Chapter 19 Part 3: Specifying Anterior-Posterior Axis

Question 1

the anterior-posterior axis is determined by the ___ ___ ___

Answer 1

zone of polarizing activity (ZPA)

Question 2

____ is expressed in the ZPA in order to induce anterior-posterior patterning

Answer 2

SHH

Question 3

Outline the effects of the SHH gradient in anterior-posterior patterning

Answer 3

cells that expressSHH become the POSTERIOR aspect of the limb (pinky), whereas cells that express lesser/least amounts of SHH become more ANTERIOR fingers (thumb)

Question 4

How does the pinky get formed?

Answer 4

Due to really high SHH via autocrine mechanisms in the posterior region of the limb bud (ZPA).
SHH expresion in the posterior region of the limb bud is really high, and in addition to being exposed to SHH by the ZPA, cells of posterior limb bud ALSO SECRETE SHH (double dose), resulting in pinky formation.

Question 5

which mammalian digits are specified by SHH?

Answer 5

digits 5 (pinky), 4 (ring), and part of three are formed from the descendants of SHH secreting cells– these fingers’ cells themselves secreted SHH

-IN the AER, once the limb bud growth is finished, cells of the AER undergo apoptotic sequences, and with nothing sustaining the reaction/diffusion mechanisms, the mesenchyme undergoes chondrogenesis and bone formation, with more posterior-located descents (shh-secreting cells) becoming bones and muscle of the posterior limb.

Question 6

IN the AER, once the limb bud growth is finished, cells of the AER undergo ____ sequences, and with nothing sustaining the ____/____ mechanisms, the mesenchyme undergoes ____ and bone formation, with more posterior-located descents (_____-secreting cells) becoming bones and muscle of the posterior limb.

Answer 6

IN the AER, once the limb bud growth is finished, cells of the AER undergo apoptotic sequences, and with nothing sustaining the reaction/diffusion mechanisms, the mesenchyme undergoes chondrogenesis and bone formation, with more posterior-located descents (shh-secreting cells) becoming bones and muscle of the posterior limb.

Question 7

digit ___ has some cells that express SHH for shorter times than those in the ring finger

Answer 7

digit 3. (middle)

Question 8

Which digit contains cells that doesn’t produce SHH, but still relies a bit on ZPA-SHH?

Answer 8

digit 2. Does not produce any SHH and relies 100% on the diffusion from the ZPA.

Question 9

SHH specifies digits posterior to anteriorly using both concentration and time dependent mechanisms. What are the two temporally distinct mechanisms of digit specification?

Answer 9

1) specifying digit identity by making SHH act as a morphogen (SHH in any given area)
2) proliferation and expansion of the limb bud mesenchyme by making SHH act as a mitogen (ZPA SHH).

Question 10

the first temporally distinct mechanism of digit specification is specifying digit identity. In this step, digit indentities are first specified by the concentration of ___ in that region of the limb bud, and THEN by the duration of exposure.

Answer 10

the first temporally distinct mechanism of digit specification is specifying digit identity. In this step, digit indentities are first specified by the concentration of SHH in that region (a given region) of the limb bud, and THEN by the duration of exposure to SHH.

Question 11

SHH in a given region of the limb bud establishes digit identity through the ___ pathway

Answer 11

SHH establishes digit identity through the BMP pathway

Question 12

Actions of SHH in a given region of the limb bud lead to the ____ activation of ____, which acts as a transcriptional effector. This results in ___ (BMP INHIBITOR) deactivation, and the upregaultion of ____ ACID. What does this cause?

Answer 12

Actions of SHH lead to the GRADED activation of Gli3, which acts as a transcriptional effector. This results in GREMLIN (BMP INHIBITOR) deactivation, and the upregaultion of HYALURONIC ACID.

This promotes the BMP-stimulated differentiation into cartilage.

Question 13

SHH is able to establish digit specification in a given region of a limb bud by promoting BMP-stimulated differentiation into cartilage, but how does it cause a restriction in proliferationin these cartilage progenitor cells (proliferation must first be stopped before allowing the cartilage progenitor cells to become chonrdrocytes)

Answer 13

SHH (through Gli3) also downregulates Cdk6, which is involved in proliferation.

Question 14

SHH initiates and sustains a graident of BMP proteins across the limb bud, and this BMP gradient can specify the digits. The identity of each digit is determined by ____ mesoderm.

Answer 14

interdital mesoderm, that is, by the webbing between the digits (the region of mesenchyme that will shortly undergo apoptosis).

The positional value of the webbing can be altered by changing the BMP level. Different levels of BMP signalling in the interdigital webbing regulates the recruitment of progress zone mesenchymal cells into the nodules that make the digits.

Question 15

the interdigit mesoderm specifies the ___ digit to it .

Answer 15

specifies the digit anterior to it. If you were to remove webbing between the cartilagenous condensations forming chick hindlimbs digits 2+3, the 2nd digit will change into a copy of digit 1.

the different levels of BMP signalling in the interdigital webbing due to SHH gradients regulate the recruitment of progress zone mesenchymal cells (PZ mesenchyme that is proliferating) into the nodules that make the digit.

Question 16

After SHH acts in a given area of a limb bud as a morphogen to regulate BMP in the interdigit mesenchyme to specify digits, what is the second mechanism (temporally distinct) that SHH is involved in?

Answer 16

SHH can also act as a mitogen to enhance proliferation and expansion of limb bud mesenchymal cells. This allow helps to shape the limb bud. In this case, this SHH comes from the ZPA.

First, the digits are specified and then as the limb bud grows, the ZPA is esablished and another loop is created for increased prliferation.

Question 17

To maintain proliferation of mesenchyme (in addition to fgf8 and fgf10 mechanisms), what does SHH of the ZPA do?

Answer 17

when the ZPA is established, it secretes SHH which ACTIVATES gremlin, which inhibits BMPS (BMPS usually repress fgf8s in the AER), thus promoting the maintenance of fgf8 and fgf10 expression and continued limb bud outgrowth.

Question 18

Explain the feedback loops established by SHH from the ZPA in order to sustain proliferation and limb bud growth

Answer 18

When fgf10 in mesenchyme activates fgf8 in the above AER, the first positive feedback loop is established so that the limb bud formation and growth occurs, and so that the AER can go to regulate limb patterning (like proximal distal patterning)

When the ZPA forms, SHH is secreted which forms the basis of the second positive feedback loop.
First, SHH in the ZPA activates gremlin, inhibiting any BMPS that may block FGF synthesis in the AER. This promotes fgf8 synthesis in the AER. Fgf8 signals INHIBITS etv4 and etv5 (both in the POSTERIOR region of the limb bud, ensuring that SHH stays in the posterior region), which are all repressors of SHH so that SHH stays in the posterior region.

*in the anterior region, Fgf8 ACTIVATES etv4/5, inhibiting SHH and continues to allow SHH to be expressed in the posterior region.

As fgf in AER continues to climb, it eventually reaches a threshold where it inhibits Gremlin, thus allowing BMPS to begin repressing the AER FGFs. As more cells multiple in the area, and are not expressing gremlin, the gremlin signal near the AER is too weak to prevent the BMPs from repressing the FGFs. The AER FGF thus get repressed by BMP, and the AER disappears, removing the signals that stablize the ZPA. The ZPA then also disappears, resulting in the END of the ebmryonic phase of limb development.

Question 19

When the ZPA forms, SHH is secreted which forms the basis of the second positive feedback loop.
First, SHH in the ZPA activates gremlin, inhibiting any BMPS that may block FGF synthesis in the AER. AER thus can produce fgfs. What are the two feedback loops that link the ZPA and the AER?

Answer 19

1) fgf8 produced by the AER (because SHH inhibits BMPS which inhibit AER) can inhibit Gremlin at low concentrations. Because Gremlin is inhibited, the AER continues to be sustained and allowing for proliferation and expansion of limb bud.
2) the fgfs in the AER (4, 9, 17) can activate SHH, stabilizing the ZPA.

Question 20

How is it ensured that ZPA SHH doesn’t migrate to the anterior region of the limb bud?

Answer 20

Fgf8 in the AER activates Etv4/5 in the anterior limb region, which are repressors of SHH. This makes sure that SHH isn’t being expressed too anteirorly. Fgf8 however does NOT activate etv4/5 in the posterior region.

Question 21

When does the embryonic phase of limb development done?

Answer 21

When SHH allows for AER to be maintained because SHH inhibits BMPS through gremlin upgregulation, Fgf8 concentrations from AER continues to grow. When it reaches a certain threshold, it can INHIBIT GREMLIN. Wihthout gremlin, BMPS can now repress AER. Thus, as the cells multiply in the mesenchyme, and none of them have Gremlin, the BMPs repress Fgfs in AER, ultimately removing the effects of AER. With the AER gone, the Fgfs (even at lower concentrations) cannot induce SHH and thus the ZPA is not stabilized. The ZPA then also disappears, and there is an ATTENUATION OF EXPRESSION OF ALL PLAYERS.

Question 22

HOX GENES IN DIGIT SPECIFICATION:

• hox genes are critical for specifying fates along each axis of the limb, and their expression, especially that of the Hoxd cluster, functions in 2 phases:

Answer 22

1) first phase is involved in the specification of the stylopod and zeugopod
2) later phase helps specify the autopod.

these Jox genes are usually WHAT MAKE THE ZPA SHH

Question 23

2 regulatory regions involved in the early phase of digit specification that work to activate Hoxd genes (in a temporal and spatial array)

Answer 23

These regions are composed of enhancers to activate Hoxd genes

1) early limb control regulatory ELCR: activaets transcription in a time-dependent manner.
2) POST posterior restriction: imposes spatial restrictions on the expression of Hoxd genes.

Question 24

What is the ELCR

Answer 24

early limb control regulatory region. Activates transcription of hox genes in a time-dependent manner.

Question 25

What is the POST

Answer 25

posterior restriction regulatory region: imposes spatial restrictions on the expression of Hoxd genes, such that the genes CLOSEST to this region have the most restricted expression domains,
starting from the posterior margin of the limb bud

Question 26

How do Hox genes create the ZPA?

Answer 26

The POST region imposes spatial restrictions of Hoxd genes starting from the posterior margin of the limb bud. Creating this pattern of Hoxd proteins activates the LONG RANGE ENHANCER (ZRS) of the SHH gene, thereby activating SHH expression in the posterior limb bud mesoderm and forming the ZPA.

Question 27

The first phase of Hoxd expression is initiated as the limb bud forms. The ___ ____ ____ ____ (ELCR) element activates those genes closest to it (Hox10) earlier than those genes away from it (hox13), whereas the ____regulatory element acts nega- tively to restrict the anterior expression of these genes in the opposite direction.. Ie, POST activates Hox13 before Hox10.

This results in expression domains such that Hoxd13 expression is confined to the most posterior region, whereas Hoxd12 is allowed to expand more anteriorly. The 5′ Hoxd (Hox11, 12, 13) genes activate the long-range Shh enhancer (ZRS), thereby creating the ZPA in the posterior limb mesoderm.

Answer 27

The first phase of Hoxd expression is initiated as the limb bud forms. The early limb control regulatory (ELCR) element activates those genes closest to it earlier than those genes away from it, whereas the POST regulatory element acts nega- tively to restrict the anterior expression of these genes in the opposite direction. (B) This results in expression domains such that Hoxd13 expression is confined to the most posterior region, whereas Hoxd12 is allowed to expand more anteriorly. The 5′ Hoxd genes activate the long-range Shh enhancer (ZRS), thereby creating the ZPA in the posterior limb mesoderm.

Question 28

Essentially, ELCR (top down) and POST (bottom up) work to activate Hox gene expression, with moving from Hox10 anteriorly to Hox 13 posteriorly.

The more posterior Hox genes can activate the ____ enhancers, creating the ZPA in the posterior limb mesoderm.

Answer 28

ssentially, ELCR (top down) and POST (bottom up) work to activate Hox gene expression, with moving from Hox10 anteriorly to Hox 13 posteriorly.

The more posterior Hox genes can activate the ZRS enhancers, ALLOWING FOR SHH EXPRESSION AND creating the ZPA in the posterior limb mesoderm.

Question 29

After Hoxd genes are induced by ELCR and POST, they activate ZRS enhancers so that SHH gets expressed inthe posterior region, forming the ZPA. These Hox genes were also involved in making the stylopod and zeugopod regions. What is the later phase for autopod specification?

Answer 29

In the second phase, the ZPA now acts to alter the Hoxd gene expression patterns. SHH expressed from the posterior margin activates a GCR (global control region).
The Hox genes closest to the GCR are expressed most broadly. This expression inverts the original pattern of Hoxd10–13 expression such that Hoxd13 is expressed at the highest level and extends most anteriorly.

Hoxd12, 11, and 10 are expressed in slightly narrowere domains so that the most anterior digit expresses Hoxd13 but no other Hox gene.

Thus, the first phase of Hoxd gene expression helps specify the ZPA, while in the second phase of Hoxd expression the ZPA instructs the expression patterns, and these patterns define the identities of the digits.

Question 30

Thus, the first phase of Hoxd gene expression helps specify the )____, while in the second phase of Hoxd expression the ____ instructs the expression ___, and these patterns define the identities of the digits.

Answer 30

Thus, the first phase of Hoxd gene expression helps specify the ZPA, while in the second phase of Hoxd expression the ZPA instructs the expression patterns, and these patterns define the identities of the digits.