Development of the

vertebrate forelimb
BY:

ALANA NELSON – 816006272

CASSIDY JAIKARAN – 816016446

KHADIJAH JAMES - 816019971

NATASHA CHARLES - 815011222

RANISA LEWIS - 816013326 GROUP 28

OBJECTIVES

• To explain how the forelimb of vertebrates are developed.

• To state what embryonic tissues are involved in the development process.

• To describe the roles of genes and morphogens in forelimb development.

• To explain how limb deformities and limb axis mutations occurs

 What are limbs?
• Limbs are mesoderm with an ectoderm housing
• They have boat shaped compositions that
moderately develop outwards
• The most recognized modification is the growth
of substantial margin vein (blood vessel)
• This is positioned beneath a solidifying part of
the ectoderm at the top of the limb bud referred
STAGE 13
to as the AER (Apical Ectodermal Ridge) (https://embryology.med.unsw.edu.au/embryology/index.php/Le
cture_-_Limb_Development)
• Limb placement is far away from the first
position.
(https://embryology.med.unsw.edu.au/embryology/index.php/Lecture_-
_Limb_Development)
Higher and Secondary Limb
• Limb growth takes place at
distinct times for hindlimbs
and forelimbs
• In the middle of week four,
the human higher limb
develops
• Almost two days after, the
secondary limbs develop
Growth in the middle of Week 4
(https://embryology.med.unsw.edu.au/e
mbryology/index.php/Lecture_-
_Limb_Development)
Human limb growth throughout week six
(https://embryology.med.unsw.edu.au/embryology/index
.php/Lecture_-_Limb_Development)
Human limb growth throughout week eight
(https://embryology.med.unsw.edu.au/embryology/index
.php/Lecture_-_Limb_Development)
Development •Beginning of the limb
and Limb Axis:
•Proximodistal axis
•Dorsoventral axis
•Anteroposterior axis
 Development
BEGINNING OF THE LIMB LIMB RECOGNITION
• Fibroblast growth factor
(FGF) coated beads can
produce extra limbs
• FGF10 is revealed in lateral
mesoderm plates prior to bud
creation, revealing the FGF8 in
the top portion of the
(https://embryology.med.unsw.edu.au/embryology/index.php/Lecture_-
ectoderm _Limb_Development)
• FGF8 produces continuous
development in the prime • Hindlimb (mouse) and forelimb identification are
mesoderm, creating a positive managed by Tbx genes in which these genes are a part
(https://embryology.med.unsw.edu.au/em response loop of transcription elements.
bryology/index.php/Lecture_-
_Limb_Development)
• The anterior end of the Hoxc6 ➢Forelimb Tbx5 is indicated
appearance agrees with the ➢Hindlimb Tbx4 is indicated
location of forelimb growth ➢In both limbs, Tbx2 and Tbx3 are indicated
 Morphogens and Axes
Proximodistal axis
• Distal to proximal (tip of the
limb to where the limb is
connected to the body
• Apical ectodermal ridge (AER)
is created at the point of FGF10
installation
• Later, the AER produces FGF8
and FGF4
• FGF’s join membrane tyrosine
kinase receptors and there are
22 of these in humans
https://embryology.med.unsw.edu.au/embryology/index.ph
p/Lecture_-_Limb_Development
Anteroposterior axis
• Head boundary to opposite •
boundary of tail or body
• This is the area of polarized
activity referred to as the
ZPA
• It is a mesoderm posterior of
the limb where there is
discharge sonic hedgehog
(SHH)
Dorsoventral axis
Back to front (spiral column to belly)
• This is essential for shaped muscles:
ventral muscles – flexors and dorsal
muscles – extensors.
• D/V signaling point occupies the
dorsal ectoderm
• Wnt7 is a diffusible morphogen that
is discharged by ectoderm cells
• Wnt7 produces the appearance pf the
homeobox gene Lmx1 in the prime
mesoderm close to the dorsal side
• The engrailed gene (En1) is revealed
in the contrary ventral ectoderm
The Time Axis
• Active growth and profane gene appearance

➢Distinct Hox genes are revealed at separate hours in the growing limb
bud and design the limb’s fine form

➢Compositions are resolved in a proximal > distal order with time, that
is, proximal forms like the humerus bone are initially transferred.
 Pre-patterning (Hox genes)
• Defined as the determination or predictive planning
of how structures are supposed to develop
• Hox genes (specifically Hoxd and Hoxa genes) play a
role in pre-patterning in limb development
• Hoxd 9 and 10 paralogues identify the stylopod
development
• Hoxd 11 paralogues identify the zeugopod
development
• Hoxd 12 and 13 paralogues identify the autopod
(https://bastiani.biology.utah.edu/courses/3230/DB%20Lecture/Lectures/b14Li
mb.html)
development
Limb pattern
• The bones of any tetrapod limb, arm or
leg, wing, or flipper, consist of a
stylopod, zeugopod and autopod
• The proximal stylopod
(humerus/femur) is adjacent to the
body wall
• The Zeugopod (radius-ulna/tibia-
fibula) is in the middle region
https://www.researchgate.net/figure/Schematic-representation-of-the-skeletal-
elements-of-a-human-limb-and-autopod-A_fig2_257125896 • The distal Autopod - (carpals-
fingers/tarsals-toes)
 Deletion of Hox genes • As discovered in a 2003 experiment by Wellik and
Capecchi- when paralogues of Hoxd genes are
removed it results in malformation of limb bone.
• In the image, we observe the wild type which is a
properly formed forelimb of a mouse and the
mutated types where single deletion or compound
deletion of Hox genes were done to produce
mutant mouse forelimb with altered skeletal
structure.

(https://www.researchgate.net/figure/A-Coordinate-expression-of-
Abdominal-B-related-HoxA-and-HoxD-genes-in-the-
developing_fig2_13979604)
 Role of genes and morphogens in Limb development
Growth along the proximal/distal axis

• The FGF family is a series of structurally related signaling related proteins

• Some of the members of this group are FGF4, FGF8 and FGF10 play a role in limb
bud formation and limb growth

• FGF10 is in mesenchymal cells and this signals the formation of the AER which
involves the thickening of the ectodermal region of selected areas

• The AER secretes growth factors FGF4 and FGF8 which maintains proliferation and
survival of the undifferentiated cells in the progress zone thus, driving the
outgrowth limb bud

• FGF4 is expressed in the posterior part of the AER meanwhile FGF8 is expressed
along the full anteroposterior length of the AER

• The action of the FGFs involves an intercellular signaling cascade which is heavily (Ornitz and Itoh 2015)
regulated
 Role of genes and morphogens in Limb development
Growth along the dorsal/ventral axis

• Engrailed 1 (En-1) is a homeobox transcription factor and it is expressed in the ventral

ectoderm of the budding limb

• It is key in establishing the dorsal/ventral patterning of the forelimb

• The radical fringe (r-Fng) is a signaling enzyme which aids in the positioning of the AER
at the dorsoventral boundary of the limb and it is expressed in the dorsal ectoderm

• R-Fng expression is repressed by En-1 so that the dorsal pattern could be formed

• Wnt7a is also repressed by En-1 so that AER formation could occur with the aid of r-Fng

• When Wnt7a is not repressed it induces Lmx1 in the underlying mesenchyme (https://www.cell.com/fulltext/S0092-8674(00)80364-5)

• Lmx1 is a transcription factor that is expressed in the dorsal mesenchymal mesodermal

cells.
 Role of genes and morphogens in Limb development
Growth in the ZPA:

• The ZPA impacts the overlying AER

• In this area, retinoic acid is produced which initiates the production of Sonic Hedgehog (SHH)

• SHH plays a role in which regulates the anterior-posterior axis.

• SHH upregulates gremlin 1, which is a glycoprotein of the Dan family and it inhibits bone morphogenic
protein (BMP) activity and maintains FGF
The progress zone model
• States that the cell fate of cells along the P/D axis is
determined by the time spent in a region of distal
mesenchyme found close to the AER called the progress zone
• As the fibroblast growth factor (FGF) releases, a signal
accumulates and directs the formation of limb buds in the
progressive zone where mesenchyme proliferates rapidly
• Cells that exit the progressive zone early are proximal while
cells that exit later are distal
(https://www.cell.com/fulltext/S0092-8674(00)80364-
• The extent of how distal cells become is dependent on the time 5?scriptOff=tru)
spent
• In the absence of AER, growth in the PD axis stops
 • Cells that exit the progress zone possess the
positional information along all three axes

• The grafting of the ZPA alters the cell fate on both

Post progress the proximal distal axis

zone • This showed an interdependence between the Shh

and the FGF and is associated with ZPA’s to alter
cell fate only in proximity of AER
 Cartilage and bone
• They are derived from replicating
mesenchyme originating in the somatic
lateral plate of the mesoderm.

• Two Bone Morphogenic Proteins (BMP2

and BMP4) are needed for the cartilage (https://embryology.med.unsw.edu.au/embryology/index.php/Lecture_-_Limb_Development)
development through the condensation
of mesenchyme (chondrogenesis).
Limb muscle and dermis in vertebrates
• Vertebrate limbs originate from both the
lateral plate and somatic mesoderm

• Limb buds originate from the lateral plates

through flank proliferation

• The limb muscles originate from migration

of cells into the limb from the lateral edges
of neighboring somite

(https://embryology.med.unsw.edu.au/embryology/index.
php/Lecture_-
_Limb_Development#Limb_cartilage_and_bone)
 Muscle formation • There are muscle progenitors which are influenced
by Pax 3 and Pax7
• Determination occurs by Myf5 and MyoD

• An activated myoblast is formed and finally

differentiation occurs under the expression of
myogenin.
• WNT from the body wall ectoderm stimulates lateral
myotome to express MyoD for the formation of
(https://europepmc.org/article/PMC/6105407)
anterior and limb musculature
The regions
sculpted by cell • The cells in these zones die via both apoptosis
death. and neurotic cell death.
• Apoptosis is initiated by a signal from BMP
proteins in sauropods.
• Interdigital necrotic zone.
• Apoptosis signaling by BMP is blocked in these
• Interior neurotic zone.
regions in the presence of BMP 2, 4 and 7. This in
• Anterior neurotic zone.
turn blocks digit growth.
• Posterior neurotic zone.
Formation of digits
• In the areas between the formation of free digits there are interdigital necrotic
zones, and these areas help to separate tissue by inducing cell death

• At this point the AER ceases to influence mesoderm and FGF8 gets down
regulated

• The BMP proteins (BMP2, BMP4 and BMP7) signal for the process of apoptosis
in the autopod.
 • Cell death plays an important role in the
Digit and Joints development and shaping of limbs. In vertebrates
formation activation or deactivation) are programmed
genetically and have been selected for through
evolution.
At the interdigital zone:
• In chick embryo presence of cell death between
the digit cartilage in limbs will produce digits.
• While in ducks the minimal cell death produces
webbed feet.

https://www.ncbi.nlm.nih.gov/books/NBK10048/figure
/A3977/?report=objectonly
 • BMPs play an important role in limb
development.
• Depending on the stage they induce
mesenchymal cells to undergo either apoptosis
BMP or become cartilage-producing chondrocytes.
These are also used to form joints
• BMP 7 is made up of perichondrial cells these
surround condensing chondrocytes and inturn
promotes cartilage formation.
• Joints are formed when proteins BMP 2 and 5 are
(https://www.ncbi.nlm.nih.gov/books/NBK10048/figure/A3977/?report=objectonly)
expressed between bones.
LIMB AXIS MUTATIONS AND LIMB
ABNORMALITIES
• Syndactyly -Syndactyly arise because of the lack
of apoptosis in the interdigital mesenchyme.
The "webbing" in this abnormality shows
how preservation of the developmental tissues
that would be in normal development are
removed by the apoptosis which is
.programmed cell death
 Polydactylies

• Have a distinct appearance of supernumerary digits that show as a complete

duplication of a whole limb or single digits

• The two forms are preaxial polydactyly type-IV (PPD-IV) and postaxial polydactyly

• These arise because of a defect in Sonic Hedgehog (Shh, which helps with the A-P
patterning of the limb.)

• Expression due to point mutations found limb-specific regulatory element from the
SHH gene

• Pre-axial polydactyly has extra digits found on the side of the hand or thumb
while postaxial polydactyly where the extra digit is found on the side of the hand or
foot
Ectrodactyly / split hand/foot
malformation (SHFM)
• Seen as an embryonic absence of the central rays
causing a severe median cleft of hand/ foot.
• In limb development, the median apical
ectodermal ridge (AER) is not maintained causing
to an absence of its developmental regional
signaling when the hand/foot forms.
 OTHER ABNORMALITIES
• Brachydactylies- defined by shortened digits and are characterized by a high degree of phenotypic variability. Type-B brachydactylies are associated
to mutation in the Ror2 gene, and Ror2 mutations are also associated with the Robinow syndrome in which brachydactyly is a common feature

• Developmental Hip Dysplasia - Incorrect development of hip during the antenatal period that causes the hip to slip from the socket

• Phocomelia - A bad P-D arrest of the developing limb bud. Results in Short arm bones, fused fingers, missing thumbs, often lacks thigh
bones, abnormally small hands/feet or stumps because of their close attachment to the body.

• Amelia - Failure of formation of limb buds, gives absent limbs. Caused as a result of an autosomal recessive disorder as a result of mutations of the
WNT3 gene

• Talipes Equinovarus - An Abnormality of the lower limb that leads to the foot being turned inward and downward when the child is born
• Trisomy 21 (Down Syndrome) : a limb reduction that gives short broad hands with curved fingers, hyperextensible finger joints, spacious toes
and acquired hip dislocation

• Diastrophic dysplasia - a limb reduction as a result of a mutation in the DTD sulphate transporter gene which causes severe short limbed dwarfism
and spinal and joint defects
References
• Jin, L., Wu, J., Bellusci, S., & Zhang, J. (2019). Fibroblast Growth Factor 10 and Vertebrate Limb Development. Frontiers in Genetics, 9.
doi:10.3389/fgene.2018.00705

• Johnson, R. L., & Tabin, C. J. (1997). Molecular Models for Vertebrate Limb Development. Cell, 90(6), 979-990. doi:10.1016/s0092-8674(00)80364-5

• Ornitz, D. M., & Itoh, N. (2015). The Fibroblast Growth Factor signaling pathway. Wiley interdisciplinary reviews. Developmental biology, 4(3), 215–266.
https://doi.org/10.1002/wdev.176

• Papaioannou V. E. (2014). The T-box gene family: emerging roles in development, stem cells and cancer. Development (Cambridge, England),
141(20), 3819–3833. https://doi.org/10.1242/dev.104471

• Pownall, M.E. and Isaacs, H.V. (2010). FGF Signalling in Vertebrate Development. San Rafael (CA): Morgan & Claypool Life Sciences.
https://www.ncbi.nlm.nih.gov/books/NBK53162/

• Stathopoulos, A., & Iber, D. (2013). Studies of morphogens: keep calm and carry on. Development (Cambridge, England), 140(20), 4119–4124.
https://doi.org/10.1242/dev.095141

• Takeuchi, J. K., Koshiba-Takeuchi, K., Suzuki, Y., Kamimura, M., Ogura, K., & Ogura, T. (2003). Tbx5 and Tbx4 trigger limb initiation through
activation of the Wnt/Fgf signaling cascade. Development, 130(12), 2729-2739. doi:10.1242/dev.00474

• https://www.researchgate.net/figure/Schematic-representation-of-the-skeletal-elements-of-a-human-limb-and-autopod-A_fig2_257125896
 Group member’s responsibility
• Alana nelson – 816006272 (slides 15-18 information), slides 20, 22-23 (information & power point)

• Cassidy Jaikaran – 816016446 (slides 3-8, information)

• Khadijah James – 816019971 (slide 10 -information and power point), 3-8, 12-19, 24-27 power point)

• Natasha Charles – 815011222 (slides 2, 9 ,11 & 28- information & power point), 12-14, 19 & 21 (information)

• Ranisa Lewis – 816013326 (slides 24-27 information)