Throw another adipocyte on the fire

Humans are able to live in so many different climates, in a wide range of temperatures and yet our inner core body temperature remains nearly constant.  This ability to thermoregulate has something to do, of course, with clothing and the ability to cool and heat our living spaces, but our bodies also offer many adaptations to regulate body temperature.  If it’s too hot, we sweat, releasing excess heat through evaporative cooling.  If it’s too cold, we shiver, producing heat in our working muscles.  The production of heat through physiological mechanisms is called thermogenesis and also includes a non-shivering version.  Today’s paper is about non-shivering thermogenesis, which is when our fat cells produce heat.

Non-shivering thermogenesis

To understand how non-shivering thermogenesis works, we need to take a step back and discuss cellular respiration.  The cells of our body store energy from food in the chemical bonds of a molecule called ATP.  During cellular respiration, a cell will convert glucose or fat into carbon dioxide, while slowly tapping into the energy in those food molecules in order to make ATP.  The final step of cellular respiration is that the energy from the electrons in glucose are passed from protein to protein, releasing energy that is used to pump protons into a membrane-bound cellular space.  You can think of these protons as a form of potential energy, like stuffing a closet full of balls.  When you open up the closet door, all the balls come tumbling out, releasing their potential energy in the process.  During cellular respiration, this potential energy is used by an enzyme to make ATP.  During non-shivering thermogenesis, though, the potential energy stored in all those protons stuffed into a small space is released by the cell as heat.  Thus, the energy from food is used to heat the body rather than being stored in ATP.

The main type of cell that does non-shivering thermogenesis is brown adipocytes, or fat cells.  Brown fat is very common in infants, but is also found in adult humans in the upper chest and neck.  The purpose of brown fat is to provide heat for the body.  Thus, non-shivering thermogenesis is activated by a drop in body temperature.  The cold temperature is sensed by the brain, which activates the sympathetic nervous system (the “fight or flight” response), which signals to the brown fat cells to express the genes necessary to bypass ATP production and release heat instead.  In a recent paper published in PNAS, Ye et al. describe how a different type of fat cell is able to skip all the nervous system steps and sense the cold directly (red arrow in diagram).  It is pretty cool that the fat cells are able to sense temperature, as if they were neurons, and can act autonomously to remedy the situation.  No need for a brain here!

Independent thermogenesis

Through a series of experiments, the authors demonstrate that a particular type of fat cell will express genes necessary for non-shivering thermogenesis when exposed to cold, independent of sympathetic nervous system activation.

In one experiment, they grew fat cells at different temperatures and measured gene expression using a technique called quantitative PCR (qPCR).  The idea behind this technique is that if a gene is highly expressed, there will be a lot of mRNA in the cell (remember the “central dogma” of molecular biology) and qPCR is a method for measuring the concentration of mRNA for a particular gene.  They focused their measurements on thermogenic genes that are known to be part of the non-shivering thermogenesis mechanism, such as Ucp1, which is the enzyme that actually allows the protons to fall back across the membrane, thereby releasing their energy as heat.  They found that these fat cells that were exposed to the cold expressed more Ucp1 mRNA, even in the absence of any nervous system.  These are just cells in a dish, so this must be an intrinsic property of fat cells.

It wasn’t just any fat cell that had this response.  In fact, brown adipocytes did not express more Ucp1 in the cold.  It was a different type of fat cell called a white adipocyte.  What is white fat?  The majority of fat in our body is white fat and its purpose is to store fat for energy (for cellular respiration) and to act as a thermal insulator, so we don’t lose as much heat through our skin.  There is one subtype of white fat that has been shown to do non-shivering thermogenesis and it was this type that could express thermogenic genes, like Ucp1, in the cold, independent of the nervous system.

Okay, so these white fat cells don’t need input from the nervous system, but do they still use the same intracellular pathway to turn on expression of these genes?  Normally, when a fat cell is activated by the sympathetic nervous system, it sets off a molecular cascade of events inside the cell, which involves activation of molecules in a pathway called the cAMP pathway (as shown in the diagram).  The authors inhibited this pathway in various ways and found that the cells could still respond to the cold as before, so this effect must use a different pathway.

There are still a number of open questions, such as: how do fat cells sense temperature?  Do they use the same types of receptors as temperature-sensitive neurons?  Why are some white fat cells independent, but brown fat cells need the nervous system to activate thermogenesis?  One thing that is clear, however, is that white fat cells are clearly important for temperature regulation as well as fat storage.  The authors suggest that tapping into thermogenesis might be a good way to help obese patients get rid of excess energy storage by releasing it as heat.  This pathway that is independent of the sympathetic nervous system could allow medications to target only the fat cells without involving the sympathetic nervous system which controls so many other functions in the body.

Something to think about as the cold Bay Area summer sets in.

(Insert mildly provocative title here)

Ever seen a pair of pigeons going at it?  And did you notice a penis on the male pigeon?  The answer is no, because most birds do not have external genitalia large enough for penetration.  And yet birds reproduce via internal fertilization.  Why would evolution favor male genitalia too small to actually enter into the female?  This just seems so inefficient. 

There are a few birds that do have well developed phalluses, such as the duck and goose.  What happened during evolution that caused some birds to retain a phallus, whereas most other birds lost it?  A paper appeared this week in Current Biology by Herrera et al., which addresses these questions from a developmental point of view.

Developmental arrest

The authors started this study by comparing the development of the phallus in embryos of two different birds.  They chose to look at (1) chick embryos, which are part of the galliformes group of birds and have reduced phalluses and (2) duck embryos, which are part of the anseriforms group, which have well developed, penetrating penises.  They followed the growth of the genital tubercle, the tissue that will form the penis.  As the duck and chick embryos grow, so do their genital tubercles, with no noticeable difference between the two species during the early stages of development.  At a later time period, though, the tubercle stops growing and regresses in the chicks, while the duck keeps on growing.  This shows that the tissue that makes the two different types of phalluses has the same developmental origin.

Why does the genital tubercle stop growing in the chick?

From a molecular stand point, the chick embryos could either lose the “growth” signal or they could gain expression of a “stop” signal not present in ducks.  From work in other animals, the authors knew that there are two major growth signals responsible for guiding the development of the external genitalia – Sonic Hedgehog (Shh) [see my other post about this protein] and Hox13.  These two genes are strongly expressed in the duck genital tubercle throughout embryonic development, as expected.  Surprisingly, though, they are also strongly expressed in the chick embryos.  This means that the chickens haven’t lost the growth signal.

The authors then investigated if there is some sort of a “stop” signal in the chicks.  They found that in chicks and quails, with reduced phalluses, there is a lot of cell death in the genital tubercle in the later stages of development.  This could account for the regression of the genital tubercle.  They then found that the chicks highly express a protein called BMP4 at the tip of the tubercle, which induces cell death, whereas ducks do not. 

In fact, by overexpressing BMPs in the duck, they induced cell death in the genital tubercle.  In the opposite experiment, they inhibited BMPs in the chick and their genital tubercles increased growth, as if they were ducks.

In summary:

            Chicken: + BMP --> increased cell death --> reduced phallus

            Duck:       - BMP --> no cell death, so continued tissue development --> large phallus

Evolution of reduced phallus

So what does this mean?  Chicks and quails have reduced phalluses, because during development, they express BMP4, which tells the developing cells of the penis to die off.  One really cool thing that the authors did next was to look at cell death in the closest relative to birds-- the alligator.  Yah, they got alligator embryos for this research!  Alligators have developed phalluses and they show hardly any cell death in the genital tubercle.  From this work, they could create an evolutionary tree, which shows that chicks and quails most likely evolved the BMP4 signal after their group separated from ducks.  Although the authors didn’t test any members (ha ha) from the neoaves group, which includes most other birds, we can presume that they also have a similar cell death mechanism to reduce the development of their phalluses.

Phylogenetic tree of birds, showing when the BMP signal evolved. (Adapted from Herrera et al., 2013)

This still begs the question of why would natural selection favor a reduced phallus so much so that it evolved independently in different lineages?  The authors propose two different theories, both of which may have occurred:

1) Sexual selection – sure, it may not be favorable for the males to have reduced phalluses, but it might be advantageous for the females.  In order for insemination to occur in these species, the female has to be a willing participant to allow the male to shimmy up next to her and release the sperm in very close proximity.  This gives the females the power to select their mates.  As opposed to species with large penises, where the male could basically rape the female and still successfully pass on his genes to the next generation.

2) Pleiotropy – this term refers to when a single gene mutation can lead to multiple noticeable changes in the body.  BMPs are a major signal during development of animals.  BMPs are involved in a number of bird-only innovations such as feathers and beaks.  Maybe increased BMP expression gave an advantage to these birds, but also lead to reduced phalluses, as a secondary effect.  This may have occurred first in evolution, but sexual selection may have stabilized this characteristic in the population.

This article was so clear and interesting.  I’m sure it will catch people’s attention because of the subject matter, but it’s a great example of using development to solve an evolutionary question.  Plus it gives reviewers and bloggers a great opportunity to think up clever titles and puns for their articles.  The review that was published alongside this article was titled “Cock-a-doodle-don’t”. How can I compete with that?

Stop seizures with a brain graft

There are two types of neurons in the brain: excitatory and inhibitory neurons.  They do exactly what you think they would.  Excitatory neurons release chemical messengers, which activate other neurons, which may eventually lead to some sort of perception or action.  Inhibitory neurons release chemicals that silence other neurons.  Why would you want inhibitory neurons in your brain?  Well, if all your neurons were excitatory and interconnected, all your neurons would be active all the time and the signals would be meaningless.  In fact, this sort of overactivation in the brain can lead to seizures.  It’s been shown in numerous cases of epilepsy that there is some sort of dysfunction of the inhibitory neurons.  The excitatory neurons have free reign and go crazy, leading to a seizure.

How is epilepsy treated?  Medications that potentiate the inhibitory neurons can help, but they activate all inhibitory neurons throughout the brain, when maybe the problem is more localized to one spot.  Just as all excitatory neurons is a bad thing, too much inhibition is also bad and can lead to cognitive side effects.  Another treatment is to open up the patient’s head, try to find the overactive area and cut it out or zap those neurons with a laser.  Destroying brain cells is always a last resort, though.

In a recent paper published in Nature Neuroscience by Hunt et al., the authors propose another potential treatment: adding new inhibitory neurons into the epileptic brain.  Like all new medical ideas, the story starts with mice.  They can create a model of human epilepsy in these mice by treating them with a potent drug.  These epileptic mice have seizures just like humans do.

Where do you get new inhibitory neurons?

The researchers obtained progenitor cells from mice embryos.  In other words, these weren’t inhibitory neurons yet, but they were destined to turn into them as the mice developed.  They grafted these progenitors into adult epileptic mice in the hippocampal region of the brain (a common area for seizures).   Amazingly, these pre-neurons migrated throughout the brain region, as far as 1.5 mm (that’s a lot… think about how small a mouse brain is).  Then the progenitors differentiated into inhibitory neurons, as if they were in a normal developing brain.  One week later, the epileptic mice with extra inhibitory neurons had hardly any seizures, whereas the untreated mice were having about 2 a day.  Not only that, but the treated mice showed cognitive improvements compared to the untreated epileptic mice. 

So they seemed to “cure” the epileptic mice by giving them some new inhibitory neurons that were able to make functional connections with the existing neurons.  This isn’t as invasive as brain surgery and it’s much more localized than medication.  If the epilepsy were focused in a different part of the brain, then they could transplant the cells there instead.

Is this possible to try in humans?  Maybe so, but the first problem is that we can’t take inhibitory progenitor cells from human embryos.  There are some ethical issues with growing clones to harvest parts from them.  However, you could use embryonic stem cells, or induced pluripotent stem cells.  Pluri-what?  Recent technology allows researchers to take a skin biopsy, do some genetic engineering to these cells and push them back in developmental time to a stem cell.  Pluripotent means that these stem cells have the potential to become any type of cell, like an inhibitory neuron.  All it takes is turning on the right genes in these cells to push them to a particular fate, and if that isn’t already known for inhibitory neurons, I bet it’s not too far off.  Plus there’s the benefit that the transplanted cells will have the same genome as all the patient’s other cells, because they originated from their skin cells.  Just wait, regenerative medicine is moving ahead at lightning speed.

Go go gadget extendo filopodia

I’m back from an intense semester of learning and teaching Developmental Biology.  One theme that emerged from my studies was that the development of organisms is centered around gene expression and cell to cell signaling.  Often times, one cell will differentiate into its mature form, and then release a signaling protein that tells neighboring cells what to develop into.  For instance, the nervous system is induced by signals released from the embryonic backbone.  There are a number of common signals that are used over and over throughout development, like BMP, Wnt and Shh.

A recent paper by Sanders et al., published in Nature, looked at how distant cells can signal to each other via the Shh pathway.  Unfortunately for Developmental Biology teachers everywhere, Shh stands for Sonic Hedgehog.  Oftentimes, strange or humorous gene names like this can be blamed on the fruit fly researchers who first discovered the gene, but in this case everyone is to blame.  This gene was originally discovered by researchers studying fruit fly embryonic development; they named the gene hedgehog because the mutant embryos had lots of tiny bristles all over, kind of like a hedgehog.  The mammalian researchers took it to the next ridiculous level, by naming the mammalian version of this gene Sonic Hedgehog.  The Shh protein is a secreted signal that binds receptors on other cells, which activate gene expression in the receiving cell.  Shh signaling is important for specifying many different cell fates, such as the different neurons in the spinal cord, the cells that become the vertebrae, as well as the formation of the digits of the hand.

Although Shh is secreted from the cell, it has chemical modifications that make it stick to the plasma membrane that surrounds the cell that released Shh.  How then can Shh induce the development of cells that are located at a distance?  Well, the answer is by stretching out long cellular extensions with Shh localized at the tip.

Shh Filopodia

Sanders et al. did live imaging of cells in the developing limb of the chicken using fluorescent proteins.  They did some genetic trickery so only a few cells were labeled in red and others in green.  This way they could detect individual cells in a sea of unlabeled cells and examine their structure in real time.  They observed individual cells extending long protrusions, called filopodia, from the cell bodies.  These filopodia could stretch long distances (150 micrometers, like 3-5 cell widths) and were dynamic-- retracting and growing over time. 

How to think about filopodia?  Imagine a stretchy balloon with a stick inside of it.  If you could push that stick into the wall of the balloon, the balloon would protrude from that one spot as the stick pushes it out.  That is like a filopodia, where the balloon wall is the plasma membrane and the stick is a protein called Actin.  Actin forms long chains that can grow, pushing out the membrane in front of it. 

The thin, string-like extensions from this cell are filopodia and are filled with Actin.  Image from proteopedia.org

The authors then labeled the Shh protein with another fluorescent marker and saw that it localized to the tips of filopodia.  Not only that, but the filopodia expressing Shh were more stable and did not retract as often.  In order for Shh to act as a signaling molecule, it has to bind a receptor on another cell.  Using a different color, the authors observed two co-receptors for Shh localized to filopodia from other cells.  They even saw filopodia from two different cells make contact with each other, where one cell expressed Shh and the other expressed the receptors.

This is amazing!  Instead of releasing a signal out of the cell with the hope that it goes to the right place and isn’t degraded, the cells literally grow to the right place with the signal on their membranes.  This is like hand delivering a note to your coworker, rather than making the note into a paper airplane and throwing it in the direction of their desk.

This is how I imagine this working.  Two cells that are located at a distance, reach out extensions and meet somewhere in the middle. The Shh signal would bind the receptor, causing changes to the pink cell.

A study like this could not have been done before recent innovations in live imaging and molecular biology to introduce the fluorescent proteins into the cells.  The filopodia are not preserved during the more traditional, static method of fixing cells with formaldehyde and then staining them.  Who knows what other tricks live cells use during embryonic development.  I suspect this is only the beginning.