GOOD AFTERNOON, EVERYONE.
WELCOME TO THE WEDNESDAY
AFTERNOON LECTURE SERIES.
I'M PAM ROBY, CHIEF OF SKELETAL
BIOLOGY SECTION AT THE NATIONAL
INSTITUTE OF DENTAL AND
CRANIOFACIAL RESEARCH.
OUR SPEAKER TODAY, DR. CLIFFORD
TABIN, GEORGE JACOB AND
JACQUELINE HAZEL LEDER PROFESSOR
OF GENETICS AND CHAIR OF THE
DEPARTMENT OF GENETICS, HARVARD
MEDICAL SCHOOL.
DR. TABIN RECEIVED HIS
UNDERGRADUATE DEGREE FROM
UNIVERSITY OF CHICAGO IN 1976,
HIS Ph.D. FROM M.I.T. IN 1984.
HE THEN UNDERTOOK POSTDOCTORAL
FELLOWSHIPS AT HARVARD
UNIVERSITY AND MASSACHUSETTS
GENERAL HOSPITAL BEFORE
SETTLINGS AT HARVARD MEDICAL
SCHOOL, ACCEPTED A POSITION OF
ASSISTANT PROFESSOR THERE IN
1989, BECAME A FULL PROFESSOR IN
1997.
CONCURRENTLY HE BECAME AN
ADJUNCT PROFESSOR AT M.I.T, A
POSITION HE STILL MAINTAINS,
BECAME CHAIRMAN OF THE
DEPARTMENT OF GENETICS AT
HARVARD IN 2007.
DR. TABIN MADE SEMINAL
DISCOVERIES ALONG WITH PHIL
GRAHAM AND ANDREW MAYHEM OF
HEDGE HOG GENES IN VERTEBRATES,
FORM AND FUNCTION IN EMBRYONIC
VERTEBRATE, AND TIME TO GENERATE
STORIED DIVERSITY OF ANIMAL
FORMS ON THIS PLANET.
THEY COMBINED CLASSICAL METHODS
OF EXPERIMENTAL EMBRYOLOGY WITH
MODERN MOLECULAR IMAGING,
GENETIC, GENOMIC TECHNIQUES FOR
INTERROGATING GENE FUNCTION.
IN HIS EVOLUTIONARY WORK
ADDRESSED QUESTIONS OF BEHAVIOR
AND METABOLIC EVOLUTION USING
DEVELOP MEMORIAL AND GENETIC
APPROACHES.
IN TODAY'S TALK HE TURNS TO
BIRDS, AND THEIR REMARKABLE
ABILITY TO VOCALIZE WITH AN
ORGAN CALLED THE SYRINX,
PRODUCING SOUNDS WITHOUT VOCAL
FOLDS MAMMALS HAVE.
DR. TABIN HAS BEEN A MEMBER OF
THE NATIONAL ACADEMY OF SCIENCES
SINCE 2007, ALSO IS ELECTED TO
THE AMERICAN ACADEMY OF ARTS AND
SCIENCES, EUROPEAN MOLECULAR
BIOLOGY ORGANIZATION, ROYAL
SOCIETY OF LONDON AS A FOREIGN
MEMBER.
HE HAS BEEN AWARDED THE EDWIN
CONKLIN MEDAL, MARCH OF DIMES
PRIZE IN DEVELOPMENTAL BIOLOGY
AND HAS WON NUMEROUS AWARDS FOR
HIS TEACHING AT HARVARD MEDICAL
SCHOOL AND IT'S REALLY A
PLEASURE TO HAVE HIM HERE TODAY.
THE TITLE OF THE TALK IS "HOW
DID BIRDS EVOLVE THE CAPACITY TO
VOCALIZE."
DR. TABIN, WELCOME, AND THANK
YOU SO MUCH FOR BEING WITH US
TODAY.
>> THANK YOU VERY MUCH FOR THE
KIND INTRODUCTION.
I WAS AMUSED, ONE GETS CME
MEDICAL CREDIT FOR LISTENING TO
A TALK ON BIRD VOCALIZATION.
I'M NOT SURE WHY THAT'S
IMPORTANT FOR CONTINUING MEDICAL
EDUCATION.
BUT IN ANY CASE, I'M TALKING TO
YOU RIGHT NOW WITH MY THROAT
THAT IS TO SAY MY LARYNX.
MOST TETRAPODS, ANIMALS THAT
CRAWL ON LAND, USE THE LARYNX TO
SPEAK, INCLUDING REPTILES LIKE
ALLIGATORS, AMPHIBIANS LIKE
PROGRESSES, ALL MAMMALS,
ULTRASONIC SQUEAK A MOUSE MAKES
IS WITH THE LARYNX.
WE'RE ALL SPEAKING WITH OUR
LARYNX.
LARYNX IS THE VOICE BOX IN THE
THROAT.
HAS VOCAL CORDS SHOWN IN THE
REGIONS IN GREEN, CROSS-SECTION.
BIRDS HAVE A LARYNX, IT'S AN
IMPORTANT PART OF THEIR ANATOMY
FOR REGULATING AIR FLOW IN AND
OUT OF THEIR MOUTH.
THERE'S NO VIBRATORY TISSUE,
DON'T HAVE VOCAL CORDS IN THE
THROAT.
INSTEAD, PERHAPS SURPRISINGLY,
BIRDS HAVE EVOLVED A SEPARATE
ORGAN, THE SYRINX, IN THE CHEST
AT THE JUNCTION HERE BETWEEN THE
TRACHEA AND BRONCHI, IN THE
CHEST WHERE THEY HAVE A
CARTILAGE BOX AS IN THE LARYNX
AND THAT LOCATION THEY PUT VOCAL
FOLDS.
THE SYRINX, AS AN ASIDE, WAS THE
NAME COINED BY THOMAS HUXLEY IN
1872, AND HE NAMED THE SYRINX
FROM GREEK MYTHOLOGY, SYRINX WAS
A WATER NYMPH WHO PAN WAS
ENAMORED OF, PAN TRIED TO TRIED
HER IN THE RIVER, TURNING HER
TO REEDS, THE PLUCKED HER AND
MADE THE FIRST PAN PIPES, IN
GREEK, THE SYRINX, MAKES
BEAUTIFUL SOUNDS, A SIMILAR
SHAPE, HENCE THE NAME FROM
HUXLEY.
THE SYRINX IS SHOWN IN THIS
BIRD, IN THIS PARTICULAR BIRD,
IS NOT THE SAME IN ALL BIRDS.
THIS IS SOMETHING I'M NOT GOING
TO ADDRESS TODAY BUT IT'S AN
ASIDE.
MY LARYNX, OF AN ALLIGATOR, OF A
FROG, ALL PRETTY MUCH LOOK THE
SAME.
THE SYRINX HOWEVER CAN BE
DRAMATICALLY DIFFERENT FROM BIRD
TO BIRD.
THIS CORRELATES WITH THEM MAKING
DIFFERENT SOUNDS.
A WOOD PIGEON SOUNDS LIKE THIS
...
WHITE THROATED SPARROW SOUNDS
LIKE THIS ...
VERY DIFFERENT STRUCTURES OF THE
SYRINX.
INCLUDING, FOR EXAMPLE,
EVOLUTION OF DIFFERENT SETS OF
MUSCLES TO REGULATE AND PRODUCE
DIFFERENT SOUNDS.
AND DIFFERENT LOCATIONS OF THE
VOCAL FOLDS, EITHER ON THE
TRACHEA SIDE OF THE SYRINX, WITH
A SINGLE PAIR, OTHER THAN -- OR
TWO PAIRS IN THE CASE OF SONG
BIRDS, SOME GROUPS, PARROTS AS
WELL.
THE REASON HAVING TWO PAIRS IS
INTERESTING IS HERE'S A WOOD
THRUSH, ITS SOUND ...
BEAUTIFUL COMPLEX SOUNDS.
THE WOOD THRUSH MAKES THESE
SOUNDS SINGING SEPARATELY FROM
THE TWO VOCAL CORDS.
IT CAN HARMONIZE WITH ITSELF,
HARMONIZING HITTING DIFFERENT
NOTES, SLOWING IT DOWN ...
SO IT'S A BEAUTIFUL AND DIVERSE
ORGAN.
THERE IS WORK IN MY LAB TO TRY
TO UNDERSTAND DIVERSIFICATION OF
THE SHIRKS.
SYRINX.
REPTILES AND AMPHIBIANS HAVE A
LARYNX, AS WE DO AS WELL.
THAT'S REPRESENTED BY THIS BLACK
DOT AND THE BARS MEAN IT'S A
FUNCTIONAL LARYNX THAT SINGS.
THEY DO NOT HAVE A SYRINX.
MODERN BIRDS HAVE A LARYNX DOES
NOT SING BUT A SYRINX THAT DOES
SING.
BETWEEN CROWN BIRDS AND
REPTILES, IN THIS NON-AVIAN
DINOSAUR LINEAGE, SYRINX MUST
HAVE EVOLVED.
IT'S INTERESTING TO THINK ABOUT
HOW IT EVOLVED.
THAT IS, TWO POSSIBILITIES.
EITHER YOU FIRST LOST THE LARYNX
AND YOU HAD A QUIET ANIMAL THAT
HAD TO INVENT THE SYRINX TO TALK
OR FUNCTIONAL OVERLAP, AT THE
SAME TIME, AND WHY YOU HAD THE
TRANSITION FROM ONE TO ANOTHER.
THIS IS SOMETHING WE'RE
EXPLORING WITH A COLLEAGUE OF
OURS, JULIA CLARK, UNIVERSITY OF
TEXAS AUSTIN, PALEONTOLOGIST,
TRYING TO FIND EVIDENCE FOR
EARLIEST SYRINXES, ESPECIALLY IN
A DINOSAUR LINEAGE.
AS FAR BACK AS SHE'S GOTTEN SO
FAR HOWEVER, IT'S A DEFINITIVE
CROWN FOWL, IT HAS A SYRINX,
LIKE DUCKS AND GEESE.
TRYING TO UNDERSTAND FROM A
DEVELOPMENTAL STANDPOINT, HOW DO
YOU MAKE A NOVEL STRUCTURE?
THE SYRINX IS AN EXAMPLE OF
SOMETHING NEW.
IT'S NOT LIKE A BIRD'S WING
WHICH IS HOMOLOGOUS TO
NON-FLYING ANIMAL'S LIMB.
IN THIS CASE EVOLUTION OF
STRUCTURE, YES IT'S BUILT ON
PARTS THAT WERE ALREADY THERE
BUT NO PREEXISTING SYRINX, NO
PREEXISTING VALVES, SO WHERE DID
IT COME FROM?
SO WE'RE GOING TO TALK ABOUT IT
IN TWO PARTS.
FIRST, THE BUILDING OF THE VOICE
BOX, THE CARTILAGE BOX ITSELF.
AND THEN HOW YOU GET THE VOCAL
FOLDS WITHIN IT.
SO, ONE WAY YOU CAN IMAGINE YOU
GET THIS SPECIALIZED SYRINX
WOULD BE TO BUILD ON THE
PREEXISTING CARTILAGE RINGS IN
THE TRACHEA AND BRONCHI, IN THE
SYRINX THEY ARE THICKER AND
CHANGE SHAPE SOME, BUT IMAGINE
LAY DOWN A CONTINUOUS PATTERN,
MODIFY SUBSEQUENTLY TO GET
STRUCTURE THAT'S APPROPRIATE FOR
HOUSING VOCAL CORDS.
SO, TO THINK ABOUT THAT ONE
WANTS TO TAKE A STEP BACK AND
SAY HOW DO YOU GET CARTILAGE
RINGS TO BEGIN WITH?
AND THERE'S BEEN A LOT OF WORK
DONE ON THE DEVELOPMENT OF THE
AIRWAY HOW YOU ACTUALLY GET
RING, NON-RING, RING, NOT RING,
NOT DEFINED DEVELOPMENTALLY
PREVIOUSLY BUT WE CAN THINK
ABOUT IT FROM THEORETICAL
STANDPOINT.
ONE WAY OF MODELING HOW YOU CAN
GET THIS SORT OF PARALLEL
STRIPES IS THROUGH A MODEL OF
SOMETHING LIKE A TOURING SYSTEM.
SO, WITHOUT GOING INTO DETAILS
ABOUT A TOURING MECHANISM TODAY,
SUFFICE IT TO SAY THAT TURING
MECHANIC, ONE ACTIVATOR, ONE
INHIBITORS, INDUCING EXPRESSION
OF INHIBITOR WHICH INHIBITS THE
PROGRAM.
JUST THE TWO MOLECULES COMBINED
CAN GIVE PERIODIC PATTERNS,
GENERATE A RANGE OF SHAPES IN
SILICO THAT MIMICS NATURE,
INCLUDING PARABLE STRIPES.
PARABLE STRIPES.
THAT'S WHAT HAPPENS AT THE
JUNCTION.
WHEN YOU HAVE THE COLLISION OF A
TURING PATTERN DOWN THE TRACHEA
AND TURING PATTERNS COMING UP
THE BRONCHI.
YOU MODEL, YOU GET A MESS.
YOU DON'T GET CLEAN STRIPES AT
THE JUNCTION.
IF YOU LOOK AT THE TRACHEAL
BRONCHIAL JUNCTION IN HUMANS OR
MICE THAT'S WHAT YOU SEE.
THIS FIGURE OVER HERE ON THE
LEFT TAKEN FROM AN 1891
MONOGRAPH, INTERESTING PAPER,
ENTIRE MONOGRAPH IS 86 HUMAN
CADAVER ANALYSES DRAWING JUST
THE CADAVER'S TRACHEA AND
BRONCHIAL JUNCTION.
THESE ARE NOT MEN WEARING WEIRD
GYM SHORTS.
THESE ARE BRONCHI IN THE
TRACHEA, YOU CAN SEE THE POINT
THEY WERE MAKING WAS NO TWO
JUNCTIONS ARE ALIKE.
WE ALL HAVE RINGS ABOVE AND
BELOW, BUT THE JUNCTION YOU GET
A MESS.
WE GET THE SAME THING IN THE
MOUSE.
REMINISCENT OF WHAT YOU GET
HERE.
THAT'S FINE IF ALL YOU'RE TRYING
DO IS GET CARTILAGE SUPPORT FOR
AIRWAY.
IF YOU WANT TO MAKE A CAREFUL
STRUCTURE TO SUPPORT SINGING OR
SPEAKING, VOICE BOX, YOU CAN'T
AFFORD TO HAVE THIS TYPE OF MESS
GOING ON.
SO HOW DO YOU AVOID THAT?
SO, A POSTDOC IN MY LAB EVAN
KINGSLEY DECIDED TO LOOK INTO
THAT AND WATCHED THE ANTOGENY OF
THE RINGS OVER TIME.
YOU HAVE AN AIRWAY, TRACHEA
BRONCHIAL JUNCTION, BASICALLY NO
RINGS.
A DAY LATER THERE'S THE START OF
RINGS.
DAY LATER YOU HAVE THE RINGS.
THEY BASICALLY ALL COME UP AT
ONCE.
THAT CONTRAST, I SHOULD SAY
UNLIKE SOMITS ADDED ONE AT A
TIME THIS SEGMENTATION IS VERY
CONSISTENT WITH THE REACTION TO
FUSION MECHANISM THAT TURING WAS
PROPOSING.
BUT YOU GET A DIFFERENT
ONTOLOGICAL PATTERN IN THE
CHICKEN, OR ANY OTHER BIRD, YOU
FIND INITIALLY THERE'S NO
CARTILAGE, BUT CARTILAGE OF THE
SYRINX COMES UP FIRST.
FIRST YOU DEVELOP THE SYRINGEAL
CARTILAGE, THEN YOU DEVELOP THE
OTHER CARTILAGE.
THIS MAKES PERFECT SENSE.
YOU AVOID HAVING THIS MESS AT
THE INTERSECTION AND THEN BUILD
RINGS ABOVE AND BELOW.
MAKES PERFECT SENSE BUT HOW DO
YOU GET THIS TO DEVELOP BEFORE
THE OTHER RINGS?
TO GET A HANDLE WE DID AN
RNAseq ANALYSIS WHERE WE
COMPARED ALLIGATOR WHERE
EVERYTHING IS COMING UP AT ONCE,
TRACHEOBRONCHIAL JUNCTION WHERE
THE SYRINX WOULD FORM WITH THE
TRACHEA IN BOTH THE MESENCHYME
AND EPITHELIUM, SAME IN CHICKEN,
ENDODERM AND MISODERMAL LAYERS,
WHAT IS NOT DIFFERENTIALLY
EXPRESSED IN THE ALLIGATOR
WHERE PATTERNS ARE THE SAME?
IF YOU DO THAT WHAT YOU FIND
VARIOUS MEMBERS OF THE HEDGE HOG
SIGNALING PATHWAY.
SO YOU CAN LOOK AT THAT, THAT'S
BY RNA ANALYSIS, VERIFIED BY IN
SITU HYBRIDIZATION.
THIS IS A LITTLE BIT CONFUSES,
THE FIRST PANEL HERE, BECAUSE
THE DISSECTION DIDN'T SEPARATE
THE GUT FROM THE TRACHEA.
SO THE ENTIRE AIRWAY FORMS THIS
BUDDING OFF OF THE GUT.
YOU CAN SEE BEHIND IT THE GUT.
SO THIS STRIPE HERE, THIS DOWN
HERE ARE EXPRESSION IN THE GUT.
IN THE AIRWAY, YOU HAVE A
CONTINUOUS EXPRESSION IN THE
TRACHEA AT THE JUNCTION AND
BRONCHI OF SONIC HEDGE HOG.
THEN IT DISAPPEARS, SONIC
HEDGEHOG GOES AWAY WHERE THE
SYRINX IS GOING TO FORM AND GOES
AWAY EVERYWHERE ELSE,
TIMING-WISE CORRELATES WELL.
FIRST SONIC GOES AWAY.
THEN YOU GET CARTILAGES AT THE
SYRINX.
THEN SONIC GOES AWAY IN THE REST
OF THE TUBE.
THEN YOU GET THE REST OF THE
TRACHEA, REST OF THE CARTILAGES.
THAT'S A CORRELATION.
TO SEE WHERE THE SONIC ACTSES,
THE ARGUMENT IS SONIC IS
INHIBITING FORMATION OF THE
CARTILAGE, SONIC DISAPPEARS, YOU
GET CARTILAGE.
TO TEST THAT, WENT IN VITRO.
YOU CAN CULTURE AVIAN AIRWAY
FROM STAGE WHEN THERE IS NO CART
RANCH, TO AFTER THE STAGE, FIVE
DAYS LATER, CARTILAGE IS FORMED,
FORMED PERFECTLY AS IN VIVO.
YOU CAN PUT IN BEADS, SONIC
HEDGEHOG OR OTHER PROTEINS, AND
SAY WHAT DOES THAT DO THE TO THE
PATTERN.
WHAT YOU FIND IS IF YOU PUT IN A
CONTROL BEAD HERE IN
CROSS-SECTION OF THE CARTILAGES,
THE SYRINX, IF YOU PUT IN SONIC
ON THE SIDE WITH THE SONIC BEAD
CARTAGES DISAPPEAR.
OR DIFFERENT TYPE EXPERIMENT
USING AGONIST TO BLOCK HEDGEHOG
SIGNALING, BLOCK GLOBALLY,
CARTILAGE FAILS TO FORM
GLOBALLY.
NOW, IF YOU APPLY SONIC HEDGEHOG
EARLY WHEN THE SYRINX IS
FORMING, IF YOU LET THE SYRINX
FORM AND APPLY HEDGEHOG AT LATER
STAGE, WHERE IT'S GOING THROUGH
THE TRACHEA, NOW YOU SEE THE
SYRINX HAS BEEN LAID DOWN
CONTINUOUS TO FORM, TRACHEAL
RINGS NO LONGER FORM.
IF HEDGEHOG IS THERE YOU DON'T
GET THE CARTILAGE RINGS.
ALL RIGHT.
WE HAVE A BIT OF A CLUE AS TO
HOW YOU GET THESE CARTILAGE
RINGS TO FORM WITHOUT INTERFERES
FROM THE TURING PATTERN BY
TAKING THIS INHIBITOR, SONIC,
REMOVING FIRST HERE AND THEN
EVERYWHERE ELSE.
WHY IS SONIC HANGING AROUND AS
INHIBITOR IN THE FIRST PLACE,
ANCESTRAL BEFORE THE EVOLUTION
OF THE SYRINX, WHAT WAS IT DOING
ON AN ALLIGATOR?
A CLUE, OTHER THAN BEING A
TIMING MECHANISM, COMES FROM
LOOKING AT THE EXPRESSION OF
HEDGEHOG, IN SECTION.
IF YOU SECTION THROUGH,
SECTIONING THROUGH AT THE LEVEL
OF SYRINX, AT THE LEVEL OF THE
TRACHEA, SONIC IS EXPRESSED IN
THE STRIPE, IT'S ACTUALLY ONLY
EXPRESSED IN CROSS-SECTION IN
THE DORSAL MOST ASPECT OF THE
TRACHEA.
HERE IS THE TRACHEAL ENDODERM.
DORSAL MOST EXPRESSES SONIC,
MESODERM, GRADIENT, HIGH
PATCHED, PATCH EXTENDS TO THE BOTTOM.
SAME TRUE ON BRONCHI, DORSAL,
NOT VENTRAL.
WHY WOULD YOU HAVE A GRADIENT IN
THIS MANNER?
WELL, LET'S GO BACK TO THINKING
ABOUT TURING MECHANISMS AGAIN, I
WAS FLIPPANT WHEN I TALKED ABOUT
IT BEFORE.
THEY ARE GREAT GENERATING ALL
SORTS OF PATTERNS FOUND IN
NATURE.
IT CAN FORM MAZES, DOTS,
CIRCLES, POLYGONS.
ONE THING THEY ARE NOT VERY GOOD
AT IS FORMING PARALLEL STRIPES.
TOM HITCHCOCK UNIVERSITY OF
ABERDEEN SHOWED IN 2015 TO GET A
TURING PATTERN TO FORM REAL
STRIPES, TRUE STRIPES, REQUIRES
SOMETHING ELSE.
THEY FORM PARALLEL-ISH LINES BUT
FOR TIGHT STRIPES FOR EXAMPLE
WITH A GRADIENT IN SILICO,
PARAMETER GRADIENT, THEORETICAL,
JUST A GRADIENT OF SOMETHING
ELSE TO ORIENT THOSE STRIPES.
SO WE ASKED TOM IF HE COULD
MODEL IN THREE DIMENSIONS WHAT'S
HAPPENING IN TWO.
YOU SEE THIS.
AS IN THE TWO DIMENSIONS, THREE
DIMENSIONALLY NO MATTER HOW YOU
TWEAK THE PARAMETERS IT'S
IMPOSSIBLE TO GET REAL PARALLEL
STRIPES UNLESS YOU PUT IN A
GRADIENT.
PARAMETER GRADIENT, IF YOU PUT
IN A STEEP, STEEP GRADIENT, YOU
GET REALLY NICE PARALLEL
STRIPES.
IF YOU HAVE A SHALLOWER
GRADIENT, STILL GIVES YOU
STRIPES BUT THEY GET BRANCHING
FORMS, STRIPES WITH DEFECTS.
THAT'S GOING TO BE IMPORTANT A
COUPLE SLIDES FROM NOW.
A FLAT GRADIENT GIVES YOU
DEFECTIVE STRIPES, STEEP
GRADIENT GIVES YOU PERFECT
STRIPES.
SO IS THERE REALLY A GRADIENT
WITH HEDGEHOG IN THE DEVELOPING
TRACHEA OF THE CHICK?
THERE IS.
IF YOU'RE A SONIC HEDGEHOG, I
SHOWED YOU BEFORE, QUANTIFY THAT
GRADIENT AND SHOW FROM DORSAL TO
VENTRAL A NICE GRADIENT.
MOUSE GRADIENT IS FLATTER.
INTRIGUINGLY, WHILE IN THE CHICK
THE STRIPES ARE PERFECT, JUST
ABSOLUTELY PERFECT, AS WE EXPECT
FROM THE STEEP GRADIENT, IN THE
MOUSE AND HUMAN FOR THAT MATTER,
MAMMALS, YOU HAVE THESE PARALLEL
STRIPES BUT YOU HAVE DEFECTS IN
THE TRACHEA AND BRONCHI.
SIMILAR TO WHAT YOU EXPECT FROM
FLATTER GRADIENT.
HOW DO WE ENVISION A FLAT
GRADIENT WORKING TO GIVE YOU
PARALLEL STRIPES?
SO REMEMBER HEDGEHOG IS SHUT
DOWN OVER TIME.
IMAGINE A SITUATION WHERE
INITIALLY HEDGEHOG SHOWN IN RED
IS EXPRESSED IN THE DORSAL
ENDODERM.
IT'S GRADED THE ENTIRE MESODERM
EXPRESSING PATCH, ENOUGH TO
INHIBIT ANY CARTILAGE ANYWHERE
WITHIN THE TUBE.
AS HEDGEHOG STARTS TO DECREASE
EXPRESSION, REPRESENTED BY THE
SMALLER DOMAIN, GRADIENT
RETRACTS BECAUSE THERE'S LESS
HEDGEHOG, LESS SIGNALING, NOW
HAVE CELLS AT THE BOTTOM THAT NO
LONGER SEE THE HEDGEHOG GRADIENT
AND START MAKING CARTILAGE.
IN A SERIES OF DOTS FOLLOWING
THE TURING MECHANISM.
WHAT HAPPENS THEN, AS THIS
CONTINUES TO RETRACT, THIS
RETRACTING GRADIENT PULL ALSO
CARTILAGE INTO PERFECT RINGS.
PREDICTIONS, IN THE CHICK OR
MOUSE YOU WOULD EXPECT RINGS TO
START AS SMALL DOMAINS ON THE
VENTRAL SIDE, EXTENDING OVER
TIME.
TWO, SONIC HEDGEHOG GRADIENT
RETRACTS OVER TIME.
THREE, IF THIS IS WHAT'S GOING
ON, ANYWHERE YOU HAVE HEDGEHOG
SIGNALING YOU SHOULD NOT HAVE
CARTILAGE AND VICE VERSA, THEY
SHOULD BE COMPLEMENTARY DOMAINS.
YOU CAN LOOK IN THE CHICK.
ON THE VENTRAL SIDE YOU CAN
CATCH IT IF YOU CHECK AT THE
RIGHT TIME.
GRADIENT RETRACTS OVER TIME, ONE
EXTENDS AROUND THE FULL RING OR
ALMOST THE FULL RING IN THIS IN
SITU OVER TIME, LESS, LESS,
SMALL.
THIS IS LOOKING AT PATCH,
READOUT FOR THE GRADIENT,
HEDGEHOG ACTIVITY.
AND INDEED ANY OF THESE STAGES
IF YOU COMPARE HEDGEHOG ACTIVITY
TO CHONDRAGENESIS, IN THIS CASE
LOOKING AT HEDGEHOG REPORTER,
THIS PINK IS PATCH, THIS IS SOX
9, WE'RE LOOKING AT -- DON'T GET
CONFUSED.
PINK IS CARTILAGE, GREEN IS
HEDGEHOG.
YOU CAN SEE THERE'S A PERFECT
RECIPROCAL RELATIONSHIP BETWEEN
WHERE YOU HAVE HEDGEHOG AND
WHERE YOU HAVE CARTILAGE.
FINALLY WHEN WE PREDICT IF THIS
IS WHAT'S HAPPENING, YOU CAN
BLOCK HEDGEHOG SIGNALING, IF
IT'S HEDGEHOG SIGNALING GIVING
PERFECT RINGS RETRACTION OF THE
GRADIENT, IF YOU BLOCK HEDGEHOG
SIGNALING UNIFORMLY GET RIDDLE
RID -- RID OF ALL, RETRACT FROM
VENTRAL SIDE UP, SHOULD BE
GETTING CHONDRAGENESIS
EVERYWHERE.
SOX9, YOU HAVE THE START OF THE
CARTILAGE RINGS, THIS IS IN THE
CONTROL.
IF YOU BLOCK HEDGEHOG SIGNALING
EVERYWHERE, YOU NOW GET THE
STARTS OF RINGS IN THE VENTRAL
SIDE, DORSAL SIDE, IN BETWEEN,
CARTILAGE EVERYWHERE BECAUSE YOU
DON'T HAVE INHIBITION OF
HEDGEHOG.
AND I ALLUDED TO THE IDEA THIS
RECIPROCAL RELATIONSHIP BETWEEN
CARTILAGE AND HEDGEHOG SIGNALING
IN THE PROCESS OF GIVING YOU THE
SYRINX, SUGGESTING PREEXISTING
SYSTEM FOR FORMING CARTILAGE
RINGS IN THE REPTILE.
THIS IS ALLIGATOR.
ONCE AGAIN YOU HAVE HEDGEHOG
BEING RECIPROCAL OF
CHONDRAGENESIS IN THE ALLIGATOR
AS IN THE CHICK.
FOCUSING ON CARTILAGE, I TRIED
TO TELL YOU THAT YOU GET
CARTILAGE RINGS IN THE TRACHEA
AND BRONCHI THROUGH HAVING
HEDGEHOG RETRACTION, OVER TIME
MORE CAPACITY FOR
CHONDRAGENESIS, DRAWING THIS
INITIAL LINEAR TURING PATTERN
INTO PERFECTLY PARALLEL RINGS.
BY HAVING THIS PROCESS TAKE
PLACE EARLIER, WHERE THE SYRINX
IS GOING TO FORM, YOU GET RID OF
THE INTERFERENCE PATTERNS FROM
COLLIDING TURING MECHANISMS,
FORMING PERFECT CARTILAGE FOR
SYRINX BEFORE FORMING BRONCHIAL
AND TRACHEAL RINGS.
NOW WE HAVE A VOICE BOX, THINK
ABOUT VIBRATORY TISSUE.
HOMOLOGY CONTEXT, THE SYRINX AND
LARYNX ARE ANALOGOUS, USED TO
MAKE SOUNDS.
BUT TO WHAT EXTENT ARE THEY THE
SAME ANIMAL?
HUXLEY REFERRED TO THEM AS BEING
THE SAME.
BIRDS POSSESS A LARYNX IN THE
ORDINARY POSITION, USE ANOTHER
APPARATUS, HE CALLED LOWER
LARYNX OR SYRINX, REFERRING TO
THE SYRINX AS A SECOND LARYNX
FURTHER DOWN SUGGESTING SOME
SORT OF -- IMPLYING HOMOLOGY.
HOW HOMOLOGOUS ARE THEY?
A GRADUATE STUDENT PICKED THIS
UP.
HE LOOKED AT THE TISSUE LEVEL,
SIMILAR TISSUES.
CERTAINLY IN TERMS OF
EXTRACELLULAR MATRIX, THEY ARE.
SYRINX EXPRESSES A VARIETY OF
EXTRACELLULAR MATRIX GIVING
VIBRATORY PROCESS, LARYNX
EXPRESSES SAME MOLECULES,
HYALURONIC ACID, A NUMBER OF
OTHERS.
THAT RAISES AT LEAST THREE
POSSIBILITIES HOW OF SYRINX AND
LARYNX HAVE SIMILAR TISSUE
PROBLEMS.
CONVERGENCE, THEY HAVE TO
VIBRATE AND MAKE SOUND,
CONSTRAINS WITH MECHANICAL
PROPERTIES, MAYBE THESE ARE THE
OPTIMAL EXTRACELLULAR MATRIX
PROTEINS, AND INDEPENDENTLY BUT
THROUGH PARALLEL EVOLUTION
EVOLVE IN THE SYRINX AND LARYNX.
OR, BOTTOM, THEY COULD BE
HOMOLOGOUS, MAYBE PROGENITOR
CELLS THAT MAKE THE VOCAL FOLDS
IN MAMMAL AND AVIAN SYSTEM ARE
THE SAME TISSUE.
A LOT OF MIGRATORY CELLS IN THE
EMBRYO, MAYBE THE CELLS MIGRATE
TO THE SYRINX IN A BIRD, REALLY
HOMOLOGOUS OR PROCESS HOMOLOGY
OR DEEP HOMOLOGY, REFERRING TO A
CASE WHERE YOU HAVE STRUCTURES
THAT EVOLVE INDEPENDENTLY, YOU
COULD IMAGINE GENE REGULATORY
NETWORK STOLEN BY EVOLUTION,
CO-OPTED TO MAKE A SYRINX
BUILDING IT IN A DIFFERENT PLACE
AND DIFFERENT WAY.
FIRST QUESTION IS ARE THEY THE
SAME -- REALLY HOMOLOGOUS, ARE
TISSUES THE SAME THING?
WE CAN ELIMINATE THAT QUICKLY.
OTHER INVESTIGATORS, TIBLER ET
AL. IN WALLINGFORD'S LABORATORY,
MAP INCOME THE MOUSE, VOCAL
CORDS OF NEURAL CREST ORIGIN, A
CELL TYPE THAT MIGRATES AROUND
AND USES MIGRATORY CELLS
ORIGINATING IN THE ECTODERM, THE
JUNCTION OF THE NEURAL CREST.
THESE ARE MIGRATORY CELLS.
IF NEURAL CREST WAS CELLS MADE
UP OF THE SYRINX VOCAL CORDS,
THAT MODEL MIGHT HOLD.
SO WE DID TRANSPLANT
EXPERIMENTS, NEURAL CREST COMES
FROM THE TO BE OF THE NEURAL
TUBE, CUT OUT THE NEURAL TUBE
FROM WILDTYPE CHICKEN, GRAFT GFP
NEURAL TUBE, AND IN THIS
CONFIGURATION, A REAL EMBRYO,
THE CELLS WILL INTEGRATE INTO
THE HOST, PLAY THE ROLES THEY
WOULD HAVE PLAYED IN THE NORMAL
SETTING.
YOU CAN ASK WHAT TISSUES DOES
THAT GIVE RISE TO?
THERE'S PLENTY OF CREST ARRANGED
THE NEURAL SYRINX, ALL THIS
TISSUE SHOWN IN PURPLE IS NEURAL
CREST.
IT'S ALL INNERVATION.
THE SYMPATHETIC NERVOUS SYSTEM,
CRANIAL NERVES AND SO ON.
WHAT YOU DON'T SEE IS PURPLE IN
THE SYRINX OR TRACHEA OR BRONCHI
FOR THAT MATTER.
SYRINX ITSELF IS NOT MADE OF
NEURAL CREST.
ANOTHER TRANSPLANT FROM LATERAL
PLATE MESODERM.
THEY COME FROM DIFFERENT
TISSUES.
THEREFORE THEY ARE NOT
HOMOLOGOUS.
WHAT ABOUT PROCESS HOMOLOGY?
ARE THE SAME REGULATORY NETWORKS
USED TO MAKE BOTH SYRINX AND
LARYNX?
TO KNOW THAT YOU HAVE TO KNOW
WHAT SORT OF REGULATORY
CIRCUMSTANCES THERE ARE MAKING
SYRINX OR LARYNX.
THERE'S BEEN A LOT OF WORK ON
THE MAMMALIAN AIRWAY AND
VOCALIZATION OF BIRDS AT THE
LEVEL OF NEURAL CIRCUITRY,
REALLY NOTHING WAS KNOWN ABOUT
GENE REGULATORY NETWORKS THAT
GIVE YOU A VOCAL CORD.
SO TO GET A HANDLE ON THAT
CHARLIE DID SINGLE CELL RNAseq
EXPERIMENTS, DISASSOCIATE CELLS
AT STAGES, SINGLE CELL
SEQUENCING, CLUSTERED ON GENE
EXPRESSION.
EACH CLUSTER, EACH COLOR,
LABELING CLUSTERS THAT HAVE
DIFFERENT SORT OF
TRANSCRIPTIONAL PROFILES, YOU
CAN RECOGNIZE, I'VE LABELED WHAT
THE CLUSTERS WOULD RELATE TO.
FOR EXAMPLE, PRE-CARTILAGE
CELLS, CARTILAGE CELLS,
ENDODERM, SO ON, BASED ON
MARKERS.
YOU CAN TELL WHICH ARE CARTILAGE
BECAUSE THEY EXPRESS VERY
SPECIFICALLY CARTILAGE-SPECIFIC
GENES LIKE COLLAGEN 2A 1 AND 9A
1.
WHAT WE'RE LOOKING FOR ARE
THE -- IS THE CLUSTER THAT MIGHT
BE RELEVANT FOR VOCAL CORDS.
AND THE ONE THAT POPPED OUT IS
THIS ONE HERE.
IT'S SHOWN ON THIS DIAGRAM RA+,
IF YOU LOOK AT EXTRACELLULAR
MATRIX FOR GENE EXPRESSION OF
EXTRACELLULAR MATRIX PROTEINS,
THEY ARE SPECIFICALLY FOUND IN
THE SYRINX ITSELF.
WITHIN THE VOCAL TISSUE.
THOSE ARE VERY HIGHLY EXPRESSED
TOGETHER, CLUSTER 10.
INDEPENDENTLY EXPRESSED IN OTHER
CLUSTERS BUT THE ONLY PLACE YOU
HAVE BOTH AS WELL AS OTHER
EXTRACELLULAR MATRIX PROTEINS
ARE CLUSTER 10.
STRIKINGLY CLUSTER 10 IS
INTRIGUING, EXPRESSES A NUMBER
OF GENES, SYNTHESIS PATHWAY,
RETINOIC ACID IN GENES
UPREGULATED BY RETINOIC ACID,
CONVERSELY NOTICEABLE
DOWNREGULATION OF RNA RETINOIC'S
A IT ANTAGONIST SUCH AS CRABP-1.
THE SYRINX PRIMORDIAN HAS
SIGNIFICANT AMOUNT OF RETINOIC
SIGNALING.
LOOKING AT LOCI, SHOWING THE
CASE BY VERIFYING THE CLUSTERING
RESULTS IN VIVO, HERE'S
EXPRESSION OF GENE, ONE OF THE
GENES MARKED IN RETINOIC ACID
ACTIVITY AT THE SYRINX, TRACHEA
UP HERE, SYRINX HERE, BETWEEN
THE TWO RETINOIC ACID ACTIVITY,
DEGRADATION ABOVE AND BELOW,
DECREASE AT THE SYRINX.
THAT'S WHERE YOU FIND VARIOUS
EXTRACELLULAR MATRIX PROTEINS
SUCH AS ALCIAN BLUE STAINING,
STAINS FOR ACIDIC GLYCANS.
TO SHOW THIS ANOTHER WAY, YOU
HAVE -- YOU CAN SHOW IF YOU PLOT
BACK TO THE CLUSTERS, CELLS THAT
ARE EXPRESSING, FOR EXAMPLE,
CARTILAGE MARKER, COLLAGEN
MARKER, RETINOIC ACID, TO WHAT
EXTENT DO THEY SUPPRESS AN
EXTRACELLULAR MATRIX PROTEIN,
LUMICAN IS EXPRESSED AS IS
FIBRONECTIN AS IS HYALURONIC
ACID.
RETINOIC ACID IS THE SAME PLACE
AS EXTRACELLULAR PROTEINS.
ANOTHER GENE THAT POPPED OUT,
SOX9.
SOX9 IS EXPRESSED IN CARTILAGE
DEVELOPMENT.
YOU CAN SEE IT IN CARTILAGE
RINGS.
IT'S NOT A CHONDROGENIC -- IT'S
A TRANSCRIPTION FACTOR, SOX9 IN
VALVES OF THE HEART, FIRST PLACE
IT WAS WELL CHARACTERIZED IN THE
TESTES, KEY GENE FOR
ESTABLISHING PHENOTYPE.
IN ADDITION TO THESE EXPRESSION
OF CARTILAGE RINGS, HIGH
EXPRESSION OF SOX9 ADJACENT TO
THE PLACE WITH HYALURONIC ACID.
SO, EXTRACELLULAR MATRIX
PROTEINS, TRANSCRIPTIONAL
REGULATOR, SIGNALING PATHWAY,
ARE THEY ALL RELATED TO EACH
OTHER?
FIRST OF ALL WE CAN SHOW
RETINOIC ACID ACTS UPSTREAM OF
SOX9 AND EXTRACELLULAR MATRIX,
CONTROL SYRINX HERE IS SHOWING
SOX9 IN GENE, HABP IS BINDING
PROTEIN, A PROBE, LABELED
BINDING PROTEIN IN VITRO THAT
USES A PROBE TO DETECT
HYALURONIC ACID IN VIVO.
IF YOU TWEAK PARALLEL CULTURE
SYRINX, WITH RETINOIC'S
RETINOIC ACID INHIBITOR.
UPREGULATE RETINOIC ACID, GET
HYALURONIC ACID, DO NOT GET
SOX9.
RETINOIC ACID IS NECESSARY FOR
SOX9 AND EXTRACELLULAR MATRIX,
SUFFICIENT -- NOT FOR THE
EXPRESSION OF SOX9.
YOU CAN QUANTIFY THIS
DEPENDENCE, THIS IS WITH
RETINOIC ACID INHIBITOR, OR
CONTROL, SHOWING YOU HAVE
INCREASE IN BOTH SOX9, DECREASE
IN SOX9 SIGNALING AND DECREASE
IN HYALURONIC ACID BINDING
PROTEIN SIGNALING WHEN YOU TREAT
WITH INHIBITOR OF RETINOIC ACID.
SO, WE HAVE AT LEAST A START OF
A REGULATION, WHERE RETINOIC
ACID IS UPSTREAM OF SOX9 AND
EXTRACELLULAR MATRIX, WHAT ABOUT
SOX9?
DOES IT PLAY A ROLE IN
REGULATING EXTRACELLULAR MATRIX?
WE LOOKED AT THAT AS WELL.
IN THIS CASE START WITH UPPER
PANEL, LOOK AT THE TRACHEA.
IN THE TRACHEA, WHICH NORMALLY
DOES NOT EXPRESS SOX9 HYALURONIC
ACID BINDING PROTEIN, IF YOU
COME IN WITH RETROVIRAL VECTOR
THAT CARRIES SOX9 IN RESPONSE TO
THAT ECTOPIC SOX89 YOU HAVE
HYALURONIC ACID QUANTIFIED
WITHOUT RETINOIC ACID AND --
SORRY, WITH AND WITHOUT SOX9.
IF YOU GO TO THE SYRINX AND ADD
TO THE SYRINX VOCAL FOLDS, IN
THIS CASE NEGATIVE SOX9, VOCAL
FOLDS NORMALLY HAVE HYALURONIC
ACID.
IF YOU COME IN WITH DOMINANT
NEGATIVE SOX9, SYRINX CEASES TO
MAKE HYALURONIC ACID.
SO HYALURONIC ACID IS DOWNSTREAM
OF SOX9.
SO ARE OTHER EXTRACELLULAR
MATRIX PROTEINS, LOW LEVELS OF
ELASTIN AND VERSICAN, PUT IN
SOX9, BOTH ARE INDUCED.
YOU HAVE THIS PATHWAY, AND THAT
MAKES GOOD SENSE BECAUSE FROM AN
EVOLUTIONARY STANDPOINT IF YOU
WANT TO EVOLVE TISSUE THAT HAS
PROPERTIES OF VOCAL FOLDS,
INSTEAD OF COMING IN ONE AT A
TIME BY SELECTING INDEPENDENTLY
WHOLE SERIES OF DIFFERENT GENES
TO BUILD DIFFERENT EXTRACELLULAR
MATRIX PROTEINS, YOU CAN COME IN
WITH ONE OR TWO REGULATORY
MOLECULES, IF YOU CAN ACTIVATE
THEM IN AN ECTOPIC PLACE, IF THE
CELLS ARE COMPETENT TO RECEIVE
THE SIGNAL, YOU CAN TURN ON THE
WHOLE SET OF DOWNSTREAM CELLULAR
PROTEINS IMPORTANT FOR GIVING
TISSUE INTEGRITY.
SO, THAT IMPLIES IF THAT'S TRUE
THAT THIS SORT OF INTERACTION IS
ALSO PRESENT IN THE MAMMALIAN
LARYNX, ANCESTRAL ALLIGATOR, AS
AVIAN SYRINX.
SO THAT'S TO SAY ELIMINATED
HOMOLOGY BUT STILL HAVEN'T
DECIDED BETWEEN DEEP HOMOLOGY OR
PROCESS HOMOLOGY VERSUS
CONVERGENCE.
SO TO DO THAT WE HAVE TO LOOK AT
MAMMALS AND CROCODILES.
FIRST WE'LL LOOK AT ALLIGATORS
TO SEE IF THEY HAVE THE SAME
GENES.
WE DON'T HAVE FUNCTIONAL DATA,
NOT MUCH, BUT WE HAVE SOME
EXPRESSION DATA.
SO YOU CAN SEE IN THE
ALLIGATORS, ALLIGATOR AS IN THE
CHICK, VERY SPECIFICALLY
EXPRESSES SOX9 AND RETINOIC
ACID SIGNALING AT VOCAL FOLDS,
NOT IN THE TRACHEA OR SYRINX,
THESE ARE IN THE LARYNX.
LARYNGEAL VOCAL FOLDS, THE ONES
I'M TALKING TO YOU WITH, DURING
THEIR ONTOGENY EXPRESS THE SAME
REGULATORS AS SYRINX DOES IN ITS
ONTOGENY.
YOU CAN SEE THE SAME SORT OF
THING IN THE MOUSE, HERE SHOWING
YOU THE CHICK SYRINX, AGAIN SOX
9.
YOU HAVE THE SAME EXPRESSION OF
MATRIX PROTEIN, SAME REGULATORS
ON RETINOIC ACID.
ONE FUNCTIONAL ANALYSIS, DOES
SOX9 HAVE THE SAME CAPABILITY
TURNING ON IN THE MAMMAL AS
BIRD?
EXPRESSING IN TRACHEAL
MESENCHYME SOX9 TURNS ON
HYALURONIC ACID, YOU CAN SEE IT
QUANTIFIED HERE.
AGAIN HERE.
NOT JUST HYALURONIC ACID BUT
VERSICAN.
MAMMALS AND CROCODILES EVOLVED
TO TAKE PLACE IN THE FORMATION
OF THE VOCAL FOLDS OF THE
LARYNX.
IN BIRDS, AS NEW VOCAL ORGAN WAS
FORMED, IT'S FORMED OUT OF
TOTALLY DIFFERENT TISSUE, WE
THINK IT CO-OPTED THIS
REGULATORY PATHWAY, AND BY
CO-OPTING UPPER END, GOT THE
DOWNSTREAM PARTS COMING FOR SHOW
WITHOUT HAVING TO INDEPENDENTLY
SELECT FOR EACH OF THE MOLECULES
AGAIN THAT HAVE BEEN OPTIMIZED
FOR VOCAL TISSUE.
NOW, I TOLD YOU WE DON'T KNOW
MUCH, BEFORE WE STARTED WORKING
ON THIS, NOTHING WAS KNOWN ABOUT
REGULATION OF VIBRATORY
MESENCHYME BUT ONE MORE PARALLEL
TO DRAW FOR YOU, NOT OF THE
VOCAL FOLD BUT EPITHELIUM, THAT
LIES ABOVE THE VOCAL FOLD.
SO, TWO DIFFERENT REPORTS IN THE
MOUSE HAVE SHOWN THAT THE
DEVELOPING VOCAL FOLD EPITHELIUM
NUMBER ONE IS STRATIFIED, YOU
CAN HIGHLIGHT THAT
STRATIFICATION WITH P63 IN A WAY
THE REST OF THE AIRWAY
EPITHELIUM IS NOT STRATIFIED.
AND THAT STRATIFIED EPITHELIUM
VERY SPECIFICALLY EXPRESSES --
ACTIVATED PHOSPHO-YAP.
THAT'S ALSO IN THE TRUE IN THE
SYRINX.
P63, SIMPLE EPITHELIUM,
STRATIFIED SPECIFICALLY AT THE
SYRINX LEVEL.
IN ADDITION, PHOSPHORYLATED IN
THE CHICK VOCAL FOLD EPITHELIUM.
NOW, PHOSPHORYLATION OF YAP IS
NEXT TO THE CELLS EXPRESSING
RETINOIC ACID, PHOSPHORYLATION
MIGHT BE DEPENDENT ON RETINOIC
ACID.
IF YOU TREAT WITH INHIBITOR,
PHOSPHO-YAP GOES AWAY.
WE THINK PHOSPHO-YAP IS -- SO
THIS PART OF THE -- WE THINK BY
BRINGING IN RETINOIC ACID,
UPSTREAM REGULATOR, MAY BE ABLE
TO SIMULTANEOUSLY AFFECT THE
VOCAL TISSUE AND OVERLYING
EPITHELIUM MIMICKING THE PROGRAM
IN THE LARYNX, BRINGING IT TO
THE SYRINX.
SO RETINOIC ACID UPSTREAM FOR
PRELIMINARY, WE THINK WE HAVE
THIS NETWORK HERE IN THE LARYNX,
IN THE SYRINX, WE HAVEN'T SHOWN
RETINOIC ACID IS UPSTREAM IN THE
HOUSE WHERE -- MOUSE WHERE IT
IS IN THE CRICK SYRINX.
THIS IS THE UNDERLYING DEEP
HOMOLOGY IN EVOLUTION OF THE
SYRINX.
THE NEXT LITTLE BIT IS JUST THE
TWO SECONDS I WANT TO BE
SPECULATIVE HERE.
WE ARGUED SYRINX EVOLVED FROM
THE LARYNX BY CO-OPTING SOME
REGULATORY NETWORKS THAT WERE
THERE.
WHETHER DID THE REGULATORY
NETWORKS OF THE LARYNX COME
FROM?
WE WANT TO RAISE THE POSSIBILITY
THAT WAS BUILDING ON SOME EVEN
EARLIER REGULATORY NETWORKS,
WERE IT NOT FOR VIBRATORY FISH,
FLOPPY SORT OF MOVABLE TISSUE,
THAT'S IN THE HEART VALVES.
I SAY THAT BECAUSE IF YOU LOOK
IN THE CHICK, THE CHICK HEART
VALVE HAS EXPRESSION OF
EXTRACELLULAR MATRIX PROTEINS
AND SOX9, SIMILAR FASHION
REMINISCENTS OF WHAT WE SHOWED
IN VOCAL FOLDS OF CHICK AND
MOUSE, MOREOVER IN ZEBRAFISH
FROM OTHER PEOPLE'S WORK WE KNOW
RETINOIC ACID IS REQUIRED IN
HEART VALVE PRECURSORS, SOX9 IS
UPSTREAM OF RETINOIC ACID.
THEY SEEM TO BE PRESENT IN HEART
AND LARYNX, BOTH ALREADY AROUND
IN THE ANCESTOR OF BIRDS, THESE
WERE CO-OPTED, REGULATORY
NETWORK TO GIVE YOU THE SYRINX.
SO, FINALLY, TO JUST TELL YOU
THE SORT OF HIGHLIGHT -- TO
HIGHLIGHT WHAT YOU TOLD YOU, HOW
DID BIRDS EVOLVE CAPACITY TO
VOCALIZE, THEY BUILT A VOCAL
ORGAN FROM SCRATCH, OUT OF
CARTILAGE IN THE CHEST, GETTING
AROUND THE MAYHEM THAT NORMALLY
HAPPENS WHEN TURING MECHANISMS
SMASH INTO EACH OTHER, THROUGH
THE PROCESS OF RETRACTION OF
HEDGEHOG GRADIENT.
CO-OPTED FROM A VOCAL FOLD
PROGRAM IN THE LARYNX TO MAKE
VIBRATORY TISSUE.
THESE PROCESSES TOGETHER SORT OF
THE BEGINNING OF THE INROAD TO
TRY TO UNDERSTAND HOW YOU MAKE A
TRULY NOVEL STRUCTURE IN
EVOLUTION OF VOCALIZATION OF
BIRDS.
I WANT TO FINISH BY THANKENING
THE PEOPLE -- THANKING THE
PEOPLE WHO DID THE WORK.
EVAN KINGSLEY DID WORK ON THE
CART LAN, CHARLIE LONGTINE ON
TISSUE, JULIA CLARK AND FRANZ
GOLLER, BOTH WORKING ON OTHER
ASPECTS OF THE EVOLUTION OF THE
SYRINX AND FUNCTION OF THE
SYRINX.
I DIDN'T HAVE TIME TO TALK ABOUT
THAT.
FINALLY A SHOUT OUT TO MIKE
DYER, AT ST. JUDE IN TENNESSEE.
EVAN WAS A POSTDOC IN MY LAB.
AFTER HIS FIRST YEAR, HIS WIFE,
A HISTORIAN, THEY DON'T HAVE TO
DO POSTDOC, CAN GO STRAIGHT TO
FACULTY, GOT A JOB AFTER
GRADUATE SCHOOL, UNIVERSITY OF
MISSISSIPPI IN OXFORD.
THEY HAD A BABY.
EVAN DIDN'T WANT TO BE APART
FROM HIS WIFE BUT DIDN'T WANT TO
GIVE UP HIS PROJECT.
MIKE DYER DOES NOT WORK ON AVIAN
SYSTEM, HE WORKS ON MICE, ON
CANCER, GENEROUSLY GAVE US A
BENCH IN HIS LAB, ALLOWED EVAN
TO WORK AT ST. JUDE ON PROJECTS
FROM MY LAB, SATELLITE
OPERATION.
IF NOT FOR THAT GENEROSITY
COULDN'T HAVE DONE THE PROJECT.
I'LL STOP THERE AND TAKE ANY
QUESTIONS YOU WANT TO RELAY.
>> THANK YOU VERY MUCH.
THAT IS REALLY, REALLY
FASCINATING EVOLUTIONARY STORY
THERE.
BEFORE WE TAKE QUESTIONS, I'D
LIKE TO REMIND PEOPLE THAT ARE
APPLYING FOR CONTINUING MEDICAL
EDUCATION CREDITS THAT THE CME
CODE FOR TODAY IS 37935.
THAT'S 37935.
OKAY.
SO, OUR FIRST QUESTION COMES
FROM SUSAN.
SHE SAYS, HI, CLIFF, TERRIFIC
TALK.
ARE VOCAL CORDS -- FOLDS
UNIFORMLY ABSENT IN LARYNX OF
ALL BIRDS?
FOR EXAMPLE, DIVING BIRDS?
ANY CLUES ON HOW LARYNGEAL
FOLDS, CAN SOX9 INDUCE THEM?
>> SOX9 -- ONE AT A TIME.
ALL BIRDS THAT WE'VE LOOKED AT
HAVE -- ALL BIRDS DESCRIBED HAVE
A SYRINX AND NOT A LARYNX.
MY OWN LAB WE'VE DONE WORK ON
SOME LIKE OSTRICHES AND EMUS,
ONLY DIVING BIRD IS PENGUIN.
WE HAVE DATA ON BIRDS FROM MY
LAB, IT'S VERY WELL ESTABLISHED,
THE SYRINX -- BIRDS HAVE A
FUNCTIONAL SYRINX AND
NON-FUNCTIONAL LARYNX,
THROUGHOUT THE CROWN BIRDS.
WHETHER YOU CAN MAKE THE
LARYNX -- INDUCE THE LARYNX TO
HAVE VOCAL FOLDS, YOU CAN INDUCE
GENE EXPRESSION AT THE LEVEL OF
LARYNX BUT SORT OF DIDN'T
EMPHASIZE THE POINT, YOU GET
CELLULAR PROPERTIES WITHIN THE
TRACHEA, MISEXPRESS SOX 9 OR
RETINOIC ACID, THERE'S SWELLING,
TISSUE GETS THICKER AND NOT
SURPRISING, MAKE HYALURONIC
ACID, AND WE HAVE CHANGES IN
EPITHELIUM, STRATIFIED
EPITHELIUM, THE YAP,
PHOSPHO-YAP, CAN BE ACTIVATED.
YOU CAN DO ALL THAT WITHIN THE
LARYNGEAL REGION BUT THEY DO NOT
MAKE VIBRATORY TISSUE.
EVERYTHING I'VE TOLD YOU ABOUT
THIS REGULATION IS SUFFICIENT TO
GIVE YOU A THICKENED MESENCHYME,
SORT OF THE SOURCE OF THE
VIBRATORY TISSUE, SUBSEQUENT
DEVELOPMENTAL STEPS ARE NEEDED
TO TAKE THAT PATTERN TISSUE AND
TURN IT INTO VIBRATORY TISSUE.
WE'VE NOT DONE THAT IN THE
TRACHEA OR LARYNX OF THE BIRD.
HI TO YOU AS WELL, SUSAN.
HOPE TO TALK TO YOU SOON.
>> OKAY.
HERE IS A QUESTION FROM MARION
MARIAN YOUNG.
IS THE STRICTURE OF THE PHARYNX
DIFFERENT, THEIR -- I'M SORRY,
THE SCREEN IS CHANGING.
>> I'M TRYING TO GET OUT OF
SHARE SCREEN.
NOT SUCCESSFUL.
>> YEAH, MADE MY SCREEN BLINK
FOR A MOMENT.
MEN OFTEN HAVE LOWER VOICES THAN
WOMEN.
IS THE STRUCTURE OF THE SYRINX
DIFFERENT IN MALES AND FEMALES
IN THE BIRDS OR THEIR RESPONSE
TO RETINOIC ACID?
>> SO, ANOTHER PROJECT THAT'S
GOING ON IN MY LAB, I'M GOING TO
LEAVE IT LIKE THIS IF THAT'S
OKAY, ANOTHER PROJECT IN MY LAB
IS FOCUSING ON THE SYRINX OF THE
DUCK.
SO SHORT ANSWER, IF YOU LOOK IT
THE CHICK, NO DIFFERENCE BETWEEN
MALE AND FEMALE CHICK, OF COURSE
CHICKS WHEN THEY ARE BORN ARE
NOT SEXUALLY DIMORPHIC UNTIL
TESTOSTERONE HITS AT A LATER
STAGE, OR ESTROGEN KICKS IN.
EVEN IN ADULT SYRINX IT'S
SLIGHTLY DIFFERENT IN SIZE
BECAUSE ROOSTER IS BIGGER THAN
THE HEN, THEY ARE NOT DIFFERENT
IN FORM AT ALL.
IN THE DUCK, IT'S A VERY
INTERESTING STRUCTURE CALLED THE
BULLA, LIKE THIS OUTPOCKETING OF
THE SYRINX JUST ON ONE SIDE, ON
THE RIGHT-HAND SIDE, ONLY IN THE
MALE.
SO IT'S SEXUALLY DIMORPHIC, USED
TO MAKE A MATING WHISTLE.
SO LIKE A BOTTLE YOU BLOW AIR
ACROSS THE TOP.
IT'S WHAT THE BULLA IS USED FOR.
IT'S THE CONVERGENCE OF THE LEFT
ASYMMETRY PATHWAY, IT TURNS OUT
THE MALE ASYMMETRY IS THE
DEFAULT.
SO LEFT IN THE DUCK.
IF YOU DIDN'T HAVE ANY SEX
STEROIDS, YOU WOULD GET
ASYMMETRIC SYRINX, NO OTHERS
HAVE THAT EXCEPT DUCKS AND
RELATIVES GEESE AND SO ON.
IN THIS GROUP, MAKES ASYMMETRIC
SYRINX, THERE'S A CONVERGENCE,
THAT'S ONE CASE WITH SEXUAL
DIMORPHISM.
OTHER BIRDS ARE SEXUALLY
DIMORPHIC, JUST IN THICKNESS AND
SIZE OF TISSUE.
MALE AND FEMALE PARROTS HAVE
DIFFERENT SIZES, FOR INSTANCE.
BUT THEY DON'T HAVE THE
LEFT-RIGHT SYMMETRY OR
DIFFERENCE IN MORPHOLOGICAL
CHANGE.
>> OKAY.
ANOTHER QUESTION FROM DAVID
LEVIN, TERRIFIC TALK, HOW DOES
THE DEVELOPMENT, I'M NOT GOING
TO SAY THIS CORRECTLY, CECTASIAN
PHONIC LIPS COMPARE WITH SYRINX
OR LARYNX, IS IT A THIRD PATTERN
OR DERIVED FROM THE LARYNX?
>> I HAVE NO IDEA.
>> OKAY.
LET'S SEE.
FROM CHARLES SNYDERMAN, DO
TERASAURS A PHARYNX AND LARYNX.
>> THESE ARE CARTILAGE NOWS, NOT
BONE.
THEY DON'T FOSSILIZE WAY.
A FEW CASES YOU CAN GET
FOSSILIZED CARTILAGE, I SHOWED
YOU A CRUSTACEOUS BIRD.
OUR COLLABORATOR IS TRYING TO
FIND THAT.
GETTING A TERASAURS, ONE THING
THAT IMPLIES, DOES FLIGHT HAVE
SOMETHING TO DO WITH CHANGE IN
STRUCTURES.
IT'S POSSIBLE.
THERE ARE A NUMBER OF ARGUMENTS
FOR WHY THE LARYNX CHANGED USE,
REPLACED BY THE SYRINX, ONE OF
THE ARGUMENTS HAS TO DO WITH
SORT OF THE PHYSIOLOGY WHERE
SOME OF THE DINOSAUR AND
EARLYBIRDS HAD LONG NEXT, MAKING
A LOUD SOUND FROM SOMETHING WITH
THAT HIGH A STANDING WIND AIR
COLUMN WOULD BE DIFFICULT.
ONE OF THE OTHER ARGUMENTS, YOU
DO HAVE A CHANGE IN LOCATION OF
THE HYLOID BONE, CHANGE IN
BREATHING, FLIGHT ADAPTATIONS
THAT MAY HAVE MADE IT DIFFICULT
TO USE THE LARYNX FOR
VOCALIZATION IN WHICH CAUSE THE
TERASAUR MIGHT HAVE COME UP WITH
A YET A THIRD SOLUTION BUT WE
DON'T HAVE RECORDS OF THE AIRWAY
OF TERASAURS OR NON-AVIAN
DINOSAURS.
>> A QUESTION FROM CHRIS.
FASCINATING TALK.
IS THERE ANYTHING UNIQUE ABOUT
BIRD BIOLOGY SUCH AS FEATHERS
THAT WOULD HAVE STIFFENED THE
LARYNX VOCAL CORDS OTHERWISE
PREVENT THEM FROM EVOLVING TO
VIBRATE, OR IS THE SYRINX
ACTUALLY BETTER AT PRODUCING THE
RANGE OF SOUNDS IMPORTANT FOR
BIRD MATING?
>> SO, WHEN YOU SAY BETTER, I
THINK THE BIRDS NEED TO BE LOUD.
BECAUSE THEY HAVE TO BE LOUDER
THAN MAMMALS, BECAUSE THEY HAVE
TO COMMUNICATE OVER MUCH LONGER
DISTANCES, BECAUSE THEY CAN FLY
SO QUICKLY BETWEEN PLACES.
SO ONE OF THE THINGS THE SYRINX
HAS TO DO IS BE EFFICIENT.
MUCH MORE SO THAN THE LARYNX.
BUT THE -- BEYOND THAT, THERE'S
NOTHING ABOUT THE LARYNX
BECOMING STIFFER OR NOT WORKING,
BASICALLY EVERY ANIMAL THAT HAS
A LARYNX, EVERY LARYNX THAT HAS
A VOCAL CORD, VOCAL CORDS WORK
PERFECTLY WELL.
OPTIMIZE FOR THE ENVIRONMENT,
UNDER WATER, IN THE AIR, WHAT
HAVE YOU.
THEN YOU HAVE THE AVIAN LINEAGE,
TOTAL ABSENCE OF VOCAL CORDS, IN
THE LARYNX.
YOU COULD HYPOTHESIZE THAT YOU
CHANGE THE STRUCTURE IN THE
LARYNX AND THAT'S WHY IT BECAME
USELESS, AFTER IT WAS STIFF IT
WAS THEN LOST.
I'M NOT SURE HOW YOU TEST THAT.
BUT GIVEN -- THEY MAKE PERFECTLY
GOOD VIBRATING TISSUE IN THE
SYRINX, THERE'S NO REASON WHY
ANYTHING ABOUT A FEATHER PROGRAM
OR ANYTHING ELSE WOULD INTERFERE
WITH MAKING THE SOFT TISSUE IN
ONE PLACE AND NOT THE OTHER.
I WOULDN'T EXPECT THAT TO BE A
PROBLEM.
BUT THERE'S NO WAY TO TEST THAT.
>> REALLY FASCINATING ASPECT OF
YOUR TALK IS THE TURING
MECHANISM FORMING THE BANDS OF
CARTILAGE.
ARE YOU AWARE OF ANY OTHER
SYSTEM IN BIOLOGY THAT USES SUCH
A MECHANISM?
>> SO, A NUMBER OF CASES WHERE
YOU HAVE TURING MECHANISMS
PROPOSED FOR PATTERNS,
ESPECIALLY CODE PATTERNS, CICLID
FISH, ZEBRA STRIPING.
PERFECTLY PARALLEL STRIPES
PEOPLE HAVE NOT BEEN ABLE TO GET
OUT OF PURE TURING MECHANISMS,
THERE'S -- SHOOT.
BLOCKING ON SOMEONE'S NAME, IT'S
IMPORTANT, GIVE ME HALF A
SECOND.
I DEEPLY APOLOGIZE, BLOCKING ON
SOMEONE'S WORK AT PRINCETON,
UNPUBLISHED WORK, IT WOULDN'T
MAKES STRIPES BUT REQUIRED
ORTHOGONAL GRADIENT.
WORKS DIFFERENTLY THAN OURS
DOES, GROUND SQUIRRELS, SAME
RESULT.
>> OKAY.
>> LANERO LAB AT PRINCETON.
>> COULD YOU SAY THAT AGAIN.
>> MULINARO LAB AT PRINCETON.
>> GREAT.
>> SECOND INSTANCE WHERE
PARALLEL STRIPES WERE DERIVED
FROM TURING MECHANISM COULDN'T
DO IT WITHOUT A REFERENCE
GRADIENT, ALTHOUGH THE REFERENCE
GRADIENT IS DIFFERENT IN THAT
CASE.
>> ONE LAST QUESTION FROM TOM
SCHNEIDER.
DO VOICES DIFFER BECAUSE OF THE
IRREGULAR RINGS?
>> NO, BECAUSE THE IRREGULAR
RINGS ARE BELOW THE LARYNX.
THE LARYNX DOES NOT HAVE
IRREGULAR LINKS.
THEY ARE THE BRONCHIO TRACHEAL
JUNCTION, STILL HOLD THE AIRWAYS
OPEN.
ALL THAT MATTERS TO GET TO THE
LARYNX, SMOOTH LAMINAR AIR FLOW
THROUGH THE AIRWAY.
THE LARYNX ITSELF DOESN'T HAVE
THAT PROBLEM.
IT'S A LONG WAY FROM THE
BRONCHI.
>> OKAY.
LET'S SEE.
I THINK WE'RE OUT OF TIME, AT
THE MOMENT.
AND I WANT TO THANK YOU AGAIN.
THERE IS A FEW QUESTIONS IN THE
CHAT BOX THAT WE CAN SEND TO YOU
THAT PERHAPS YOU'LL HAVE A
CHANCE TO ANSWER.
BUT THANK YOU SO MUCH AGAIN FOR
JOINING US.
AND WE HOPE THAT YOU HAVE A GOOD
REST OF YOUR WEDNESDAY
AFTERNOON.
>>THANK FOR FOR INVITING ME.