (II). Between E17 and P7 (III), FSH+ gonadotropes upregulate FSHβ and lose TSHβ expression and start to express GnRHR (green). Adapted, with permission, from (284).


Figure 16. Ablation efficiency comparison between systemic and intracerebroventricular DT injections in POMC‐IRES‐Cre/R26‐DTR/R26‐LacZ mice. (A) Hypothalamic POMC+ neurons visualized by β‐gal staining in POMC/R26‐LacZ intracerebroventricular‐treated controls (left), and both IP‐treated (central) and ICV‐treated (right) POMC/R26‐DTR/R26‐LacZ, 15 days after DT treatment. (B) Bilateral hypothalamic POMC+ cell counting. (C) Relative pituitary POMC mRNA levels (%). Notably, pituitary POMC mRNA expression is unaffected following icv injection but reduced by 72% in systemic treatment. POMC: pro‐opiomelanocortin. Adapted, with permission, from (222).


Figure 17. Overview of the main classes of optogenetic tools. (A) Light‐gated opsins are naturally found in microbial organisms. ChR2, an excitatory cation‐conducting light‐gated channel was identified in the unicellular algae Chlamydomonas reinhardtii (left) and responds to blue light. VChR1, a cation‐conducting channelrhodopsin from the algae Volvox carteri (center), elicits large depolarizing photocurrents at red‐shifted wavelengths. Halorhodopsin (right) identified in haloarchea Natronomonas pharaonis (NpHR) elicit inward hyperpolarizing Cl photocurrents in response to yellow light. (B) Chimeric opsin‐GPCRs, called “optoXRs” were developed to achieve optical control of G‐coupled intracellular signaling. Adapted, with permission, from (299).


Figure 18. (A) Structural alignment between bacteriorhodopsin (BR) and channelrhodopsin‐2 (ChR2) based on homology modeling. (B) ChR2 photocycle model [details in (22,286)]. (C) Photocurrents elicited by single pulses of blue light, showing kinetics over time. tinact: inactivation time, intrinsic switching to the closed state during stimulation. tdeact: deactivation time. trec: recovery time in the dark pulses. (D) Retinal‐binding pocket of BR showing the most relevant residues in proton transfer and the ChR2 residues critical for gating and cation transfer, based on sequence homology with BR. The opsins light‐sensing ability is due to the formation of a covalent complex with the all‐trans‐retinal rhodopsin. A noncovalent interaction between a conserved lysine within the 7TM structure and the all trans‐retinal constitutes a retinal Schiff base (RSB) critical for signal transduction. In type I opsins, photon absorption by the retinal molecule causes its isomerization from all trans‐retinal to 13 cis‐retinal. Following isomerization, the RSB undergoes dipole shift triggering conformational changes overall the 7TM structure, allowing gating and ions conductance. The residues lining the retinal‐binding pocket strongly influence the ionic environment and the biochemical properties of the RSB, defining the identity of any single channel (in terms of ions conductance, gating kinetics, light sensitivity, and wavelength tuning). Adapted, with permission, from (300).
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Michael Candlish, Roberto De Angelis, Viktoria Götz, Ulrich Boehm. Gene Targeting in Neuroendocrinology. Compr Physiol 2015, 5: 1645-1676. doi: 10.1002/cphy.c140079