The Role of the Indigenous Microbiota in Zebrafish Gastrointestinal Tract Development. Karen Guillemin University of Oregon

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1 The Role of the Indigenous Microbiota in Zebrafish Gastrointestinal Tract Development Karen Guillemin University of Oregon

2 human cells GI tract bacteria cells 25,000 genes ~500 species 1,000,000 genes

3 How is the microbiota established during development? What role does the microbiota play in host development? How does the host perceive and respond to the presence of the microbiota?

4 Advantages of a germ-free zebrafish model Ex-utero development facilitates sterilization of embryos Transparency of larvae allows visualization of internal organs and bacterial interactions Zebrafish GI tract development and physiology is similar to that of mammals Zebrafish have an adaptive and innate immune system similar to mammals Zebrafish are amenable to genetic analysis

5 Anatomy of a zebrafish larva esophageal-intestinal junction intestinal bulb distal intestine vent 8 dpf DAPI

6 Important events in zebrafish development macrophages functional lymphocytes in thymus first V(D)J recombination thymus mature onset of humoral immunity 21 28

7 Bacteria on the zebrafish chorion 10 µm

8 Bacterial distribution during development DAPI Eubacteria 35 hpf 4 dpf 5 dpf 8 dpf

9 Bacteria in the adult gut lumen are sampled by host cells Aeromonas veronii 16S probe Flavobacterium johnsoniae 16S probe

10 Bacterial load during development based on qpcr of 16S rdna E E E+08 Copies 16S rdna/fish E E E E E E E E Adult 12 GI Age (days post fertilization)

11 8 dpf 4 dpf The microbiota changes during development Eubacteria Aeromonas Eubacteria Pseudomonas

12 Important events in zebrafish development macrophages functional lymphocytes in thymus first V(D)J recombination establishment of microbiota ` thymus mature onset of humoral immunity 21 28

13 Raising germfree zebrafish larvae bleach treat newly laid embryos larvae grown in sterile media CV d0 CV d8 CV d8 GF d8 - control - control + control Assay for sterility by: plating PCR with pan bacterial 16S primers microscopy negative control positive bleached untreated control

14 SEM of the surface of untreated and bleached embryos 10 µm Conventionally reared 10 µm Bleach treated

15 Quantification of bacterial 16S rdna S rdna copy number water GF CV

16 Reconstitution of the microbiota in germ-free zebrafish larvae bleach treat newly laid embryos grow in sterile media Day 5 Day 8 transfer to media from conventionally reared fish ex-germ-free larvae

17 Quantification of bacterial 16S rdna S rdna copy number water GF CV XGF

18 Germ-free and ex-germ-free zebrafish CV GF XGF Eubacteria autofluorescence

19 The germ-free intestine fails to up-regulate alkaline phosphatase activity CV 2 dpf CV 3 dpf CV 5 dpf CV 8 dpf GF 8 dpf XGF 8 dpf Alkaline phosphatase activity

20 Quantification of intestinal alkaline phosphatase activity 1.6 ng Pi/min/mg tissue CV 5dpf CV 8dpf GF 8dpf XGF 8dpf

21 The germ-free intestine fails to down-regulation Galα1,3Gal expression CV 5 dpf CV 8 dpf GF 5 dpf GF 8 dpf XGF 8 dpf distal proximal Isolectin B-4 autofluorescence

22 Quantification of Galα1,3Gal expression at the esophogeal-intestinal junction 700 IB4 staining (arbitrary units) CV 5dpf CV 8dpf GF 8dpf XGF 8dpf

23 The germ-free intestine contains fewer goblet cells CV 5 dpf CV 8 dpf GF 5 dpf XGF 8 dpf

24 Quantification of goblet cells 250 goblet cells/intestine CV 5 dpf CV 8 dpf GF 8 dpf XGF 8 dpf

25 The germ-free intestine fails to absorb protein CV GF XGF HRP protein after 24 hour feeding

26 Regular peristalsis is established by 6 dpf 4.5 dpf 5.5 dpf 4.5 dpf 5.5 dpf 6 dpf Julie Kuhlman Judith Eisen

27 Peristalsis is faster in germ-free larvae GF CV XGF Average time between contractions

28 Monoassociation of germfree zebrafish larvae bleach treat newly laid embryos grow in sterile media Day 5 Day 8 Introduce bacterial culture into flask with a sterile needle Monoassociated larvae

29 Monoassociations with Aeromonas sobria and Pseudomonas putida AMA Eubacteria PMA Aeromonas Merge Eubacteria Psedomonas Merge

30 Monoassociation with Aeromonas and Pseudomonas spp. reverses GF phenotypes 1.6 Alkaline phosphatase activity 700 Galα1,3Gal expresison ng Pi/min/mg tissue IB4 staining (arbitrary units) CV 8dpf GF 8dpfXGF XGF 0 8dpfAMA AMA 8dpfPMA PMA 8dp CV CV 8dpfGF GF 8dpfXGF XGF 8dpf AMA AMA 8dpf PMA PMA 8d

31 Heat-killed bacteria are not sufficient to reverse all GF phenotypes 1.6 Alkaline phosphatase activity 700 Galα1,3Gal expresison ng Pi/min/mg tissue IB4 staining (arbitrary units) CV GF XGF AMA PMA HK CV 8dpfGF 8dpfXGF 8dpf AMA 8dpf PMA 8dpf HK 8dpf 0 CV CV 8dpf GF GF 8dpf XGF XGF 8dpf AMA AMA 8dpf PMA PMA 8dpf HK HK 8dp

32 LPS is sufficient to induce intestinal alkaline phosphatase activity 1.6 Alkaline phosphatase activity 700 Galα1,3Gal expresison ng Pi/min/mg tissue IB4 staining (arbitrary units) CV CV8dpf GF GF 8dpf XGF XGF 8dpf AMAPMA AMA 8dpf PMA 8dpf HK HK 8dpfLPS LPS CV CV 8dpf GF GF 8dpf XGF XGF 8dpf AMA AMA 8dpf PMA PMA 8dpf HK HK 8dp

33 The GI microbiota is established after hatching The microbiota is required for the maturation of patterns of expression of glycans and intestinal alkaline phosphatase activity The microbiota is required for normal goblet cell numbers The microbiota is required for protein absorption in the distal intestine and rates of peristalsis Heat-killed bacteria reverse the GF alkaline phosphatase phenotype but not the expression of Gala1,3Gal glycans.

34 Toll like receptors recognize generic microbial patterns Akira (2003) JBC 278:

35 TLR signaling is required to protect against intestinal injury Susceptibility to dextran sulfate sodium Rakoff-Nahoum et al. (2004) Cell 118:229-41

36 Microbial patterns are necessary and sufficient to protect against intestinal injury Antibiotic depletion of microbiota Addition of microbial molecules (LPS, LTA) Rakoff-Nahoum et al. (2004) Cell 118:229-41

37 The mouse intestinal epithelium expresses glycoconjugates in response to the microbiota Hooper et al. (1999) PNAS 96:

39 Erika Mittge Jennifer Bates Sarah Cheesman Katy Baden Julie Kuhlman

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION DOI: 10.1038/ncb3000 Supplementary Figure 1 Lgr5 expression in the Lgr5-LacZ reporter knock-in mouse. P1: Lgr5-LacZ expression on the ovary surface (black arrow) and sub-surface (arrowheads), as well as