>> HI, EVERYONE.

GREAT SURE TO WELCOME KAREN

GUILLEMIN TO GIVE -- TO THE NIH

TODAY.

SHE WILL PRESENT TODAY THE ROLA

EUGENE LECTURE.

IT WAS FIRST GIVEN IN 1951 BY

GEORGE BEETLE.

IT HONORS DR. DIRE WHO IN

ADDITION TO NIH DIRECTOR

CONDUCTED IN THE FIELD OF

INFECTIOUS DISEASE.

A MAJOR FOCUS LED TO HOW ENDEMIC

TYPE IS SPREAD AND DEVELOPMENT

OF VACCINE TO PROTECT AGAINST

THE DISEASE.

KAREN HAS A DIVERSE SCIENTIFIC

BACKGROUND POSITIONED HER TO

FURTHER OUR UNDERSTANDING OF THE

INTERACTIONS BETWEEN MICROBES

AND THEIR HOSTS IN MODEL

ORGANISMS.

AS A GRADUATE STUDENT AT

STANFORD UNIVERSITY MARK

(INAUDIBLE) LAB SHE STUDIED

DEVELOPMENT OF DROSOPHILA

RESPIRATORY SYSTEM.

SHE LATER STUDIED BACTERIAL

PATHOGENESIS AS A POST DOC IN

STANLEY FALLCO'S LAB IN

STANFORD.

AFTER THAT SHE STARTED HER

INDEPENDENT RESEARCH CAREER AT

THE UNIVERSITY OF OREGON.

SHE MODELED HER INTEREST IN

MICROBES AN BEGAN TO STUDY THE

MICROBIOME OF THE ZEBRAFISH

INTESTINE.

THERE ARE MANY ADVANTAGES TO

THIS APPROACH.

YOU CAN STUDY LARGE NUMBER OF

ANIMAL, YOU CAN VISUALIZE THE

INSIDE OF THE INTESTIN IN LIVE

ANIMALS, YOU CAN CONTROL THE

GENETIC PACK GROUND OF THE

ANIMAL -- BACK GROWN OF THE AB

MALL AND MICROBES THE ANIMAL

COMES IN CONTACT WITH.

THESE ADVANTAGES TO ALLOW KAREN

THE ADDRESS MANY IMPORTANT

QUESTIONS REGARDING THE

INTERACTIONS BETWEEN MICROBES

AND THEIR HOSTS THAT ARE VERY

HARD TO ADDRESS IN HUMANS OR

OTHER VERTEBRATES.

AND THIS IS PARTICULARLY

RELEVANT AS MANY OF YOU KNOW,

THERE WAS COMMON FUND PROJECT

CALLED THE HUMAN MICROBIOME

PROJECT AND THIS LET US TO

REALIZE THERE ARE MANY CASES

DISEASE INDIVIDUALS HAVE

DIFFERENT MICROBIOMES THAN

NON-DISEASED INDIVIDUALS AND ONE

OF THE OBVIOUS QUESTIONS WAS IS

THE DISEASE STATE CAUSING THE

MICROBIOME CHANGES, THE

MICROBIOME CHANGE CAUSING THE

DISEASE STATE?

AND I THINK MODEL ORGANISMS LIKE

THE SYSTEM KAREN WILL TELL US

ABOUT TODAY ARE A GOOD WAY TO

ADDRESS THIS.

AND KAREN HAS RECEIVED MANY

HONORS INCLUDING THE AMERICAN

SOCIETY FOR MICROBIOLOGY MERCK

IRVINE S. SEE GAL MEMORIAL AWARD

AND EARLY CAREER AN INVESTIGATOR

AAWARD SO A PLEASURE TO WELCOME

INSIGHTS FROM THE ZEBRAFISH

INTESTINE.

>> THANK YOU SO MUCH.

IT'S AN HONOR TO BE HERE TODAY

AND ESPECIALLY AN HONOR TO BE

GIVING THIS LECTURE FOR

MICROBIOLOGY.

AND I THINK IT'S ESPECIALLY

EXCITING BECAUSE WE'RE IN SUCH A

EXCITING TIME IN THE HISTORY OF

MICROBIOLOGY SO THINKING TO THE

BIRTH AS A FIELD WAS REALLY

ENABLED BY THE DISCOVERY OF

METHODS TO AMPLIFY AND VISUALIZE

MICROCELLS WITH INNOVATION OF

MICROSCOPY.

AND I THINK ONE OF THE THINGS

THAT WAS INTERESTING ABOUT THAT

MOMENT WHEN (INDISCERNIBLE)

DISCOVERED THE EXISTENCE OF

THESE EMACULES THAT HE WAS ABLE

TO VISUALIZE WITH HIS

MICROSCOPE.

HIS IMMEDIATE 'ACTION WAS --

REACTION WAS TO ASK WHAT ARE

THESE EMACULES ASSOCIATED WITH

MYSELF SO HE LOOKED AT HIS

SALIVA AND TEARS AND ASKED CAN

WE FIND MICROORGANISMS, THESE

CREATURES OF THE MICROSCOPIC

SCALE ASSOCIATED WITH OURSELVES.

I THINK NOW WE'RE AT THIS MOMENT

IN TIME WITH THE ADVENT OF NEXT

GENERATION SEQUENCING THAT'S

GIVING US THIS WHOLE NEW INSIGHT

INTO MICROBES IN OUR WORLD WE'RE

TURNING THIS ATTENTION ON TO

OURSELVES IN AN EFFORT SUCH AS

NIH FUNDED HUMAN MICROBIOME

PROJECT, WE'RE ASKING WHAT ARE

THE MICROBIAL ASSOCIATES THAT

EXIST WITH US.

THERE'S AN EXCITING FLOOD OF

INFORMATION ABOUT THESE COMMUNE

TINTIVES THAT ARE ASSOCIATED

WITH TO THE POINT WHERE THE

HUMAN I THINK IS ONE OF THE MOST

BEST DESCRIBED MICROBIAL

ECOSYSTEM ON OUR PLANET AT THIS

POINT.

IT RAISES A LOT OF FUNDAMENTAL

QUESTIONS ABOUT THESE MICROBIAL

COMMUNITIES.

SO WE LOOK WITHIN OURSELVES HERE

LOOKING INTO A GASTRO INTESTINAL

TRACK AT THESE MICROBES.

AND ASK QUESTIONS ABOUT HOW

THESE COMMUNITIES ASSEMBLE IN

ASSOCIATION WITH HOSTS.

AND WHETHER THESE COMMUNITIES

CHANGE OVER TIME, AND WHAT OTHER

KINDS OF FACTORS, ENVIRONMENTAL,

DIET, HOST GENETICS MIGHT

DETERMINE THE COMPOSITION OF

THESE COMMUNITIES THAT WE

CO-EXIST WITH.

AND ULTIMATELY WHAT DO -- HOW DO

THESE COMMUNITIES AFFECT THE

HOST BIOLOGY.

WHAT I WOULD LIKE TO PUT FORTH

IS THE IMPORTANT OF USING MODEL

ORGANISMS TO ADDRESS THESE

QUESTIONS.

AND SHOWING A FEW EXAMPLES OF

POWERFUL MODEL ORGANISM SYSTEMS

THAT ARE USED FOR STUDYING POST

MICROBIAL INTERACTIONS INCLUDING

A SIMPLE ASSOCIATION BETWEEN A

SIMPLE CONSORTIA OR AN EXAMPLE

OF VERTEBRATES THAT HAVE COMPLEX

MICROBIAL COMMUNITIES AND WE

WOULD LIKE TO UNDERSTAND THIS,

SOMETHING OF VALUE OF THESE

PROPERTIES IN IMMUNITIES.

AND I'LL TELL YOU ABOUT THE

MODEL SYSTEM OF ZEBRAFISH THE

ADDRESS THESE FUP MENTAL

QUESTIONS.

I WOULD LIKE TO FURTHER PUT

FORTH THE IDEA THAT WE NEED TO

THINK ABOUT THESE QUESTIONS OF

HOST MICROBE ASSOCIATIONS IN THE

FRAMEWORK OF SYSTEMS BIOLOGY

WHERE WE'RE THINKING OF SYSTEMS

OF INTERACTING MICROBIAL CELLS,

AND THEN BUILDING ON TO THAT

FRAMEWORK THINKING ABOUT THE

SYSTEMS OF INTERACTIONS BETWEEN

HOSTS AN MICROBIAL CELLS, HOW

THESE TWO INTERACTING SYSTEMS

WORK TOGETHER AND ULTIMATELY

THINKING ABOUT HOW POPULATIONS

OF THESE HOST MICROBES SUPER

ORGANISMS, HOW THEY INTERACTION.

AND HOW THEY EVOLVE THROUGH

TIME, WHAT ARE TYPE OS OF

SELECTIVE PRESSURES THAT

MAINTAIN THESE COMMUNITIES AN

ALLOW THEM TO CHANGE.

SO I'M GOING TO TELL YOU TODAY

ABOUT THREE DIFFERENT KINDS OF

SYSTEMS LEVEL PROPERTIES OF HOST

MICROBE SYSTEMS, WILL BE

THINKING HOW THESE SYSTEMS

ASSEMBLE.

WHAT ARE THE DYNAMICS OVER TIME,

AND AT THE END I'LL TELL YOU

ABOUT OUR INVESTIGATIONS ON HOW

THESE SYSTEMS EVOLVE IN ANIMAL

HISTORY.

AND THE ZEBRAFISH MODEL IS A

REALLY USEFUL ONE FOR DOING THIS

BECAUSE WE CAN MODULATE THE

COMPLEXITY OF THESE SYSTEMS.

WE HAVE METHODS WHERE WE CAN

DERIVE GERM-FREE OR ENTIRELY

STERILE ANIMALS AND THEN WE CAN

USE THAT AS A PLATFORM TO BUILD

UP INCREASINGLY COMPLEX SYSTEMS

FROM MONOASSOCIATED SYSTEMS TO

THE EXTENT WE CAN LOOK AT THE

RECAPITULATION OF AN ENTIRELY

COMPLEX COMMUNITY WITH A NATURAL

INOCULUM.

SO WITH THIS SCALABLE COMPLEXITY

WE CAN REALLY GET INSIGHTS INTO

THE SYSTEMS PROPERTIES OF

ASSEMBLY.

THIS IS ALSO -- THE FISH IS

AMAZING MODEL FOR LOOKING AT

DYNAMICS OF THESE SYSTEMS.

ZEBRAFISH LARVAE ARE TRANSPARENT

SO WHAT YOU'RE LOOKING AT IS

INDIVIDUAL BACTERIAL CELLS

WITHIN A ZEBRAFISH LARVA, THIS

IS AN INDIVIDUAL ZEBRAFISH

IMMUNE CELL AN NEUTROPHIL

HANGING AND LOOKING AT THESE

MICROBES HERE.

WE CAN WATCH THE DYNAMICS OF THE

BEHAVIORS OF THESE INDIVIDUALS

BACTERIAL CELLS, YOU CAN SEE

FROM THESE SWIMMING HERE WE CAN

LOOK AT INTERACTIONS BETWEEN

HOST AN MICROBIAL CELLS,

IMPORTANTLY WE CAN GENETICALLY

MANIPULATE THE HOST AN MICROBES

SO WE CAN DO THINGS LIKE HAVE

THEM EXPRESS FLUORESCENT

PROTEINS TO VISUALIZE THEM.

THEN AT THE END OF THE TALK

WE'LL TALK HOW WE'RE USING

METHODOLOGIES, THOSE NOTABLE

METHODOLOGIES AND TRANSPORTING

THOSE TO OTHER FISH SYSTEMS

WHERE WE CAN ASK QUESTIONS ABOUT

EVOLUTION OF THESE HOST MICROBE

SYSTEMS SO THAT IN PARTICULAR WE

HAVE BEEN FOCUSING ON THE

ECOLOGICAL AN EVOLUTIONARY MODEL

FISH, THE THREE SPINE SICKLE

BACK WHICH HAS DIVERSIFIED AND

THERE'S A LOT OF REALLY

INTERESTING DATA ON DIFFERENT

POPULATIONS OF THESE FISH AND

WEAR STARTING TO ASK QUESTIONS

ABOUT HOW DIFFERENCES IN THE

GENETICS OF THE HOST VARY WITH

DIFFERENCES IN THE MICROBIOTA.

SO I'M GOING TO START TODAY'S

TALK TALKING ASSEMBLY OF HOST

MICROBE SYSTEMS AND THIS IS WORK

FUNDED THROUGH THE INSTITUTE OF

GENERAL MEDICINE AND IT'S A

COLLABORATIVE EFFORT WITH MY

COLLEAGUE BRENDEN MOHANON AN

ECOLOGIST AT UNIVERSITY OF

OREGON AND MY CO-SPIRTOR IN ALL

THINGS GERM FREE ZEBRAFISH, JOHN

RALES, INVESTIGATOR AT UNC

INVOLVED IN A NUMBER OF TALENTED

GRADUATE STUDENTS, IN PARTICULAR

I WANT TO HIGHLIGHT ZACK

STEVENS, A DRIVING FORCE BEHIND

A LOT OF WHAT I'M GOING TO TALK

ABOUT TODAY, BOTH AN EXCEPTIONAL

EMPERIMENTALLIST AND BECOMING A

TALENTED BIOINFLOORTITION.

SO -- INFORMATICIAN.

WHEN WE STARTED THIS PROJECT CE

WE STARTED THINKING ASSEMBLY OF

THE ZEBRAFISH MICROBIOTA, HOW DO

COMMUNITIES ASSEMBLE IN THE

MODEL SYSTEM?

WE WANTED TO THINK ABOUT HOW

THESE COMMUNITIES ASSEMBLING THE

INITIAL ASSOCIATION OF THE HOST,

MICROBES THAT OCCURS AT HATCHING

WHEN THE STERILE EMBRYO ENTERS

INTO A MICROBIAL WORLD AND

STARTED TO THINK ABOUT DIFFERENT

EVENTS IN THE DEVELOPMENTAL

HISTORY OF THESE ANIMALS THAT

MIGHT INFLUENCE THE MICROBIAL

COMMUNITIES WHICH THEY'RE

ASSOCIATED.

SO WE DECIDED TO DO A

LONGITUDINAL SURVEY ACROSS

ZEBRAFISH DEVELOPMENT, THIS IS

OUR LITTLE ZMP PROAPOLOGETIC

WHERE WE'RE LOOKING AT

POPULATIONS OF FISH, WE HAVE

REPLICATE POPULATIONS.

AND WE DECIDED TO SAMPLE

DIFFERENT TIME POINTS AT

DIFFERENT EVENTS.

SO FOR EXAMPLE FOUR DAYS WHEN

THE FISH ARE FIRST -- THE ENTIRE

GUT INTESTINAL TRACK IS OPEN TO

THE ENVIRONMENT IS FIRST

COLONIZED, AT TEN DAYS AFTER

THEY HAVE BEEN FEEDING, AND THEN

AROUND THE PERIOD WHERE THE FISH

ELABORATED ADAPTIVE IMMUNE

RESPONSE.

EARLY IN THE DEVELOPMENT OF

THESE FISH, THEY'RE RELYING ON

INNATE IMMUNITY TO CONFER IMMUNE

PROTECTION.

BUT AT AROUND FOUR WEEKS OF AGE

IS WHEN THE FUNCTIONAL ADAPTIVE

IMMUNE RESPONSE, TIME POINTS

BRACKET THAT AND WE LOOK AT A

TIME POINT AFTER WHICH THEY

REACH SEXUAL MATURITY AND WE

SAMPLE THE WATER, THE SIZE AND

BOTTOM OF THE TANK, FOOD

SOURCES, THEY PARENTS, AND WE'RE

JUST NOW ANALYZING ALL THIS

DATA, THIS WAS DATA GENERATED

USING ALUMINA SEQUENCING, A VERY

RICH DATA SET BUT WHAT I WANTED

TO SHOW, TELL YOU ONE STORY

THAT'S EMERGING FROM THIS IS A

STORY ABOUT HOW MICROBIAL

COMMUNITIES ARE CHANGING

DRAMATICALLY OVER DEVELOPMENTAL

TIME.

IN THIS MODEL SYSTEM.

SO I'M GOING TO SHOW YOU THIS

USING REFERENCE MUTATION OF

PRINCIPAL COMPONENT ANALYSIS

WHERE EVERY DATA SAMPLE IS

PLACED ON THIS SLOT BETWEEN TWO

PRINCIPLE COMPONENTS THAT

REPRESENT THE MAXIMAL SPACE OF

VARIATION OF DIFFERENT MICROBIAL

COMMUNITIES.

SO THIS FIRST POINT HERE

REPRESENTS THE PARENT, THE ADULT

PARENTS THAT GAVE RISE TO IT,

ALL THE PROGENY USED FOR THIS

STUDY.

AND THEN UP HERE YOU SEE THE

FOUR DAYTIME POINTS THAT ARE

CLUSTERED AT ONE POSITION SPHAIS

AND THEN AS WE GO THROUGH TIME,

WE START TO SEE THAT THESE

COMMUNITIES ARE SHIFTING IN A

RATHER STEREOTYPED MANNER.

AND SO NOW 28 DAYS THIS IS A

POINT WHERE IT'S NOW A

FUNCTIONAL ADAPTIVE IMMUNE

SYSTEM AND IF WE LOOK AT FEW

DAYS AFTER THAT WE SEE AGAIN

MORE A SHIFT AND IF WE LOOK AT

ADULTS IN A VERY DIFFERENT PLACE

CLUSTERING WITH THOSE PARENTS.

SO THERE'S A DRAMATIC CHANGE IN

THE COMMUNITY STRUCTURE WHICH IS

REPRESENTED WHEN WE LOOK AT

DIFFERENT PHYLA THAT ARE PRESENT

IN THESE FISH AT DIFFERENT TIME

POINTS.

WHAT'S STRIEBING TO US IS WE

KNEW QUITE A BIT ABOUT THE

LARVAL STAGES DOMINANTED BY

GAMMA BACTERIA, WE ALSO KNEW

ABOUT THE ADILL ZEBRAFISH BUT

WHAT WE WERE STRUCK BY HOW

DIDN'T MIC AND DIFFERENT THESE

CHANGES ARE THROUGH THEIR LIFE

HISTORY, WE REALLY DIDN'T KNOW

THERE WAS OTHER BACTERIAL GROUPS

ONE SEES AT DEVELOPMENTAL TIME

POINTS.

SOMETHING THEY'RE PLANNING FOR

US TO THINK ABOUT IS HOW

ASSOCIATIONS WITH YOUR MICROBES

ARE CHANGING WITH DEVELOPMENTAL

HISTORY.

THIS IS LOOKING AT THE NATURAL

COMPLEXITY OF THE FISH, AND ONE

QUESTION THAT WE HAD IS, IS THAT

-- THE DIFFERENCE BETWEEN

INDIVIDUAL, IS THAT DIVERGE OVER

TIME, AND ONE HYPOTHESIS WE HAVE

IS THAT WITH ACQUISITION OF

ADAPTIVE IMMUNITY YOUR PREVIOUS

HISTORY OF EXPOSURE TO MICROBES

ARE INFLUENCING YOUR

IMMUNOLOGICAL STATE, WOULD THAT

INCREASE YOUR INDIVIDUALALTY OF

YOUR MICROBIOTA.

WE HAVE EVIDENCE TO SUGGEST

THAT.

SO THIS IS LOOKING AT HOW

DIFFERENT THE INDIVIDUAL SAMPLES

ARE FROM EACH OTHER BY ONE

METRIC, ONE WAY TO DESCRIBE

THIS, WOULD BE THE BETA

DIVERSITY OF SAMPLES SO IF WE

LOOK AT THE ADULT FISH AS 75

DAYS THEY'RE MUCH MORE

DIFFERENCE THAN EACH OTHER THAN

THESE FISH AT FOUR DAYS.

SO THAT WOULD SUGGEST THERE IS

SOMETHING AS INDIVIDUALS GO ON

THEIR LIFE TRAJECTORY, THEY'RE

ASSEMBLING MORE INDIVIDUAL

COMMUNITIES.

WE ALSO IN THE STUDY WERE ABLE

TO SAMPLE ASPECTS OF THE

IMMUNOLOGY OF THESE INDIVIDUALS,

SO ONE MEASURE WE LOOKED AT THE

AMOUNT OF SECRETED

IMMUNOGLOBULIN TRANSCRIPTS FROM

EACH INDIVIDUAL IN THE STUDY AND

I I'M SHOWING YOU THAT DATA FOR

THESE DIFFERENT TIME POINTS, AN

THESE ARE COLOR CODED FOR THE

DIFFERENCE TANKS OF THE FISHCH

WHAT WAS STRIEBING TO US IS HOW

MUCH VARIATION ONE SEES BETWEEN

INDIVIDUALS IN THE LEVELS OF

EXPRESSED SECRETING

IMMUNOGLOBULIN TRANSCRIPT.

AND THERE WERE TRENDS CERTAIN

TANK LIKES THIS TANK, THIS GREEN

TANK HERE LOOKED LIKE LEVELS IN

GENERAL MUCH LOWER THAN SOME OF

THE OTHER TANKS BUT WE ALSO SEE

AS ONE SEES IN THIS TANK A GREAT

AMOUNT OF INTERINDIVIDUAL

VARIATION WITHIN A TANK.

SO WE'RE LOOKING HOW THESE KINDS

OF MEASUREMENTS OF DIVERSITY IN

THE IMMUNE RESPONSIVENESS OF THE

HOST MIGHT CORRELATE WITH

DIVERSITY IN THE ASSOCIATED

MICROBES.

AND I THINK THESE KIND OF DATA

ARE GOING TO TELL US A LOT ABOUT

SOME OF THE IMMUNOLOGICAL

INFLUENCES ON COMMUNITY

MEMBERSHIP.

SO WE'RE ALSO INTERESTED IN

LOOKING AT ASSEMBLY IN VERY

SUFFER L SYSTEM M SO NOW I'M

GOING TO TALK TO YOU ABOUT WHAT

WE HAVE BEEN DOING LOOKING AT

EARLY EVENTS OF FISH

COLONIZATION LOOK AT SIMPLE

SYSTEM WHERE IS WE'RE

MONOASSOCIATING FISH.

ONE THING WE WANT TO DO IS

ESTABLISH THE RATE OF

COLONIZATION, IF WE INOCULATE

NOVEL GERM FREE FISH WITH A

DOMINANT MEMBER OF THE STRAIN WE

WORK WITH, WE'RE ABLE TO SEE

CHARACTERISTIC TRENDS WHERE 24

HOURS AFTER COLONIZATION YOU

HAVE A MAXIMAL CAPACITY OF 10 TO

THE 4TH COLONY FORMING UNITS IN

THE SPECTRUM PER GUT.

NOTE THAT AT 6 1/2 HOURS POST

INNOCULATION YOU'RE NOT AT THAT

FULL CAPACITY.

SO THEY'RE STILL IS ROOM TO

COLONIZE.

AND THEN MY STUDENT JENNIFER

HAMPTON DID AN INTERESTING

EXPERIMENT WHERE SHE JUST DID A

TIME SERIES OF INNOCULATION WITH

TWO IDENTICAL STRAINS OF THIS

AIR MOW NOWS THAT WERE

DIFFERENTIALLY MARKED.

WHAT SHE FOUND WHEN SHE

PERFORMED THE EXPERIMENT WAS THE

EXPERIMENT WAS TO INOCULATE FISH

AND WAIT SIX HOURS, INTRODUCE

ANOTHER DIFFERENTIALLY MARKED

STRAIN AND THEN WAIT AND LOOK AT

24 HOURS LATER.

AND LOOK AT THE SUCCESS OF THE

SECOND STRAIN IN COLONIZING AND

WHAT SHE FOUND WHICH WAS

REMARKABLE TO US AT A 5 AND 7

DAY PERIOD, THE SECOND STRAIN

WAS LARGELY EXCLUDED AFTER THE 6

HOUR TIME PERIOD.

THOSE THESE ARE IDENTICAL

STRAINS BUT JUST DIFFERENTIALLY

MARKED.

THIS IS SIX MOWR TIME PONT WE

HAD TO ESTABLISH WAS NOT -- THE

GUT IS NOT AT FULL CARRYING

CAPACITY.

SO WE WANTED TO LOOK AT THAT

FURTHER AND LOOK AT REALLY THE

DYNAMICS OF COLONIZATION.

SO NOW I'M GOING TO INTRODUCE

YOU TO MY COLLEAGUE

(INDISCERNIBLE) WHO HAS BEEN

COLLABORATING WITH US ON A

APPROACH TO IMAGING DOING

ZEBRAFISH USING A TECHNOLOGY

CALLED LIGHT SHEET MYCRO

CROSSSCOPY WHICH IS A SIMPLE

STRATEGY BUT REQUIRE AS CUSTOM

MICROSCOPE TO PERFORM.

AND WHAT IT DOES IS INVOLVES

ILLUMINATEING A SAMPLE

ASSESSMENT WITH A SHEET OF LIGHT

AND IMAGING PERPENDICULAR TO

THAT LIGHT.

THIS HAS A LOT OF ADVANTAGES

IMAGE FOR LONG PERIODS OF TIME

ON TISSUES.

THIS IS ALSO GREAT COLLABORATION

WITH TWO PHYSICS GRADUATE

STUDENTS, MIKE AND MATT

J,REMITA.

SO THE FIRST EMPERIMENT WE DID F

WE INOCULATED GERM FREE

ZEBRAFISH WITH DIFFERENTIALLY

MARKED POPULATIONS, NOW WE'RE

MARKING WITH DIFFERENT

FLUROPHORES AN IMAGED OVER 24

HOURS, WE WANTED TO LOOK AT

COLONIZATION LOOKS LIKE AND WE

END UP WITH INTESTINES THAT ARE

COME RECOGNIZED BY RED AND GREEN

-- COLONIZED BY E REDND GREEN

CELLS BY EQUAL PROPORTIONS AND

WE CAN LOOK AT THE DISTRIBUTIONS

OF POPULATIONS OF O MICROBIAL

CELLS ALONG THE LENGTH OF THE

GU. EACH OF THESE IMAGING

EXPERIMENTS INVOLVES 300

GIGABYTES OF INFORMATION.

SO THIS IS A VAST AMOUNT OF

INFORMATION THAT WE'RE

GENERATING SO WE'RE THINKING OF

WAYS TO PROCESS IT.

THIS IS A WAY WE USE THE ACCESS

OF THE GUT AS ONE ACCESS AND WE

COLLAPSE DOWN THE FLUORESCENCE

INTENSITY INFORMATION ALONG THAT

ACCESS AND LOOK ACROSS TIME AND

YOU CAN START TO GET THESE

IMPRESSIONS HOW THERE'S QUITE A

LOT OF DYNAMICS OF COLONIZATION

AND HOW NON-UNIFORM IT IS THAT

WE SEE THESE DIFFERENT

DISTRIBUTIONS OF MICROBIAL

CELLS.

THEN WE PERFORM SOME EXPERMITS

DOING SERIAL INNOCULATIONS SO

THIS IS AN EXPERIMENT TO

INOCULATE FIRST WITH ONE STRAIN

AND A SECOND STRAIN THREE HOURS

LATER.

AND WHAT WE FOUND HERE WAS THAT

WE DID SEE SOME OF THE GREEN

CELLS BUT THERE WAS ACTUALLY A

MORE PREDOCUMENT INNOCENCE OF

RED CELLS AT THIS POINT --

PREPREDOMINANCE.

IF WE REPLICATED SIX HOURS AFTER

THE SECOND INOCULUM WE SAW AGAIN

AS WE OBSERVED IN THE PLATING

DATA WE REALLY -- MOSTLY THERE'S

THE GREEN CELLS ARE PRESENT AND

NOT THE RED.

AND WE HAVE REVERSED THE TWO

COLORS.

SO WE'RE FINING THERE'S AN

INTERESTING„7a EXCLUSION OF

SUBSEQUENT MEMBERS COLONIZING SO

THERE'S REAL STRONG PRIORITY

EFFECTS AS TO WHO COLONIZES

FIRST.

EVEN WHEN TALKING TWO IDENTICAL

STRAINS.

THAT FOR US RAISES THE

POSSIBILITY THAT MAYBE THERE ARE

WAYS IN WHICH THE ACTUAL

COLONIZATION TO THE HOST IS

CHANGING THE ENVIRONMENT TO MAKE

IT MORE RESTRICTIVE ENVIRONMENT

SUBSEQUENT COLONIZATION.

ONE OF THE THINGS WE'RE EXCITED

TO DO IS LOOK AT IN REAL TIME

DURING COLONIZATION AT INNATE

IMMUNE RESPONSES TO THE

COLONIZATION EVENT.

SO THIS IS SOME IMAGES WE HAVE

TAKEN FROM A TRANSGENIC

ZEBRAFISH LINE FOR THE REPORTER

INNATE IMMUNE PATHWAY THE NF

KAPPA B SIGNALING PATHWAY

DEVELOPED WHICH OUR COLLEAGUE

JOHN RALES AND WE'RE ABLE TO SEE

AT A CELLULAR RESOLUTION WHICH

ARE ACTIVATING INNATE IMMUNE

SIGNALING AND CAREFULLY OVER

TIME WE CAN FOLLOW CERTAIN

CHANGES OF INTENSITY OF THE

SIGNALING IN CERTAIN CELLS.

SO WE'RE EXCITED TO SEE WHETHER

THERE'S CORRELATION OF INCREASE

ACTIVITY INNATE IMMUNE SIGNALING

IN THIS PERIOD TO RESTRICTION OF

COLONIZATION DYNAMICS.

WE'RE INTERESTED IN ACTIVITIES

OF DIFFERENT IMMUNE CELLSCH THIS

IS A TRANSGENIC ZEBRAFISH LINE

WHERE YOU CAN VISUALIZE THE

NEUTROPHILS ALONG THE INTESTIN.

THIS IS THE INTESTINE.

THE NEUTROPHILS EXPRESS GREEN TO

US.

SO YOU CAN SEE THE ACTIVE

NEUTROPHILS EXPLORING THE

INTESTINAL ENVIRONMENT AND WE

IMAGINE THE NEUTROPHILS ARE

CREEPING ALONG, EXTENDING

PROCESSES DOING SO IN RESPONSE

TO MICROBIAL CONSTITUENTS AND WE

LOOK AT THOSE INTERACTIONS

BETWEEN HOST IMMUNE CELLS AND

RESIDENT MICROBIAL CELLS.

WE'RE ALSO INTERESTED IN

THINKING DYNAMICS NOT JUST

SIMPLE COLONIZATION BUT WE THINK

ABOUT MORE COMPLEX ASSEMBLED

COMMUNITIES HOW ARE THOSE

COMMUNITIES INTERACTING WITH THE

HOST AND HOW DOES THAT BALANCE

BETWEEN THE HOST AND THE

MICROBIAL COMMUNITY GET

ESTABLISHED HERE ARE TWO SHORT

VIGNETTES OF INSIGHTS THAT WE

HAVE GAINED FROM LOOKING,

COMPARING THE COLONIZED STATE OF

THE ZEBRAFISH WITH GERM FREE

ANIMALS.

AND LEARNING SOMETHING ABOUT THE

DIALOGUES THAT GO ON BETWEEN THE

MICROBIAL AN HOST CELLS.

SO THE FIRST ONE, THIS IS ONE OF

OUR -- FIRST FINDINGS WE MADE

WHEN LOOKING CAREFULLY AT GERM

FREE ZEBRAFISH, AND ASKING

WHETHER THERE WERE ANY KINDS OF

DEVELOPMENTAL, DIFFERENCES OR

MATURATION DIFFERENCES IN THE

INTESTINE, AND WHAT WE FOUND IS

THAT IN GERM FREE ANIMALS

COMPARED TO CONVENTIONALLY

REARED ANIMAL THERE'S A PAUCITY

OF THIS BRUSH BORDER ENZYME

ALKALINE PHOSPHOTASE THAT WE CAN

VISUALIZE WITH THE STAINING HERE

SHOWN IN BLUE.

AND WE COULD RESTORE ALKALINE

PHOSPHOTASE BACK IF WE ADD BACK

INOCULUM FROM THE CONVENTIONAL

CONTROLS.

THAT'S WHAT WE CALL THE X GERM

FREE ANIMALS.

WE COULD QUANTIFY THAT ENZYMATIC

ACTIVITY NICELY, SO HERE YOU CAN

SEE AGAIN THE GERM FREE ANIMALS

HAVE LESS ENZYMEATIC ACTIVITY

THAN CONVENTIONAL ANIMALS AND

THEN ASK WHAT KINDS OF SIGNALS

ARE FR THE MICROBES ARE INDUCING

THIS ACTIVITY.

FIRST WE SHOWED THAT WE CAN

RESTORE ACTIVITY WITH A COMPLEX

MICROBIAL COMMUNITY OR IF WE USE

THE SINGLE GRAM NEGATIVE BACK

BACTERIUM BUT NOT GRAM POSITIVE

BACTERIUM.

SO THEN WE TESTED A MAYOR

COMPONENT OF THE BACTERIAL CELL

WALL OF GRAM NEGATIVE CELLS WITH

A POLYSACCHARIDE AND FOUND THAT

SUFFICIENT TO INDUCE ACTIVITY IN

GERM FREE ANIMALS AND INCREASE

ACTIVITY WHEN ADDED BACK TO

CONVENTIONAL ANIMALS.

SO WE WHEN TO SAY SOMETHING

ABOUT THE REGULATION OF ALKALINE

PHOSPHOTASE ACTIVITY BY LPS BUT

WE'RE PUZZLED TO THINK WHY THIS

BACTERIAL CELL WALL COMPONENT

MIGHT BE INCREASING ALKALINE

PHOSPHOTASE IN THE INTESTIN

DURING COLONIZATION.

WHAT WE HAVE SEEN THAT WAS

INTERESTING TO FINE IN THE

LITERATURE, LPS CAN ACTUALLY BE

A SUBSTRATE FOR ALKALINE

PHOSPHOTASES AND THESE ENZYMES

REMOVE THE PHOSPHATES ON THE

LIPID A MOIETY OF LPS.

THIS IS THE ENDOTOXIC PORTION OF

THE MOLECULE THAT IS WHAT CAUSES

ENDOTOXIC SHOCK IF YOU EXPOSE AN

ANIMAL TO LARGE QUANTITIES OF

LPS.

SO WE WONDERED WHETHER ALKALINE

PHOSPHOTASE IN THE INTESTINES

MIGHT BE FUNCTIONING6:– TO DETOXIFY

LEVELS OF LPS PRESENT BECAUSE OF

THE MICROBIOTA.

SO WE WERE ABLE TO ESTABLISH LPS

IS ENDOTOXIC ACTIVITY IF WE ADD

TO THE WATER, WE CAN SEE A

SPECIFIC KILLING CURVE.

FURTHERMORE IF WE MAYBE

ZEBRAFISH MODIFIED SO THEY LACK

FOS TOTASE ACTIVITY NOW THEY'RE

HYPERSENSITIVE TO LPS KILLING.

FOR THE GERM FREE FISH WHICH

HAVE ALKALINE PHOSPHOTASE

ACTIVITY.

SO ALKALINE PHOSPHOTASE HAS AN

IMPORTANT ROLE IN PROTECTING

AGAINST EXOGENOUSLY AD LPS BUT

WE WERE CURIOUS WHETHER THIS

ENZYME MIGHT HAVE A FUNCTION IN

MODULATING RESPONSES TO LPS

ASSOCIATED WITH THE MICROBIOTA.

FOR THAT WE NEED A MORE SUBTLE

PHENOTYPE AND WE LOOK AT

RECRUITMENT OF NEUTROPHIL INTO

THE INTESTINE AS A MARKER FOR

INTESTINAL INFLAMMATION.

WECIAL VISUALIZE THESE CELLS,

WITH A HISTOLOGICAL STAIN FOR

MYELOID PEROXIDASE ACTIVITY,

THESE ARE THESE LITTLE BROWN

CELLS IN THE INTESTINE AND WE

FOUND IN CONVENTIONAL ANIMALS

THERE'S A LOW NUMBER OF THESE

CELLS.

IN CONTRAST IN GERM FREE

ANIMALS, THERE IS COMPLETE

ABSENSE OF THOSE CELLS SO THAT

TOLD US THAT THE STATE, THE

NORMAL HOMEOSTATIC LEVEL OF

NEUTROPHILS IN A CONVENTIONALLY

REARED ANIMAL IS SET BY PRESENCE

OF MICROBIOTA.

WE COULD INCREASE THE NUMBER OF

NEUTROPHILS THAT ARE PRESENT

EXPOSE THEM TO POLYSACCHARIDE

AND OBSERVE IN FISH THAT WERE

GENETICALLY ENGINEERED TO HAVE

REDUCE PHOSPHOTASE ACTIVITY THEY

ALSO HAD AN INCREASE OF THESE

CELLS.

IT'S ACTUALLY THE CASE THAT WE

SHOULD ALLEVIATE THIS

INFLAMMATION IF WE RENOVEMBER

MICROBIOTA WHICH WE'RE

HYPOTHESIZING IS A SOURCE OF

LPS.

SO THESE ARE LOOKING IN CROSS

SECTIONS OF ZEBRAFISH INTESTINES

AND A CONVENTIONALLY REARED

ANIMAL LACKING INTESTINAL ALPHA

PHOSPHOTASE.  THERE'S INFLUX OF

NEUTROPHILS BUT THOSE ARE ABSENT

IN THE GERM FREE ANIMALS.

THAT'S JUST QUANTIFIED ON THIS

SLIDE HERE.

SO THIS WAS TELLING US THAT

THERE'S THIS DIALOGUE GOING ON

BETWEEN MICROBIOTA THAT THROUGH

THE PRESENCE OF LPS ARE INDUCING

THIS ENZYME IN THE HOST

INTESTINAL ALPHA PHOSPHOTASE

WHICH ACTS TO DETOXIFY LPS

ASSOCIATED WITH THE MICROBIOTA

AND THAT WOULD PROMOTE AN

APPROPRIATE REACTION TO THIS

IMMUNITY.

SO WE SAW THIS AS A IMPORTANT

BALANCE IN THE DYNAMICS OF THE

HOST MICROBIAL INTERACTION.

WE STARTED TO THINK ABOUT OTHER

WAYS IN WHICH YOU COULD HAVE AN

IMBALANCE SET UP IN THIS

DIALOGUE.

ONE THING WE WONDERED IS WHETHER

THERE MIGHT BE SITUATIONS WHERE

YOU HAVE ALTERED GUT PHYSIOLOGY

THAT COULD GIVE RISE TO AN

ALTERED MICROBIOTA THAT MIGHT BE

FUNCTIONALLY DIFFERENT AND

INCREASE INFLAMMATION.

IN THE INTESTINE.

SO THIS WOULD BE A DIFFERENT

DYNAMIC, A MORE PATHOLOGICAL

DYNAMIC TO DRIVE INFLAMMATION.

SO TO TELL YOU NOW COLLABORATION

WITH MY COLLEAGUE JUDITH ISAN

AND A NUMBER OF TALENTED

INVESTIGATORS AND I PARTNERED

WITH JUDITH BECAUSE SHE WAS

INTERESTED IN THE NERVOUS SYSTEM

THAT ENTERINVESTIGATES THE GUT

AND CONTROLS GUT MOTILITY.

AND SHE WAS STUDYING MUTANT IN

TRANSCRIPTION FACTOR SOX 10

WHICH IS REQUIRED FOR THE

SPECIFICATION OF ENTERIC NEURONS

THAT INNER INVESTIGATE THE --

INTER-- INNERVATE THE GUT.

IF WE LOOK IN THE SOX 10 ANIMALS

THEY LACK ENTERIC NEURONS AND

ALSO LACK THE STRONG PARASTALL

TICK WAVES YOU SAW.

SO THERE'S A LITTLE MOVEMENT BUT

NOT ABLE TO PUSH CONTENT THROUGH

THE GUT.

P

AND YOU CAN SEE IN THE

FUNCTIONAL ASSAY WE CAN FEED OUR

FISH A PULSE CHASE EMPERIMENT, A

GREEN AND THEN A PULSE OF RED

FOOD AND VISUALIZE AFTER AT THAT

TIME POINT AND WILD TYPE FISH

ARE EFFICIENT AT CLEARING OUT

FOOD THROUGH THEIR GUT.

SO IN THIS WILD TYPE YOU SEE THE

RED FOOD AT THE END OF THE

TREATMENT, WHEREAS IN THE SOX 10

MUTANT FISH YOU CAN SEE A BOLUS

OF GREEN FOOD THAT STILL

MAINTAINED IN THESE ANIMALS.

SO WE WONDERED WHETHER IT'S THIS

KIND OF CHANGE IN THE ECOLOGY OF

INTESTINE MIGHT HAVE FUNCTIONAL

CONSEQUENCES, WE FOUND INDEED

THESE SOX 10 MUTANT ANIMALS WHEN

WE QUANTIFY NEUTROPHILS IN

INTESTINES HAD HIGHER LEVEL OF

INFLAMMATION BY THIS METRIC THAN

WILD TYPE SIBLINGS.

THESE ARE EMPERIMENTS WHERE

WE'RE REARING THE FOX 10 MUTANT

AND WILD TYPE ANIMALS IN THE

SAME DISH.

THE SAME INNO, MA'AM LUM AND

THEY HAVE DIFFERENT -- O

INOCULANT.

AND DIFFERENT ENZYME RESPONSES.

SO WE CAN SHOW THIS THIS WASN'T A

RESPONSE TO THE MICROBIOTA BY

SHOWING WHEN WE DI RIEFED SOX 10

MUTANTS UNDER GERM FREE WE SAW

LOW LEVELS OF INFLAMMATION

DISTINGUISHABLE FROM WILE TYPE.

SO WE WONDERED WHETHER

FLAINMATION IN THE MUTANTS IS

DUE TO A FUNCTIONALLY DIFFERENT

MICROBAYOU TA. THE WAY TO TEST

THAT IS TO DO A MICROBIOTA

TRANSPLANTATION EXPERIMENT

TAKING THE GUT CONTENT OF AN

INFLAMED DONOR AND THENNEN KNOCK

LATE THAT INTO A DISH WITH GERM

FREE WILD TYPE RECIPIENTS AND WE

CAN DISTINGUISH WHETHER THAT

INFLAMMATION ASSOCIATED WITH

THIS MUTANT MICROBIOTA IS

TRANSMISSIBLE TO GERM FREE WILD

TYPE ANIMALS.

WE FOUND WHEN WE USED MICROBIOTA

DONOR FROM THE SOX 10 MUTANT

ANIMALS WE DID SEE INCREASE

INFLAMMATION AS COMPARED TO THE

WILD TYPE SIB LLINGS FROM THAT

SAME DISH.

ALSO IMPORTANTLY IF WE

TRANSPLANTED MICROBIOTA FROM OUR

INTESTINAL ALPHA PHOSPHOTASE

DEFICIENT FISH WE DIDN'T SEE

INCREASED INNAMATION AND IN THIS

CASE SOME INFLAMMATION IS DUE

MORE TO THE PRESENCE OF LPS BUT

NOT A SHIFT IN THE MICROBIOTA.

THIS IS TELLING US UNDER CERTAIN

SITUATIONS YOU CAN HAVE A

MICROBIOTA THAT IS PRO

PROINFLAMTORY.

WE THINK THIS SITUATION WITH A

MODAL GUT THE SITUATION WHERE WE

HAVE THE MICROBIOTA DRIVING

INTESTINAL INFLAMMATION, SO WE

THINK THAT'S ANOTHER HOW THE

DYNAMICS BETWEEN THE HOST AND

THE MICROBIOTA ARE PLAYING OUT

IN THIS CASE DRIVING DISEASE

STATE.

AS WE THOUGHT MORE ABOUT THESE

KINDS OF QUESTIONS, IT BECAME

FASCINATING TO THINK ABOUT THE

PARADIGM HOW HOST ENGINE TYPE OR

TRAIT MAY INFLUENCE MICROBIOTA

COMPOSITION AND PLAY BACK ON THE

HOST PHENOTYPE AND YOU COULD GET

THIS CONTINUAL DIALOGUE BETWEEN

THESE TWO ENTERACTING SYSTEMS.

ONE WAY WE WOULD LIKE TO LOOK AT

THAT IS LOOK AT HOW THESE

DIFFERENT ASSOCIATIONS EVOLVE

OVER TIME.

WHAT THE HOST GENES CHANGING IN

EVOLUTIONARY TIME THAT MAY

INFLUENCE DIFFERENT TYPES OF

ASSOCIATIONS.

SO THIS IS WORK WITH MY

COLLEAGUE, BILL PRESCO

UNIVERSITY OF OREGON WHO HAS

BEEN A PIONEER DEVELOPING THE

FISH SYSTEM THE THREE BIND

SICKLE L BACK AS A GREAT SYSTEM

FOR ASKING EVOLUTIONARY

QUESTIONS.

THIS WAS A GREAT SEEM OF PEOPLE

WHO WENT UP, THIS IS WHERE WE'RE

CATCHING SICKLE BACK FROM STREAM

JUST IN EUGENE, TO SAMPLE

MICROBIOTA.

AND SICKLE BACK HAS REALLY

TURNED OUT TO BE A GREAT MODEL

SYSTEM FOR ASKING QUESTIONS

ABOUT EVOLUTION.

IT'S QUITE RELEVANT WHEN

THINKING ABOUT THINKING OF

SICKLE BACK AS A MODEL FOR

QUESTIONS ABOUT HUMAN EVOLUTION,

BECAUSE THEY HAVE QUITE SIMILAR

POPULATION STRUCTURES WHERE

THERE'S BEEN IN THE CASE OF A

FISH THE INTESTINAL POPULATION

FROM THE OCEAN WAS ISOLATE

DURING RECESSION OF THE GLACIERS

INTO VARIOUS DIFFERENT

FRESHWATER POPULATIONS AND

THERE'S BEEN EVOLUTIONARY EVENTS

OCCURRING DURING THAT TIME

PERIOD THAT HAVE DIFFERENTIATED

THESE DIFFERENT POPULATIONS.

SO BILL'S LAB HAS BEEN

INSTRUMENTAL IN DEVELOPING

TECHNIQUES TO USE THOSE

DIFFERENCES BETWEEN OCEAN AND

FRESHWATER FISH TO MAP, DO S-2

MAPPING USING USING TECHNOLOGY

DEVELOPED BY MY COLLEAGUE ERIC

JOHNSON UNIVERSITY OF OREGON

SEQUENCING TO IDENTIFY LOCI IN

THE GENOME THAT CORRELATE WITH

DIFFERENT TRAITS.

SO FOR EXAMPLE, THEY HAVE BEEN

ABLE TO MAP TRACE ASSOCIATED

WITH THE PRESENCE OR LOSS OF

BODY ARMER IN THE FISH.

OUR IDEA IS WE CAN LOOK AT

ASSOCIATED MICROBIAL COMMUNITIES

IN THESE POPULATIONS AND LOOK

WHETHER WE FIND ASSOCIATED GENES

THAT SEGREGATE WITH THESE

DIFFERENT POPULATIONS.

SO OUR INITIAL SURVEY OF OCEAN

AND FRESHWATER POPULATIONS WE

HAVE FOUND POPULATIONS IN

MICROBIAL COMMUNITIES SO JUST

ONE EXAMPLE.

SO WE'RE NOW MOVING FORWARD ON

AN EXPERIMENT WHERE WE HAVE WHAT

WE'RE CALLING A COMMON GARDEN

EXPERIMENT WE INOCULATE OUR --

AND MAINTAIN BOTH POPULATIONS OF

OCEAN AND FRESH WEAR POPULATIONS

IN THE SAME TANK SO THEY'RE

SHARING THE SAME MICROBIAL

ENVIRONMENT AND WE'RE LOOKING AT

WHAT COMMUNITIES ASSEMBLE UNDER

SHARED CONDITIONS BUT WE MADE

ADVANCES IN TRANSPORTING OUR

ZEBRAFISH BIOLOGY TECHNIQUES TO

CIRCLE BACK AN THIS IS SHOWING

YOU THAT WE CAN A SICKLE BACK

GASTROINTESTINAL TRACT AND THESE

ARE CROSS SECTIONS SHOWING YOU

THIS, WE CAN VISUALIZE BACK BACTERIA

IN A CONVENTIONALLY REARED

SICKLE BACK AN DERIVE GERM FREE

SICKLE BACK AND WE CAN LOOK AT

THE NEUTRAFILL INFLUX IN THESE

DIFFERENT SICKLE BACK AND WHAT

WE FIND IN ONE STRAIN, WE LOOK

AT ABUNDANT NEUTROPHILS IN THE

CONVENTIONAL STATE AND MANY FEW

FEWER IN THE GERM FREE STATE.

SO THOSE DATA WERE DERIVED FROM

THE AN SES CENTRAL MOTION

POPULATIONS WHERE YOU SEE A

DIFFERENCE BETWEEN THE GERM FREE

AND CONVENTIONALLY REARED FISH

AND THE NUMBER OF NEUTROPHILS,

BUT WE FOUND IN THE FRESHWATER

POPULATIONS THERE WAS LITTLE

DIFFERENCE BETWEEN GERM FREE AND

CONVENTIONAL STATE AND IT WAS

OVERALL A MUTED INFLAMMATORY

RESPONSE.

SO WE'RE REALLY FASCINATED TO

THINK ABOUT HOW VARIATION IN THE

STRENGTH OF INNATE IMMUNE

RESPONSE TO THE RESIDENT

MICROBIOTA MIGHT PLAY OUT IN

WAYS THAT COULD SHAPE THE

COMMUNITY ASSEMBLY OF THESE

COMMUNITIES.

SO WE'RE LOOKING AT THESE -- THE

IMMUNITY STRUCTURE OF THESE

DIFFERENCE FISH THAT WERE REARED

IN THE INOCULUM.

WE'RE ANALYZING OUR DATA FROM

PROFILING BUT WE ALSO HAVE

PROFILING FROM CULTURE BASED

ANAL SEIZE AND THAT'S WHAT I'M

SHOWING YOU HERE, WITH 10

FRESHWATER INDIVIDUALS ON TOP

AND TEN MARINE INDIVIDUALS ON

THE BOTTOM.

AND WHAT AT FIRST GLANCE WHAT

STRIKES US IS THAT IT LOOKS LIKE

THE FRESHWATER INDIVIDUALS HAVE

A MORE RANDOM ASSEMBLAGE OF

COMMUNITY MEMBERS IN COMPARISON

TO THE MARINE.

WE WONDER IF THIS COULD BE

CORRELATED WITH MORE MUTED

IMMUNE RESPONSE WE'RE SEEING IN

THESE ANIMALS TO THEIR

MICROBIOTA.

SO WE DECIDED TO CONTINUE TO

EXPLORE THESEKINE OF QUESTIONS.

SO WE HAVE -- WHAT OUR WORK IS

SHOWING IS THERE ARE SOME

INTERESTING WAYS IN WHICH CROSS

TALK BETWEEN HOST N PARTICULAR

THE HOST IMMUNE SYSTEM AND

MICROBIOTA CAN REALLY HAVE

PROFOUND INFLUENCES ON THE

COMPOSITION OF THIS MICROBIAL

CELL SYSTEM AS WELL AS THE SUPER

SYSTEM OF THE HOST COLONIZED BY

MICROBIOTA.

SO SOME EXAMPLES OF THAT THAT I

TOUCHED ON TODAY ARE LOOKING AT

OUR PROFILES OF MICROBIOTA

COMMUNITY ASSEMBLY OVER TIME IN

THE ZEBRAFISH AND WE SEE THERE'S

DRAMATIC CHANGE IN THE COMMUNITY

STRUCTURES OVER DEVELOPMENTAL

TIME.

AND THEN WE ALSO SEE THAT

THERE'S CORRELATION WITH THE

SITUATION OF ADAPTIVE IMMUNITY

AND MORE INDIVIDUALALTY TO THE

B.

MICROBIOTA.

IT'S SOMETHING WE'RE EAGER TO

EXPLORE.

THAT LACK ADAPTIVE IMMUNE SYSTEM

AND ASK IF WE FAIL TO SEE THAT

INDIVIDUALALTY E ELABORATED.

IN THOSE LIFE TRAJECTORIES.

WE'RE INTRIGUED BY PRIORITY

EFFECTS THAT WE'VE STARTED TO

UNCOVER WHEN WE HAVE DONE THESE

SIMPLE ASSEMBLY EXPERIMENTS,

COLONIZING GERM FREE FISH WITH

JUST A SINGLE STRAIN OF

BACTERIA.

AND WONDERING WHETHER IMMEDIATE

INNATE IMMUNE RESPONSES TO THE

MICROBIOTA MIGHT BE PART OF WHAT

CREATES THESE SORTS OF PRIORITY

EFFECTS WE'RE DETECTING SO

THAT'S ANOTHER AREA WHERE THE

DIALOGUE BETWEEN THE MICROBIOTA

AND THE HOST MIGHT HAVE

IMMEDIATE CONSEQUENCES BUT COULD

HAVE LONG LASTING HISTORICAL

CONSEQUENCES.

WHAT TYPES OF COMMUNITIES

ASSEMBLE.

FUNCTION OF INTESTINAL ALPHA

PHOSPHOTASE, TOLD US GIVEN AN

EXAMPLE HOW SIGNALING AND

MOLECULAR DIE LOAG BETWEEN THE

MICROBIOTA AND THE HOST CAN TUNE

THE IMMUNE RESPONSIVENESS OF THE

HOST SO WE IMAGINE AND EXPLORING

THE POSSIBILITY THAT THE

PHOSPHOTASE IS REALLY IMPORTANT

FOR MAINTAINING AN APPROPRIATE

INFLAMMATORY RESPONSE TO

ASSOCIATED MICROBIOTA.

SO IF YOU HAVE AN ADEQUACY IN

THAT ACTIVITY AS WE SHOWED AN

EXCESSIVE INFLAMMATORY RESPONSE

TO MICROBIOTA, HAVING TOO MUCH

OF THIS ENZYMEATIC ACTIVITY

WOULD MAKE THE HOST REALLY DEAF

TO THE PRESENCE OF IMPORTANT

SIGNALS FOR PATHOGEN NAS ARE

THERE TO PRIME THE INNATE IMMUNE

SYSTEM SO THERE MIGHT BE AN

APPROPRIATE LEVEL OF TUNING THAT

NEEDS TO HAPPEN AND THAT'S

ESTABLISHED THROUGH DIALOGUE

WITH THE MICROBIOTA.

AND WE HAVE I TOLD YOU ABOUT THE

EXAMPLE OF THE SOX-10 AMODAL

INTESTIN WHERE IS WE SEE

ELABORATION OF A PERTURBED

MICROBIOTA THAT IS FUNCTIONALLY

CAPABLE OF DRIVING EXCESSIVE

INFLAMMATION WHEN TRANSPLANTED

INTO A WILD TYPE RECIPIENT.

SWOA THINK THAT'S GOING TO BE A

REALLY EXCITING MODEL TO EXPLORE

THE KINDS OF SITUATIONS IN

INFLAMMATION IN DISEASE STATES.

FINALLY I INTRODUCED YOU TO A

NEW MODEL WE'RE EXPLORING OF THE

THREE SPINE SICKLE BACK WHICH IS

A POWERFUL WAY FOR IDENTIFYING

HOW DIFFERENCES IN MICROBIOTA

ACROSS INDIVIDUALS AN

POPULATIONS WHAT TYPES OF

GENETIC LOCI ARE ASSOCIATED BUT

ALSO EXPLORING THE POSSIBILITY

THAT FUNCTIONAL DIFFERENCES

BETWEEN INDIVIDUALS SUCH AS

THEIR IMMUNE RESPONSIVE TONS

THEIR MICROBIOTA MIGHT

CONTRIBUTE TO ASPECTS OF

MICROBIOTA COMPOSITION.

SO WITH THAT I WOULD LIKE TO

THANK MANY REALLY IMPORTANT

COLLABORATORS, RAGU

(INDISCERNIBLE) COLLABORATING ON

LIGHT SHEET MICROSCOPY AN

IMAGING, JUDY ISEN ON THE

ENTERIC NERVOUS SYSTEM.

BILL CRESCO WHO IS INTRODUCING

US TO THIS SICKLE BACK SYSTEM

FOR ASKING EVOLUTIONARY

QUESTIONS.

BRENDAN MOHANON AND JESSICA

GREEN ARE GREAT COLLEAGUES

UNIVERSITY OF OREGON THINKING

ABOUT ECOLOGY AND JOHN ROLLS WHO

ALONG WITH BRENDAN IS PI ON MY

FUNDING FROM NIGMS AND THE WORK

I TOLD YOU ABOUT TODAY IN ALPHA

PHOSPHOTASE IS FUNDD THROUGH

NIDDK.

THESE ARE FUNDED THROUGH GREAT

MEMBERS IN MY LAB, IN PARTICULAR

I WANT TO HIGHLIGHT ZACK STEVEN

WHOSE IS INVOLVED IN ALL THE

PROJECTS I TALKED ABOUT TODAY.

I'M HAPPY TO ANSWER QUESTIONS.

[APPLAUSE]

>> IF YOU HAVE QUESTION COME TO

THE MICROPHONE.

I HAVE ONE QUESTION THE START

WITH ABOUT STEMS MICKING THE TWO

GENETICALLY IDENTICAL BACTERIA

WITH DIFFERENT TIMING.

HAVE YOU TRIED -- IF YOU TAKE

LPS AND ADD WHEN YOU ADDED THE

FIRST AND THEN THE SECOND

SPECIESINGS DO YOU SEE THE SAME

EFFECT?

AND WHEN YOU HAD THE RESISTANCE,

IF YOU ADD RIFAMPOSIN AND WINT

RECESSTANT AT THE SAME TIME DO

YOU SEE THE SAME EFFECT?

>> THOSE ARE BOTH GOOD QUESTIONS

AND WE HAVEN'T -- THOSE ARE GOOD

SUGGESTIONS.

WE HAVEN'T DONE EITHER OF THOSE

BUT THOSE WOULD BE I THINK THOSE

WOULD BE INTERESTING IDEAS TO

TEST.

SO LOOKING FIRST PRIMING THE

INNATE IMMUNE RESPONSE

PREVIOUSLY WOULD CREATE

(INAUDIBLE) AND ALSO

COMPROMISING THE ABILITY OF THE

FIRST COCOLLYZE BY ADDING

ANTIBIOTIC.

(OFF MIC)

>> THOSE ARE BOTH (INAUDIBLE)

EXPERIMENTS.

>> I HAVE A SIMILAR QUESTION

ABOUT THE PRIORITY EFFECT.

YOU SHOWED WITH SIMILAR STRAINS

THE FIRST STRAIN THAT GETS A

FOOT HOLE HAS MAINTAINS THAT

FOOTHOLD AND YOU SHOWED THAT IN

YOUR SOX 10 MUTANT.

SOMETHING HAPPENS TO MAKE THE

MICROBIOTA MORE INFLAMMATORY.

HAVE YOU CONSIDERED THOSE

MICROBIOTA DOING THE SAME PULSE

CHASE AND SEEING IF THEY CAN

OUTPETE THE ORIGINAL POPULATION?

>> THAT'S ANOTHER GREAT IDEA.

OUR INITIAL PRIORITY EFFECT HAVE

BEEN VERY SIMPLE WITH SINGLE

STRAIN.

IT WOULD BE INTERESTING TO SEE

WHAT WE CAN SELECT OUT OF THAT

DIFFERENT COMMUNITY.

THAT MIGHT BE AN INTERESTING

ENRICHMENT FOR CERTAIN MEMBERS.

SO WE'RE LOOKING AT USING

CULTURE INDEPENDENT METHODS TO

PROFILE THE SOX 10 COMMUNITIES

IN IN THIS BIOTIC SITUATION.

BUT WE'RE ALSO INTERESTED IN

ACTUALLY ENRICHING CULTURING

DIFFERENCE MEMBERS THAT WE CAN

WORK WITH SO THAT MIGHT BE A

NEAT WAY TO DO THAT.

>> ANOTHER QUESTION.

DO YOU NEED A ZEBRAFISH GUT TO

SEE THIS?

DOES THIS HAPPEN IN A BROTH

CULTURE OR ON A P-3 PLATE?

>> GREAT QUESTION.

SO WE CAN RECREATE IT IF WE HAVE

TO AND WE DOPE GET THE SAME

EFFECT.

>> REALLY GLAD TO HEAR THAT.

>> SO THIS IS ANOTHER PRIORITY

QUESTION I GUESS YOU'RE GOING TO

GET A LOT OF THESE.

SO BACTERIA THE SAME GENETICALLY

IDENTICAL BACTERIA, OF COURSE,

CAN GROW IN VERY DIFFERENT WAYS

DEPENDING UPON ITS ENVIRONMENT.

I'M WONDERING IF YOU LOOKED

SIMILAR TO SEE WHETHER SIMPLY

THE BEING INTRODUCED INTO THE

GUT IT CHANGES THE MOAD OF

GROWTH -- MODE OF GROWTH, CHANGE

IT IS BACTERIA PHYSIOLOGICALLY

IN A WAY THEY'RE MORE LIKELY OR

LESS LIKELY TO RECOLONIZE.

>> THAT'S ALSO AN EXCELLENT

QUESTION WE'VE -- SO WE HAVEN'T

ADDRESSED IT BUT IT'S A

HYPOTHESIS THAT WE THOUGHT

ABOUT.

THAT'S CERTAIN LAY LOT OF

EXAMPLES OF PATHOGENS WHICH THAT

MODEL HAS BEEN VALIDATED.

SO IT'S A GOOD ONE TO THINK

ABOUT.

>> I HAVE A NON-PRIORITY

QUESTION.

>> OKAY.

>> DIET OFTEN DICTATE IT IS TYPE

OF BACTERIA IN YOUR GUT.

HAVE YOU LOOKED INTO WHETHER YOU

CAN TEASE APART THE TYPE OF DIET

IS IT A COMMON FISH FOOD FOR THE

OCEAN GOING FISH VERSUS

FRESHWATER THAT YOU'RE GETTING,

CAN YOU SWITCH DIET AND SWITCH

THE MICROBIOTA COMPOSITION?

>> WE'RE VERY INTERESTED IN THE

QUESTION OF DIET THAT'S A FOCUS

OF OF MY COLLEAGUE JOHN ROLL'S

WORK.

ONE NICE THING ABOUT

EXPERIMENTAL SYSTEMS IS WE CAN

MANIPULATE THESE THINGS.

SO EXPERIENCE LOOKING AT

COMMUNITY IN THE SICKLE BACK

POPULATION, THEY'RE ALL

RECEIVING THE SAME DIET.

THEY'RE IN THE SAME ENVIRONMENT.

SO WE CAN CONTROL FOR ALL OF

THOSE THINGS.

BUT THEY'RE NATURAL DIET COULD

BE QUITE DIFFERENCE.

THAT'S ONE OF THE DRIVING FORCES

OF WHY THEY'VE EVOLVED THESE

DIFFERENT EXTERNAL MORPHOLOGIES

IS THEY HAVE DIFFERENT KINDS OF

PREDATORS, STRATEGIES.

BUT ALSO THE QUESTION OF DIET IS

RELEVANT FOR THE STUDY THAT WE

DID ON THE LONGITUDINAL STUDY OF

ZEBRAFISH.

THEY'RE ACTUALLY RECEIVING

DIFFERENT DIETS AT DIFFERENT

TIMES.

WE OUR FIRST PASS WAS TO PROFILE

THEM AS THEY ARE NORMALLY REARED

IN OUR FACILITY WHICH MEANS

THEY'RE -- REARED ON A DIET

INITIALLY OF CARAMESIA AND AS

THEY'RE BIG ENOUGH THEY'RE GIVEN

A DIET OF BRIAN SHRIMP AND

THAT'S FURTHER SUPPLEMENTS AS

ADULTS SO THAT'S A PARAMETER

THAT THEN NOW THAT WE HAVE THAT

BASELINE WE CAN COMPARE AS WE

MANIPULATE DIETS.

>> WHEN YOU GROW SAL AND

FRESHWATER TOGETHER DO YOU GROW

THEM IN SALT OR FRESHWATER?

>> INTERMEDIATE.

AND -- THAT'S ONE OF THE

ADVANTAGES OF THE SYSTEM IS YOU

CAN GROW THEM IN THE SAME

ENVIRONMENT.

>> IF YOU HAVE INDIVIDUAL

QUESTIONS WE'LL BE HAPPY TO

ENTERTAIN THEM.

THANK YOU FOR COMING.