L7 - Development of the Hypothalamus

how are innate behaviours controlled?

• primarily controlled by neurons in the basal forebrain particularly the hypothalamus

• genetic and environmental factors influence these behaviours

how do genes affect behaviour?

• by regulating hypothalamic development

• specifically by controlling how hypothalamic progenitor cells differentiate into specific neuronal subtypes

• this in turn shapes the neural circuits that drive innate behaviours

what does characterising and categorising neurons help us understand?

• helps to understand how genes influence innate behaviours by identifying which cell types are affected by mutations and the functional consequences of those changes

what is the first stage of hypothalamic development that occurs as the embryo matures?

Induction of hypothalamic floor plate like (Hyp-FP) cells

• at the neural tube stage, the earliest hypothalamic progenitors are specified

• the first hypothalamic cells arise at the ventral midline of the anterior neural tube

• these cells are called the hypothalamic floor plate like (Hyp-FP) cells, and they represent the earliest definitive hypothalamic progenitors

what is the second stage of hypothalamic development?

Regional specification of hypothalamic progenitors

• distinct progenitor domains are established

• this allows identification of mammillary, paraventricular and tuberal progenitors

• each progenitor type occupies a specific spatial region of the developing hypothalamus

what is the third stage of hypothalamic development?

Neurogenesis and gliogenesis

• the regional progenitors generate distinct classes of neurons and glial cells

• these neurons and glial cells populate defined territories that prefigure future hypothalamic nuclei

what is the fourth stage of hypothalamic development?

Axon guidance and circuit formation

• once neurons are specified they extend axons and form synaptic connections

• this establishes functional hypothalamic circuitry required for behaviour and homeostasis

what is the key goal of studies into hypothalamic development?

• to characterise stem, progenitor and differentiated cell types in the hypothalamus and the molecular mechanisms that direct their formation and function

what did studies (before 2020) focus on?

• focus on earliest stages when first hypothalamic cells are induced/identified

• OR

• late progenitors that had gone through almost their final divisions and were about to differentiate

—> for example, cells begin to express Islet1 just before they turn into Pomc neurons or cells begin to express Lhx9 before they turn into hypocretin neurons

what are the firs identified cells?

• the earliest hypothalamic progenitors are hypothalamic floor plate like (Hyp-FP) cells

where do the hypothalamic floor plate (Hyp-FP) like cells arise?

• the Hyp-FP like cells arise at the ventral midline of the anterior forebrain during early neural tube development

• the Hyp-FP like cells overlie the prechordal mesoderm and are induced by signals from this tissue (rather than the notochord)

why are Hyp-FP like cells termed floor plate like?

• because they express classical FP markers such as Shh

why are Hyp-FP like cells molecularly distinct from true FP cells?

• as they express additional TFs not found in the FP

• one key marker is Nkx2.1 - which is an early TF essential for hypothalamic development

• KO of TF Nkx2.1 results in complete absence of the hypothalamus

what does the prechordal mesoderm induce?

hypothalamic floor plate (Hyp-FP) like cells

how does the prechordal mesoderm induce hypothalamic floor plate (Hyp-FP) like cells?

• its inductive ability is partly due to its expression of Shh which is a key ventralising factor signal

• Shh from the prechordal mesoderm acts on anterior neural tube, which has a distinct competence leading to specification of hypothalamic FP progenitors

• in addition to Shh, the prechordal mesoderm secretes other signalling molecules that act together with Shh to induce specialised molecular and transcriptional characteristics to hypothalamic floor plate like cells

—> prechordal mesoderm induces the overlying anterior neural tube cells to become Hyp-FP like cells by sending signals like Shh and others

what induces and maintains hypothalamic floor plate (Hyp-FP) like cells?

• a Gene Regulatory Network (GRN) of signals and transcription factors

how was the hypothalamic gene regulatory network (GRN) worked out through experimental work?

• loss of function studies

what did Shh KO studies show?

• Shh was identified as a key ventralising signal in vertebrates

• KO of Shh in mice - resulted in loss of ventral forebrain identity

• causing defects such as cyclopia/holoprosencephaly - where a single eye forms and the forebrain fails to seperate into two hemispheres

• interestingly, the posterior spinal cord development is less affected highlighting the anterior neural tube’s unique sensitivity to Shh in establishing hypothalamic and forebrain structures

what is observed in human Shh KO/loss?

• cyclopia and holoprosencephaly is also observed when Shh signalling is disrupted

• producing a phenotype of a single cyclopic eye, midline defects and underdeveloped forebrain structures (same as in mice)

• it is also linked to spontaneous miscarriages

• highlighting the importance of Shh in early forebrain and hypothalamic development

what were most studies on early induction of Hyp-FP like cells focused on?

• performed in chick and mouse

• focusing on how these earliest progenitors are specified

how were late hypothalamic progenitors studied?

• studied in mouse and zebrafish

• by examining TFs known for spinal cord progenitors or by using targeted screens

• in situ hybridisation often used to determine whether a selected gene is expressed in hypothalamic progenitors

• in situ was used to see if TFs known to be expressed in developing spinal cord were also expressed in developing hypothalamus

what did the study by Blackshaw lab (2010) identify?

• nine distinct hypothalamic progenitor types

• progenitors were classified according to the regions they would populate, tuberal, mammillary, or PVN nuclei

• for example, ARCN progenitors give rise to neurons in the arcuate nucleus, while VMH progenitors produce neurons in the ventromedial nucleus (VMN)

what were most studies on the birth of the first hypothalamic progenitor (Hyp-FP) cells performed in?

chick embryos

what were investigations into the stages by which the 9 progenitors types give rise to specific hypothalamic neurons mainly done in?

mouse embryos

what is the scRNA-seq approach (single cell RNA sequencing)?

• begins by isolating individual cells and fusing each to a barcoded bead allowing the transcriptional profile of every single cell to be tracked

• this generates thousands of DNA barcoded single cell transcriptomes showing which mRNAs are expressed in each cell

• after bioinformatics and quality control, cells are clustered based on their transcriptional signatures

• each cell’s unique gene expression allows to categorise cells into distinct types based on specific transcriptional features

how was scRNA-seq used in studying hypothalamic development?

• chick embryos collected at the stage when Hyp-FP like cells are first induced

• neural tube isolated to focus on neural tissue

• region containing Hyp-FP cells carefully dissected out for further analysis

what did the analysis of scRNA-seq identify?

• cell clusters based on similarity in mRNA expression

• at stage 8, two major clusters were observed,

• 1. diencephalic-like cells (prethalamus like expressing follistatin)

• 2. hypothalamic FP like cells (Hyp-FP)

→ the diencephalic like cells surround the Hyp-FP like cells, the closer the transcriptional profiles of cells, the closer they appear on the clustering plot