Expression Analysis and Lineage Tracing in <i>Relb-Cre-Katushka</i> Reporter Mice

Expression Analysis and Lineage Tracing of RelB in Relb-Cre-Katushka Reporter Mice

Authors

Christian Engelmann¹, Marc Riemann¹, Swen Hartfiel¹, Randy Grimlowski¹, Nico Andreas¹, Ievgen Koliesnik¹, Elke Meier¹, Phillip Austerfield¹, Konrad Lehmann², Jürgen Bolz², Falk Weih^1,‡, Ronny Haenold^1,3,#

1 Leibniz Institute on Aging − Fritz-Lipmann-Institute (FLI), Beutenbergstrasse 11, 07745 Jena, Germany

2 Institute for General Zoology and Animal Physiology − Friedrich Schiller University Jena, Erbertstrasse 1, 07743 Jena, Germany

3 Department of Genetics, Friedrich Schiller University Jena, Philosophenweg 12, 07743 Jena, Germany

^‡ Deceased

^# Corresponding author: Ronny Haenold, Leibniz Institute on Aging − Fritz-Lipmann-Institute (FLI), Beutenbergstrasse 11, 07745 Jena, Germany; Phone: +49-3641-656022; Fax: +49-3641-656335; E-mail: ronny.haenold@leibniz-fli.de

Abstract

Noncanonical NF-κB signaling by activation of the transcription factor RelB acts as key regulator of cell lineage determination and differentiation in various tissues including the immune system. In contrast to constitutive presence of canonical NF-κB components, RelA and p50, synthesis of RelB is tightly controlled and activation of the noncanonical pathway requires preceding transcriptional upregulation of Relb. Thus, knowledge on cellular expression patterns of Relb provides crucial insights into the commitment of noncanonical NF-κB signaling in organ development and disease. To elucidate temporal and spatial aspects of Relb expression, we generated a BAC transgenic knock-in mouse expressing the far-red fluorescent protein Katushka and the enzyme Cre recombinase under control of the murine Relb promoter (Relb^Cre-Kat mice). Cellular co-expression of Katushka and RelB protein in stimulated fibroblast cultures and tissues of transgenic mice revealed highly specific reporter functions of the transgene. Moreover, crossing Relb^Cre-Kat mice with ROSA26R reporter mice that allow for consecutive β-galactosidase or YFP expression upon Cre-mediated recombination of LacZ or YFP reporter genes revealed temporarily restricted Relb expression patterns and proved highly useful for cell tracing of Relb in developing and mature mice. Here, highly specific expression patterns were found, besides thymus and spleen, in neural tissue (brain, spinal cord, spinal ganglia, retina), as well as other non-immune organs including skin, bone and kidney. To assess the role of RelB in the nervous system, we generated mice with neuro-ectodermal Relb deletion (Relb^CNS-KO mice). Relb^CNS-KO mice developed normal but acquired impairments in visual acuity in later life, indicating a so far unknown role of RelB for neuronal functions. In summary, our results demonstrate the usability of Relb^Cre-Kat reporter mice for the detection of de novo and temporarily restricted Relb expression. Moreover, Relb lineage tracing in non-immune organs suggests, beyond immunity, important functions of this transcription factor in development and tissue maturation.

This image gallery is a collection of histological serial sections from a 2-days-old Relb^Cre-Kat;ROSA26R (LacZ) reporter mouse to depict tissue-specific and cellular Relb expression patterns in the whole organism.

Methods

Transgenic mice

To generate high-fidelity Relb^Cre-Kat reporter mice, a bacterial artificial chromosome (BAC) of 93 kb (bMQ-258k08) containing the complete Relb locus with 45 kb of upstream region was modified by introducing the gene coding for a fusion protein of Cre recombinase and Katushka, both linked by a self-cleaving P2A peptide (Kim, Lee et al. 2011), into the first exon of the Relb gene just downstream of the translation initiation codon (Relb–Cre–P2A–Katushka). The vector was injected into pro-nuclei from fertilized C57BL/6J oocytes and three founder mice were obtained, which transmitted the transgene to the progeny. In one of the three founder lines transgene expression satisfactorily reflected that of endogenous Relb (termed Relb^Cre-Kat mice). Transgenic mice were generated by PolyGene (Rümlang, Switzerland). For cell tracing of Relb expression in developing and mature organs Relb^Cre-Kat mice were crossed with ROSA26-LacZ mice (Soriano 1999) that allow for continuous expression of beta-galactosidase after Cre-mediated excision of a loxP-flanked STOP sequence (suppl. Fig. 1). Double-transgenic mice heterozygous for Relb^Cre-Kat and ROSA26-LacZ transgenes (termed Relb^Cre-Kat;LacZ mice), and single transgenic controls were used for analysis of beta-galactosidase activity.

Suppl. Figure 1: Scheme of reporter protein expression in double-transgenic Relb^Cre-Kat;Rosa26R reporter mice.
In Relb^Cre-Kat reporter mice, expression of the Cre-P2A-Katushka transgene is driven by the murine Relb promoter.
Inducible expression of Cre recombinase leads to constitutive expression
of β-galactosidase (detected by X-gal staining) in Relb^Cre-Kat;LacZ double transgenic mice.

Immunohistochemistry

For whole body sections of postnatal mice (P2 or P7), animals were sacrificed and snap-frozen in methyl-butane. Mice were embedded in Tissue Tec for preparation of serial sagittal cryo-sections with a slice thickness of 16 µm at a distance of approximately 100 µm on a cryostat-microtome (Leica CM3000; Wetzlar, Germany) (suppl. Fig. 2). Tissue sections were postfixed for 5 min in 4% buffered paraformaldehyde (PFA, pH 7.4), rinsed in PBS and incubated in X-gal staining solution (Roche, Mannheim, Germany) for 24 hrs at 37 ̊C. Section were counterstained with eosin to depict anatomical structures. For anatomical orientation, sections 1, 8, 17, and 35 were solely stained with H&E. Sections were scanned under transmission light using Olympus Virtual Slide microscope at 20x magnification (numerical aperture [NA] 0.75) or 40x (NA 0.9) magnification. Focus plane and stitching of the tiles were set automatically. Scanned tiles were aligned to obtain the final images.

Suppl. Figure 2: Preparation of whole body cryo-sections from postnatal mice (shown for P7).
(A) Sacrificed animals were snap-frozen and immediately mounted on a cryostat-microtome.
Mice were embedded in layers with Tissue Tec and 16 µm cryo-sections were prepared using
a standard microtome razor blade (B). Quality of sections was validated by H&E staining (C).

Images

Please select an image

Section 1
Hematoxylin & Eosin
Cranial
Caudal
Elbow joint
Paw
Section 2
X-Gal & Eosin
Elbow joint
Paw
Striated muscle
Section 3
X-Gal & Eosin
Paw
Skin
Elbow joint
Section 4
X-Gal & Eosin
Earcup
Shoulder
Paw
Elbow joint
Section 5
X-Gal & Eosin
Earcup
Shoulder
Paw
Elbow joint
Costal arch
Costal arch
Section 6
X-Gal & Eosin
Inner ear
Brain
Cranial cartilage
Shoulder
Paw
Hand bone
Eye
Lung
Liver
Section 7
X-Gal & Eosin
Liver
Lung
Brain
Eye
Hind leg
Inner ear
Shoulder
Paw
Hand bone
Section 8
Hematoxylin & Eosin
Liver
Lung
Skin
Stomach
Brain (Cerebrum)
Retina
Eye
Striated muscle
Ear
Shoulder
Costal arch
Paw
Section 9
X-Gal & Eosin
Liver
Lung
Stomach
Instestine
Brain
Eye
Striated muscle
Ear
Shoulder joint
Section 10
X-Gal & Eosin
Liver
Lung
Stomach
Instestine
Brain
Eye
Striated muscle
Shoulder joint
Section 11
X-Gal & Eosin
Liver
Lung
Stomach
Intestine
Brain
Eye
Cartilage
Heart
Section 12
X-Gal & Eosin
Liver
Submaxillary gland
Lung
Stomach
Instestine
Brain
Eye
Retina
Vibrisses
Cartilage
Heart
Section 13
X-Gal & Eosin
Liver
Submaxillary gland
Lung
Stomach
Instestine
Brain
Eye
Vibrisses
Heart
Section 14
X-Gal & Eosin
Liver
Submaxillary gland
Lung
Stomach
Instestine
Brain
Eye
Hind leg
Vibrisses
Heart
Section 15
X-Gal & Eosin
Stomach
Heart
Submaxillary Gland
Lung
Liver
Instestine
Brain
Eye
Hind leg
Vibrisses
Section 16
X-Gal & Eosin
Backbone
Stomach
Heart
Submaxillary Gland
Lung
Liver
Kidney
Instestine
Brain
Paw
Eye
Mouth
Muscle
Brown fat tissue
Ear
Section 17
Hematoxylin & Eosin
Backbone
Stomach
Heart
Submaxillary Gland
Lung
Liver
Kidney
Instestine
Brain
Paw
Eye
Mouth
Muscle
Brown fat tissue
Ear
Section 18
X-Gal & Eosin
Backbone
Stomach
Heart
Submaxillary Gland
Lung
Liver
Kidney
Instestine
Brain
Paw
Eye
Mouth
Muscle
Brown fat tissue
Ear
Section 19
X-Gal & Eosin
Backbone
Stomach
Heart
Submaxillary Gland
Lung
Liver
Kidney
Instestine
Brain
Paw
Eye
Mouth
Muscle
Brown fat tissue
Ear
Section 20
X-Gal & Eosin
Backbone
Stomach
Thymus
Heart
Submaxillary Gland
Lung
Liver
Kidney
Instestine
Brain
Paw
Eye
Mouth
Muscle
Brown fat tissue
Ear
Section 21
X-Gal & Eosin
DRGs
Stomach
Thymus
Heart
Submaxillary Gland
Lung
Liver
Kidney
Instestine
Brain
Paw
Eye
Mouth
Muscle
Brown fat tissue
Ear
Section 22
X-Gal & Eosin
DRGs
Stomach
Thymus
Heart
Submaxillary Gland
Lung
Liver
Kidney
Instestine
Brain
Paw
Mouth
Muscle
Brown fat tissue
Ear
Section 23
X-Gal & Eosin
DRGs
Stomach
Thymus
Heart
Submaxillary Gland
Lung
Liver
Kidney
Instestine
Brain
Paw
Mouth
Muscle
Brown fat tissue
Ear
Section 24
X-Gal & Eosin
Spinal cord
DRGs
Stomach
Thymus
Heart
Submaxillary Gland
Lung
Liver
Kidney
Instestine
Bladder
Brain
Paw
Mouth
Muscle
Brown fat tissue
Section 25
X-Gal & Eosin
Spinal cord
DRGs
Stomach
Thymus
Heart
Submaxillary Gland
Lung
Liver
Kidney
Instestine
Bladder
Brain
Paw
Mouth
Muscle
Brown fat tissue
Section 26
X-Gal & Eosin
Spinal cord
DRGs
Stomach
Thymus
Heart
Submaxillary Gland
Lung
Liver
Kidney
Instestine
Bladder
Brain
Paw
Mouth
Muscle
Brown fat tissue
Section 27
X-Gal & Eosin
Spinal cord
DRGs
Stomach
Thymus
Heart
Submaxillary Gland
Lung
Liver
Kidney
Instestine
Bladder
Brain
Paw
Mouth
Muscle
Brown fat tissue
Section 28
X-Gal & Eosin
Spinal cord
DRGs
Stomach
Thymus
Heart
Submaxillary Gland
Lung
Liver
Kidney
Instestine
Bladder
Brain
Paw
Mouth
Muscle
Brown fat tissue
Section 29
X-Gal & Eosin
Spinal cord
DRGs
Stomach
Thymus
Heart
Submaxillary Gland
Liver
Kidney
Instestine
Bladder
Brain
Paw
Mouth
Muscle
Brown fat tissue
Section 30
X-Gal & Eosin
Spinal cord
DRGs
Stomach
Thymus
Heart
Submaxillary Gland
Liver
Kidney
Instestine
Bladder
Brain
Paw
Mouth
Muscle
Brown fat tissue
Section 31
X-Gal & Eosin
Spinal cord
DRGs
Stomach
Thymus
Heart
Submaxillary Gland
Liver
Kidney
Instestine
Bladder
Brain
Paw
Mouth
Muscle
Brown fat tissue
Section 32
X-Gal & Eosin
Spinal cord
DRGs
Stomach
Thymus
Heart
Submaxillary Gland
Liver
Kidney
Instestine
Bladder
Brain
Paw
Mouth
Muscle
Brown fat tissue
Section 33
X-Gal & Eosin
Spinal cord
DRGs
Stomach
Thymus
Heart
Submaxillary Gland
Liver
Kidney
Instestine
Bladder
Brain
Paw
Mouth
Muscle
Brown fat tissue
Section 34
X-Gal & Eosin
Spinal cord
DRGs
Stomach
Thymus
Heart
Submaxillary Gland
Liver
Kidney
Instestine
Bladder
Brain
Paw
Mouth
Muscle
Brown fat tissue
Section 35
Hematoxylin & Eosin
Spinal cord
DRGs
Stomach
Thymus
Heart
Submaxillary Gland
Liver
Kidney
Instestine
Bladder
Brain
Paw
Mouth
Muscle
Brown fat tissue
Section 36
X-Gal & Eosin
Spinal cord
DRGs
Stomach
Thymus
Heart
Submaxillary Gland
Liver
Kidney
Instestine
Bladder
Brain
Paw
Mouth
Brown fat tissue
Section 37
X-Gal & Eosin
Spinal cord
DRGs
Stomach
Thymus
Heart
Submaxillary Gland
Lung
Liver
Kidney
Instestine
Bladder
Brain
Mouth
Muscle
Brown fat tissue
Section 38
X-Gal & Eosin
Spinal cord
DRGs
Thymus
Heart
Submaxillary Gland
Lung
Liver
Instestine
Bladder
Brain
Mouth
Muscle
Brown fat tissue
Section 39
X-Gal & Eosin
Spinal cord
DRGs
Thymus
Submaxillary Gland
Lung
Liver
Instestine
Bladder
Brain
Mouth
Muscle
Brown fat tissue
Section 40
X-Gal & Eosin
Spinal cord
DRGs
Thymus
Submaxillary Gland
Lung
Liver
Instestine
Bladder
Brain
Mouth
Muscle
Brown fat tissue
Section 41
X-Gal & Eosin
Spinal cord
DRGs
Thymus
Submaxillary Gland
Lung
Liver
Instestine
Bladder
Brain
Mouth
Muscle
Brown fat tissue
Section 42
X-Gal & Eosin
Spinal cord
DRGs
DRGs
Thymus
Submaxillary Gland
Lung
Liver
Instestine
Bladder
Brain
Mouth
Muscle
Brown fat tissue
Section 43
X-Gal & Eosin
Spinal cord
DRGs
DRGs
Submaxillary Gland
Lung
Liver
Instestine
Bladder
Brain
Mouth
Muscle
Brown fat tissue
Section 44
X-Gal & Eosin
Spinal cord
DRGs
DRGs
Submaxillary Gland
Lung
Liver
Instestine
Bladder
Brain
Mouth
Muscle
Brown fat tissue
Section 45
X-Gal & Eosin
Spinal cord
DRGs
DRGs
Submaxillary Gland
Lung
Liver
Instestine
Bladder
Brain
Mouth
Muscle
Brown fat tissue
Section 46
X-Gal & Eosin
Kidney
DRGs
Spinal Cord
Submaxillary Gland
Lung
Liver
Instestine
Bladder
Brain
Backbone
Eye
Brown fat tissue
Shoulder
Section 47
X-Gal & Eosin
DRGs
Submaxillary Gland
Lung
Instestine
Bladder
Brain
Backbone
Eye
Shoulder

Credits

This work is dedicated to Professor Dr. Falk Weih (1959–2014). We thank Maik Baldauf (Leibniz Institute on Aging) for exceptional histological support and Dr. Lucien Frappart (Leibniz Institute on Aging) for histological advice. This work was supported by the Leibniz Association, the German Research Association (DFG grant number WE 2224/6-2), and the VELUX Foundation, Switzerland.