L11+12 - Limbs + urogenital system

Question 1

limbs

Answer 1

skeletal muscles cartilage and bone

Question 2

@hh21

Answer 2

limb buds

dorsoventrally flattened

Question 3

@hh25

Answer 3

limb buds grown away from the body = proximo distally

Question 4

proximo distal patterning

upper limb first digits last

Answer 4

humerus stylepod
ulna zengopod
radius
metacarpals antopod
digits

fgf8 from AER maintains cell proliferation and hence outgrowth and prioximo-distal patterning

rostro-caudal
anterior posterior

zone of polarising activity establiahes posterior anterior polarity

Shh is expressed in the posterior mesoderm of the limb = ZPA

grafting these cells to an anterior positio = image duplication of the limb

the graft polarises the tissue

dorso ventral patterning

nails dorsal
loss of Wnt7a or transcription factor Lmx1 = double ventral limbs
loss of En1 = double dorsal limbs

proximal distal
upper limb - digitsaxis is controlled by fgf8 from aer

rostral caudal
thumb - little finger
shh from ZPA

dorsal ventral
back -palm
Wnt7

Question 5

how does the limb become bestowed with skeletal muscle and nerves

Answer 5

pre-cartilage condensations become separate skeletal elements

Question 6

unexpected phenotypes

Answer 6

gain of fgf - few/fused digits
loss of Shh - arrest of limb development
Loss of Wnt7a = lack of posterior digits

Question 7

fgf10-wnt3a - fgf8 feedback loop

Answer 7

When a bead loaded with Fgf10 is implanted into the flank below the normal fore limb, expression of Fgf10 in the lateral mesoderm as well as expression of Wnt3 and Wnt3a and Fgf8 in the AER are induced

Fgf10 produced by the lateral mesoderm derived limb mesenchyme induces Wnt3/3a in the AER, which induces Fgf8 to drive limb outgrowth. Fgf8 from the AER signals back to the limb mesenchyme to stabilise Fgf10.​

Question 8

how does the forelimb become different from the hindlimb

Answer 8

Tbx5 specifies the forelimb and Tbx4 together with Pitx1 specifies the hind limb.​

Question 9

cartilage and bone

Answer 9

Limb bones form via endochrondral ossification that needs to be distinguished from dermal ossification of, for example, the bony plates of the skull

The limb lateral mesoderm will form mesenchymal condensations

Cells in the centre of this condensation will be driven into forming cartilage shown in red, which will become hypertrophied

Hypoxic conditions trigger the ingrow of blood vessels and the replacement of the cartilage by bone

At the end of the long bones in the growth plate, undifferentiated cells remain that allow further bone growth. Eventually, also they will ossiffy.

Question 10

how the limb gets the functional elements it needs to work in locomotion: cartilage and bone, muscle and innervation.​

Answer 10

factors that control Sox9 expression and endochondral ossification including BMP – bone morphogenetic proteins.​

​interzones consisting of densely packed, flattened, small mesenchymal cells occur that are connected by jap junctions

The outermost interzone layer becomes epiphyseal cartilage that elongates the bones, the intermediate layer becomes articular(=permanent) cartilage. ​

​The intermediate interzone undergoes cavitation filling with synovial fluid. The articular cartilages undergo morphogenesis to give rise to the interlocking shapes specific for a particular joint.

Question 11

skeletal muscle precursors recruitment

Answer 11

Limb cartilage and bone, connective tissues and tendons all stem form the lateral mesoderm-derived limb mesenchyme

Question 12

skeletal muscle precursors recruitment

Answer 12

Limb cartilage and bone, connective tissues and tendons all stem form the lateral mesoderm-derived limb mesenchyme

Limb muscle, stem from somites in the myotomes but they are far away

somitic dermomyotomes, lay down muscle in the myotomes. These myotomes grow into the ventral body wall to deliver body wall muscles- your six packs. When limbs emerged in vertebrate evolution, they were muscularised by extensions or outgrowths from the somite.

but in bony vertebrates
how migratory muscle precursors labelled with GFP migrate into the chicken limb bud
​Lbx1, together with other players, keeps the cells undifferentiated. In addition. The cells are allowed to detach and become migratory

Hox gene expression on the somites determines which somites will express Lbx1. Fgfs from the limb bud and other factors ensure that the cells migrate to the correct target.​

Question 13

innervation

Answer 13

the developing limb organises the recruitment of muscle precursors from the paraxial mesoderm and innervation from the spinal cord

Specific motor neurones develop in the spinal cord at limb levels. They are referred to as lateral motor column depending on Hox-controlled positional value

The lateral motor column does not form at flank levels; here, only medial motor column exists that innervates the dorsal muscle of the back and the ventral muscles of the body wall.

Medial and lateral LMC neurons project into a network or plexus in the proximal limb. Their axons then innervate the limb, reading the ventral and dorsal and cues set by Lmx1, respectively

Question 14

summary

Answer 14

The signalling systems controlling the patterning along the three limb axes are interdependent.​

Fore limb/ hind limb identity is regulated by the transcription factors Tbx5 and Tbx4+Pitx1.​

The limb skeleton is the key product of the lateral mesoderm-derived limb mesenchyme. It forms via endochondral ossification.​

Sox9+ cartilage precursors form continuous mesenchymal condensations. Joints form where differentiation is suppressed.​

The limb musculature stems from the trunk paraxial mesoderm, the somites. In bony vertebrates, limb muscle precursor use a specific developmental programme and actively migrate to their target sites.​

Limb muscle innervation comes from specific motor neurons located in the lateral motor column. Axons find their correct extensor or flexor muscles by reading the dorso-ventral limb pattern.​

Digits separate, because the intervening tissue is removed by Bmp-controlled apoptosis.​

Question 15

the intermediate mesoderm forms discrete structures early on.​

Answer 15

Wnt 4 is expressed in a mesenchymally organised intermediate mesoderm while lateral to it, there is a round, unstained structure where arrow points, which is an epithelially organised tubing or duct.​
lateral to the somites and medial to the lateral mesoderm

Question 16

the function of the kidney

Answer 16

Osmoregulation
Excretion of metabolic waste
Non-recyclable end products of the carbohydrate and fat metabolism are CO2, water which are non-toxic and easily disposed of​

The non-recyclable end product of the protein and nucleic acid metabolism is ammonia, which is toxic​

nitrogen excretion as uric acid or urea

Bird and reptiles typically convert ammonia into uric acid. Uric acid is poorly water soluble. When it precipitates out of solution it does not exert osmotic pressure within the embryo and can be collected in the allantois

Question 17

the renin–angiotensin system

Answer 17

The filtering activity of the kidney depends on blood pressure, which in turn depends both on the diameter of blood vessels and the volume of fluid pumped around.

Question 18

nephron structure

Answer 18

glomerulus = a filtering unit - ball-shaped structure capillary blood vessels
Bowman’s capsule with podocytes,​

proximal tubule, distal tubule and intermediate segment in between which in amniotes is Henle’s loop is between connected to a collecting duct where reabsorption and osmoregulation happens.​

Question 19

Ultrafiltration

Answer 19

Glomerular blood vessels have pores= blood can freely exit

The podocytes of the Bowman’s capsule have gaps between their pedicles, allowing for fluid to move freely into the nephron​

The blood is filtered through the basement membrane ( size-selective and restricts the passage of blood cells and large proteins​) which lies between the glomerulus and Bowman’s capsule​

the filtrate formed does not contain any blood cells, platelets or plasma proteins

Question 20

Reabsorption​

Answer 20

The tubules reabsorb all glucose, amino acids, vitamins and hormones, 80% mineral ions and water​

Mineral ions and vitamins are actively transported by protein pumps and carrier proteins respectively​

Glucose and amino acids are co-transported across the apical membrane with sodium

Water follows the movement of the mineral ions passively via osmosis​

Question 21

3 stages of amniote kidney development

Answer 21

1.the pro-nephros - forming in neck or cervical intermediate mesoderm
nephrons or tublues, draining into a nephric duct.​

The pro-nephros is a working organ in embryos and larvae
In amniotes, the pro-nephros is not functional

2. the meso-nephros

The nephric duct then grows out caudally and links to the cloaca
it induces nephron formation in the mesenchymal neighbouring intermediate mesoderm = nephrogenic cord.= functional kidney in amniote embryos + = functional kidney in adult fish and amphibians.​

3. the meta-nephros

caudal to the meso-nephros, an outgrowth from the nephric duct forms = the uretric bud.
It interacts w meta-nephrogenic mesenchyme = will filter tubules. The uretric bud branches and tubules will form around each branch = the meta-nephros kidney​

In amniotes, the meso-nephros will degenerate, with the exception of the nephric duct which in males becomes the Wolffian duct

Non-amniotes do not develop a meta-nephros

Question 22

3 stages of amniote kidney development

Answer 22

1.the pro-nephros - forming in neck or cervical intermediate mesoderm
nephrons or tublues, draining into a nephric duct.​

The pro-nephros is a working organ in embryos and larvae
In amniotes, the pro-nephros is not functional
the collecting duct of the pro-nephros, (the nephric or Wolffian duct ) induces the tubules from the neighbouring mesenchyme which encompasses the mesonephric tubules, functional in all vertebrates, permanently in non-amniotes and embryonically in amniotes

2. the meso-nephros

The nephric duct then grows out caudally and links to the cloaca
it induces nephron formation in the mesenchymal neighbouring intermediate mesoderm = nephrogenic cord.= functional kidney in amniote embryos + = functional kidney in adult fish and amphibians.​

3. the meta-nephros

caudal to the meso-nephros, an outgrowth from the nephric duct forms = the uretric bud.
It interacts w meta-nephrogenic mesenchyme = will filter tubules. The uretric bud branches and tubules will form around each branch = the meta-nephros kidney​

In amniotes, the meso-nephros will degenerate, with the exception of the nephric duct which in males becomes the Wolffian duct

Non-amniotes do not develop a meta-nephros

the nephric duct delivers the uretric bud. Without that, the meta-nephros cannot form, and there won’t be any adult amniote kidney.​

Question 23

when and how does pro-nephros development start?​

Answer 23

the rostral-most 5 somites are occipital and contribute to the base of the skull, the next are cervical and contribute to the neck.

The Pax2 expression domains begins next to the 6th somite the 1st neck somite, and then expands caudally

Question 24

how does the pro-nephros come about?

Answer 24

induced by the neck paraxial mesoderm
the somites have positional information, the somites play a role in the axial positioning of the limbs, the pro-nephros forms at upper neck levels and not next to the occipital somites

Question 25

competence factors for metaphros developments

Answer 25

outgrowing uretric bud
occurs in
neural tube, lung buds and salivary glands drive intermediate mesoderm into tubule formation
-suggesting these release similar signals

only nephric tubules are able to make nephric tubules = only the intermediate mesoderm has the competence to make nephric tubules.

Question 26

instructive induction

Answer 26

happens when the inducing signal tells the tissue what to do. This happens when cells or tissues have several options to develop into and need to be told which to pick

Question 27

Induction by a morphogen

Answer 27

the decision on which path to follow is controlled by the concentration of the inducer.

Question 28

chyme​

​

What provides the tissue with competence to generate nephric tubules

Answer 28

Hoxb4 provides general nephric competence, Hox11 genes competence to form metanephric tubules, indicating that Hox genes have roles beyond determining positional information.​

Question 29

the reciprocal, -signalling between the meta-nephrogenic mesenchyme and the ureteric bud.​

Answer 29

1. the ureteric bud growing + branching out into the surrounding mesenchyme, forming more buds. induced by mesenchyme. (the epithelium of the ureteric bud keeps the mesenchyme alive - preventing apoptosis).
2. mesenchyme condensates around the tips of the branches, forming a cap

the epithelium at the tips of the branches induces the condensed mesenchyme to form an elongated pre-tubular condensate + mesenchyme-epithelial transition and cavitation = tubule formation.​

Question 30

the reciprocal, -signalling between the meta-nephrogenic mesenchyme and the ureteric bud.​

Answer 30

1. the ureteric bud growing + branching out into the surrounding mesenchyme, forming more buds. induced by mesenchyme. (the epithelium of the ureteric bud keeps the mesenchyme alive - preventing apoptosis).
2. mesenchyme condensates around the tips of the branches, forming a cap

the epithelium at the tips of the branches induces the condensed mesenchyme to form an elongated pre-tubular condensate + mesenchyme-epithelial transition and cavitation = tubule formation.
At the same time, arterioles grow in to form the glomerulus. Finally, the mesenchyme-derived cells digest the basal lamina of the ureteric bud cells, thus linking the nephric tubule with the collecting duct​

Question 31

factors controlling the reciprocal

Answer 31

1, Gdnf, Glial-derived neurotrophic factor
- expressed in the metanephrogenic mesenchyme. Its receptor Ret is expressed in the ureteric bud.​

= support neurone survival and axon outgrowth.

+ve Gdnf = the ureteric bud grows out from the nephric duct + branches into the mesenchyme (dosage dependent)

knockout = no ureteric bud forms

Question 32

keeping the mesenchyme alive

Answer 32

matenephtogenic mesenchyme - fgf + ureteric bud = survival

Question 33

inducing

Answer 33

Wnt9b from the ureteric bud induces Wnt4 expression in the mesenchyme. ​

Wnt4 work inside the mesenchyme -autocrine fashion. stimulates the expression of epithelial Cadherin,
Calcium dependent cell adhesion molecule typical for epithelial tissues.​
​facilitates mesenchyme-to-epithelial transition and tubule formation

Question 34

where does the unrecycelable waste go

Answer 34

adult placental mammals, urine is collected in and then discharged from a urinary bladder.

reptiles and birds, there is not much more to it. They excrete uric acid as an almost semi-solid paste from the cloaca

a uro-rectal fold begins to divide the cloaca into a ventral urogenital sinus inks the ureters, in males also the Wolffian or left-over nephric duct, and the urinary bladder
and a dorsal rectum.

the ureters also separate from the Wolffian duct and shift anteriorly. The bladder and allantois will separate and the bladder will become the collecting organ for the urine.​

Question 35

two ways in which germ cells that can create the next generation are being made.

Answer 35

1, totipotent Primordial germ cells form autonomously

2. PGC - may form in response to specific induction events )lead to the expression of genes) from the caudal or posterior epiblast. = axolotl or in mammals

PCG formation in rodents is somewhat special and liked to the specific inside-out layout of the gastrulating embryo.​

Question 36

how the mouse PGC migrate to the genital ridge.​

Answer 36

PGC are specified at day 6.5 of development. At day 8.5 of development, they in an extraembryonic location at the caudal pole of the embryo

migrate from a position between the wall of the yolk sac and the allantois into the hindgut, then through the dorsal mesentery and into the genital ridge

Question 37

The intermediate mesoderm medial to the mesonephros forms the genital ridge, the target site for the migrating PGC

Answer 37

the mesonephros with its glomerulus and Bowman’s capsule and mesonephric tubule draining into the nephric duct, also known as Wolffian duct. ​

at 4 weeks of gestation, the gonad shows no differences between males and females

at 6 weeks of development. The gonad has grown in size and consists of a mixture of somatic cells and PGC. Moreover, a new duct, the Muellerian duct shown in orange, has appeared. ​

The gonad is still sexually indifferent.​

till now gonad dev is controlled by the Wilms tumour suppressor gene WT1
and SF
Now male and female gonads become different.

MALE =
The mesonephric tubule degenerates - there is a metanephros now. But the nephric duct, i.e. the Wolffian duct, remains. It will become the outlet of the testis, the epididymis and vas deferens. Testis cords appear, specialised tubular structures that shelter germ cells from exogenous signals and make the link to the efferent ducts and the vas deference.​

FEMALE
mesonephrogenic tubules disappear. Different to the male, the nephric or Wolffian duct also disappears. The Muellerian duct remains and becomes the outlet of the ovary, the oviduct. It also contributes to the uterus, cervix and upper part of the vagina. ​
oogonia become surrounded by connective tissue cells, the pre-granulosa cells, which eventually form the ovarian follicles. The developing follicle acquires a layer of connective tissue and associated blood vessels called the theca. Theca cells are then stimulated to produce testosteron

Question 38

how initially the gonad is sexually indifferent and bipotential, able to develop either into a testis or into an ovary.​

Answer 38

Controlled by estrogen from granulosa cells, the gonad will develop into a functional ovary.

Controlled by the anti-Muellerian factor, a TGFb signalling molecule
as well as testosterone, the gonad will become a functional testis.​

At 8 weeks, males and female gonads become anatomically different.​

Question 39

the ovary pathway

Answer 39

Wnt4 an autocrine signalling molecule acting in the metanephric mesenchyme and driving mesenchymal cell aggregation and tubule formation.​
as well as

expressed in the early, sexually indifferent gonad. However, expression only persists in females.​

​+ve Wnt4 =

Question 40

the ovary pathway

Answer 40

Wnt4 an autocrine signalling molecule acting in the metanephric mesenchyme and driving mesenchymal cell aggregation and tubule formation.​
as well as

expressed in the early, sexually indifferent gonad. However, expression only persists in females.​

​+ve Wnt4 = beta-Catenin can travel to the nucleus = transcription factor= upregulates Wnt4 in a positive feedback loop, and upregulates ovary specific genes and suppresses testis specific genes.

R-Spondin enhances Wnt signalling, stabilising the Lrp co-receptor by blocking its internalisation.​

ABSENCE of Wnt4 or Rspo1, ovary specific genes remain repressed
testis genes are expressed albeit transiently.

The gonad degenerates =
more to male gonad development than just the absence of Wnt signalling.​

​

Yet when in XY mice which are genetically male bCat is overexpressed and the gonad becomes female.​

Question 41

Sry gene

Answer 41

sex determining region of the Y chromosome gene.​

​ expressed in the gonad just before sexual dimorphism appears​

When the Sry gene is introduced into XX mice, their gonads become male. In humans, the condition is associated with the XXY genotype and is known as Klinefelter’s syndrome​
​
when the Sry gene is lost or non-functional, gonads develop as female. In humans, the condition is associated with the X0 genotype and is known as Turner‘s syndrome​

Sry activates the autosomal Sox9 gene.

Question 42

Sox9

Answer 42

If Sox9 is lost, a gonad becomes female even in the presence of Sry. Thus, Sox9 is required for testis formation. ​

Sry is found in mammals, Sox9 in all vertebrates. Sox9 has a widespread role in “male” determination incl . temperature dependent sex determination. Thus, Sox9 is the key male determinator.​

​

Sox9 activates testis specific genes, represses genes for ovarian development, and enhances its own expression.​

​

Question 43

dax1

Answer 43

Dax1 protein also acts antagonistically to SRY.​

So Dax1 is an anti-testis gene.

Dax1 is expressed in the developing gonad but then expression declines in males

In the absence of Sry, 1 copy of Dax1 is enough for female development​

2 copies of Dax1 override a Y chromosome and promote female development​

Question 44

endocrine disruptors

Answer 44

PCB Polychlorinated biphenyl compound can act as estrogen

Atrazine acts as an agonist of the G protein-coupled estrogen receptor 1 and stimulates the production of aromatase, the enzyme that converts androgens into estrogens