lec 12 - limb development

3 principle axes of the limb

• proximo-distal (PD)

• AP

• DV

PD axis runs from

humerus → digits

parts of the PD axis

stylopod, zeugopod, autopod

the AP axis runs from

thumb → pinkie

DV axis runs from

back of hand → palm of hand

the early limb bud consists of _____ surrounded by _____, with a ___

mesenchyme cells

an ectodermal (epithelial) layer,

thickened distal ridge called the AER

what are the 3 key signaling centers and which axes do they control

ZPA → AP

AER → PD

Dorsal ectoderm → DV

ZPA stands for

Zone of polarizing margin

the ZPA expresses ___ and specifies _____ via ____

Shh

AP digit identity

morphogen gradient

AER =

apical ectodermic ridge

the AER expresses ___ and specifies _____

FGF8

PD outgrowth and patterning

the Dorsal Ectoderm expresses ____ and specifies ____

the Ventral Ectoderm expresses ____ and specifies ____

Wnt7a; dorsal identity

BMP/Eg1; ventral identity

together pattern the DV axis

limbuds first appear as

bumps at defined positions along the AP axis

Limb bud positioning is determined by

Hox gene products in the lateral plate mesoderm

hox genes - forelimb position determed by

Hox4 and Hox5

hox genes - hindlimb position specfied by

Hox9-100

____ provide positional information interpreted by Hox proteins

signaling gradients like RA or FGF

Hox gene products activate

limb type-specific TFs

limb type-specific TF function

determine forelimb vs hindlimb identity

limb type-specific TFs - what specifies the forelimb

Tbx5

• T-box TF

limb type-specific TFs - what specifies the hindlimb

Tbx4 + Pitx1

• T-box TFs

Describe the takeuchi experiment

Ectopic Tbx5 expression in the leg → feathers normally restricted to wing appear.

Ectopic Tbx4 in the wing → four digits (like leg).

Demonstrates these TFs can re-specify some limb identity features.

describe the mingullion experiment

Forced expression of Pitx1 transgene in forelimb → elbow develops a knee-like articulation. Confirms Pitx1 contributes to hindlimb-specific morphology.

describe the limb bud initiation loop

1. Tbx4/5 induces Fgf10 in the lateral plate mesoderm/early limb bud mesenchyme via Wnt signaling

2. Fgf10 signals to the overlying ectoderm via Wnt 3a to induce Fgf8 in the AER (PD axis)

3. Fgf8 signals back to the limb bud mesenchyme to maintain Fgf10 expression, creating a self-sustaining feedback loop

4. this loop drives PD outgrowth

Fgf10 KO in mouse →

complete absence of limbs

descirbe the Cohn and Tickle experiments

an Fgf-soaked bead grafted into the interlimb flank is sufficient to induce an entire ectopic limb

cohn and tickle experiments - what was the identity of the limb they grew in the interlimb flank

identity of the limb (wing-like vs leg-like vs chimeric) depended on how close the bead was to the Tbx5/Tbx4 expression domains, which expanded into the new bud.

conclusions of the cohn/tickle experiments

Fgf induction is sufficient in inducing limb growth

Fgf does not directly induce Tbx 4/5

The AER is essential for ____ outgrowth

PD

what did Saunders test for

what would happen if you surgically removed the AER

what happens if you surgically remove the AER

timing-dependent

• remove early → only humerus forms

• the later the removal, the more distal the elements that are lost become

  • the later the removal, the more complete the skeleton

Saunders AER removal experiment - conclusion

• AER is required for PD outgrowth

• skeletal elements are laid down in a proximal-to-distal order

three models of PD patterning

progress zone model

two-signal model

signal-progress zone model

progress zone model was found by

summerbell and wolpert

describe the progress zone model

Limb patterning depends on timing/how long you stay in the progress zone

• Undifferentiated cells at the distal tip (progress zone) are maintained by AER-Fgfs

• Cells leaving early → proximal fate (stylopod)

• Cells leaving late → distal fate (zeugopod, then autopod)

• Timing relates to progressive Hox gene activation

describe the two-signal model

pattern depends on opposing gradients

• high RA from the flank specifies stylopod (proximal fate)

• high FGF from AER specifies autopod (distal fate)

• as the limb grows, both drop → zeugopod (intermediate fate)

what is the issue with the two-signal model

This implies that the proximal and distal structures are developed first, and then intermediate structures are formed.

However this contradicts the AER removal experiment and the Hox gene layout (which patterns the same way as the body forms, humerus → digits), as both show that limb patterning occurs from proximal to distal.

what is the current consensus on PD limb patterning

signal-progress zone model - limb patterning depend on both timing and morphogen concentration

describe the signal-progress zone model

high RA early

• prevents progress zone from forming

• specifies proximal stylopod before progress zone is established

• FGFs antagonize RA from AER, allowing a distal progress zone to form

• once the progress zone exists, timing of the cells leaving the progress zone specifies zeugopod and autopod

_____ are colinearly expressed along the PD axis

HoxA and HoxD gene paralogs

Hox genes in PD patterning

____ → stylopod

____ → zeugopod

____ → autopod

• Hox10

• Hox11

• Hox12/13

in what order do HoxA and HoxD genes express along the PD axis

10 → 13, 3’ → 5’, proximal → distal

what are hox paralogs

each Hox gene has a number and a letter. two genes with the same number but a different letter are paralogs

HoxA11 + HoxD11 + HoxC11 triple KO

zeugopod fails to form (both fore- and hindlimb)

HoxA13 + HoxD13 double KO

autopod cartilage fails to form (soft tissues remain but no phalanges)

Hox gene paralog KO to cause zeugopod to fail to form

HoxA11/D11/C11 triple KO

Hox gene paralog KO to remove phalanges and cartilage

HoxA13/D13 double KO

ZPA

zone of polarizing activity (aka polarizing region)

ZPA is important for ___ patterning

AP

who discovered the ZPA and how

Saunders and Gasseling

• grafted tissue from posterior margin to anterior margin of host wing bud

• result: mirror image duplications of digit pattern (321123)

  • grafts of only a few cells → only digit 1 induced

• conclusion: the region must produce a morphogenic signaling molecules in the posterior margin - ZPA

ZPA morphogen hypothesis

predicted the ZPA produced a diffusible morphogen forming a posterior → anterior gradient

• high ZPA concentration = posterior digit identity (pinkie)

• intermediate = digit 2

• low = digit 1/thumb

what is the ZPA morphogen

Shh

How do we know that Shh is the ZPA morphogen + who found out

Riddle et al

Shh-expressing cells/Shh beads grafted to the anterior margin produced concentration-dependent digit duplication - same as ZPA grafts

Shh specifies digit identity in tissue adjacent to ZPA

non-cell-autonomous

caused by external factors

what did Towers’ GFP fate mapping find

ZPA cells do not contribute to the duplicated digit skeleton.

Shh acts on host tissue in a non-cell-autonomous manner.

Shh takes 12 hours to pattern the limbs

how long does it take for Shh to specify digits 1→3 + how do we know + who did it

12 hours

towers et al

• Cyclopamine (blocks Shh pathway) applied at 4hr intervals.

• 3 administartions of cyclopamine (12 hrs) for all 3 chick wing digit identities to be specified, from digit 1→3

wolley et al

• made a mathematical model of Shh gradient, found the same timeframe

how are more than 3 digits patterned

timing - length of Shh exposure

• ZPA cells are specified as digit 4 and 5 because they were exposed to Shh the longest

Shh signaling operates through

Gli TFs

Describe the Gli3A/R gradient + what it depends on

Gli3 activator posteriorly, Gli3 repressor anteriorly, forms a gradient

depends on Shh concentration (high posterior, low anterior)

Shh/Gli3 targets

Ptch1

Gremlin1

Tbx2

Tbx3

What are the current strongest downstream Shh signaling candidates for specifying digit identity

Tbx2 - digit 4

Tbx3 - digit 3

what is AERMF

BMP antagonist secreted by Gremlin (Shh target)

explain how AP and PD patterning are coordinated

context

• AER-FGF4/8 are required for Shh expression

• BMP inhibits AER-FGF4/8

• Gremlin1 encodes BMP antagonist AERMF

gremlin feedback loop

• Shh → gremlin

• gremlin —| BMP, which → AER-FGF4/8

• AER-FGF4/8 → Shh

how is Shh only expressed in posterior limb?

ZRS (ZPA regulatory sequence) controls it

• TFs like Hand2 and HoxA/D bind to ZRS

• chromosome looping → ZRS contacts Shh promoter → TFs swithc on Shh transcription posteriorly

• transcriptional repressors also bind ZRS to prevent Shh from being expressed anteriorly

ZRS mutation + example

Shh expressed ectopically at anterior margin → polydactyly

• ex: hemingway cats wihth polydactyly on anterior margins of paw

____ controls DV polarity

the ectoderm

how do we know that the ectoderm instructs DV identity

classic chick experiment - ectoderm was removed from the mesoderm of an early chick wing bud, rotated 180 degrees (flipped DV), then replaced.

result: DV polarity of mesoderm was reversed, even though only the ectoderm was rotated - showing that the ectoderm instructs mesoderm DV identity

what is the default state - dorsal or ventral

ventral

which signaling molecules are important for DV patterning

Wnt (Wnt7a)

BMPs

explain how DV patterning is set up

• dorsal ectoderm: Wnt7a → TF Lmx1b → dorsal fates

• ventral ectoderm: BMPs → Engrailed1 —| Wnt7a ventrally → ventral fates

Wnt7a mutant mouse

Lmx1b is not induced, whole limb becomes ventral (double ventral pattern

Lmx1b mutant

same double ventral outcome as Wnt7a mutant - Wnt7a can express but cannot act without Lmx1b

Engrailed1 mutant

Wnt7a expressed in the entire ectoderm → Lmx1b induce everywhere → whole limb becomes dorsal (double dorsal)