Lecture 11 - Branching morphogenesis

Question 1

What is branching morphogenesis?

Answer 1

fundamental to function in MANY tissues, that require a high surface area for gas/fluid exchange

It often relies on the communication between different tissue layers - notably the epithelium & the mesenchyme

Question 2

Describe features of the lung

Answer 2

One of its essential features is a MASSIVE surface area - like a tree - lots & lots of branches

54, 000 branches

Question 3

Describe features of the kidney

Answer 3

Nephron - functional unit of kidney - remove waste products & excess water from the blood, which is excreted as urine - also reabsorbs other important substances back into the blood.

Collecting duct - connects nephron to ureter

Average human kidney has 1,000,000 nephrons

Question 4

How does A/P regionalisation of the main body axis occur?

Answer 4

Hox11 genes are important for determining region of intermediate mesoderm to form kidneys (lumbar/sacral boundary)

Deletion of Hoxa11/c11/d11 prevents kidney formation in the mouse

Question 5

Describe the embryonic origin of the kidney

Answer 5

Signals from the lateral plate mesoderm (BMPs) & paraxial mesoderm (somites) specify a region of intermediate mesoderm to form a kidney called the nephrogenic cords.

Marked by transcription factor expression - Pax2, Pax8 & Lim1

Question 6

What governs kidney development?

Answer 6

Intermediate mesoderm gives rise to 2 cell types that govern kidney development - Metanephric mesenchyme & the ureteric bud epithelium of the nephrogenic cords

Metanephric mesenchyme & the ureteric bud are both derived from Pax2-expressing intermediate mesoderm in the embryo.

Question 7

What is the Anterior intermediate mesoderm exposed to?

Answer 7

Anterior intermediate mesoderm is exposed to higher levels of Fgf9 & Retinoic Acid for longer & lower levels of Wnt for less time –> Ureteric Bud Epithelium

Question 8

What is the Posterior intermediate mesoderm exposed to?

Answer 8

Posterior intermediate mesoderm is exposed to lower levels of Fgf9 & Retinoic Acid for less time & higher levels of Wnt for longer –> Metanephric Mesenchyme

Question 9

What are the 3 distinct stages of kidney development?

Answer 9

• Pronephros
• Mesonephros
• Metanephros

Question 10

What occurs during pronephros?

Answer 10

Week 4 gestation in humans - primitive kidneys with no filtration ability (degrade immediately)

Question 11

What occurs during mesonephros?

Answer 11

Tubules associated with capillaries - some have some filtration ability & drain into the mesonephric ducts, also known as Wolffian & Nephric ducts

Question 12

What occurs during metanephros?

Answer 12

Definitive kidney - week 5 gestation in humans - formation involves the differentiation of the ureteric epithelium into multiple collecting ducts

Question 13

What are the key steps in the formation of the metanephros - collecting ducts of the definitive kidney?

Answer 13

1. Signal from metanephric mesenchyme causes proliferation & outgrowth of epithelial ureteric bud ‘tip cells’.
2. Leading-edge tip cell arrests its proliferation, resulting in a flattening of the bud growth
3. Lateral tip cells continue to proliferate, resulting in formation of a cleft & 2 tips
4. Lateral tip cells still surrounded by metanephric mesenchyme: process repeats

• signalling from mesenchyme is GDNF (Glial cell line-derived neurotrophic factor)
• Receptor for GDNF on the ureteric bud is a receptor tyrosine kinase called Ret
• GDNF induces Wnt 11 in ureteric bud in tip cells, which by paracrine signalling maintains GDNF levels and positive feedback between mesoderm & epithelium

Question 14

What occurs after signals come from the mesenchyme?

Answer 14

First, signals come from a different tissue (the mesenchyme). Then, signals from within the epithelial ureteric bud act locally to govern gene expression. These lead to changes in transcription factor gene expression that intrinsically/autonomously lead to changes in cell behaviours.

Question 15

What are the cellular responses that occur?

Answer 15

• local proliferation
• oriented cell division
• cell migration
• cell adhesion
• cell shape
• ECM remodelling

Question 16

What results in the formation of multiple branches?

Answer 16

Repeated process. These will go on to differentiate into the multiple collecting ducts of each kidney

Question 17

Describe the formation of the nephron from renal vesicles.

Answer 17

The ureteric bud signals & induces local metanephric mesenchyme to condense around the bud & undergo a MESENCHYMAL-TO-EPITHELIAL CELL TRANSITION, and form renal epithelium, then renal vesicles.

The renal vesicles proliferate &
differentiates to form a nephron.

Distally they will fuse with the collecting duct, proximally they will attract endothelial capillaries.

Number of nephrons exceeds the number of collecting ducts

Question 18

What are the ‘take home messages’ regarding nephron formation?

Answer 18

1. The signals that initiate the mesenchymal to epithelial transition
2. The TFs included in the renal epithelium, that cause it to undergo cell-shape changes & form the renal vesicle
3. The molecular pathways that govern renal vesicle proliferation & formation of an S-shaped body
4. The molecular pathways that determine how the S-shaped body becomes polarized, so that its distal & proximal ends have different properties.

Proximal ends: provide local signals for angiogenesis - so capillaries form

Distal end: fuses with collecting duct by selective apoptosis

Question 19

What is a 2nd example of branching morphogenesis?

Answer 19

Lungs

Question 20

How are lungs similar to kidneys?

Answer 20

They are derived from 2 tissues - endoderm & mesoderm

Question 21

What does endoderm give rise to?

Answer 21

Epithelial lining of trachea, larynx, bronchi, alveoli, through branching morphogenesis

Question 22

What does mesoderm give rise to?

Answer 22

Will give rise to cartilage, muscle & connective tissue

Question 23

What arises from a bud called the respiratory divericulum?

Answer 23

Formation of trachea, bronchi, bronchioles, alveoli

The bronchial tip of the respiratory diverticulum then begins to undergo branching morphogenesis

Question 24

What do lung buds then undergo?

Answer 24

Lungs buds then undergo further branching. A sac of mesoderm surrounds each lung bud

Question 25

What drives branching & why is it limited to the tips?

Answer 25

Step 1: Epithelial cells, expressing FGF receptor (FGFR), respond to the secretion of FGF from nearby mesenchyme & form buds that extends towards the FGF source. Exposure of tip cells to a high concentration of FGF10 induces the expression of secondary genes in the tip, including genes that code for signals - in particular BMP4, Shh, thus turning the tips of the bronchial branches into signalling centres (again - emphasizing importance of mesenchymal-epithelial signalling. Step 2: BMP4 is expressed at highest levels in the 'leading edge' tip cells & autonomously inhibit epithelial cell proliferation, limiting branch extension - this causes a flattening of the bud. At the same time, Shh expressed by the tip cells diffuses to the mesenchyme & inhibits FGF10 expression in the mesenchyme nearest the tip. This splits FGF10 expression promoting the next round of branching Sprouty2 limits the action of FGF10, so that branching is restricted and can only occur at the tip.

Question 26

Describe Sprouty & negative feedback loops

Answer 26

1. FGF10 induces expression of genes that direct growth & proliferation. 2. Over a slighlty longer time-frame, FGF induces expression of another gene - Sprouty. 3. Sprouty inhibits FGF signalling 4. This is an example of a negative feedback loop, where a signal induces its own inhibitor, to limit the time of its own action. This is a commonly-deployed mechanism in developmental biology. Other examples are Shh-Ptc

Question 27

Summarize branching morphogenesis

Answer 27

1. Branching morphogenesis is essential to enable the physiological functions of many different tissues, especially where there is a need for a high SA:V in order to maximize the efficiency of gas, fluid or solute exchange, secretion or excretion. 2. Many organs display remarkably similar branching patterns. 3. Branching morphogenesis in the mammalian kidney is driven by GDNF signalling from mesenchyme to ureteric epithelium via the RET (Receptor Tyrosine Kinase). 4. Branching morphogenesis of the mammalian lung is driven by FGF10 signalling by endothelial endodermal cells that induces Shh & BMP4 signalling in mesenchymal cells. 5. This branching is attenuated by Fgf signalling inducing its own inhibitor Sprouty2.